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COMPOSITIONS OF ANTIBODY CONSTRUCT-AGONIST CONJUGATES AND
METHODS OF USE THEREOF
CROSS REFERENCE
[0001] This application claims the benefit of U.S. Provisional Patent
Application No. 62/371,141
filed on August 4, 2016, U.S. Patent Application No. 15/173,075, filed on June
3, 2016, and U.S.
Provisional Patent Application No. 62/264,260, filed on December 7, 2015, each
of which is
incorporated herein by reference in its entirety.
BACKGROUND
[0002] One of the leading causes of death in the United States is cancer. The
conventional
methods of cancer treatment, like chemotherapy, surgery, or radiation therapy,
tend to be either
highly toxic or nonspecific to a cancer, or both, resulting in limited
efficacy and harmful side
effects. However, the immune system has the potential to be a powerful,
specific tool in fighting
cancers. In many cases tumors can specifically express genes whose products
are required for
inducing or maintaining the malignant state. These proteins may serve as
antigen markers for the
development and establishment of more specific anti-cancer immune response.
The boosting of
this specific immune response has the potential to be a powerful anti-cancer
treatment that can be
more effective than conventional methods of cancer treatment and can have
fewer side effects.
INCORPORATION BY REFERENCE
[0003] All publications, patents, and patent applications mentioned in this
specification are
herein incorporated by reference to the same extent as if each individual
publication, patent, or
patent application was specifically and individually indicated to be
incorporated by reference.
SUMMARY
[0004] The composition described herein relates to different embodiments of a
conjugate. In
various embodiments, a conjugate comprises a) an immune-stimulatory compound;
b) an
antibody construct comprising an antigen binding domain and an Fc domain,
wherein said
antigen binding domain binds to a first antigen and wherein a Kd for binding
of said Fc domain to
an Fc receptor in the presence of said immune-stimulatory compound is no
greater than about
100 times a Kd for binding of said Fc domain to said Fc receptor in the
absence of the immune
stimulatory compound; and c) a linker, wherein said linker attaches said
antibody construct to
said immune-stimulatory compound. In some aspects, said antigen binding domain
binds said
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first antigen in a presence of said immune-stimulatory compound. In some
aspects, a Kd for
binding of said antigen binding domain to said first antigen in a presence of
said immune-
stimulatory compound is less than about 100 nM and no greater than about 100
times a Kd for
binding of said antigen binding domain to said first antigen in the absence of
said immune-
stimulatory compound. In some aspects, said Kd for binding of said antigen
binding domain to
said first antigen in the presence of said immune-stimulatory compound is less
than about 100nM
and is no greater than about 10 times the Kd of the binding of the antigen
binding domain to said
first antigen in the absence of the immune-stimulatory compound; and said Kd
for binding of said
Fc domain to said Fc receptor in the presence of said immune-stimulatory
compound is no
greater than about 10 times said Kd for the binding of said Fc domain to said
Fc receptor in the
absence of said immune stimulatory compound. In some aspects, a molar ratio of
immune-
stimulatory compound to antibody construct is less than 9, less than 8, less
than 7, less than 6,
less than 5, less than 4, less than 3, or less than 2.
[0005] In some aspects, said conjugate further comprises a targeting binding
domain, wherein
said targeting domain is attached to said antibody construct. In some aspects,
said targeting
binding domain binds a second antigen. In some aspects, said targeting binding
domain is
attached to said antibody construct at a C-terminal end of said Fc domain.
[0006] In some aspects, said antigen binding domain is from an antibody or non-
antibody
scaffold.
[0007] The conjugate of any of claims 1-9, wherein said antigen binding domain
is at least 80%
homologous to an antigen binding domain from an antibody or non-antibody
scaffold. In some
aspects, said non-antibody scaffold is a DARPin, affimer, avimer, knottin,
monobody, or affinity
clamp. In some aspects, said antigen binding domain is at least 80% homologous
to an antigen
binding domain from a DARPin, affimer, avimer, knottin, monobody, or affinity
clamp.
[0008] In some aspects, said antigen binding domain recognizes a single
antigen. In some aspects,
said antigen binding domain recognizes two or more antigens. In some aspects,
said first antigen
is a tumor antigen. In some aspects, said first antigen that is at least 80%
homologous to CD5,
CD19, CD20, CD25, CD37, CD30, CD33, CD45, CAMPATH-1, BCMA, CS-1, PD-L1, B7-H3,
B7-DC, HLD-DR, carcinoembryonic antigen, TAG-72, EpCAM, MUC1, folate-binding
protein,
A33, G250, prostate-specific membrane antigen, ferritin, GD2, GD3, GM2, Leg,
CA-125, CA19-
9, epidermal growth factor, p185HER2, IL-2 receptor, de2-7 EGFR, fibroblast
activation protein,
tenascin, metalloproteinases, endosialin, vascular endothelial growth factor,
avB3, WT1, LMP2,
HPV E6 E7, EGFRvIII, Her-2/neu, idiotype, MAGE A3, p53 nonmutant, NY-ESO-1,
PMSA,
GD2, CEA, MelanA/MART1, Ras mutant, gp100, p53 mutant, PR1, bcr-abl,
tyronsinase,
survivin, PSA, hTERT, Sarcoma translocation breakpoints, EphA2, PAP, ML-IAP,
AFP, ERG,
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NA17, PAX3, ALK, androgen receptor, cyclin Bl, polysialic acid, MYCN, RhoC,
TRP-2,
fucosyl GM1, mesothelin, PSCA, MAGE Al, sLe(animal), CYP1B1, PLAV1, GM3,
BORIS, Tn,
GloboH, ETV6-AML, NY-BR-1, RGS5, SART3, STn, Carbonic anhydrase IX, PAX5, OY-
TESL Sperm protein 17, LCK, HMWMAA, AKAP-4, 55X2, XAGE 1, B7H3, Legumain, Tie
3,
Page4, VEGFR2, MAD-CT-1, PDGFR-B, MAD-CT-2, ROR2, TRAILl, MUC16, MAGE A4,
MAGE C2, GAGE, EGFR, CMET, HER3, MUC1, MUC15, MSLN, CA6, NAPI2B, TROP2,
CLDN18.2, RON, LY6E, FRA, DLL3, PTK7, LIV1, ROR1, MAGE-A3, or Fos-related
antigen 1.
In some aspects, wherein said first antigen is expressed on an immune cell. In
some aspects, said
first antigen is expressed on an antigen-presenting cell. In some aspects,
said first antigen is
expressed on a dendritic cell, a macrophage, or a B-cell. In some aspects,
wherein said first
antigen is CD40. In some aspects, said antigen binding domain is a CD40
agonist.
[0009] In some aspects, said antibody construct is an antibody. In some
aspects, said antibody
construct is a human antibody or a humanized antibody. In some aspects, said
antibody construct
comprises a light chain sequence that is at least 80%, 90%, or 100% homologous
to SEQ ID NO:
4, at least 80%, 90%, or 100% homologous to SEQ ID NO: 26, or at least 80%,
90%, or 100%
homologous to SEQ ID NO: 34. In some aspects, said antibody construct
comprises a light chain
variable domain sequence that is at least 80%, 90%, or 100% homologous to SEQ
ID NO: 6. In
some aspects, said antibody construct comprises: a) a heavy chain sequence
that is at least 80%,
90%, or 100% homologous to SEQ ID NO: 15; b) a heavy chain sequence that is at
least 80%,
90%, or 100% homologous to SEQ ID NO: 16; c) a heavy chain sequence that is at
least 80%,
90%, or 100% homologous to SEQ ID NO: 17; d) a heavy chain sequence that is at
least 80%,
90%, or 100% homologous to SEQ ID NO: 18; e) a heavy chain sequence that is at
least 80%,
90%, or 100% homologous to SEQ ID NO: 22; or f) heavy chain sequence that is
at least 80%,
90%, or 100% homologous to SEQ ID NO: 30. In some aspects, said antibody
construct
comprises a heavy chain variable domain that is at least 80%, 90%, or 100%
homologous to SEQ
ID NO: 20. In some aspects, said antibody binding domain comprises at least
80%, 90%, or
100% homology to: a) HC CDR1 comprising an amino acid sequence of SEQ ID NO:
23, HC
CDR2 comprising an amino acid sequence of SEQ ID NO: 24, a HC CDR3 comprising
an amino
acid sequence of SEQ ID NO: 25, LC CDR1 comprising an amino acid sequence of
SEQ ID NO:
27, LC CDR1 comprising an amino acid sequence of SEQ ID NO: 28, and LC CDR3
comprising
an amino acid sequence of SEQ ID NO: 29; or b) HC CDR1 comprising an amino
acid sequence
of SEQ ID NO: 31, HC CDR2 comprising an amino acid sequence of SEQ ID NO: 32,
a HC
CDR3 comprising an amino acid sequence of SEQ ID NO: 33, LC CDR1 comprising an
amino
acid sequence of SEQ ID NO: 35, LC CDR1 comprising an amino acid sequence of
SEQ ID NO:
36, and LC CDR3 comprising an amino acid sequence of SEQ ID NO: 37.
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[0010] In some aspects, said Fc domain is from an antibody. In some aspects,
said Fc domain is
at least 80% homologous to an Fc domain from an antibody. In some aspects,
said Fc domain
binding to said Fc receptor in the presence of said immune-stimulatory
compound results in Fc-
receptor-mediated signaling. In some aspects, said Fc domain binding to said
Fc receptor in the
presence of said immune-stimulatory compound results increased antigen
presentation on an
immune cell. In some aspects, said Fc domain is a human Fc domain. In some
aspects, said Fc
domain is selected from a group consisting of a human IgG1 Fc domain, a human
IgG2 Fc
domain, a human IgG3 Fc domain, and a human IgG4 Fc domain. In some aspects,
wherein said
Fc domain is an Fc domain variant comprising at least one amino acid residue
change as
compared to a wild type sequence of said Fc domain. In some aspects, said Fc
domain binds said
Fc receptor with altered affinity as compared to a wild type Fc domain. In
some aspects, wherein
said Fc receptor is selected from a group consisting of CD16a, CD16b, CD32a,
CD32b, and
CD64. In some aspects, said Fc receptor is a CD16a F158 variant or a CD16a
V158 variant. In
some aspects, said Fc domain binds said Fc receptor with higher affinity than
a wild type Fc
domain. In some aspects, said Fc receptor is selected from a group consisting
of: CD16a, CD16b,
CD32a, CD32b, or CD64. In some aspects, said Fc receptor is a CD16a F158
variant or a CD16a
V158 variant. In some aspects, said Fc domain has at least one amino acid
residue change as
compared to wildtype, wherein said at least one amino acid residue change is:
a) F243L, R292P,
Y300L, L235V, and P396L, wherein numbering of amino acid residues in said Fc
domain is
according to the EU index as in Kabat; b) S239D and 1332E, wherein numbering
of amino acid
residues in said Fc domain is according to the EU index as in Kabat; or c)
S298A, E333A, and
K334A, wherein numbering of amino acid residues in said Fc domain is according
to the EU
index as in Kabat.
[0011] In some aspects, said immune-stimulatory compound is a damage-
associated molecular
pattern molecule or a pathogen associated molecular pattern molecule In some
aspects, said
immune-stimulatory compound is a toll-like receptor agonist, STING agonist, or
RIG-I agonist.
In some aspects, said immune-stimulatory compound is a TLR1 agonist, a TLR2
agonist, a TLR3
agonist, a TLR4 agonist, a TLR5 agonist, a TLR6 agonist, a TLR7 agonist, a
TLR8 agonist, a
TLR9 agonist, or a TLR10 agonist. In some aspects, said immune-stimulatory
compound is
selected from a group consisting of: S-27609, CL307, UC-IV150, imiquimod,
gardiquimod,
resiquimod, motolimod, VTS-1463G5-9620, G5K2245035, TMX-101, TMX-201, TMX-202,
isatoribine, AZD8848, MEDI9197, 3M-051, 3M-852, 3M-052, 3M-854A, S-34240,
KU34B, and
CL663.
[0012] In some aspects, said immune-stimulatory compound comprises one or more
rings
selected from carbocyclic and heterocyclic rings. In some aspects, said linker
is covalently
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attached to said antibody construct. In some aspects, said linker is
covalently attached to said
immune-stimulatory compound. In some aspects, said linker is not attached to
an amino acid
residue of said Fc domain selected from a group consisting of: 221, 222, 224,
227, 228, 230, 231,
223, 233, 234, 235, 236, 237, 238, 239, 240, 241, 243, 244, 245, 246, 247,
249, 250, 258, 262,
263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274, 275, 276, 278,
280, 281, 283, 285,
286, 288, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 302, 305,
313, 317, 318, 320,
322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335 336, 396,
or 428, wherein
numbering of amino acid residues in said Fc domain is according to the EU
index as in Kabat. In
some aspects, said linker is attached to an amino acid residue of said
antibody construct by a
THIOMAB linker, or a Sortase A-catalyzed linker. In some aspects, said linker
is attached to said
antibody construct via a sulfhydryl group, a primary amine, a hinge cysteine,
a CL lysine, an
engineered cysteine in a light chain, an engineered light chain glutamine, or
an unnatural amino
acid engineered into a light chain or heavy chain. In some aspects, said
linker does not interfere
with said Fc domain binding to said Fc receptor when said linker is attached
to said antibody
construct at an amino acid residue. In some aspects, said linker does not
interfere with Fc-
receptor-mediated signaling resulting from said Fc domain binding to said Fc
receptor when said
linker is attached to said Fc domain at an amino acid residue. In some
aspects, said linker is
attached to said immune-stimulatory compound via an exocyclic nitrogen or
carbon atom of said
immune-stimulatory compound. In some aspects, said immune-stimulatory compound
is
covalently attached to said linker by a bond to an exocyclic carbon or
nitrogen atom on said
immune-stimulatory compound.
[0013] In some aspects, wherein said linker is a peptide. In some aspects,
said linker is a
cleavable linker. In some aspects, said linker selected from a group
consisting of: a) a valine-
citrulline linker; b) a valine-citrulline linker containing a
pentafluorophenyl group; c) a valine-
citrulline linker containing a succinimide group; d) a valine-citrulline
linker containing a para
aminobenzoic acid group; e) a valine-citrulline linker containing a para
aminobenzoic acid group
and a pentafluorophenyl group; and f) a valine-citrulline linker containing a
para aminobenzoic
acid group and a succinimide group.
[0014] In some aspects, said linker is a non-cleavable linker. In some
aspects, said linker selected
from a group consisting of: a) a maleimidocaproyl linker; b) a combination of
a
maleimidocaproyl group and one or more polyethylene glycol molecules; c) a
maleimide-PEG4
linker; d) a maleimidocaproyl linker containing a succinimide group; e) a
maleimidocaproyl
linker containing a pentafluorophenyl group; f) a combination of a
maleimidocaproyl linker
containing a succinimide group and one or more polyethylene glycol molecules;
and g) a
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combination of a maleimidocaproyl linker containing a pentafluorophenyl group
and one or more
polyethylene glycol molecules.
[0015] In some aspects, said conjugate induces the secretion of cytokine by an
antigen presenting
cell. In some aspects, said cytokine is IFN-y, IL-8, IL-12, IL-2, or a
combination thereof. In some
aspects, said conjugate induces antigen presentation on an antigen presenting
cell.
[0016] In some aspects, said conjugate is formulated to treat tumors.
[0017] In some aspects, wherein said conjugate is in a pharmaceutical
formulation.
[0018] In some embodiments, a pharmaceutical composition comprises said
conjugate of any of
the proceeding embodiments and a pharmaceutically acceptable carrier.
[0019] In some embodiments, a method of producing the conjugate of any of the
preceding
embodiments, comprises: a) selecting an antibody construct; b) selecting an
immune-stimulatory
compound; and c) attaching said antibody construct to said immune-stimulatory
compound,
wherein said immune-stimulatory compound is attached to said antibody
construct via a linker
and said antibody construct comprises an antigen binding domain and an Fc
domain, wherein
said antigen binding domain specifically binds an antigen in the presence of
said immune-
stimulatory compound and said Fc domain specifically binds an Fc receptor in
the presence of
said immune-stimulatory compound.
[0020] In some embodiments, a method of producing the conjugate of any of the
preceding
embodiments, comprises: a) selecting an antibody construct; b) selecting an
immune-stimulatory
compound; c) selecting a targeting binding domain; d) attaching said targeting
binding domain to
said antibody construct; and e) attaching said antibody construct to said
immune-stimulatory
compound, wherein said immune-stimulatory compound is attached to said
antibody construct
via a linker, wherein said antigen binding domain specifically binds a first
antigen in the presence
of said immune-stimulatory compound and said targeting binding specifically
binds a second
antigen in the presence of said immune-stimulatory compound.
[0021] In some embodiments, a method for treating a subject in need thereof,
comprises
administering a therapeutic dose of said conjugate of any one of the preceding
embodiments or
said pharmaceutical composition of any of the preceding embodiments. In some
aspects, said
subject has cancer. In some aspects, said composition is administered
intravenously, cutaneously,
subcutaneously, or injected at a site of affliction.
[0022] In some embodiments, a kit comprises said conjugate of any of the
preceding
embodiments.
[0023] A composition comprising a light chain sequence that is at least 80%,
90%, or 100%
homologous to SEQ ID NO: 4 and heavy chain sequence that is at least 80%, 90%,
or 100%
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[0024] A composition comprising:
a) a light chain sequence that is at least 80%, 90%, or 100% homologous to SEQ
ID NO: 4 or
at least 80%, 90%, or 100% homologous to SEQ ID NO: 26; and
b) a heavy chain sequence that is at least 80%, 90%, or 100% homologous to SEQ
ID NO:
16, at least 80%, 90%, or 100% homologous to SEQ ID NO: 17, or at least 80%,
90%, or
100% homologous to SEQ ID NO: 18.
[0025] The composition of claim 75, wherein said composition binds to an Fc
receptor with
greater affinity than an antibody comprising a heavy chain sequence of SEQ ID
NO: 15 or SEQ
ID NO: 22.
[0026] In some aspects, the present disclosure provides a compound represented
by the structure
of Formula (I):
0 X1
/ p ,
-LJ
/ B1
k
iy\
0
0
B2 , /0 N(R1)2
v-p
/ 0
X2
(I)
or a pharmaceutically acceptable salt thereof, wherein:
Xl is selected from -0R2 and -SW;
X2 is selected from -0R3 and -Re;
B1 and B2 are independently selected from optionally substituted nitrogenous
bases;
Y is selected from -0124, -NR4R4, and halogen;
121, R2, R3 and R4 are independently selected at each occurrence from
hydrogen, -
c(=o)Rioo, _
C(=0)0Rim and -C(=0)NRim; Ci_io alkyl, C2-10 alkenyl, C2-10 alkynyl, each of
which is independently optionally substituted at each occurrence with one or
more substituents
selected from halogen, -ORm , -swoo, _N(Rioo)2, _s(0)Rioo, -S(0)2R' ,
_c(0)Rioo, _
C(0)0Rim,
-0C(0)Rim, -NO2, =0, =s, =N(Rioo), _
P(0)(0R1m)2, -0P(0)(0R1 )2, -CN, C3-10 carbocycle and
3- to 10-membered heterocycle; and C3-10 carbocycle and 3- to 10-membered
heterocycle,
wherein each C3-10 carbocycle and 3- to 10-membered heterocycle in 121, R2, R3
and R4 is
independently optionally substituted with one or more substituents selected
from halogen, -
()R' , -SR100, _N(R100)2, _s(o)R100, _s(0)2R100 _c(o)R100, _
C(0)ORM, -0C(0)R' , -NO2, =0,
=s, =N(Rioo), _
P(0)(0R1m)2, -0P(0)(0Rim)2, -CN, C1_6 alkyl, C2_6 alkenyl, and C2_6 alkynyl;
and
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Rm at each occurrence is independently selected from hydrogen; and C1_10
alkyl, C2-10
alkenyl, C2-10 alkynyl, C3-10 carbocycle, and 3- to 10-membered heterocycle
each of which is
independently optionally substituted at each occurrence with one or more
substituents selected
from halogen, -CN, -NO2, =0, =S, and haloalkyl.
[0027] In some embodiments, the compound of Formula (I) is represented by
Formula (IA):
X1
0 1
¨0
VP
\
Y 0 B1
0
B2 0 N(R1)2
0¨p
/ 0
X2
(IA),
or pharmaceutically acceptable salts thereof.
[0028] In an alternative embodiment, the compound of Formula (I) is
represented by Formula
(TB):
X1
0 I
P-0
Z
B1
i-- 0
2
NH(R1)2c
0"----
/F)
x2 0 (IB) ,
or a pharmaceutically acceptable salt thereof.
[0029] In various embodiments, B1 and B2 are independently selected from
optionally substituted
purines, such as optionally substituted adenine, optionally substituted
guanine, optionally
substituted xanthine, optionally substituted hypoxanthine, optionally
substituted theobromine,
optionally substituted caffeine, optionally substituted uric acid, and
optionally substituted
isoguanine. In a preferred embodiment, B1 and B2 are independently selected
from optionally
substituted adenine and optionally substituted guanine.
[0030] In some embodiments, B1 and B2 are independently optionally substituted
with one or
more substituents, wherein the optional substituents on B1 and B2 are
independently selected at
each occurrence from halogen, =0, =S, -ORE) , _swoo, _N(Rio0)2, _s(0)Rioo, -
S(0)2R' , _
C(0)R' , _
C(0)0Rioo, -0C(0)R' , _
NO2,oo.2 _
-P(0)(0R1 ), OP(0)(0R1 )2 and -CN; C1_10 alkyl,
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C2_10 alkenyl, C2-10 alkynyl, each of which is independently optionally
substituted at each
occurrence with one or more substituents selected from halogen, -0Rioo,
_sizioo, _N(Rio)2, _
S(0)R' , -S(0)2R' , _c(0)Rioo, _
C(0)0Rioo, -0C(0)Rim, -NO2, =0, =S,
=N(Rioo),
-P(0)(0R1m2, -0P(0)(0R1
) )2, -CN, C3-10 carbocycle and 3- to 10-membered
heterocycle; and C3_10 carbocycle and 3- to 10-membered heterocycle, wherein
each C3_10
carbocycle and 3- to 10-membered heterocycle is independently optionally
substituted with one
or more substituents selected from halogen, -0Rioo, _sizioo, _N(Rio)2, ioo,
_s(0)2Rioo _
C(0)R' , _
C(0)0Rioo, -0C(0)Rim, -NO2, =0, =S,
=N(Rioo),
-P(0)(0R1m2, -0P(0)(0R1
) )2, -CN, C1_6 alkyl, C2_6 alkenyl, and C2_6
alkynyl. In a
preferred embodiment, B1 and B2 are independently optionally substituted with
one or more
substituents, wherein the optional substituents on B1 and B2 are independently
selected at each
occurrence from halogen, =0, =S, -ORM, -SR100, _N(R100)2, _s(o)R100,
_s(o)2R100, _
C(0)R' ,
_
C(0)0R100, -0C(0)R' , _
NO2,oos2 _
-P(0)(0R1 ), OP(0)(0Rin2, ¨CN and C1_10 alkyl.
In some embodiments, B1 is an optionally substituted guanine. In some
embodiments, B2 is an
optionally substituted guanine.
[0031] In various embodiments, Xl is selected from ¨OH and ¨SH. For example,
Xl may be ¨
OH. In various embodiments, X2 is selected from ¨OH and ¨SH. For example, X2
may be ¨OH.
[0032] In various embodiments, Y is selected from ¨OH, ¨0-Ci_10 alkyl,
¨NH(Ci_10 alkyl), and ¨
NH2. For example, Y may be ¨OH.
[0033] In various embodiments, Rm is independently selected at each
occurrence from hydrogen
and C1_10 alkyl optionally substituted at each occurrence with one or more
substituents selected
from halogen, -CN, -NO2, =0, and =S.
[0034] In various embodiments, the compound of Formula (I) is represented by
Formula (IC):
0
0 PH
N
p..... AH
Z 0µ
Ho!, k , N N NH2
0
H2N,I*NI.Ni 0 NH2
HN I
0-p
/ 0
N (IC)
HO
0 ,
or a pharmaceutically acceptable salt thereof. In some embodiments, the
compound of
Formula (IC) is represented by Formula (ID):
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0.9H 1\1AFi
PO
HO 0 N N N
01
H2NNN ,0 NH2
I yr
H N 0
/ 0
HO (ID)
0
or a pharmaceutically acceptable salt thereof.
[0035] In various embodiments, the compound is a pharmaceutically acceptable
salt. The
compound or salt may agonize a stimulator of interferon genes (STING).
[0036] In some aspects, the present disclosure provides an antibody drug
conjugate, comprising a
compound or salt previously described, an antibody, and a linker group,
wherein the compound
or salt is linked to the antibody through the linker group. The linker group
may be selected from
a cleavable or non-cleavable linker.
[0037] In some aspects, the present disclosure provides a compound represented
by the structure
of Formula (II):
0 X1
/
p-o
Bi
Y 0
0
0 Z
B2
0-p
/
x2
(II)
or a pharmaceutically acceptable salt thereof, wherein:
Xl is selected from ¨0R2 and ¨SW;
X2 is selected from ¨0R3 and ¨Re;
B1 and B2 are independently selected from optionally substituted nitrogenous
bases, wherein each optional substituent is independently selected from
halogen, -ORm , -
slew; _N(Rioo)2; _s(0)Rioo; _s(0)2Rioo; _c(0)Rioo; _
C(0)0Rim, -0C(0)R1 , -NO2, =0, =S,
=N(Rioo); _cN; R6; and _)(3;
Y is selected from ¨0124, ¨5124,¨NR4R4, and halogen;
Z is selected from ¨0R5' ¨Re, and ¨NR5R5;
121, R2, R3, R4, and R5 are independently selected from a -X3; hydrogen, -
c(=o)Rioo; _
C(=0)0Rim and -C(=0)NRim; Ci_io alkyl, C2-10 alkenyl, C2-10 alkynyl, each of
which is independently optionally substituted at each occurrence with one or
more
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substituents selected from halogen,-0Rioo, _sRioo, _N( )2, Rioos _
S(0)Rim, -
S(0)2R' , _c(0)Rioo, _
C(0)0Rioo, -0C(0)Rim, -NO2, =0, =S,
=N(Rioo),
-P(0)(0R1m2, -0P(0)(0R1
) )2, -C N, C3-10 carbocycle and 3- to 10-
membered
heterocycle; and C3-10 carbocycle and 3- to 10-membered heterocycle, wherein
each C3-10
carbocycle and 3- to 10-membered heterocycle in Ri, R2, R3, R4, and R5 is
optionally
substituted with one or more substituents selected from halogen, -0Rioo,
_sRioo, _N(Rioo)2, _
S(0)R' , _s(0)2Rioo _c(0)Rioo, _
C(0)0Rioo, -0C(0)Rim, -NO2, =0, =S,
=N(Rioo),
-P(0)(0R1m2, -0P(0)(ORM)2,
) -C N, Ci_6 alkyl, C2lk
-6 aenyl, C2_6 alkynyl;
R6 is independently selected from -C(=o)Rioo, _
C(=0)0Rim and -C(=0)NRin Ci_10 alkyl,
C2_10 alkenyl, C2-10 alkynyl, each of which is independently optionally
substituted at each
occurrence with one or more substituents selected from halogen, -0Rioo,
_sRioo, _N(Rio)2, _
S(0)R' , -S(0)2R' , _c(0)Rioo, _
C(0)0Rioo, -0C(0)Rim, -NO2, =0, =S,
=N(Rioo),
-P(0)(0R1m2, -0P(0)(0R1
) )2, -C N, C3-10 carbocycle and 3- to 10-
membered
heterocycle; and C3-10 carbocycle and 3- to 10-membered heterocycle, wherein
each C3-10
carbocycle and 3- to 10-membered heterocycle in R6 is optionally substituted
with one or
more substituents selected from halogen, -0Rioo, _sRioo, _N(Rioo)2, _s(0)Rioo,
_s(0)2Rioo _
C(0)R' , _
C(0)0Rioo, -0C(0)Rim, -NO2, =0, =S,
=N(Rioo),
-P(0)(0R1m2, -0P(0)(ORM)2,
) -C N, C1_6 alkyl, C2lk
-6 aenyl, C2_6 alkynyl;
Rm at each occurrence is independently selected from hydrogen; and C1_10
alkyl,
C2_10 alkenyl, C2-10 alkynyl, C3-10 carbocycle, and 3- to 10-membered
heterocycle each of
which is independently optionally substituted at each occurrence with one or
more
substituents selected from halogen, -CN, -NO2, =0, =S, and haloalkyl; and
X3 is a linker moiety, wherein at least one of Ri, R2, R3, R4, Rs, xl, )(2, a
b-1
substituent and a
B2 substituent is -X3.
[0038] In various embodiments, the compound of Formula (II) is represented by
a structure of
Formula (IA):
xi
0 1
B1
p-0
Z \
Y 0
0
B2 /0 N(R1)2
0-p
/ 0
X2
(IIA),
or pharmaceutically acceptable salts thereof.
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[0039] In various embodiments, the compound of Formula (II) is represented by
a structure of
Formula (JIB):
X1
0 I
P-0
/
ON
B- -2 N----131CH(R1)2 0
0-- -7-.
/P
X2 0 (IIB),
or a pharmaceutically acceptable salt thereof.
[0040] In various embodiments, B1 and B2 are independently selected from
optionally substituted
purines. B1 and B2 may be each, independently selected from one another,
adenine, guanine, and
derivatives thereof. B1 and B2 may be independently selected from optionally
substituted adenine,
optionally substituted guanine, optionally substituted xanthine, optionally
substituted
hypoxanthine, optionally substituted theobromine, optionally substituted
caffeine, optionally
substituted uric acid, and optionally substituted isoguanine. In a preferred
embodiment, B1 and B2
are independently selected from optionally substituted adenine and optionally
substituted guanine.
[0041] In various embodiments, B1 is substituted by X3 and optionally one or
more additional
substituents independently selected from halogen, -ORloo, _swoo, _N(Rioo)2, _
S(0)R1 , -
S(0)2R' , _c(0)Rioo, _
C(0)0Rioo, -0C(0)R' , -NO2, =0, =s, =N(Rioo), _
CN, and R6. For
HN,X3
1\1)\1
N N
example, B1 may be represented by: =,-1, , and wherein B1 is optionally
further substituted
by one or more substituents.
[0042] In various embodiments, B2 is substituted by X3 and optionally one or
more additional
substituents independently selected from halogen, -ORloo, _swoo, _N(Rioo)2, _
S(0)R1 , -
S(0)2R' , _c(0)Rioo, _
C(0)0Rioo, -0C(0)R' , -NO2, =0, =s, =N(Rioo), _
CN, and R6. For
HN, X3
1\136
N N
example, B2 may be represented by: ¨1-- , and wherein B2 is optionally
further substituted
by one or more substituents.
[0043] In various embodiments, Xl is selected from -0- X3 and-S-X3. In some
embodiments, Xl
is selected from ¨OH and ¨SH.
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[0044] In various embodiments, X2 is selected from -0- X3 and-S-X3. In some
embodiments, X2
is selected from ¨OH and ¨SH.
[0045] In various embodiments, Y is selected from -NR4X3, -S-X3, and -0- X3.
In some
embodiments, Y is selected from ¨OH, -SH, ¨0-Ci_io alkyl, ¨NH(Ci_io alkyl),
and ¨NH2.
[0046] In various embodiments, Z is selected from -NR4X3, -S-X3, and -0- X3.
In some
embodiments, Z is selected from ¨OH, -SH, ¨0-Ci_io alkyl, ¨NH(Ci_io alkyl),
and ¨NH2.
[0047] In various embodiments, ¨X3 is a represented by the formula:
0 j(
N peptide
[0048] In some embodiments, ¨X3 is represented by the formula:
oil
N peptide¨RX
, wherein RX comprises a reactive moiety, such a maleimide.
[0049] In some embodiments, ¨X3 is represented by the formula:
oil
N peptide¨RX¨Antibody
*
, wherein RX is a reactive moiety that has reacted
with a moiety on an antibody to form an antibody drug conjugate.
[0050] In some embodiments, ¨X3 is represented by the formula:
RX
1-4 H , wherein RX is
a reactive moiety, such as
a maleimide.
[0051] In some embodiments, ¨X3 is represented by the formula:
0 0
0 0
RX* Antibody
/1-4 H , wherein RX* is a reactive
moiety that has reacted with a moiety on an antibody to form an antibody drug
conjugate.
[0052] In some embodiments, the compound is represented by the formula:
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o
NINH
I *I,
)(P N N NH2
P-0
HO ,
0 H
0 0
0-1D, _
H2N y,;(1 1 N d x2 o 0 o
HN I
O , or a pharmaceutically
acceptable salt thereof. The compound may be represented by the formula:
NI NH
*1,
HOP N N NI-12
, P-0
7___.
0 r0 u,s, H
hr,..,,,,
H2NNiirtN, 0 OH 0 0
HN 0
N
O , or a pharmaceutically
acceptable salt thereof. The compound may be represented by the formula:
<,112y...H
HS Q N N NH2
P-
/ Cc_04 H
(:jt?0 01(ro HN-1(\0/\00 NN
H2N),;
HN(11.N\ 0 SH 0 0
0
Nr
O , or a pharmaceutically
acceptable salt thereof.
[0053] In some embodiments, the compound is represented by the formula:
o
NNH
</I *I,
x1P N N NH2
FLO
HO 7
j__,0 IcLC H cH3 0 H 0
4.--0-* ,0 Ny,N)5cy.wwii
0_,R---2
H2Ny,;(1.N HN
d x o
o,...1
HN I > 0
0 , or a pharmaceutically
acceptable salt
thereof. The compound may be represented by the formula:
<J\1115,1H
HOP N N NH2
r P-0
H1,0_4 L0_? H cH3y
r -1-cre HN 40 Nilr'N 1....3N
H2N y,:r\r1I N y) 0 H 0
0 OH
HN I /> 0 0
N
0 , or a pharmaceutically
acceptable salt
thereof. The compound may be represented by the formula:
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<P1/12::õNLH
HS. P N N NH2
õ....P-0
Hillo 'V,L?:) H g H3j5....,Ei
7 HN 40 NY'N Y.'....3
N
H2N NrIN 0-EL y)
o SH
HN I 0 0
N
O , or a pharmaceutically acceptable salt
thereof.
[0054] In some embodiments, the compound is represented by the formula:
o
<PIT'IIIIH
xP N N NH2
OH F F
0--r
H2N ¨, 0
_::' F
yfi 1( ris L (1;x2 0 0 F F
H1.1
N I N/
F
O , or a pharmaceutically acceptable
salt thereof. The compound may be represented by the formula:
<,N1 2:õ...NLH
HOP N N NH2
,P-0
OH ..-
F
1473(
H2N N Nj n 1 v ,..07K,
\..,..==O,.,...-^Ø,,C)0., C)
\,. II 0 F
0 .1k
NV /> 0 OH 0 0F F
H N
F
O , or a pharmaceutically acceptable
salt thereof. The compound may be represented by the formula:
HS. P N N NH2
,P-0
OH
F
1473( ,..07K, ,0 F
n 1 v --ir\,,.f.cr,...(:),õ.^..cr.,.C),õ,,,rr II 0
H2N N N0 .1k
NV /> 0 SH 0 0 F F
H N
F
O , or a pharmaceutically acceptable
salt thereof.
[0055] In some embodiments, the compound is represented by the formula:
o
.NH Fi2Nyo
x1P N N NH2 1-IN
F;-0
C
HO ,
j_.4) 1cLo_? H g 0 H o
(-cr.-1,e HN,yo 41110 NN
H2N
H2N N 0 /
d X
HVI\S 0 0
N
O , or a pharmaceutically acceptable salt
thereof. The compound may be represented by the formula:
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H 2 N ,t 0
HOP N N NH2 HN )
El
H/ PO HO, 'VI IL?:) 1\
,..N...,.,,,
o_F(70 HN Nll.i Ny.,..,).3
..)ro (1101 0 H
H2NN r N
6. OH 0
I 1 0 0
HNx.
N
O , or a pharmaceutically acceptable salt
thereof. The compound may be represented by the formula:
H 2 N Nr. 0
HS. P N N NH2 HN
H/ PO HO, IciL?:) '1\
.N.....,NH
H2N Ir...õ.....,..,..,......).3
0_10 HN Ny.
o
IN 0 H
;I N 0
I I 0 SH
HN
0 0
N
O , or a pharmaceutically acceptable salt
thereof.
[0056] In some embodiments, the compound is represented by the formula:
o
tr
xP N N NH2
FLO
OI-417
0
C1:7r
H2 N Nd x2 o o
HV NI 0
N
O , or a pharmaceutically acceptable
salt thereof. The compound is represented by the formula:
HOP N N NH2
OFAr P-Ckic
H2N.rytN N \
00H 0 0 0
HN N/I
O , or a pharmaceutically acceptable
salt thereof. The compound may be represented by the formula:
HS. P N N NH2
OH r P-Cklc4
H2N N
y.,N
0 SH 0 0 0
HNt\ N'I
O , or a pharmaceutically acceptable
salt thereof.
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[0057] In some embodiments, the compound is represented by the formula:
o
o o
HN=0(:)0(:)N).1\1-jc
H ...,
Nf.,Ki 0
' I
)( P N N
, FLO
1-1C4LAi
0 OH
H2N);NrIN 0 /
.---FLX2
HN I
O , or a pharmaceutically
acceptable salt thereof. The compound may be represented by the formula:
HN N
H
q,N1.-"L. N
1 o
Hog N N
0 p OH
H2N N N 0-, /
Nv \ S- OH
H Nj
O , or a pharmaceutically
acceptable salt thereof. The compound may be represented by the formula:
/
' 1 0
HS. P N N
P- 0
H. IcLO_
o1 S H
H2N
NV /> lx- OH
H N
O , or a pharmaceutically
acceptable salt thereof.
[0058] In some embodiments, the compound is represented by the formula:
o
o o
FINjOC)0 N).L.N-jc
NI/IN 0
</ 1
X19 N N
P-0
1-1(4Z:kr
cLO_
OH/0
H2Ny:NirlIN o"----, /
HN I N/> cilD'X2
O , or a pharmaceutically
acceptable salt thereof. The compound may be represented by the formula:
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iNi...3N /
I 0
HOP N N
P-0
7_._. 1cLC:_i
O OH ,0
H2N.TA)x
li..N
HN I I \i P'
0 OH
O , or a pharmaceutically
actable salt thereof. The compound may be represented by the formula:
iNi...3N /
I 0
HS. P N N
P-0
7_._. 1cLC:_i
O OH ,0
H2N NN
HN1.N
); \
S'SH
iff
O , or a pharmaceutically
acceptable salt thereof.
[0059] In some embodiments, the compound is represented by the formula:
o
-...,
N
H 0
X1P N N
FLO
o-p,----"
H2NX) N N d x2
F :N
O , or a pharmaceutically acceptable
salt thereof. The compound may be represented by the formula:
-õ,
HNJLO 110
N Lc Nis.... ENi ,..ss..........Z
NI/IN
Ir'H
I H 0 0
HOP N N
7_3r( P-hc0
O OH
0_ Ft------ 0
H2NN N d OH
HNYT
N
O , or a pharmaceutically acceptable
salt thereof. The compound may be represented by the formula:
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O o
--,..-
HN---k0
H 1rN
H 0
HS, P N N
IHC4L0
077
0-P----"
H2N,r1;11.N
0 SH
HN )N'>
NI>
O , or a pharmaceutically acceptable
salt thereof.
[0060] In some embodiments, the compound is represented by the formula:
o o
\1
--,--
HN)L N0 iii EN,
1 -..r---
I-1 I.:El 0
xip N N
HN
1-1C4L0Z:k'
H2N0
o77
o-p,----"
H2Ny.,NrIN d x2
HNI I NP
O , or a pharmaceutically acceptable
salt thereof. The compound may be represented by the formula:
o
...,--
HNIO io N H 1
1....õ......,,,,..........õp
NXL NY'N
I H 0 H 0
HOP N N
HN
H2N 0
0R__------- V
H2N ,N1
0 OH
H)N'j I \
N
O , or a pharmaceutically acceptable
salt thereof. The compound may be represented by the formula:
o
...,--
HNIO io N H 1
1....õ......,,,,..........õp
NXL N
NY'
I H 0H 0
HS. P N N
HN
H2N 0
0 OH
H2N
oik-----
,N1
0 SH
V;(1j N
N
O , or a pharmaceutically acceptable
salt thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0061] The novel features of the disclosure are set forth with particularity
in the appended
claims. A better understanding of the features and advantages of the present
disclosure will be
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obtained by reference to the following detailed description that sets forth
illustrative aspects, in
which the principles of the disclosure are utilized, and the accompanying
drawings of which:
[0062] FIGURE 1A illustrates a DNA sequence (SEQ ID NO: 1) of a light chain of
a human
CD40 monoclonal antibody SBT-040. Furthermore, SEQ ID NO: 1 illustrates a DNA
sequence
containing a signal sequence (SEQ ID NO: 2) as shown in FIGURE 1B and a
variable domain
sequence (SEQ ID NO: 3) as shown in FIGURE 1C.
[0063] FIGURE 1B illustrates a DNA sequence of a signal sequence (SEQ ID NO:
2) of a light
chain of a human CD40 monoclonal antibody SBT-040.
[0064] FIGURE 1C illustrates a DNA sequence of a variable domain (SEQ ID NO:
3) in a light
chain of a human CD40 monoclonal antibody SBT-040.
[0065] FIGURE 2A illustrates an amino acid sequence (SEQ ID NO: 4) of a light
chain of a
human CD40 monoclonal antibody SBT-040. Furthermore, SEQ ID NO: 4 illustrates
an amino
acid sequence containing a signal sequence (SEQ ID NO: 5) as shown in FIGURE
2B and a
variable domain sequence (SEQ ID NO: 6) as shown in FIGURE 2C.
[0066] FIGURE 2B illustrates an amino acid sequence of a signal sequence (SEQ
ID NO: 5) of
a light chain of a human CD40 monoclonal antibody SBT-040.
[0067] FIGURE 2C illustrates an amino acid sequence of a variable domain (SEQ
ID NO: 6) in
a light chain of a human CD40 monoclonal antibody SBT-040.
[0068] FIGURE 3A illustrates a DNA sequence (SEQ ID NO: 7) of a wildtype IgG2
isotype
heavy chain of a human CD40 monoclonal antibody SBT-040, wherein this heavy
chain of the
SBT-040 antibody can also be referred to as SBT-040-G2. Furthermore, SEQ ID
NO: 7
illustrates a DNA sequence containing a signal sequence (SEQ ID NO: 12) as
shown in FIGURE
3F and a variable domain sequence (SEQ ID NO: 13) as shown in FIGURE 3G.
[0069] FIGURE 3B illustrates a DNA sequence (SEQ ID NO: 8) of a wild type IgG1
isotype
heavy chain of a human CD40 monoclonal antibody SBT-040, wherein this heavy
chain of the
SBT-040 antibody can also be referred to as SBT-040-G1WT. Furthermore, SEQ ID
NO: 8
illustrates a DNA sequence containing a signal sequence (SEQ ID NO: 12) as
shown in FIGURE
3F and a variable domain sequence (SEQ ID NO: 13) as shown in FIGURE 3G.
[0070] FIGURE 3C illustrates a DNA sequence (SEQ ID NO: 9) of an IgG1 isotype
heavy
chain of a human CD40 monoclonal antibody SBT-040 containing DNA nucleotide
modifications corresponding to L235V, F243L, R292P, Y300L, and P396L amino
acid residue
modifications of a wild type IgG1 Fc domain, wherein this heavy chain of the
SBT-040 antibody
can also be referred to as SBT-040-G1VLPLL. The modified DNA nucleotides
corresponding to
the L235V, F243L, R292P, Y300L, and P396L amino acid residue modifications are
in bold.
Furthermore, SEQ ID NO: 9 illustrates a DNA sequence containing a signal
sequence (SEQ ID
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NO: 12) as shown in FIGURE 3F and a variable domain sequence (SEQ ID NO: 13)
as shown in
FIGURE 3G.
[0071] FIGURE 3D illustrates a DNA sequence (SEQ ID NO: 10) of an IgG1 isotype
heavy
chain of a human CD40 monoclonal antibody SBT-040 containing DNA nucleotide
modifications corresponding to 5239D and 1332E amino acid residue
modifications of a wild
type IgG1 Fc domain, wherein this heavy chain of the SBT-040 antibody can also
be referred to
as SBT-040-G1DE. The modified DNA nucleotides corresponding to the 5239D and
1332E
amino acid residue modifications are in bold. Furthermore, SEQ ID NO: 10
illustrates a DNA
sequence containing a signal sequence (SEQ ID NO: 12) as shown in FIGURE 3F
and a variable
domain sequence (SEQ ID NO: 13) as shown in FIGURE 3G.
[0072] FIGURE 3E illustrates a DNA sequence (SEQ ID NO: 11) of an IgG1 isotype
heavy
chain of human CD40 monoclonal antibody SBT-040 containing DNA nucleotide
modifications
corresponding to 5298A, E333A, and K334A amino acid residue modifications of a
wild type
IgG1 Fc domain, wherein this heavy chain of the SBT-040 antibody can also be
referred to as
SBT-040-G1AAA. The modified DNA nucleotides corresponding to the 5298A, E333A,
and
K334A amino acid residue modifications are in bold. Furthermore, SEQ ID NO: 11
illustrates a
DNA sequence containing a signal sequence (SEQ ID NO: 12) as shown in FIGURE
3F and a
variable domain sequence (SEQ ID NO: 13) as shown in FIGURE 3G.
[0073] FIGURE 3F illustrates a DNA sequence of a signal sequence (SEQ ID NO:
12) of a
heavy chain of a human CD40 monoclonal antibody SBT-040.
[0074] FIGURE 3G illustrates a DNA sequence of a variable domain (SEQ ID NO:
13) in a
heavy chain of a human CD40 monoclonal antibody SBT-040.
[0075] FIGURE 4A illustrates an amino acid sequence (SEQ ID NO: 14) of a
wildtype IgG2
isotype heavy chain of a human CD40 monoclonal antibody SBT-040, wherein this
heavy chain
of the SBT-040 antibody can also be referred to as SBT-040-G2. Furthermore,
SEQ ID NO: 14
illustrates an amino acid sequence containing a signal sequence (SEQ ID NO:
19) as shown in
FIGURE 4F and a variable domain sequence (SEQ ID NO: 20) as shown in FIGURE
4G.
[0076] FIGURE 4B illustrates an amino acid sequence (SEQ ID NO: 15) of a wild
type IgG1
isotype heavy chain of a human CD40 monoclonal antibody SBT-040, wherein this
heavy chain
of the SBT-040 antibody can also be referred to as SBT-040-G1WT. Furthermore,
SEQ ID NO:
15 illustrates an amino acid sequence containing a signal sequence (SEQ ID NO:
19) as shown in
FIGURE 4F and a variable domain sequence (SEQ ID NO: 20) as shown in FIGURE
4G.
[0077] FIGURE 4C illustrates an amino acid sequence (SEQ ID NO: 16) of an IgG1
isotype
heavy chain of a human CD40 monoclonal antibody SBT-040 containing L235V,
F243L, R292P,
Y300L, and P396L amino acid residue modifications of a wild type IgG1 Fc
domain, wherein
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this heavy chain of the SBT-040 antibody can also be referred to as SBT-040-
G1VLPLL. The
amino acid residues corresponding to the L235V, F243L, R292P, Y300L, and P396L
amino acid
residue modifications are in bold. Furthermore, SEQ ID NO: 16 illustrates an
amino acid
sequence containing a signal sequence (SEQ ID NO: 19) as shown in FIGURE 4F
and a variable
domain sequence (SEQ ID NO: 15) as shown in FIGURE 4G.
[0078] FIGURE 4D illustrates an amino acid sequence (SEQ ID NO: 17) of an IgG1
isotype
heavy chain of a human CD40 monoclonal antibody SBT-040 containing 5239D and
1332 amino
acid residue modifications of a wild type IgG1 Fc domain, wherein this heavy
chain of the SBT-
040 antibody can also be referred to as SBT-040-G1DE. The amino acid residues
corresponding
to the 5239D and 1332E amino acid residue modifications are in bold.
Furthermore, SEQ ID NO:
17 illustrates an amino acid sequence containing a signal sequence (SEQ ID NO:
19) as shown in
FIGURE 4F and a variable domain sequence (SEQ ID NO: 20) as shown in FIGURE
4G.
[0079] FIGURE 4E illustrates an amino acid sequence (SEQ ID NO: 18) of an IgG1
isotype
heavy chain of a human CD40 monoclonal antibody SBT-040 containing 5298A,
E333A, and
K334A amino acid residue modifications of a wild type IgG1 Fc domain, wherein
this heavy
chain of the SBT-040 antibody can also be referred to as SBT-040-G1AAA. The
amino acid
residues corresponding to the 5298A, E333A, and K334A amino acid modifications
are in bold.
Furthermore, SEQ ID NO: 11 illustrates an amino acid sequence containing a
signal sequence
(SEQ ID NO: 19) as shown in FIGURE 4F and a variable domain sequence (SEQ ID
NO: 20) as
shown in FIGURE 4G.
[0080] FIGURE 4F illustrates an amino acid sequence of a signal sequence (SEQ
ID NO: 19) of
a heavy chain of a human CD40 monoclonal antibody SBT-040.
[0081] FIGURE 4G illustrates an amino acid sequence of a variable domain (SEQ
ID NO: 20)
in a heavy chain of a human CD40 monoclonal antibody SBT-040.
[0082] FIGURES 5A, 5B, & 5C illustrate a CLUSTAL 0(1.2.1) multiple DNA
sequence
alignment of the DNA sequences of SBT-040-G1VLPLL (SEQ ID NO: 9), SBT-040-
G1AAA
(SEQ ID NO: 11), SBT-040-G1WT (SEQ ID NO: 8), and SBT-040-G1DE (SEQ ID NO:
10).
The SBT-040-G1VLPLL sequence is a DNA sequence of an IgG1 isotype heavy chain
of a
human CD40 monoclonal antibody SBT-040 containing DNA nucleotide modifications
corresponding to L235V, F243L, R292P, Y300L, and P396L amino acid residue
modifications of
a wild type IgG1 Fc domain. The modified DNA nucleotides corresponding to the
L235V,
F243L, R292P, Y300L, and P396L amino acid residue modifications are in bold.
The SBT-040-
G1AAA sequence is a DNA sequence of an IgG1 isotype heavy chain of a human
CD40
monoclonal antibody SBT-040 containing DNA nucleotide modifications
corresponding to
5298A, E333A, and K334A amino acid residue modifications of a wild type IgG1
Fc domain.
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The modified DNA nucleotides corresponding to the S298A, E333A, and K334A
amino acid
residue modifications are boxed. The SBT-040-G1WT sequence is a DNA sequence
of an IgG1
isotype heavy chain of a human CD40 monoclonal antibody SBT-040. The SBT-040-
G1AAA
sequence is a DNA sequence of an IgG1 isotype heavy chain of a human CD40
monoclonal
antibody SBT-040 containing DNA nucleotide modifications corresponding to
S239D and 1332E
amino acid residue modifications of a wild type IgG1 Fc domain. The modified
DNA nucleotides
corresponding to the S239D and 1332E amino acid residue modifications are in
bold italics.
FIGURE 5A shows the start of the sequence alignment. FIGURE 5B shows the
middle of the
sequence alignment as a continuation of FIGURE 5A. FIGURE 5C shows the end of
the
sequence alignment as continuation of FIGURE 5C.
[0083] FIGURE 6 illustrates a CLUSTAL 0(1.2.1) multiple amino acid sequence
alignment of
the amino acid sequences of SBT-040-G1VLPLL (SEQ ID NO: 16), SBT-040-G1AAA
(SEQ ID
NO: 18), SBT-040-G1WT (SEQ ID NO: 15), and SBT-040-G1DE (SEQ ID NO: 17). The
SBT-
040-G1VLPLL sequence is an amino acid sequence of an IgG1 isotype heavy chain
of a human
CD40 monoclonal antibody SBT-040 containing L235V, F243L, R292P, Y300L, and
P396L
amino acid residue modifications of a wild type IgG1 Fc domain. The L235V,
F243L, R292P,
Y300L, and P396L amino acid residue modifications are in bold. The SBT-040-
G1AAA
sequence is an amino acid sequence of an IgG1 isotype heavy chain of a human
CD40
monoclonal antibody SBT-040 containing 5298A, E333A, and K334A amino acid
residue
modifications of a wild type IgG1 Fc domain. The 5298A, E333A, and K334A amino
acid
residue modifications are italics. The SBT-040-G1WT sequence is an amino acid
sequence of an
IgG1 isotype heavy chain of a human CD40 monoclonal antibody SBT-040. The SBT-
040-
G1AAA sequence is an amino acid sequence of an IgG1 isotype heavy chain of a
human CD40
monoclonal antibody SBT-040 containing 5239D and 1332E amino acid residue
modifications
bold italics. Additionally, the hinge region of each amino acid sequence is
differentiated from
other regions of the amino acid sequence by brackets. The left bracket
indicates the upper portion
of the hinge region (UH). The four residues between the brackets are the
middle portion of the
hinge region. The right bracket indicates the lower portion of the hinge
region (LH).
[0084] FIGURE 7 illustrates a schematic of an antibody. An antibody contains
two heavy chains
as shown in gray and two light chains as shown in light gray. A portion of the
heavy chains
contain Fc domains (705 and 720). An antibody contains two antigen binding
sites (710 and
715).
[0085] FIGURE 8 illustrates a schematic of an exemplary conjugate. An antibody
construct is an
antibody, which contains two heavy chains as shown in gray and two light
chains as shown in
light gray. The antibody comprises two antigen binding sites (810 and 815),
and a portion of the
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heavy chains contain Fc domains (805 and 820). The immune-stimulatory
compounds (830 and
840) are conjugated to the antibody by linkers (860 and 870).
[0086] FIGURE 9 illustrates a schematic of an exemplary conjugate. An antibody
construct is an
antibody, which contains two heavy chains as shown in gray and two light
chains as shown in
light gray. The antibody comprises two antigen binding sites (910 and 915),
and a portion of the
heavy chains contain Fc domains (905 and 920). The immune-stimulatory
compounds (930 and
940) are conjugated to the antibody by linkers (960 and 970). Targeting
binding domains are
conjugated to the antibody (980 and 985).
[0087] FIGURE 10 illustrates a schematic of an exemplary conjugate. An
antibody construct
contains the Fc region of an antibody with the heavy chains shown in gray, and
two scaffolds as
shown in light gray. The antibody construct comprises two antigen binding
sites (1010 and 1015)
in the scaffolds, and a portion of the heavy chains contain Fc domains (1005
and 1020). The
immune-stimulatory compounds (1030 and 1040) are conjugated to the antibody
construct by
linkers (1060 and 1070).
[0088] FIGURE 11 illustrates a schematic of an exemplary conjugate. An
antibody construct
contains the Fc region of an antibody with the heavy chains shown in gray, and
two scaffolds as
shown in light gray. The antibody construct comprises two antigen binding
sites (1110 and 1115)
in the scaffolds, and a portion of the heavy chains contain Fc domains (1105
and 1120). The
immune-stimulatory compounds (1130 and 1140) are conjugated to the antibody
construct by
linkers (1160 and 1170). Targeting binding domains are conjugated to the
antibody construct
(1180 and 1185).
[0089] FIGURE 12 illustrates a schematic of an exemplary conjugate. An
antibody construct
contains the F(ab')2 region of an antibody with heavy chains shown in gray and
light chains
shown in light gray, and two scaffolds as shown in dark gray. The antibody
construct comprises
two antigen binding sites (1210 and 1215), and a portion of two scaffolds
contain Fc domains
(1220 and 1245). The immune-stimulatory compounds (1230 and 1240) are
conjugated to the
antibody construct by linkers (1260 and 1270).
[0090] FIGURE 13 illustrates a schematic of an exemplary conjugate. An
antibody construct
contains the F(ab')2 region of an antibody with heavy chains shown in gray and
light chains
shown in light gray, and two scaffolds as shown in dark gray. The antibody
construct comprises
two antigen binding sites (1310 and 1315), and a portion of two scaffolds
contain Fc domains
(1320 and 1345). The immune-stimulatory compounds (1330 and 1340) are
conjugated to the
antibody construct by linkers (1360 and 1370). Targeting binding domains are
conjugated to the
antibody construct (1380 and 1385).
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[0091] FIGURE 14 illustrates a schematic of an exemplary conjugate. An
antibody construct
contains two scaffolds as shown in light gray and two scaffolds as shown in
dark gray. The
antibody construct comprises two antigen binding sites (1410 and 1415), and a
portion of the two
dark gray scaffolds contain Fc domains (1420 and 1445). The immune-stimulatory
compounds
(1430 and 1440) are conjugated to the antibody construct by linkers (1460 and
1470).
[0092] FIGURE 15 illustrates a schematic of an exemplary conjugate. An
antibody construct
contains two scaffolds as shown in light gray and two scaffolds as shown in
dark gray. The
antibody construct comprises two antigen binding sites (1510 and 1515), and a
portion of the two
dark gray scaffolds contain Fc domains (1520 and 1545). The immune-stimulatory
compounds
(1530 and 1540) are conjugated to the antibody construct by linkers (1560 and
1570). Targeting
binding domains are conjugated to the antibody construct (1580 and 1585).
[0093] FIGURE 16 is the two-dimensional structure of the heavy chain of
Dacetuzumab.
[0094] FIGURE 17 is the two-dimensional structure of the light chain of
Dacetuzumab.
[0095] FIGURE 18 is the two-dimensional structure of the heavy chain of
Bleselumab.
[0096] FIGURE 19 is the two-dimensional structure of the light chain of
Bleselumab.
[0097] FIGURE 20 is the two-dimensional structure of the heavy chain of
Lucatumumab.
[0098] FIGURE 21 is the two-dimensional structure of the light chain of
Lucatumumab
[0099] FIGURE 22 is the two-dimensional structure of the heavy chain of ADC-
1013.
[0100] FIGURE 23 is the two-dimensional structure of the light chain of ADC-
1013.
[0101] FIGURE 24 is the two-dimensional structure of the heavy chain of
humanized rabbit
antibody APX005.
[0102] FIGURE 25 is the two-dimensional structure of the light chain of
humanized rabbit
antibody APX005.
[0103] FIGURE 26 is the two-dimensional structure of the heavy chain of Chi
Lob 7/4.
[0104] FIGURE 27 is the two-dimensional structure of the light chain of Chi
Lob 7/4.
[0105] FIGURE 28 shows HPLC analysis of SBT-040-G1WT conjugated to a Cys-
targeted drug
linker tool compound.
[0106] FIGURE 29 shows HPLC analysis of SBT-040-G1WT conjugated to ATAC2.
[0107] FIGURE 30 shows HPLC analysis of SBT-040-G2WT conjugated to ATAC2.
[0108] FIGURE 31A shows the concentration of IL-12p70 produced by dendritic
cells (DCs)
from donor 358 after incubation with SBT-040-WT-ATAC23 or SBT-040-WT-ATAC17 as
compared with SBT-050-WT.
[0109] FIGURE 31B shows the concentration of IL-12p70 produced by DCs from
donor 363
after incubation with SBT-040-WT-ATAC23 or SBT-040-WT-ATAC17 as compared with
SBT-
050-WT.
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[0110] FIGURE 31C shows the concentration of TNFa produced by DCs from donor
358 after
incubation with SBT-040-WT-ATAC23 or SBT-040-WT-ATAC17 as compared with SBT-
050-
WT.
[0111] FIGURE 31D shows the concentration of TNFa produced by DCs from donor
363 after
incubation with SBT-040-WT-ATAC23 or SBT-040-WT-ATAC17 as compared with SBT-
050-
WT.
[0112] FIGURE 32A shows the concentration of IL-12p70 produced by DCs after
incubation
with SBT-040-WT-ATAC4, SBT-040-WT-ATAC3, SBT-040-G2-ATAC4, SBT-040-G2-
ATAC3, SBT-040-AAA-ATAC22, SBT-040-VLPLL-ATAC22, SBT-040-WT-ATAC1, SBT-
040-G2-ATAC1, SBT-040-WT-ATAC12, SBT-040-G2-ATAC12, SBT-040-WT-ATAC30,
SBT-040-G1AAA-ATAC11, SBT-040-VLPLL-ATAC11, SBT-040-VLPLL-ATAC12, SBT-
040-AAA-ATAC12, SBT-040-VLPLL-ATAC23, and SBT-040-AAA-ATAC23 as compared
with SBT-050-G2 or CD40 ligand.
[0113] FIGURE 32B shows the concentration of IL-6 produced by DCs from donor 2
after
incubation with SBT-040-WT-ATAC4, SBT-040-WT-ATAC3, SBT-040-G2-ATAC4, SBT-040-
G2-ATAC3, SBT-040-AAA-ATAC22, SBT-040-VLPLL-ATAC22, SBT-040-WT-ATAC1,
SBT-040-G2-ATAC1, SBT-040-WT-ATAC12, SBT-040-G2-ATAC12, SBT-040-WT-ATAC30,
SBT-040-AAA-ATAC11, SBT-040-VLPLL-ATAC11, SBT-040-VLPLL-ATAC12, SBT-040-
AAA-ATAC12, SBT-040-VLPLL-ATAC23, and SBT-040-AAA-ATAC30 compared with SBT-
050-G2 or CD40 ligand. Results are shown for the immune stimulatory cytokines
IL-12p70 and
IL-6.
[0114] FIGURE 33A shows a dose dependent increase in CD86 expression on
dendritic cells
after treatment with SBT-040-WT-ATAC23, SBT-040-WT-ATAC17, SBT-040-VLPLL-
ATAC22, SBT-040-AAA-ATAC23 as compared to treatment a control SBT-050-WT or
staining
with an isotype control.
[0115] FIGURE 33B shows a dose dependent increase in CD83 expression on
dendritic cells
after treatment with SBT-040-WT-ATAC23, SBT-040-WT-ATAC17, SBT-040- VLPLL-
ATAC23, SBT-040-AAA-ATAC23 as compared to treatment a control SBT-050-WT or
staining
with an isotype control.
[0116] FIGURE 33C shows a dose dependent increase in MHC class II expression
on dendritic
cells after treatment with SBT-040-WT-ATAC23, SBT-040-WT-ATAC17, SBT-040-VLPLL-
ATAC23, SBT-040-AAA-ATAC23 as compared to treatment a control SBT-050-WT or
staining
with an isotype control.
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DETAILED DESCRIPTION
[0117] Additional aspects and advantages of the present disclosure will become
apparent to those
skilled in this art from the following detailed description, wherein
illustrative aspects of the
present disclosure are shown and described. As will be realized, the present
disclosure is capable
of other and different aspects, and its several details are capable of
modifications in various
respects, all without departing from the disclosure. Accordingly, the drawings
and description are
to be regarded as illustrative in nature, and not as restrictive.
[0118] Cancer is one of the leading causes of death in the United States.
Conventional methods
of cancer treatment like chemotherapy, surgery or radiation therapy, can be
limited in their
efficacy since they are often nonspecific to the cancer. In many cases tumors,
however, can
specifically express genes whose products are required for inducing or
maintaining the malignant
state. These proteins may serve as antigen markers for the development and
establishment of
efficient anti-cancer treatments.
[0119] As used herein, "homologous" or "homology" can refer to the similarity
between a DNA,
RNA, nucleotide, amino acid, or protein sequence to another DNA, RNA,
nucleotide, amino
acid, or protein sequence. Homology can be expressed in terms of a percentage
of sequence
identity of a first sequence to a second sequence. Percent (%) sequence
identity with respect to a
reference DNA sequence can be the percentage of DNA nucleotides in a candidate
sequence that are
identical with the DNA nucleotides in the reference DNA sequence after
aligning the sequences.
Percent (%) sequence identity with respect to a reference amino acid sequence
can be the percentage
of amino acid residues in a candidate sequence that are identical with the
amino acid residues in the
reference amino acid sequence after aligning the sequences and introducing
gaps, if necessary, to
achieve the maximum percent sequence identity, and not considering any
conservative substitutions
as part of the sequence identity.
[0120] As used herein, the term "antibody" can refer to an immunoglobulin
molecule that
specifically binds to, or is immunologically reactive toward, a specific
antigen. Antibody can
include, for example, polyclonal, monoclonal, genetically engineered, and
antigen binding
fragments thereof. An antibody can be, for example, murine, chimeric,
humanized,
heteroconjugate, bispecific, diabody, triabody, or tetrabody. The antigen
binding fragment can
include, for example, Fab', F(ab')2, Fab, Fv, rIgG, and scFv.
[0121] As used herein, "recognize" can refer to the association or binding
between an antigen
binding domain and an antigen.
[0122] As used herein, a "tumor antigen" can be an antigenic substance
associated with a tumor
or cancer cell, and can trigger an immune response in a host.
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[0123] As used herein, an "antibody construct" can refer to a construct that
contains an antigen
binding domain and an Fc domain.
[0124] As used herein, an "antigen binding domain" can refer to an antigen
binding domain from
an antibody or from a non-antibody that can bind to the antigen.
[0125] As used herein, a "Fe domain" can be an Fc domain from an antibody or
from a non-
antibody that can bind to an Fc receptor.
[0126] As used herein, a "target binding domain" can refer to a construct that
contains an antigen
binding domain from an antibody or from a non-antibody that can bind to the
antigen.
[0127] The term "salt" or "pharmaceutically acceptable salt" refers to salts
derived from a
variety of organic and inorganic counter ions well known in the art.
Pharmaceutically acceptable
acid addition salts can be formed with inorganic acids and organic acids.
Inorganic acids from
which salts can be derived include, for example, hydrochloric acid,
hydrobromic acid, sulfuric
acid, nitric acid, phosphoric acid, and the like. Organic acids from which
salts can be derived
include, for example, acetic acid, propionic acid, glycolic acid, pyruvic
acid, oxalic acid, maleic
acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid,
benzoic acid, cinnamic
acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-
toluenesulfonic acid, salicylic
acid, and the like. Pharmaceutically acceptable base addition salts can be
formed with inorganic
and organic bases. Inorganic bases from which salts can be derived include,
for example, sodium,
potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper,
manganese, aluminum,
and the like. Organic bases from which salts can be derived include, for
example, primary,
secondary, and tertiary amines, substituted amines including naturally
occurring substituted
amines, cyclic amines, basic ion exchange resins, and the like, specifically
such as
isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine,
and ethanolamine.
In some embodiments, the pharmaceutically acceptable base addition salt is
chosen from
ammonium, potassium, sodium, calcium, and magnesium salts.
[0128] The term "Cx_y" when used in conjunction with a chemical moiety, such
as alkyl, alkenyl,
or alkynyl is meant to include groups that contain from x to y carbons in the
chain. For example,
the term "Cx_yalkyl" refers to substituted or unsubstituted saturated
hydrocarbon groups,
including straight-chain alkyl and branched-chain alkyl groups that contain
from x to y carbons
in the chain, including haloalkyl groups such as trifluoromethyl and 2,2,2-
trifluoroethyl, etc.
[0129] The terms "Cx_yalkenyl" and "Cx_yalkynyl" refer to substituted or
unsubstituted
unsaturated aliphatic groups analogous in length and possible substitution to
the alkyls described
above, but that contain at least one double or triple bond respectively.
[0130] The term "carbocycle" as used herein refers to a saturated, unsaturated
or aromatic ring in
which each atom of the ring is carbon. Carbocycle includes 3- to 10-membered
monocyclic rings,
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6- to 12-membered bicyclic rings, and 6- to 12-membered bridged rings. Each
ring of a bicyclic
carbocycle may be selected from saturated, unsaturated, and aromatic rings. In
an exemplary
embodiment, an aromatic ring, e.g., phenyl, may be fused to a saturated or
unsaturated ring, e.g.,
cyclohexane, cyclopentane, or cyclohexene. Any combination of saturated,
unsaturated and
aromatic bicyclic rings, as valence permits, is included in the definition of
carbocyclic.
Exemplary carbocycles include cyclopentyl, cyclohexyl, cyclohexenyl,
adamantyl, phenyl,
indanyl, and naphthyl.
[0131] The term "heterocycle" as used herein refers to a saturated,
unsaturated or aromatic ring
comprising one or more heteroatoms. Exemplary heteroatoms include N, 0, Si, P,
B, and S
atoms. Heterocycles include 3- to 10-membered monocyclic rings, 6- to 12-
membered bicyclic
rings, and 6- to 12-membered bridged rings. Each ring of a bicyclic
heterocycle may be selected
from saturated, unsaturated, and aromatic rings wherein at least one of the
rings includes a
heteroatom. In an exemplary embodiment, an aromatic ring, e.g., pyridyl, may
be fused to a
saturated or unsaturated ring, e.g., cyclohexane, cyclopentane, morpholine,
piperidine or
cyclohexene. The term "heteroaryl" includes aromatic single ring structures,
preferably 5- to 7-
membered rings, more preferably 5- to 6-membered rings, whose ring structures
include at least
one heteroatom, preferably one to four heteroatoms, more preferably one or two
heteroatoms.
The term "heteroaryl" also include polycyclic ring systems having two or more
cyclic rings in
which two or more carbons are common to two adjoining rings wherein at least
one of the rings
is heteroaromatic, e.g., the other cyclic rings can be aromatic or non-
aromatic carbocyclic, or
heterocyclic. Heteroaryl groups include, for example, pyrrole, furan,
thiophene, imidazole,
oxazole, thiazole, pyrazole, pyridine, pyrazine, pyridazine, and pyrimidine,
and the like.
[0132] The term. "substituted" refers to moieties having substituents
replacing a hydrogen on one
or more carbons or substitutable heteroatoms, e.g., NH, of the structure. It
will be understood that
"substitution" or "substituted with" includes the implicit proviso that such
substitution is in
accordance with permitted valence of the substituted atom and the substituent,
and that the
substitution results in a stable compound, i.e., a. compound which does not
spontaneously
undergo transformation such as by rearrangement, cyclization, elimination,
etc. In certain
embodiments, substituted refers to moieties having substituents replacing two
hydrogen. atoms on
the same carbon atom, such as substituting the two hydrogen atoms on a single
carbon with an
oxo, imino or thioxo group. As used herein, the term "substituted" is
contemplated to include all
permissible substituents of organic compounds. In a broad aspect, the
permissible substituents
include acyclic and cyclic, branched and unbranched, carbocyclic and
heterocyclic, aromatic and
non-aromatic substituents of organic compounds. The permissible substituents
can be one or
more and the same or different for appropriate organic compounds. For purposes
of this
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disclosure, the heteroatifins such as nitrogen may have hydrogen substituents
and/or any
permissible substituents of organic compounds described herein which satisfy
the valences of the
heteroatoms.
[0133] In some embodiments, substituents may include any substituents
described herein, for
example: halogen, hydroxy, oxo (=0), thioxo (=S), cyano (-CN), nitro (-NO2),
imino (=N-H),
oximo (=N-OH), hydrazino (=N-
NH2), -Rb-ORa, -Rb-OC(0)-Ra, -Rb-OC(0)-0Ra, -Rb-OC(0)-N(Ra)2, -Rb_N(Ra)2,
_Rb_c(0)Ra, _Rb
-C(0)0Ra, -Rb-C(0)N(R1)2, -Rb-O-Rc-C(0)N(Ra)2, -Rb-N(Ra)C(0)0Ra, -Rb-
N(Ra)C(0)Ra, -Rb-N
(Ra)S(0)Ra (where t is 1 or 2), -Rb-S(0)tRa (where t is 1 or 2), -Rb-S(0)tORa
(where t is 1 or 2),
and -Rb-S(0)tN(R1)2 (where t is 1 or 2); and alkyl, alkenyl, alkynyl, aryl,
aralkyl, aralkenyl,
aralkynyl, cycloalkyl, cycloalkylalkyl, heterocyclo alkyl,
heterocycloalkylalkyl, heteroaryl, and
heteroarylalkyl any of which may be optionally substituted by alkyl, alkenyl,
alkynyl, halogen,
haloalkyl, haloalkenyl, haloalkynyl, oxo (=0), thioxo (=S), cyano (-CN), nitro
(-NO2), imino
(=N-H), oximo (=N-OH), hydrazine (=N-
NH2), -Rb-ORa, -Rb-OC(0)-Ra, -Rb-OC(0)-0Ra, -Rb-OC(0)-N(R1)2, -Rb_N(R1)2,
_Rb_c(0)Ra, _Rb
-C(0)0Ra, -Rb-C(0)N(R1)2, -Rb-O-Rc-C(0)N(Ra)2, -Rb-N(Ra)C(0)0Ra, -Rb-
N(R1)C(0)Ra, -Rb-N
(Ra)S(0)Ra (where t is 1 or 2), -Rb-S(0)tRa (where t is 1 or 2), -Rb-S(0)tORa
(where t is 1 or 2)
and -Rb-S(0)tN(R1)2 (where t is 1 or 2); wherein each Ra is independently
selected from
hydrogen, alkyl, cycloalkyl, cycloalkylalkyl, aryl, aralkyl, heterocycloalkyl,
heterocycloalkylalkyl, heteroaryl, or heteroarylalkyl, wherein each Ra,
valence permitting, may
be optionally substituted with alkyl, alkenyl, alkynyl, halogen, haloalkyl,
haloalkenyl,
haloalkynyl, oxo (=0), thioxo (=S), cyano (-CN), nitro (-NO2), imino (=N-H),
oximo (=N-OH),
hydrazine (=N-
NH2), -Rb-ORa, -Rb-OC(0)-Ra, -Rb-OC(0)-0Ra, -Rb-OC(0)-N(R1)2, -Rb_N(R1)2,
_Rb_c(0)Ra, _Rb
-C(0)0Ra, -Rb-C(0)N(R1)2, -Rb-O-Rc-C(0)N(Ra)2, -Rb-N(Ra)C(0)0Ra, -Rb-
N(R1)C(0)Ra, -Rb-N
(Ra)S(0)Ra (where t is 1 or 2), -Rb-S(0)tRa (where t is 1 or 2), -Rb-S(0)tORa
(where t is 1 or 2)
and -Rb-S(0)tN(R1)2 (where t is 1 or 2); and wherein each Rb is independently
selected from a
direct bond or a straight or branched alkylene, alkenylene, or alkynylene
chain, and each Rc is a
straight or branched alkylene, alkenylene or alkynylene chain.
[0134] It will be understood by those skilled in the art that substituents can
themselves be
substituted, if appropriate. Unless specifically stated as "unsubstituted,"
references to chemical
moieties herein are understood to include substituted variants. For example,
reference to a
"heteroaryl" group or moiety implicitly includes both substituted and
unsubstituted variants.
[0135] Chemical entities having carbon-carbon double bonds or carbon-nitrogen
double bonds
may exist in Z- or E- form (or cis- or trans- form). Furthermore, some
chemical entities may exist
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in various tautomeric forms. Unless otherwise specified, chemical entities
described herein are
intended to include all Z-, E- and tautomeric forms as well.
[0136] A "tautomer" refers to a molecule wherein a proton shift from one atom
of a molecule to
another atom of the same molecule is possible. The compounds presented herein,
in certain
embodiments, exist as tautomers. In circumstances where tautomerization is
possible, a chemical
equilibrium of the tautomers will exist. The exact ratio of the tautomers
depends on several
factors, including physical state, temperature, solvent, and pH. Some examples
of tautomeric
y H
.j (A: \\.
\11\r\
H H
)
\NH2 \ N H \N \A )1/2,
N H2 1/2. N H
N., N
csss. N ciss H issr isss
ssi\J
N N'N H
N N HN N NN'
'
csss N
OH 0
equilibrium include:
[0137] The compounds disclosed herein, in some embodiments, are used in
different enriched
isotopic forms, e.g., enriched in the content of 2H, 3H, 11,,, 13
C and/or 14C. In one particular
embodiment, the compound is deuterated in at least one position. Such
deuterated forms can be
made by the procedure described in U.S. Patent Nos. 5,846,514 and 6,334,997.
As described in
U.S. Patent Nos. 5,846,514 and 6,334,997, deuteration can improve the
metabolic stability and or
efficacy, thus increasing the duration of action of drugs.
[0138] Unless otherwise stated, structures depicted herein are intended to
include compounds
which differ only in the presence of one or more isotopically enriched atoms.
For example,
compounds having the present structures except for the replacement of a
hydrogen by a
deuterium or tritium, or the replacement of a carbon by 13C- or 14C-enriched
carbon are within
the scope of the present disclosure.
[0139] The compounds of the present disclosure optionally contain unnatural
proportions of
atomic isotopes at one or more atoms that constitute such compounds. For
example, the
compounds may be labeled with isotopes, such as for example, deuterium (2H),
tritium (3H),
iodine-125 (1251) or carbon-14 (..,) Isotopic substitution with
2H, HC, 13C, 14C, 15C, 12N, 13N,
'6N, 16 17 '4F '5F,
'6F, '7F,
'8F, 33S,
34S, 35S,
36S, 35C1,
37 79 81 125
N, N, 0, 0, F, F, F, F, F, S, S, S, S, Cl, Cl, Br, Br, I are all
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contemplated. All isotopic variations of the compounds of the present
invention, whether
radioactive or not, are encompassed within the scope of the present invention.
[0140] In certain embodiments, the compounds disclosed herein have some or all
of the 1H atoms
replaced with 2H atoms. The methods of synthesis for deuterium-containing
compounds are
known in the art and include, by way of non-limiting example only, the
following synthetic
methods.
Deuterium substituted compounds are synthesized using various methods such as
described in:
Dean, Dennis C.; Editor. Recent Advances in the Synthesis and Applications of
Radiolabeled
Compounds for Drug Discovery and Development. [In: Curr., Pharm. Des., 2000;
6(10)[ 2000,
110 pp; George W.; Varma, Rajender S. The Synthesis of Radiolabeled Compounds
via
Organometallic Intermediates, Tetrahedron, 1989, 45(21), 6601-21; and Evans,
E. Anthony.
Synthesis of radiolabeled compounds, J. Radioanal. Chem., 1981, 64(1-2), 9-32.
[0141] Deuterated starting materials are readily available and are subjected
to the synthetic
methods described herein to provide for the synthesis of deuterium-containing
compounds. Large
numbers of deuterium-containing reagents and building blocks are available
commercially from
chemical vendors, such as Aldrich Chemical Co.
[0142] Compounds of the present invention also include crystalline and
amorphous forms of
those compounds, pharmaceutically acceptable salts, and active metabolites of
these compounds
having the same type of activity, including, for example, polymorphs,
pseudopolymorphs,
solvates, hydrates, unsolvated polymorphs (including anhydrates),
conformational polymorphs,
and amorphous forms of the compounds, as well as mixtures thereof.
[0143] The phrases "parenteral administration" and "administered parenterally"
as used herein
means modes of administration other than enteral and topical administration,
usually by injection,
and includes, without limitation, intravenous, intramuscular, intraarterial,
intrathecal,
intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal,
transtracheal, subcutaneous,
subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and
intrasternal injection and
infusion.
The phrase "pharmaceutically acceptable" is employed herein to refer to those
compounds,
materials, compositions, and/or dosage forms which are, within the scope of
sound medical
judgment, suitable for use in contact with the tissues of human beings and
animals without
excessive toxicity, irritation, allergic response, or other problem or
complication, commensurate
with a reasonable benefit/risk ratio.
[0144] The phrase "pharmaceutically acceptable excipient" or "pharmaceutically
acceptable
carrier" as used herein means a pharmaceutically acceptable material,
composition or vehicle,
such as a liquid or solid filler, diluent, excipient, solvent or encapsulating
material. Each carrier
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must be "acceptable" in the sense of being compatible with the other
ingredients of the
formulation and not injurious to the patient. Some examples of materials which
can serve as
pharmaceutically acceptable carriers include: (1) sugars, such as lactose,
glucose and sucrose; (2)
starches, such as corn starch and potato starch; (3) cellulose, and its
derivatives, such as sodium
carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered
tragacanth; (5) malt;
(6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository
waxes; (9) oils, such as
peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and
soybean oil; (10)
glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol,
mannitol and
polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13)
agar; (14) buffering
agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid;
(16) pyrogen-
free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol;
(20) phosphate buffer
solutions; and (21) other non-toxic compatible substances employed in
pharmaceutical
formulations.
[0145] "Antibody drug conjugate" ("ADC") can refer to an antibody construct
immune-
stimulatory compound conjugate. An ADC can comprise any embodiment as
described herein for
an antibody construct immune-stimulatory compound conjugate. Therefore, ADC
and antibody
construct immune-stimulatory compound conjugate can be used interchangeably
herein.
[0146] An antigen can elicit an immune response. An antigen can be a protein,
polysaccharide,
lipid, or glycolipid, which can be recognized by an immune cell, such as a T
cell or a B cell.
Exposure of immune cells to one or more of these antigens can elicit a rapid
cell division and
differentiation response resulting in the formation of clones of the exposed T
cells and B cells. B
cells can differentiate into plasma cells which in turn can produce antibodies
which selectively
bind to the antigens.
[0147] In cancer, there are four general groups of tumor antigens: (i) viral
tumor antigens which
can be identical for any viral tumor of this type, (ii) carcinogenic tumor
antigens which can be
specific for patients and for the tumors, (iii) isoantigens of the
transplantation type or tumor-
specific transplantation antigens which can be different in all individual
types of tumor but can
be the same in different tumors caused by the same virus; and (iv) embryonic
antigens.
[0148] As a result of the discovery of tumor antigens, tumor antigens have
become important in
the development of new cancer treatments that can specifically target the
cancer. This has led to
the development of antibodies directed against these tumor antigens.
[0149] In addition to the development of antibodies against tumor antigens for
cancer treatment,
antibodies that target immune cells to boost the immune response have also
been developed. For
example, an anti-CD40 antibody that is a CD40 agonist can be used to activate
dendritic cells to
enhance the immune response.
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[0150] Cluster of Differentiation 40 (CD40) is a member of the Tumor Necrosis
Factor Receptor
(TNF-R) family. CD40 can be a 50 kDa cell surface glycoprotein that can be
constitutively
expressed in normal cells, such as monocytes, macrophages, B lymphocytes,
dendritic cells,
endothelial cells, smooth muscle cells, fibroblasts and epithelium, and in
tumor cells, including
B-cell lymphomas and many types of solid tumors. Expression of CD40 can be
increased in
antigen presenting cells in response to IL-10p, IFN-y, GM-CSF, and LPS induced
signaling
events.
[0151] Humoral and cellular immune responses can be regulated, in part, by
CD40. For example,
in the absence of CD40 activation by its cognate binding partner, CD40 Ligand
(CD4OL), antigen
presentation can result in tolerance. However, CD40 activation can ameliorate
tolerance. In
addition, CD40 activation can positively impact immune responses by enhancing
antigen
presentation by antigen presenting cells (APC), increasing cytokine and
chemokine secretion,
stimulating expression of and signaling by co-stimulatory molecules, and
activating the cytolytic
activity of different types of immune cells. Accordingly, the interaction
between CD40 and
CD4OL can be essential to maintain proper humoral and cellular immune
responses.
[0152] The intracellular effects of CD40 and CD4OL interaction can include
association of the
CD40 cytoplasmic domain with TRAFs (TNF-R associated factors), which can lead
to the
activation of NFKB and Jun/AP1 pathways. While the response to activation of
NFKB and
Jun/AP1 pathways can be cell type-specific, often such activation can lead to
increased
production and secretion of cytokines, including IL-6, IL-8, IL-12, IL-15;
increased production
and secretion of chemokines, including MIPla and 0 and RANTES; and increased
expression of
cellular adhesion molecules, including ICAM. While the effects of cytokines,
chemokines and
cellular adhesion molecules can be widespread, such effects can include
enhanced survival and
activation of T cells.
[0153] In addition to the enhanced immune responses induced by CD40
activation, CD40
activation can also be involved in chemokine- and cytokine-mediated cellular
migration and
differentiation; activation of immune cells, including monocytes; activation
of and increased
cytolytic activity of immune cells, including cytolytic T lymphocytes and
natural killer cells;
induction of CD40-positive tumor cell apoptosis and enhanced immunogenicity of
CD40-positive
tumors. In addition, CD40 can initiate and enhance immune responses by many
different
mechanisms, including, inducing antigen-presenting cell maturation and
increased expression of
costimulatory molecules, increasing production of and secretion of cytokines,
and enhancing
effector functions.
[0154] CD40 activation can be effective for inducing immune-mediated antitumor
responses. For
example, CD40 activation reverses host immune tolerance to tumor-specific
antigens which leads
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to enhanced antitumor responses by T cells. Such antitumor activity can also
occur in the absence
of immune cells. Similarly, antitumor effects can occur in response to anti-
CD40 antibody-
mediated activation of CD40 and can be independent of antibody-dependent
cellular cytotoxicity.
In addition to other CD40-mediated mechanisms of antitumor effects, CD4OL-
stimulation can
cause dendritic cell maturation and stimulation. CD4OL-stimulated dendritic
cells can contribute
to the antitumor response. Furthermore, vaccination strategies including CD40
can result in
regression of CD40-positive and CD40-negative tumors.
[0155] CD40 activating antibodies (e.g., anti-CD40 activating monoclonal
antibodies) can be
useful for treatment of tumors. This can occur through one or more mechanisms,
including cell
activation, antigen presentation, production of cytokines and chemokines,
amongst others. For
example, CD40 antibodies activate dendritic cells, leading to processing and
presentation of
tumor antigens as well as enhanced immunogenicity of CD40-positive tumor
cells. Not only can
enhanced immunogenicity result in activation of CD40-positive tumor specific
CD4+ and CD8+ T
cells, but further antitumor activity can include, recruitment and activation
monocytes, enhanced
cytolytic activity of cytotoxic lymphocytes and natural killer cells as well
as induction of
apoptosis or by stimulation of a humoral response so as to directly target
tumor cells. In addition,
tumor cell debris, including tumor-specific antigens, can be presented to
other cells of the
immune system by CD40-activated antigen presenting cells.
[0156] Since CD40 can be important in an immune response, there is a need for
enhanced CD40
meditated signaling events to provide reliable and rapid treatment options to
patients suffering
from diseases which may be ameliorated by treatment with CD40-targeted
therapeutic strategies.
[0157] The HER2/neu (human epidermal growth factor receptor 2/receptor
tyrosine-protein
kinase erbB-2) is part of the human epidermal growth factor family.
Overexpression of this
protein has been shown to play an important role in the progression of cancer,
for example, breast
cancer. The HER2/neu protein functions as a receptor tyrosine kinase and
autophosphorylates
upon dimerization with binding partners. HER2/neu can activate several
signaling pathways
including, for example, mitogen-activated protein kinase, phosphoinositide 3-
kinase,
phospholipase Cy, protein kinase C, and signal transducer and activator of
transcription (STAT).
Several compounds have been developed to inhibit HER2/neu including for
example, the
monoclonal antibody trastuzumab and the monoclonal antibody pertuzumab.
[0158] Immune-stimulatory molecular motifs, such as Pathogen-Associated
Molecular Pattern
molecules, (PAMPs) can be recognized by receptors of the innate immune system,
such as Toll-
like receptors (TLRs), Nod-like receptors, C-type lectins, and RIG-I-like
receptors. These
receptors can be transmembrane and intra-endosomal proteins which can prime
activation of the
immune system in response to infectious agents such as pathogens. Similar to
other protein
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families, TLRs can have many isoforms, including TLR4, TLR7 and TLR8. Several
agonists
targeting activation of different TLRs can be used in various immunotherapies,
including vaccine
adjuvants and in cancer immunotherapies. TLR agonists can range from simple
molecules to
complex macromolecules. Likewise, the sizes of TLR agonists can range from
small to large.
TLR agonists can be synthetic or biosynthetic agonists. TLR agonists can also
be PAMPs.
Additional immune-stimulatory compounds, such as cytosolic DNA and unique
bacterial nucleic
acids called cyclic dinucleotides, can be recognized by Interferon Regulatory
Factor (IRF) or
stimulator of interferon genes (STING), which can act a cytosolic DNA sensor.
Compounds
recognized by Interferon Regulatory Factor (IRF) can play a role in
immunoregulation by TLRs
and other pattern recognition receptors.
[0159] Imiquimod, a synthetic TLR7 agonist, is currently approved for human
therapeutic
applications. Contained in a cream and marketed under the brand name Aldara,
imiquimod serves
as a topical treatment for a variety of indications with immune components,
such as, actinic
keratosis, genital warts, and basal cell carcinomas. In addition, imiquimod is
indicated as a
candidate adjuvant for enhancing adaptive immune responses when applied
topically at an
immunization site.
[0160] Another type of immune stimulatory molecular motif, damage-associated
molecular
pattern molecules (DAMPs), can initiate and maintain an immune response
occurring as part of
the non-infectious inflammatory response. DAMPs can be specially localized
proteins that, when
detected by the immune system in a location other than where DAMPs should be
located,
activate the immune system. Often, DAMPs can be nuclear or cytosolic proteins
and upon release
from the nucleus or cytosol, DAMP proteins can become denatured through
oxidation. Examples
of DAMP proteins can include chromatin-associated protein high-mobility group
box 1
(HMGB1), S100 molecules of the calcium modulated family of proteins and
glycans, such as
hyaluronan fragments, and glycan conjugates. DAMPs can also be nucleic acids,
such as DNA,
when released from tumor cells following apoptosis or necrosis. Examples of
additional DAMP
nucleic acids can include RNA and purine metabolites, such as ATP, adenosine
and uric acid,
present outside of the nucleus or mitochondria.
[0161] Therapeutic application of DAMPs can focus on indications with an
immune component,
such as arthritis, cancer, ischemia-reperfusion injury, myocardial infarction
and stroke. In these
indications, the mechanism of action for DAMP therapeutic effects can include
the prevention of
DAMP release using therapeutic strategies, such as proapoptotic interventions,
platinum and
ethyl pyruvate, extracellular neutralization or blockade of DAMP release or
signaling using
therapeutic strategies such as anti-HMGB1, rasburiaspect and sRAGE, as well as
direct or
indirect blockade of DAMP receptors, and downstream signaling events, using
therapeutic
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strategies such as RAGE small molecule antagonists; TLR4 antagonists and
antibodies to
DAMP-R.
[0162] Additionally, the immune response elicited by TLR agonists can further
be enhanced
when co-administered with a CD40-agonist antibody. For example, co-
administration of a TLR
agonist such as poly IC:LC with a CD40-agonist antibody can synergize to
stimulate a greater
CD8+ T cell response than either agonist alone.
[0163] However, therapeutic use of PAMPs and DAMPs or other mechanisms of
intervention
can be limited because systemic activation of PAMP and DAMP signaling pathways
can have
life-threatening consequences due to cytokine syndrome-induced or cytokine
storm-induced toxic
shock syndrome. Accordingly, there is a critical need for therapeutic,
clinically relevant targeted
delivery of PAMP and DAMP agonists for safe and effective strategies to
enhance immune
responses. The presently described conjugate can be utilized as a safe and
effective strategy to
enhance immune responses. A conjugate can comprise an antibody construct and
an immune-
stimulatory compound.
Antibody Construct
[0164] An antibody construct can comprise an antigen binding domain. An
antigen binding
domain can be a domain that can specifically bind to an antigen. An antigen
binding domain can
be an antigen-binding portion of an antibody or an antibody fragment. An
antigen binding
domain can be one or more fragments of an antibody that can retain the ability
to specifically
bind to an antigen. An antigen binding domain can be any antigen binding
fragment. An antigen
binding domain can recognize a single antigen. An antigen binding domain can
recognize, for
example, two, three, four, five, six, seven, eight, nine, ten, or more
antigens. An antibody
construct can contain, for example, two, three, four, five, six, seven, eight,
nine, ten, or more
antigen binding domains. An antibody construct can contain two antigen binding
domains in
which each antigen binding domain can recognize the same antigen. An antibody
construct can
contain two antigen binding domains in which each antigen binding domain can
recognize
different antigens. An antigen binding domain can be in a scaffold, in which a
scaffold is a
supporting framework for the antigen binding domain. An antigen binding domain
can be in a
non-antibody scaffold. An antigen binding domain can be in an antibody
scaffold. An antibody
construct can comprise an antigen binding domain in a scaffold. The antibody
construct can
comprise a Fc fusion protein product. In some embodiments, the antibody
construct is a Fc fusion
protein product.
[0165] The antigen binding domain of an antibody construct can be selected
from any domain
that binds the antigen including, but not limited to, from a monoclonal
antibody, a polyclonal
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antibody, a recombinant antibody, or a functional fragment thereof, for
example, a heavy chain
variable domain (VH) and a light chain variable domain (VL), a DARPin, an
affimer, an avimer, a
knottin, a monobody, an affinity clamp, an ectodomain, a receptor ectodomain,
a receptor, a
cytokine, a ligand, an immunocytokine, a T cell receptor, or a recombinant T
cell receptor. The
antigen binding domain of an antibody construct can be at least 80% homologous
to an antigen
binding domain selected from, but not limited to, a monoclonal antibody, a
polyclonal antibody,
a recombinant antibody, or a functional fragment thereof, for example, a heavy
chain variable
domain (VH) and a light chain variable domain (VL), a DARPin, an affimer, an
avimer, a knottin,
a monobody, an affinity clamp, an ectodomain, a receptor ectodomain, a
receptor, a cytokine, a
ligand, an immunocytokine, a T cell receptor, or a recombinant T cell
receptor.
[0166] An antigen binding domain of an antibody construct, for example an
antigen binding
domain from a monoclonal antibody, can comprise a light chain and a heavy
chain. In one aspect,
the monoclonal antibody binds to CD40 and comprises the light chain of an anti-
CD40 antibody
and the heavy chain of an anti-CD40 antibody, which bind a CD40 antigen. In
another aspect, the
monoclonal antibody binds to a tumor antigen and comprises the light chain of
a tumor antigen
antibody and the heavy chain of a tumor antigen antibody, which bind the tumor
antigen.
[0167] An antibody construct can be an antibody. An antibody can consist of
two identical light
protein chains and two identical heavy protein chains, all held together
covalently by precisely
located disulfide linkages. The N-terminal regions of the light and heavy
chains together can
form the antigen recognition site of an antibody. Structurally, various
functions of an antibody
can be confined to discrete protein domains (i.e., regions). The sites that
can recognize and can
bind antigen can consist of three complementarity determining regions (CDRs)
that can lie within
the variable heavy chain region and variable light chain region at the N-
terminal end of the heavy
chain and the light chain. The constant domains can provide the general
framework of the
antibody and may not be involved directly in binding the antibody to an
antigen, but can be
involved in various effector functions, such as participation of the antibody
in antibody-
dependent cellular cytotoxicity, and can bind Fc receptors.
[0168] The domains of natural light and heavy chains can have the same general
structures, and
each domain can comprise four framework regions, whose sequences can be
somewhat
conserved, connected by three hyper-variable regions or CDRs. The four
framework regions can
largely adopt a (3-sheet conformation and the CDRs can form loops connecting,
and in some
aspects forming part of, the 0 -sheet structure. The CDRs in each chain can be
held in close
proximity by the framework regions and, with the CDRs from the other chain,
can contribute to
the formation of the antigen binding site.
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[0169] An antibody of an antibody construct can include an antibody of any
type, which can be
assigned to different classes of immunoglobins, e.g., IgA, IgD, IgE, IgG, and
IgM. Several
different classes can be further divided into isotypes, e.g., IgGl, IgG2,
IgG3, IgG4, IgAl, and
IgA2. An antibody can further comprise a light chain and a heavy chain, often
more than one
chain. The heavy-chain constant regions (Fc) that corresponds to the different
classes of
immunoglobulins can be a, 6, , y, and 11, respectively. The light chains can
be one of either
kappa or lc and lambda or k, based on the amino acid sequences of the constant
domains. The Fc
region can contain an Fc domain. An Fc receptor can bind an Fc domain.
Antibody constructs
can also include any fragment or recombinant forms thereof, including but not
limited to, single
chain variable fragments (scFvs), 'T-bodies', anti-calins, centyrins,
affibodies, domain
antibodies, or peptibodies.
[0170] An antibody can comprise an antigen binding domain, which can refer to
a portion of an
antibody comprising the antigen recognition portion, i.e., an antigenic
determining variable
region of an antibody sufficient to confer recognition of the antigen and
binding of the antigen
recognition portion to a target, such as an antigen, i.e., the epitope.
Examples of antibody binding
domains can include, but are not limited to, Fab, scFv, variable Fv fragment,
and other antibody
fragments, combinations of fragments or types of fragments known or knowable
to one of
ordinary skill in the art.
[0171] An antibody construct can comprise an antigen binding domain of an
antibody. An
antigen binding domain of an antibody can comprise one or more light chain
(LC) CDRs and one
or more heavy chain (HC) CDRs. For example, an antibody binding domain of an
antibody can
comprise one or more of the following: a light chain complementary determining
region 1 (LC
CDR1), a light chain complementary determining region 2 (LC CDR2), or a light
chain
complementary determining region 3 (LC CDR3). For another example, an antibody
binding
domain can comprise one or more of the following: a heavy chain complementary
determining
region 1 (HC CDR1), a heavy chain complementary determining region 2 (HC
CDR2), or a
heavy chain complementary determining region 3 (HC CDR3). As an additional
example, an
antibody binding domain of an antibody can comprise one or more of the
following: LC CDR1,
LC CDR2, LC CDR3, HC CDR1, HC CDR2, and HC CDR3.
[0172] An antibody construct can comprise an antibody fragment. An antibody
fragment can
include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL
and CHi domains;
(ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments
linked by a disulfide
bridge at the hinge region; and (iii) a Fv fragment consisting of the VL and
VH domains of a
single arm of an antibody. Although the two domains of the Fv fragment, VL and
VH, can be
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coded for by separate genes, they can be linked by a synthetic linker to be
made as a single
protein chain in which the VL and VH regions pair to form monovalent
molecules.
[0173] F(ab')2 and Fab' moieties can be produced by treating immunoglobulin
(e.g., monoclonal
antibody) with a protease such as pepsin and papain, and can include an
antibody fragment
generated by digesting immunoglobulin near the disulfide bonds existing
between the hinge
regions in each of the two H chains. The Fab fragment can also contain the
constant domain of
the light chain and the first constant domain (CH1) of the heavy chain. Fab'
fragments can differ
from Fab fragments by the addition of a few residues at the carboxyl terminus
of the heavy chain
CHi domain including one or more cysteine(s) from the antibody hinge region.
[0174] An Fv can be the minimum antibody fragment which contains a complete
antigen-
recognition and antigen-binding site. This region can consist of a dimer of
one heavy chain and
one light chain variable domain in tight, non-covalent association. In this
configuration the three
hypervariable regions of each variable domain can interact to define an
antigen-binding site on
the surface of the VH-VL dimer. A single variable domain (or half of an Fv
comprising only three
hypervariable regions specific for an antigen) can recognize and bind antigen,
although the
binding can be at a lower affinity than the affinity of the entire binding
site.
[0175] An antibody used herein can be "humanized." Humanized forms of non-
human (e.g.,
murine) antibodies can be chimeric immunoglobulins, immunoglobulin chains or
fragments
thereof (such as Fv, Fab, Fab', F(ab')2 or other target-binding subdomains of
antibodies), which
can contain minimal sequences derived from non-human immunoglobulin. In
general, the
humanized antibody can comprise substantially all of at least one, and
typically two, variable
domains, in which all or substantially all of the CDR regions correspond to
those of a non-human
immunoglobulin and all or substantially all of the FR regions are those of a
human
immunoglobulin sequence. The humanized antibody can also comprise at least a
portion of an
immunoglobulin constant region (Fc), typically that of a human immunoglobulin
consensus
sequence.
[0176] An antibody described herein can be a human antibody. As used herein,
"human
antibodies" can include antibodies having, for example, the amino acid
sequence of a human
immunoglobulin and can include antibodies isolated from human immunoglobulin
libraries or
from animals transgenic for one or more human immunoglobulins that do not
express
endogenous immunoglobulins. Human antibodies can be produced using transgenic
mice which
are incapable of expressing functional endogenous immunoglobulins, but which
can express
human immunoglobulin genes. Completely human antibodies that recognize a
selected epitope
can be generated using guided selection. In this approach, a selected non-
human monoclonal
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antibody, e.g., a mouse antibody, can be used to guide the selection of a
completely human
antibody recognizing the same epitope.
[0177] An antibody described herein can be a bispecific antibody or a dual
variable domain
antibody (DVD). Bispecific and DVD antibodies can be monoclonal, often human
or humanized,
antibodies that can have binding specificities for at least two different
antigens.
[0178] An antibody described herein can be a derivatized antibody. For
example, derivatized
antibodies can be modified by glycosylation, acetylation, pegylation,
phosphorylation, amidation,
derivatization by known protecting/blocking groups, proteolytic cleavage,
linkage to a cellular
ligand or other protein.
[0179] An antibody described herein can have a sequence that has been modified
to alter at least
one constant region-mediated biological effector function relative to the
corresponding wild type
sequence. For example, in some embodiments, the antibody can be modified to
reduce at least
one constant region-mediated biological effector function relative to an
unmodified antibody, e.g.,
reduced binding to the Fc receptor (FcR). FcR binding can be reduced by, for
example, mutating
the immunoglobulin constant region segment of the antibody at particular
regions necessary for
FcR interactions.
[0180] An antibody described herein can be modified to acquire or improve at
least one constant
region-mediated biological effector function relative to an unmodified
antibody, e.g., to enhance
FcyR interactions. For example, an antibody with a constant region that binds
FcyRIIA, FcyRIIB
and/or FcyRIIIA with greater affinity than the corresponding wild type
constant region can be
produced according to the methods described herein.
[0181] An antibody described herein can bind to tumor cells, such as an
antibody against a cell
surface receptor or a tumor antigen. An antibody described herein can bind to
CD40, such as an
antibody that can be a CD40 agonist and bind to CD40.
[0182] As used herein, the abbreviations for the natural 1-enantiomeric amino
acids are
conventional and can be as follows: alanine (A, Ala); arginine (R, Arg);
asparagine (N, Asn);
aspartic acid (D, Asp); cysteine (C, Cys); glutamic acid (E, Glu); glutamine
(Q, Gln); glycine (G,
Gly); histidine (H, His); isoleucine (I, Ile); leucine (L, Leu); lysine (K,
Lys); methionine (M,
Met); phenylalanine (F, Phe); proline (P, Pro); serine (S, Ser); threonine (T,
Thr); tryptophan (W,
Trp); tyrosine (Y, Tyr); valine (V, Val). Unless otherwise specified, X can
indicate any amino
acid. In some aspects, X can be asparagine (N), glutamine (Q), histidine (H),
lysine (K), or
arginine (R).
[0183] An antibody construct can comprise an anti-CD40 antibody. An antibody
construct can
comprise an antibody light chain. A light chain can be a light chain of an
anti-CD40 antibody
which can bind a CD40 antigen. A light chain of an anti-CD40 antibody can be
expressed from a
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DNA sequence comprising
ATGAGGCTCCCTGCTCAGCTCCTGGGGCTCCTGCTGCTCTGGTTCCCAGGTTCCAGAT
GCGACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAG
TCACCATCACTTGTCGGGCGAGTCAGGGTATTTACAGCTGGTTAGCCTGGTATCAGC
AGAAACCAGGGAAAGCCCCTAACCTCCTGATCTATACTGCATCCACTTTACAAAGTG
GGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCA
GCAGCCTGCAACCTGAAGATTTTGCAACTTACTATTGTCAACAGGCTAACATTTTCCC
GCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGAACTGTGGCTGCACCAT
CTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGT
GTGCCTGCTGAATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATA
ACGCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGAC
AGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACA
CAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGA
GCTTCAACAGGGGAGAGTGTTAG (SEQ ID NO: 1). A light chain of an anti-CD40 antibody
can be expressed from DNA sequence comprising greater than 70%, greater than
75%, greater
than 80%, greater than 85%, greater than 90%, greater than 95%, or greater
than 99% homology
to SEQ ID NO: 1. A variable region of a light chain of an anti-CD40 antibody
can be expressed
from a DNA sequence comprising
GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGAGACAGAGTC
ACCATCACTTGTCGGGCGAGTCAGGGTATTTACAGCTGGTTAGCCTGGTATCAGCAG
AAACCAGGGAAAGCCCCTAACCTCCTGATCTATACTGCATCCACTTTACAAAGTGGG
GTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGC
AGCCTGCAACCTGAAGATTTTGCAACTTACTATTGTCAACAGGCTAACATTTTCCCGC
TCACTTTCGGCGGAGGGACCAAGGTGGAGATCAA (SEQ ID NO: 3). A variable region of
a light chain of an anti-CD40 antibody can be expressed from a DNA sequence
comprising
greater than 70%, greater than 75%, greater than 80%, greater than 85%,
greater than 90%,
greater than 95% or greater than 99% homology to SEQ ID NO: 3. Additionally,
anti-CD40
antibodies expressed from SEQ ID NO: 1, or expressed from a DNA sequence
comprising
greater than 70% homology to SEQ ID NO: 1 can have a dissociation constant
(Kd) for CD40
that is less than lOnM. Anti-CD40 antibodies expressed from SEQ ID NO: 1, or
expressed from a
DNA sequence comprising greater than 70% homology to SEQ ID NO: 1 can have a
dissociation
constant (Kd) for CD40 that is less than 1 nM, less than 100 pM, less than 10
pM, less than 1 pM,
or less than 0.1 pM. The anti-CD40 light chain can be expressed with any anti-
CD40 heavy chain
or fragment thereof. The anti-CD40 light chain can also expressed with any
anti-CD40 heavy
chain or fragment thereof to form an anti-CD40 antibody or fragment thereof.
The anti-CD40
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antibody or fragment thereof can be purified, and can be combined with a
pharmaceutically
acceptable carrier. The anti-CD40 antibody can be an antibody construct.
[0184] A light chain of an anti-CD40 antibody can comprise an amino acid
sequence
MRLPAQLLGLLLLWFPGSRCDIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQK
PGKAPNLLIYTASTLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQANIFPLTFGGG
TKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQ
ESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID
NO: 4). A light chain of an anti-CD40 antibody can comprise an amino sequence
with greater
than 70%, greater than 75%, greater than 80%, greater than 85%, greater than
90%, greater than
95% or greater than 99% homology to SEQ ID NO: 4. A variable region of a light
chain of an
anti-CD40 antibody can comprise an amino acid sequence
DIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPGKAPNLLIYTASTLQSGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQANIFPLTFGGGTKVEIK (SEQ ID NO: 6). A
variable region of a light chain of an anti-CD40 antibody can comprise an
amino acid sequence
with greater than 70%, greater than 75%, greater than 80%, greater than 85%,
greater than 90%,
greater than 95% or greater than 99% homology to SEQ ID NO: 6. Additionally,
anti-CD40
antibodies comprising SEQ ID NO: 4, or comprising an amino acid sequence with
greater than
70% homology to SEQ ID NO: 4 can have a dissociation constant (Kd) for CD40
that is less than
lOnM. Anti-CD40 antibodies comprising SEQ ID NO: 4, or comprising an amino
acid sequence
with greater than 70% homology to SEQ ID NO: 4 can have a dissociation
constant (Kd) for
CD40 that is less than 1 nM, less than 100 pM, less than 10 pM, less than 1
pM, or less than 0.1
pM. The anti-CD40 light chain can be purified, and can be combined with a
pharmaceutically
acceptable carrier. The anti-CD40 light chain can be combined with any anti-
CD40 heavy chain
or fragment thereof. The anti-CD40 light chain can also be combined with any
anti-CD40 heavy
chain or fragment thereof to form an anti-CD40 antibody or fragment thereof.
The anti-CD40
antibody or fragment thereof can be purified, and can be combined with a
pharmaceutically
acceptable carrier. The anti-CD40 antibody can be an antibody construct.
Additionally, one
skilled in the art would recognize that these same concepts could apply to
anti-CD40 antibodies
created for use in the veterinary sciences and/or in laboratory animals.
[0185] An antibody construct can comprise an antibody light chain. A light
chain can be a light
chain of an anti-CD40 antibody which can bind a CD40 antigen. A light chain of
an anti-CD40
antibody can be SBT-040 VL-Ck. SBT-040 VL-Ck can comprise an amino acid
sequence
DIQMTQSPSSVSASVGDRVTITCRASQGIYSWLAWYQQKPGKAPNLLIYTASTLQSGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQANIFPLTFGGGTKVEIKRTVAAPSVFIFPPSD
EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL
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SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 26). SBT-040 VL-Ck can
comprise an amino acid sequence with greater than 70%, greater than 75%,
greater than 80%,
greater than 85%, greater than 90%, greater than 95% or greater than 99%
homology to SEQ ID
NO: 26.
[0186] A light chain of an anti-CD40 antibody can comprise a CDR. A light
chain of an anti-
CD40 antibody can comprise a CDR with an amino acid sequence QGIYSW (SEQ ID
NO: 27).
A light chain of an anti-CD40 antibody can comprise a CDR with an amino acid
sequence TAS
(SEQ ID NO: 28). A light chain of an anti-CD40 antibody can comprise a CDR
with an amino
acid sequence QQANIFPLT (SEQ ID NO: 29). A light chain CDR of an anti-CD40
antibody can
comprise an amino acid sequence with greater than 70%, greater than 75%,
greater than 80%,
greater than 85%, greater than 90%, greater than 95% or greater than 99%
homology to SEQ ID
NO: 27. A light chain CDR of an anti-CD40 antibody can comprise an amino acid
sequence with
greater than 70%, greater than 75%, greater than 80%, greater than 85%,
greater than 90%,
greater than 95% or greater than 99% homology to SEQ ID NO: 28. A light chain
CDR of an
anti-CD40 antibody can comprise an amino acid sequence with greater than 70%,
greater than
75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or
greater than
99% homology to SEQ ID NO: 29.
[0187] An antibody construct can comprise an antibody heavy chain. A heavy
chain can be a
heavy chain of an anti-CD40 antibody which can bind a CD40 antigen. A heavy
chain of an anti-
CD40 antibody can be an IgG1 isotype. A heavy chain of an anti-CD40 antibody
can be
Dacetuzumab. Dacetuzumab can comprise an amino acid sequence
EVQLVESGGGLVQPGGSLRLSCAASGYSFTGYYIHWVRQAPGKGLEWVARVIPNAGGT
SYNQKFKGRFTLSVDNSKNTAYLQMNSLRAEDTAVYYCAREGIYWWGQGTLVTVSSA
STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSG
LYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGP
SVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY
NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR
EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK
SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 38). Dacetuzumab can
comprise an amino acid sequence with greater than 70%, greater than 75%,
greater than 80%,
greater than 85%, greater than 90%, greater than 95% or greater than 99%
homology to SEQ ID
NO: 38. A heavy chain of an anti-CD40 antibody can comprise a CDR. A heavy
chain of an anti-
CD40 antibody can comprise a CDR with an amino acid sequence GYSFTGYY (SEQ ID
NO:
39). A heavy chain of an anti-CD40 antibody can comprise a CDR with an amino
acid sequence
VIPNAGGT (SEQ ID NO: 40). A heavy chain of an anti-CD40 antibody can comprise
a CDR
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with an amino acid sequence AREGIYW (SEQ ID NO: 41). A heavy chain CDR of an
anti-
CD40 antibody can comprise an amino acid sequence with greater than 70%,
greater than 75%,
greater than 80%, greater than 85%, greater than 90%, greater than 95% or
greater than 99%
homology to SEQ ID NO: 39. A heavy chain CDR of an anti-CD40 antibody can
comprise an
amino acid sequence with greater than 70%, greater than 75%, greater than 80%,
greater than
85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID
NO: 40. A
heavy chain CDR of an anti-CD40 antibody can comprise an amino acid sequence
with greater
than 70%, greater than 75%, greater than 80%, greater than 85%, greater than
90%, greater than
95% or greater than 99% homology to SEQ ID NO: 41. The two-dimensional
structure of the
dacetuzumab heavy chain is shown in FIGURE 16.
[0188] An antibody construct can comprise an antibody light chain. A light
chain can be a light
chain of an anti-CD40 antibody which can bind a CD40 antigen. A light chain of
an anti-CD40
antibody can be dacetuzumab. Dacetuzumab can comprise an amino acid sequence
DIQMTQSPSSLSASVGDRVTITCRSSQSLVHSNGNTFLHWYQQKPGKAPKLLIYTVSNRF
SGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCSQTTHVPWTFGQGTKVEIKRTVAAPSVFI
FPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLS
STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 42). Dacetuzumab
can comprise an amino acid sequence with greater than 70%, greater than 75%,
greater than 80%,
greater than 85%, greater than 90%, greater than 95% or greater than 99%
homology to SEQ ID
NO: 42. A light chain of an anti-CD40 antibody can comprise a CDR. A light
chain of an anti-
CD40 antibody can comprise a CDR with an amino acid sequence QSLVHSNGNTF (SEQ
ID
NO: 43). A light chain of an anti-CD40 antibody can comprise a CDR with an
amino acid
sequence TVS (SEQ ID NO: 44). A light chain of an anti-CD40 antibody can
comprise a CDR
with an amino acid sequence SQTTHVPWT (SEQ ID NO: 45). A light chain CDR of an
anti-
CD40 antibody can comprise an amino acid sequence with greater than 70%,
greater than 75%,
greater than 80%, greater than 85%, greater than 90%, greater than 95% or
greater than 99%
homology to SEQ ID NO: 43. A light chain CDR of an anti-CD40 antibody can
comprise an
amino acid sequence with greater than 70%, greater than 75%, greater than 80%,
greater than
85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID
NO: 44. A
light chain CDR of an anti-CD40 antibody can comprise an amino acid sequence
with greater
than 70%, greater than 75%, greater than 80%, greater than 85%, greater than
90%, greater than
95% or greater than 99% homology to SEQ ID NO: 45. The two-dimensional
structure of the
Dacetuzumab light chain is shown in FIGURE 17.
[0189] An antibody construct can comprise an antibody heavy chain. A heavy
chain can be a
heavy chain of an anti-CD40 antibody which can bind a CD40 antigen. A heavy
chain of an anti-
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CD40 antibody can be an IgG4 isotype. A heavy chain of an anti-CD40 antibody
can be
Bleselumab. Bleselumab can comprise an amino acid sequence
QLQLQESGPGLLKPSETLSLTCTVSGGSISSPGYYGGWIRQPPGKGLEWIGSIYKSGSTYH
NPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCTRPVVRYFGWFDPWGQGTLVTVS
SASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFEGGPS
VFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNS
TYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEE
MTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSR
WQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 46). Bleselumab can comprise
an amino acid sequence with greater than 70%, greater than 75%, greater than
80%, greater than
85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID
NO: 46. A
heavy chain of an anti-CD40 antibody can comprise a CDR. A heavy chain of an
anti-CD40
antibody can comprise a CDR with an amino acid sequence GGSISSPGYY (SEQ ID NO:
47). A
heavy chain of an anti-CD40 antibody can comprise a CDR with an amino acid
sequence
IYKSGST (SEQ ID NO: 48). A heavy chain of an anti-CD40 antibody can comprise a
CDR with
an amino acid sequence TRPVVRYFGWFDP (SEQ ID NO: 49). A heavy chain CDR of an
anti-
CD40 antibody can comprise an amino acid sequence with greater than 70%,
greater than 75%,
greater than 80%, greater than 85%, greater than 90%, greater than 95% or
greater than 99%
homology to SEQ ID NO: 47. A heavy chain CDR of an anti-CD40 antibody can
comprise an
amino acid sequence with greater than 70%, greater than 75%, greater than 80%,
greater than
85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID
NO: 48. A
heavy chain CDR of an anti-CD40 antibody can comprise an amino acid sequence
with greater
than 70%, greater than 75%, greater than 80%, greater than 85%, greater than
90%, greater than
95% or greater than 99% homology to SEQ ID NO: 49. The two-dimensional
structure of the
bleselumab heavy chain is shown in FIGURE 18.
[0190] An antibody construct can comprise an antibody light chain. A light
chain can be a light
chain of an anti-CD40 antibody which can bind a CD40 antigen. A light chain of
an anti-CD40
antibody can be Bleselumab. Bleselumab can comprise an amino acid sequence
AIQLTQSPSSLSASVGDRVTITCRASQGISSALAWYQQKPGKAPKLLIYDASNLESGVPSR
FSGSGSGTDFTLTISSLQPEDFATYYCQQFNSYPTFGQGTKVEIKRTVAAPSVFIFPPSDEQ
LKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK
ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 50). Bleselumab can comprise
an amino acid sequence with greater than 70%, greater than 75%, greater than
80%, greater than
85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID
NO: 50. A
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light chain of an anti-CD40 antibody can comprise a CDR. A light chain of an
anti-CD40
antibody can comprise a CDR with an amino acid sequence QGISSA (SEQ ID NO:
51). A light
chain of an anti-CD40 antibody can comprise a CDR with an amino acid sequence
DAS (SEQ ID
NO: 52). A light chain of an anti-CD40 antibody can comprise a CDR with an
amino acid
sequence QQFNSYPT (SEQ ID NO: 53). A light chain CDR of an anti-CD40 antibody
can
comprise an amino acid sequence with greater than 70%, greater than 75%,
greater than 80%,
greater than 85%, greater than 90%, greater than 95% or greater than 99%
homology to SEQ ID
NO: 51. A light chain CDR of an anti-CD40 antibody can comprise an amino acid
sequence with
greater than 70%, greater than 75%, greater than 80%, greater than 85%,
greater than 90%,
greater than 95% or greater than 99% homology to SEQ ID NO: 52. A light chain
CDR of an
anti-CD40 antibody can comprise an amino acid sequence with greater than 70%,
greater than
75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or
greater than
99% homology to SEQ ID NO: 53. The two-dimensional structure of the bleselumab
light chain
is shown in FIGURE 19.
[0191] An antibody construct can comprise an antibody heavy chain. A heavy
chain can be a
heavy chain of an anti-CD40 antibody which can bind a CD40 antigen. A heavy
chain of an anti-
CD40 antibody can be an IgG1 isotype. Lucatumumab can comprise an amino acid
sequence
QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWVAVISYEESNR
YHADSVKGRFTISRDNSKITLYLQMNSLRTEDTAVYYCARDGGIAAPGPDYWGQGTLV
TVSSASTKGPSVFPLAPASKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV
LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPE
LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR
EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL
PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT
VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 54). Lucatumumab
can comprise an amino acid sequence with greater than 70%, greater than 75%,
greater than 80%,
greater than 85%, greater than 90%, greater than 95% or greater than 99%
homology to SEQ ID
NO: 54. A heavy chain of an anti-CD40 antibody can comprise a CDR. A heavy
chain of an anti-
CD40 antibody can comprise a CDR with an amino acid sequence GFTFSSYG (SEQ ID
NO:
55). A heavy chain of an anti-CD40 antibody can comprise a CDR with an amino
acid sequence
ISYEESNR (SEQ ID NO: 56). A heavy chain of an anti-CD40 antibody can comprise
a CDR
with an amino acid sequence ARDGGIAAPGPDY (SEQ ID NO: 57). A heavy chain CDR
of an
anti-CD40 antibody can comprise an amino acid sequence with greater than 70%,
greater than
75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or
greater than
99% homology to SEQ ID NO: 55. A heavy chain CDR of an anti-CD40 antibody can
comprise
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an amino acid sequence with greater than 70%, greater than 75%, greater than
80%, greater than
85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID
NO: 56. A
heavy chain CDR of an anti-CD40 antibody can comprise an amino acid sequence
with greater
than 70%, greater than 75%, greater than 80%, greater than 85%, greater than
90%, greater than
95% or greater than 99% homology to SEQ ID NO: 57. The two-dimensional
structure of the
lucatumumab heavy chain is shown in FIGURE 20.
[0192] An antibody construct can comprise an antibody light chain. A light
chain can be a light
chain of an anti-CD40 antibody which can bind a CD40 antigen. A light chain of
an anti-CD40
antibody can be Lucatumumab. Lucatumumab can comprise an amino acid sequence
DIVMTQSPLSLTVTPGEPASISCRSSQSLLYSNGYNYLDWYLQKPGQSPQVLISLGSNRAS
GVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQARQTPFTFGPGTKVDIRRTVAAPSVF
IFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL
SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 58).
Lucatumumab can comprise an amino acid sequence with greater than 70%, greater
than 75%,
greater than 80%, greater than 85%, greater than 90%, greater than 95% or
greater than 99%
homology to SEQ ID NO: 59. A light chain of an anti-CD40 antibody can comprise
a CDR with
an amino acid sequence LGS (SEQ ID NO: 60). A light chain of an anti-CD40
antibody can
comprise a CDR with an amino acid sequence MQARQTPFT (SEQ ID NO: 61). A light
chain
CDR of an anti-CD40 antibody can comprise an amino acid sequence with greater
than 70%,
greater than 75%, greater than 80%, greater than 85%, greater than 90%,
greater than 95% or
greater than 99% homology to SEQ ID NO: 59. A light chain CDR of an anti-CD40
antibody can
comprise an amino acid sequence with greater than 70%, greater than 75%,
greater than 80%,
greater than 85%, greater than 90%, greater than 95% or greater than 99%
homology to SEQ ID
NO: 60. A light chain CDR of an anti-CD40 antibody can comprise an amino acid
sequence with
greater than 70%, greater than 75%, greater than 80%, greater than 85%,
greater than 90%,
greater than 95% or greater than 99% homology to SEQ ID NO: 61. The two-
dimensional
structure of the lucatumumab light chain is shown in FIGURE 21.
[0193] An antibody construct can comprise an antibody heavy chain. A heavy
chain can be a
heavy chain of an anti-CD40 antibody which can bind a CD40 antigen. A heavy
chain of an anti-
CD40 antibody can be an IgG1 isotype. A heavy chain of an anti-CD40 antibody
can be ADC-
1013. ADC-1013 can comprise an amino acid sequence
EVQLLESGGGLVQPGGSLRLSCAASGFTFSTYGMHWVRQAPGKGLEWLSYISGGSSYIF
YADSVRGRFTISRDNSENALYLQMNSLRAEDTAVYYCARILRGGSGMDLWGQGTLVTV
SSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQ
SSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLG
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GPSVFLFPPKPKDTLMISRTPEVTCNAVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQ
YNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR
DELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK
SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 62). ADC-1013 can
comprise an amino acid sequence with greater than 70%, greater than 75%,
greater than 80%,
greater than 85%, greater than 90%, greater than 95% or greater than 99%
homology to SEQ ID
NO: 62. A heavy chain of an anti-CD40 antibody can comprise a CDR. A heavy
chain of an anti-
CD40 antibody can comprise a CDR with an amino acid sequence GFTFSTYG (SEQ ID
NO:
63). A heavy chain of an anti-CD40 antibody can comprise a CDR with an amino
acid sequence
ISGGSSYI (SEQ ID NO: 64). A heavy chain of an anti-CD40 antibody can comprise
a CDR
with an amino acid sequence ARILRGGSGMDL (SEQ ID NO: 65). A heavy chain CDR of
an
anti-CD40 antibody can comprise an amino acid sequence with greater than 70%,
greater than
75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or
greater than
99% homology to SEQ ID NO: 63. A heavy chain CDR of an anti-CD40 antibody can
comprise
an amino acid sequence with greater than 70%, greater than 75%, greater than
80%, greater than
85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID
NO: 64. A
heavy chain CDR of an anti-CD40 antibody can comprise an amino acid sequence
with greater
than 70%, greater than 75%, greater than 80%, greater than 85%, greater than
90%, greater than
95% or greater than 99% homology to SEQ ID NO: 65. The two-dimensional
structure of the
ADC-1013 heavy chain is shown in FIGURE 22.
[0194] An antibody construct can comprise an antibody light chain. A light
chain can be a light
chain of an anti-CD40 antibody which can bind a CD40 antigen. A light chain of
an anti-CD40
antibody can be ADC-1013. ADC-1013 can comprise an amino acid sequence
QSVLTQPPSASGTPGQRVTISCTGSSSNIGAGYNVYWYQQLPGTAPKLLIYGNINRPSGVP
DRFSGSKSGTSASLAISGLRSEDEADYYCAAWDKSISGLVFGGGTKLTVLGQPKAAPSVT
LFPPSSEELQANKATLVCLISDFYPGAVTVAWKADSSPVKAGVETTTPSKQSNNKYAAS
SYLSLTPEQWKSHRSYSCQVTHEGSTVEKTVAPTECS (SEQ ID NO: 66). ADC-1013 can
comprise an amino acid sequence with greater than 70%, greater than 75%,
greater than 80%,
greater than 85%, greater than 90%, greater than 95% or greater than 99%
homology to SEQ ID
NO: 66. A light chain of an anti-CD40 antibody can comprise a CDR. A light
chain of an anti-
CD40 antibody can comprise a CDR with an amino acid sequence SSNIGAGYN (SEQ ID
NO:
67). A light chain of an anti-CD40 antibody can comprise a CDR with an amino
acid sequence
GNI (SEQ ID NO: 68). A light chain of an anti-CD40 antibody can comprise a CDR
with an
amino acid sequence AAWDKSISGLV (SEQ ID NO: 69). A light chain CDR of an anti-
CD40
antibody can comprise an amino acid sequence with greater than 70%, greater
than 75%, greater
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than 80%, greater than 85%, greater than 90%, greater than 95% or greater than
99% homology
to SEQ ID NO: 67. A light chain CDR of an anti-CD40 antibody can comprise an
amino acid
sequence with greater than 70%, greater than 75%, greater than 80%, greater
than 85%, greater
than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 68. A
light chain
CDR of an anti-CD40 antibody can comprise an amino acid sequence with greater
than 70%,
greater than 75%, greater than 80%, greater than 85%, greater than 90%,
greater than 95% or
greater than 99% homology to SEQ ID NO: 69. The two-dimensional structure of
the ADC-1013
light chain is shown in FIGURE 23.
[0195] An antibody construct can comprise an antibody heavy chain. A heavy
chain can be a
heavy chain of an anti-CD40 antibody which can bind a CD40 antigen. A heavy
chain of an anti-
CD40 antibody can be the humanized rabbit antibody APX005. APX005 can comprise
an amino
acid sequence
QVQLVESGGGVVQPGRSLRLSCAASGFSFSS TYVCWVRQAPGKGLEWIACIYTGDGTN
YSASWAKGRFTISKDSSKNTVYLQMNSLRAEDTAVYFCARPDITYGFAINFWGPGTLVT
VSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPEL
LGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPRE
EQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLP
PSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTV
DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 70). APX005 can
comprise an amino acid sequence with greater than 70%, greater than 75%,
greater than 80%,
greater than 85%, greater than 90%, greater than 95% or greater than 99%
homology to SEQ ID
NO: 70. A heavy chain of an anti-CD40 antibody can comprise a CDR. A heavy
chain of an anti-
CD40 antibody can comprise a CDR with an amino acid sequence GFSFSSTY (SEQ ID
NO:
71). A heavy chain of an anti-CD40 antibody can comprise a CDR with an amino
acid sequence
IYTGDGTN (SEQ ID NO: 72). A heavy chain of an anti-CD40 antibody can comprise
a CDR
with an amino acid sequence ARPDITYGFAINFW (SEQ ID NO: 73). A heavy chain CDR
of an
anti-CD40 antibody can comprise an amino acid sequence with greater than 70%,
greater than
75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or
greater than
99% homology to SEQ ID NO: 71. A heavy chain CDR of an anti-CD40 antibody can
comprise
an amino acid sequence with greater than 70%, greater than 75%, greater than
80%, greater than
85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID
NO: 72. A
heavy chain CDR of an anti-CD40 antibody can comprise an amino acid sequence
with greater
than 70%, greater than 75%, greater than 80%, greater than 85%, greater than
90%, greater than
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95% or greater than 99% homology to SEQ ID NO: 73. The two-dimensional
structure of the
APX005 heavy chain is shown in FIGURE 24.
[0196] An antibody construct can comprise an antibody light chain. A light
chain can be a light
chain of an anti-CD40 antibody which can bind a CD40 antigen. A light chain of
an anti-CD40
antibody can be the humanized rabbit antibody APX005. APX005 can comprise an
amino acid
sequence
DIQMTQSPSSLSASVGDRVTIKCQASQSISSRLAWYQQKPGKPPKLLIYRASTLASGVPSR
FSGSGSGTDFTLTISSLQPEDVATYYCQCTGYGISWPIGGGTKVEIKRTVAAPSVFIFPPSD
EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL
SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 74). APX005 can comprise
an amino acid sequence with greater than 70%, greater than 75%, greater than
80%, greater than
85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID
NO: 74. A
light chain of an anti-CD40 antibody can comprise a CDR. A light chain of an
anti-CD40
antibody can comprise a CDR with an amino acid sequence QSISSR (SEQ ID NO:
75). A light
chain of an anti-CD40 antibody can comprise a CDR with an amino acid sequence
RAS (SEQ ID
NO: 76). A light chain of an anti-CD40 antibody can comprise a CDR with an
amino acid
sequence QCTGYGISWP (SEQ ID NO: 77). A light chain CDR of an anti-CD40
antibody can
comprise an amino acid sequence with greater than 70%, greater than 75%,
greater than 80%,
greater than 85%, greater than 90%, greater than 95% or greater than 99%
homology to SEQ ID
NO: 75. A light chain CDR of an anti-CD40 antibody can comprise an amino acid
sequence with
greater than 70%, greater than 75%, greater than 80%, greater than 85%,
greater than 90%,
greater than 95% or greater than 99% homology to SEQ ID NO: 76. A light chain
CDR of an
anti-CD40 antibody can comprise an amino acid sequence with greater than 70%,
greater than
75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or
greater than
99% homology to SEQ ID NO: 77. The two-dimensional structure of the APX005
light chain is
shown in FIGURE 25.
[0197] An antibody construct can comprise an antibody heavy chain. A heavy
chain can be a
heavy chain of an anti-CD40 antibody which can bind a CD40 antigen. A heavy
chain of an anti-
CD40 antibody can be Chi Lob 7/4. Chi Lob 7/4 can comprise an amino acid
sequence
EVQLQQSGPDLVKPGASVKISCKTSGYTFTEYIMHWVKQSHGKSLEWIGGIIPNNGGTSY
NQKFKDKATMTVDKSSSTGYMELRSLTSEDSAVYYCTRREVYGRNYYALDYWGQGTL
VTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPA
VLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAP
ELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP
REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYT
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LPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKL
TVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 78). Chi Lob 7/4
can comprise an amino acid sequence with greater than 70%, greater than 75%,
greater than 80%,
greater than 85%, greater than 90%, greater than 95% or greater than 99%
homology to SEQ ID
NO: 78. A heavy chain of an anti-CD40 antibody can comprise a CDR. A heavy
chain of an anti-
CD40 antibody can comprise a CDR with an amino acid sequence GYTFTEYI (SEQ ID
NO:
79). A heavy chain of an anti-CD40 antibody can comprise a CDR with an amino
acid sequence
IIPNNGGT (SEQ ID NO: 80). A heavy chain of an anti-CD40 antibody can comprise
a CDR
with an amino acid sequence TRREVYGRNYYALDY (SEQ ID NO: 81). A heavy chain CDR
of an anti-CD40 antibody can comprise an amino acid sequence with greater than
70%, greater
than 75%, greater than 80%, greater than 85%, greater than 90%, greater than
95% or greater
than 99% homology to SEQ ID NO: 79. A heavy chain CDR of an anti-CD40 antibody
can
comprise an amino acid sequence with greater than 70%, greater than 75%,
greater than 80%,
greater than 85%, greater than 90%, greater than 95% or greater than 99%
homology to SEQ ID
NO: 80. A heavy chain CDR of an anti-CD40 antibody can comprise an amino acid
sequence
with greater than 70%, greater than 75%, greater than 80%, greater than 85%,
greater than 90%,
greater than 95% or greater than 99% homology to SEQ ID NO: 81. The two-
dimensional
structure of the Chi Lob 7/4 heavy chain is shown in FIGURE 26.
[0198] An antibody construct can comprise an antibody light chain. A light
chain can be a light
chain of an anti-CD40 antibody which can bind a CD40 antigen. A light chain of
an anti-CD40
antibody can be Chi Lob 7/4. Chi Lob 7/4 can comprise an amino acid sequence
DIQMTQTTSSLSASLGDRVTITCSASQGINNYLNWYQQKPDGTVKLLIYYTSSLHSGVPS
RFSGSGSGTDYSLTISNLEPEDIATYYCQQYSNLPYTFGGGTKLEIKRTVAAPSVFIFPPSD
EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL
SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 82). Chi Lob 7/4 can
comprise an amino acid sequence with greater than 70%, greater than 75%,
greater than 80%,
greater than 85%, greater than 90%, greater than 95% or greater than 99%
homology to SEQ ID
NO: 82. A light chain of an anti-CD40 antibody can comprise a CDR. A light
chain of an anti-
CD40 antibody can comprise a CDR with an amino acid sequence QGINNY (SEQ ID
NO: 83).
A light chain of an anti-CD40 antibody can comprise a CDR with an amino acid
sequence YTS
(SEQ ID NO: 84). A light chain of an anti-CD40 antibody can comprise a CDR
with an amino
acid sequence QQYSNLPYT (SEQ ID NO: 85). A light chain CDR of an anti-CD40
antibody
can comprise an amino acid sequence with greater than 70%, greater than 75%,
greater than 80%,
greater than 85%, greater than 90%, greater than 95% or greater than 99%
homology to SEQ ID
NO: 83. A light chain CDR of an anti-CD40 antibody can comprise an amino acid
sequence with
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greater than 70%, greater than 75%, greater than 80%, greater than 85%,
greater than 90%,
greater than 95% or greater than 99% homology to SEQ ID NO: 84. A light chain
CDR of an
anti-CD40 antibody can comprise an amino acid sequence with greater than 70%,
greater than
75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or
greater than
99% homology to SEQ ID NO: 85. The two-dimensional structure of the Chi Lob
7/4 light chain
is shown in FIGURE 27.
[0199] An antibody construct can comprise an antibody heavy chain. A heavy
chain can be a
heavy chain of an anti-CD40 antibody which can bind a CD40 antigen. A heavy
chain of an anti-
CD40 antibody can be an IgG1 isotype. A heavy chain of an anti-CD40 antibody
can be SBT-
040-G1WT. SBT-040-G1WT can be expressed from a DNA sequence comprising
ATGGACTGGACCTGGAGGATCCTCTTCTTGGTGGCAGCAGCCACAGGAGCCCACTCC
CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAA
GGTCTCCTGCAAGGCTTCTGGATACACCTTCACCGGCTACTATATGCACTGGGTGCG
ACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAACCCTGACAGTGGTG
GCACAAACTATGCACAGAAGTTTCAGGGCAGGGTCACCATGACCAGGGACACGTCC
ATCAGCACAGCCTACATGGAGCTGAACAGGCTGAGATCTGACGACACGGCCGTGTA
TTACTGTGCGAGAGATCAGCCCCTAGGATATTGTACTAATGGTGTATGCTCCTACTTT
GACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCA
TCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCGGCCCTG
GGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGC
GCTCTGACCAGCGGCGTGCACACCTTCCCAGCTGTCCTACAGTCCTCAGGACTCTAC
TCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTTCGGCACCCAGACCTACACC
TGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGACAGTTGAGCCCAA
ATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGG
ACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGAC
CCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGT
TCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAG
GAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGAC
TGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCC
CATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACA
CCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTG
GTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCC
GGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCT
CTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCAT
GCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGT
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CCCCGGGTAAATGA (SEQ ID NO: 8). SBT-040-G1WT can be expressed from a DNA
sequence comprising greater than 70%, greater than 75%, greater than 80%,
greater than 85%,
greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO:
8. A variable
region of SBT-040-G1WT can be expressed from a DNA sequence comprising
CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAA
GGTCTCCTGCAAGGCTTCTGGATACACCTTCACCGGCTACTATATGCACTGGGTGCG
ACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAACCCTGACAGTGGTG
GCACAAACTATGCACAGAAGTTTCAGGGCAGGGTCACCATGACCAGGGACACGTCC
ATCAGCACAGCCTACATGGAGCTGAACAGGCTGAGATCTGACGACACGGCCGTGTA
TTACTGTGCGAGAGATCAGCCCCTAGGATATTGTACTAATGGTGTATGCTCCTACTTT
GACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAG (SEQ ID NO: 13). A
variable region of SBT-040-G1WT can be expressed from a DNA sequence
comprising greater
than 70%, greater than 75%, greater than 80%, greater than 85%, greater than
90%, greater than
95% or greater than 99% homology to SEQ ID NO: 13. Additionally, anti-CD40
antibodies
comprising SBT-040-G1WT expressed from SEQ ID NO: 8, or expressed from a DNA
sequence
comprising greater than 70% homology to SEQ ID NO: 8 can have a dissociation
constant (Kd)
for CD40 that is less than 10 nM. Anti-CD40 antibodies comprising SBT-040-G1WT
expressed
from DNA sequence comprising SEQ ID NO: 8, or comprising greater than 70%
homology to
SEQ ID NO: 8 can have a dissociation constant (Kd) for CD40 that is less than
1 nM, less than
100 pM, less than 10 pM, less than 1 pM, or less than 0.1 pM. SBT-040-G1WT can
be expressed
with any anti-CD40 light chain or fragment thereof. SBT-040-G1WT can also be
expressed with
any anti-CD40 light chain or fragment thereof to form an anti-CD40 antibody or
fragment
thereof. The anti-CD40 antibody or fragment thereof can be purified, and can
be combined with a
pharmaceutically acceptable carrier. The anti-CD40 antibody can be an antibody
construct.
Additionally, one skilled in the art would recognize that these same concepts
could apply to
antibody constructs comprising anti-CD40 antibodies created for use in the
veterinary sciences
and/or in laboratory animals.
[0200] SBT-040-G1WT can comprise an amino acid sequence
MDWTWRILFLVAAATGAHSQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWV
RQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVY
YCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGC
LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVD
HKPSNTKVDKTVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV
VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV
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EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT
QKSLSLSPGK (SEQ ID NO: 15). SBT-040-G1WT can comprise an amino acid sequence
with
greater than 70%, greater than 75%, greater than 80%, greater than 85%,
greater than 90%,
greater than 95% or greater than 99% homology to SEQ ID NO: 15. SBT-040-G1WT
can
comprise an amino acid sequence
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSG
GTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFD
YWGQGTLVTVSS (SEQ ID NO: 20). A variable region of SBT-040-G1WT can comprise
an
amino acid sequence with greater than 70%, greater than 75%, greater than 80%,
greater than
85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID
NO: 20.
Additionally, anti-CD40 antibodies comprising SBT-040-G1WT with SEQ ID NO: 15
or with an
amino acid sequence with greater than 70% homology to SEQ ID NO: 15 can have a
dissociation
constant (Kd) for CD40 that is less than 10 nM. Anti-CD40 antibodies
comprising SBT-040-
G1WT with SEQ ID NO: 15 or with an amino acid sequence with greater than 70%
homology to
SEQ ID NO: 15 can have a dissociation constant (Kd) for CD40 that is less than
1 nM, less than
100 pM, less than 10 pM, less than 1 pM, or less than 0.1 pM. SBT-040-G1WT can
be purified.
SBT-040-G1WT can be combined with any anti-CD40 light chain or fragment
thereof to form an
anti-CD40 antibody or fragment thereof. The anti-CD40 antibody or fragment
thereof can be
purified, and can be combined with a pharmaceutically acceptable carrier. The
anti-CD40
antibody can be an antibody construct. Additionally, one skilled in the art
would recognize that
these same concepts could apply to antibody constructs comprising anti-CD40
antibodies created
for use in the veterinary sciences and/or in laboratory animals.
[0201] An antibody construct can comprise an antibody heavy chain. A heavy
chain can be a
heavy chain of an anti-CD40 antibody which can bind a CD40 antigen. A heavy
chain of an anti-
CD40 antibody can be an IgG1 isotype. A heavy chain of an anti-CD40 antibody
can be SBT-
040 VH-hIgG1 wt. SBT-040 VH-hIgG1 wt can comprise an amino acid sequence with
greater
than 70%, greater than 75%, greater than 80%, greater than 85%, greater than
90%, greater than
95% or greater than 99% homology to an amino acid sequence
QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPDSG
GTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFD
YWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTS
GVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVEPKSCDKTH
TCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV
HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQ
PREPQVYTL
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PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT
VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 22). A heavy chain of
an anti-CD40 antibody can comprise a CDR. A heavy chain of an anti-CD40
antibody can
comprise a CDR with an amino acid sequence GYTFTYY (SEQ ID NO: 23). A heavy
chain of
an anti-CD40 antibody can comprise a CDR with an amino acid sequence INPDSGGT
(SEQ ID
NO: 24). A heavy chain of an anti-CD40 antibody can comprise a CDR with an
amino acid
sequence ARDQPLGYCTNGVCSYFDY (SEQ ID NO: 25). A heavy chain CDR of an anti-
CD40 antibody can comprise an amino acid sequence with greater than 70%,
greater than 75%,
greater than 80%, greater than 85%, greater than 90%, greater than 95% or
greater than 99%
homology to SEQ ID NO: 23. A heavy chain CDR of an anti-CD40 antibody can
comprise an
amino acid sequence with greater than 70%, greater than 75%, greater than 80%,
greater than
85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID
NO: 24. A
heavy chain CDR of an anti-CD40 antibody can comprise an amino acid sequence
with greater
than 70%, greater than 75%, greater than 80%, greater than 85%, greater than
90%, greater than
95% or greater than 99% homology to SEQ ID NO: 25.
[0202] A heavy chain of an anti-CD40 antibody can be an IgG2 isotype. A heavy
chain of an
anti-CD40 antibody can be SBT-040-G2. SBT-040-G2 be expressed from a DNA
sequence
comprising
ATGGACTGGACCTGGAGGATCCTCTTCTTGGTGGCAGCAGCCACAGGAGCCCACTCC
CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAA
GGTCTCCTGCAAGGCTTCTGGATACACCTTCACCGGCTACTATATGCACTGGGTGCG
ACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAACCCTGACAGTGGTG
GCACAAACTATGCACAGAAGTTTCAGGGCAGGGTCACCATGACCAGGGACACGTCC
ATCAGCACAGCCTACATGGAGCTGAACAGGCTGAGATCTGACGACACGGCCGTGTA
TTACTGTGCGAGAGATCAGCCCCTAGGATATTGTACTAATGGTGTATGCTCCTACTTT
GACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCA
TCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCGGCCCTG
GGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGC
GCTCTGACCAGCGGCGTGCACACCTTCCCAGCTGTCCTACAGTCCTCAGGACTCTAC
TCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTTCGGCACCCAGACCTACACC
TGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGACAGTTGAGCGCAA
ATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGACCGTCAGTCTT
CCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCAC
GTGCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGTACG
TGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAAC
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AGCACGTTCCGTGTGGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAACGGC
AAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAGCCCCCATCGAGAAAAC
CATCTCCAAAACCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCAT
CCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTC
TACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTA
CAAGACCACACCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCT
CACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGC
ATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAT
GA (SEQ ID NO: 7). SBT-040-G2 can be expressed from a DNA sequence comprising
greater
than 70%, greater than 75%, greater than 80%, greater than 85%, greater than
90%, greater than
95% or greater than 99% homology to SEQ ID NO: 7. A variable region of SBT-040-
G2 can be
expressed from a DNA sequence comprising SEQ ID NO: 13. A variable region of
SBT-040-G2
can be expressed from a DNA sequence comprising greater than 70%, greater than
75%, greater
than 80%, greater than 85%, greater than 90%, greater than 95% or greater than
99% homology
to SEQ ID NO: 13. Additionally, anti-CD40 antibodies comprising SBT-040-G2
expressed from
SEQ ID NO: 7, or expressed from a DNA sequence comprising greater than 70%
homology to
SEQ ID NO: 7 can have a dissociation constant (Kd) for CD40 that is less than
10 nM. Anti-
CD40 antibodies comprising SBT-040-G2 expressed from DNA sequence comprising
SEQ ID
NO: 7, or comprising greater than 70% homology to SEQ ID NO: 7 can have a
dissociation
constant (Kd) for CD40 that is less than 1 nM, less than 100 pM, less than 10
pM, less than 1 pM,
or less than 0.1 pM. SBT-040-G2 can be expressed with any anti-CD40 light
chain or fragment
thereof. SBT-040-G2 can also be expressed with any anti-CD40 light chain or
fragment thereof
to form an anti-CD40 antibody or fragment thereof. The anti-CD40 antibody or
fragment thereof
can be purified, and can be combined with a pharmaceutically acceptable
carrier. The anti-CD40
antibody can be an antibody construct. Additionally, one skilled in the art
would recognize that
these same concepts could apply to antibody constructs comprising anti-CD40
antibodies created
for use in the veterinary sciences and/or in laboratory animals.
[0203] SBT-040-G2 can comprise an amino acid sequence
MDWTWRILFLVAAAT GAHS QVQLVQS GAE VKKPGAS VKVS C KAS GYTFT GYYMHWV
RQAPGQGLEWMGWINPDS GGTNYAQKFQGRVTMTRDT S IS TAYMELNRLRS DDTAVY
YCARDQPLGYCTNGVCS YFDYWGQGTLVTVS SAS TKGPS VFPLAPCS RS TSES TAALGC
LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVD
HKPSNTKVDKTVERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVS
HEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVS
NKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESN
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GQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLS
LSPGK (SEQ ID NO: 14). SBT-040-G2 can comprise an amino acid sequence with
greater than
70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%,
greater than 95%
or greater than 99% homology to SEQ ID NO: 14. SBT-040-G1WT can comprise an
amino acid
sequence SEQ ID NO: 20. A variable region of SBT-040-G2 can comprise an amino
acid
sequence with greater than 70%, greater than 75%, greater than 80%, greater
than 85%, greater
than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 20.
Additionally,
anti-CD40 antibodies comprising SBT-040-G2 with SEQ ID NO: 14 or with an amino
acid
sequence with greater than 70% homology to SEQ ID NO: 14 can have a
dissociation constant
(Kd) for CD40 that is less than 10 nM. Anti-CD40 antibodies comprising SBT-040-
G2 with SEQ
ID NO: 14 or with an amino acid sequence with greater than 70% homology to SEQ
ID NO: 14
can have a dissociation constant (Kd) for CD40 that is less than 1 nM, less
than 100 pM, less than
pM, less than 1 pM, or less than 0.1 pM. SBT-040-G2 can be purified. SBT-040-
G2 can be
combined with any anti-CD40 light chain or fragment thereof to form an anti-
CD40 antibody or
fragment thereof. The anti-CD40 antibody or fragment thereof can be purified,
and can be
combined with a pharmaceutically acceptable carrier. The anti-CD40 antibody
can be an
antibody construct. Additionally, one skilled in the art would recognize that
these same concepts
could apply to antibody constructs comprising anti-CD40 antibodies created for
use in the
veterinary sciences and/or in laboratory animals.
[0204] An antibody construct can comprise an antibody with modifications
occurring at least at
one amino acid residue. Modifications can be substitutions, additions,
mutations, deletions, or the
like. An antibody modification can be an insertion of an unnatural amino acid.
[0205] An antibody construct can comprise a light chain of an amino acid
sequence having at
least one, two, three, four, five, six, seven, eight, nine or ten
modifications but not more than 40,
35, 30, 25, 20, 15 or 10 modifications of the amino acid sequence relative to
the natural or
original amino acid sequence. An antibody construct can comprise a heavy chain
of an amino
acid sequence having at least one, two, three, four, five, six, seven, eight,
nine or ten
modifications but not more than 40, 35, 30, 25, 20, 15 or 10 modifications of
the amino acid
sequence relative to the natural or original amino acid sequence. A heavy
chain can be the heavy
chain of an anti-CD40 antibody which can bind to the CD40 antigen.
[0206] An antibody construct can be an IgG1 isotype. An antibody construct can
be an IgG2
isotype. An antibody construct can be an IgG3 isotype. An antibody construct
can be an IgG4
isotype. An antibody construct can be of a hybrid isotype comprising constant
regions from two
or more isotypes. An antibody construct can be an anti-CD40 antibody, in which
the anti-CD40
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antibody can be a monoclonal human antibody comprising a wild-type sequence of
an IgG1
isoform, in particular, at an Fc region of the antibody.
[0207] Additional anti-CD40 antibody sequences that can be used in the
antibody construct can
comprise any sequence as shown below in TABLE 1 or combination thereof:
TABLE 1
SEQ
Description of
ID Sequence
Sequence
NO:
Heavy Chain
86 ATGGAGTTTGGGCTGAGCTGGGTTTTCCTCGTTGCTCTTTTAAGAGGTG
DNA sequence
TCCATGTCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCC
of antibody 3.1.1
TGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGT
AGTTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGA
GTGGGTGGCAGTTATATCAAAGGATGGAGGTAATAAATACCATGCAG
ACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAATG
CGCTGTATCTGCAAATGAATAGCCTGAGAGTTGAAGACACGGCTGTGT
ATTACTGTGTGAGAAGAGGGCATCAGCTGGTTCTGGGATACTACTACT
ACAACGGTCTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCT
CAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCA
GGAGCACCTCCGAGAGCACAGCGGCCCTGGGCTGCCTGGTCAAGGAC
TACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCTCTGACC
AGCGGCGTGCACACCTTCCCAGCTGTCCTACAGTCCTCAGGACTCTAC
TCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTTCGGCACCCAG
ACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGA
CAAGACAGTTGAGCGCAAATGTTGTGTCGAGTGCCCACCGTGCCCAGC
ACCACCTGTGGCAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAA
GGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGTGGT
GGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGTACGTGG
ACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCACGGGAGGAGCAG
TTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTTGTGCACCAG
GACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGG
CCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGGCAGC
CCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAGATG
ACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTACCCC
AGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAA
CTACAAGACCACACCTCCCATGCTGGACTCCGACGGCTCCTTCTTCCTC
TACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGT
CTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCA
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GAAGAGCCTCTCCCTGTCTCCGGGTAAATGA
Heavy Chain 87 MEFGLSWVFLVALLRGVQCQVQLVESGGGVVQPGRSLRLSCAASGFTFS
protein sequence SYGMHWVRQAPGKGLEWVAVISKDGGNKYHADSVKGRFTISRDNSKNA
of Antibody LYLQMNSLRVEDTAVYYCVRRGHQLVLGYYYYNGLDVWGQGTTVTVS
3.1.1 SASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV
HTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVER
KCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE
VQFNWYVDGVEVHNAKTKPREEQFNSTFRVVS VLTVVHQDWLNGKEYK
CKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKG
FYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQG
NVFSCSVMHEALHNHYTQKSLSLSPGK
Light Chain 88 ATGAGGCTCCCTGCTCAGCTCCTGGGGCTGCTAATGCTCTGGGTCTCTG
DNA sequence GATCCAGTGGGGATATTGTGCTGACTCAGTCTCCACTCTCCCTGCCCGT
of Antibody CACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCT
3.1.1 CTTGTATAGTAATGGATACAACTTTTTGGATTGGTACCTGCAGAAGCC
AGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCC
GGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACA
CTGAAAATCAGCAGATTGGAGGCTGAGGATGTTGGGGTTTATTACTGC
ATGCAAGCTCTACAAACTCCTCGGACGTTCGGCCAAGGGACCAAGGTG
GAAATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCA
TCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGA
ATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAAC
GCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAG
CAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAG
CAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAG
GGCCTGAGCTCGCCCGTCACAAAGGCTTCAACAGGGGAGAGTGTTAG
Light Chain 89 MRLPAQLLGLLMLWVS GS S GDIVLT QSPLSLPVTPGEPASIS CRS S
QSLLYS
protein sequence NGYNFLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKISRL
of Antibody EAEDVGVYYCMQALQTPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSG
3.1.1 TASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS
TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
Mature Variable 90 CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAG
Domain of GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGTTAT
Heavy Chain GGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGT
DNA Sequence GGCAGTTATATCAAAGGATGGAGGTAATAAATACCATGCAGACTCCGT
of Antibody GAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAATGCGCTGTA
3.1.1 TCTGCAAATGAATAGCCTGAGAGTTGAAGACACGGCTGTGTATTACTG
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TGTGAGAAGAGGGCATCAGCTGGTTCTGGGATACTACTACTACAACGG
TCTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA
Mature Variable 91 QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWV
Domain of AVIS KDGGNKYHAD S VKGRFTISRDNS KNALYL QMNSLRVEDTAVYYC V
Heavy Chain RRGHQLVLGYYYYNGLDVWGQGTTVTVSS
Protein
Sequence of
Antibody 3.1.1
Mature Variable 92 GATATTGTGCTGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAG
Domain of Light AGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCTTGTATAGTA
Chain DNA ATGGATACAACTTTTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTC
Sequence of CACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGA
Antibody 3.1.1 CAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAG
CAGATTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCT
ACAAACTCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA
Mature Variable 93 DIVLT QSPLSLPVTPGEPAAIS CRS S QSLLYSNGYNFLDWYLQKPGQSPQL
Domain of Light LIYLGSNRASGVPDPYSGSGSGTDFTLKISRLEAEDVGVYYCMQALQTPR
Chain Protein TFGQGTKVEIK
Sequence of
Antibody 3.1.1
Heavy Chain 94 CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAG
DNA (variable GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGTTAT
domain 3.1.1H- GGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGT
A78T) GGCAGTTATATCAAAGGATGGAGGTAATAAATACCATGCAGACTCCGT
GAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAATACGCTGTT
CTGCAAATGAATAGCCTGAGAGTTGAAGACACGGCTGTGTATTACTGT
GTGAGAAGAGGGCATCAGCTGGTTCTGGGATACTACTACTACAACGGT
CTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA
Heavy Chain 95 QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWV
Protein (variable AVIS KDGGNKYHAD S VKGRFTISRDNS KNTLYLQMNSLRVEDTAVYYC V
domain 3.1.1H- RRGHQLVLGYYYYNGLDVWGQGTTVTVSS
A78T)
Heavy Chain 96 CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAG
DNA (variable GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGTTAT
domain 3.1.1H- GGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGT
A78T-V88A- GGCAGTTATATCAAAGGATGGAGGTAATAAATACCATGCAGACTCCGT
V97A) GAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAATACGCTGTA
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TCTGCAAATGAATAGCCTGAGAGcTGAAGACACGGCTGTGTATTACTG
TGCGAGAAGAGGGCATCAGCTGGTTCTGGGATACTACTACTACAACGG
TCTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCA
Heavy Chain 97 QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWV
Protein (variable AVISKDGGNKYHADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCA
domain 3.1.1H- RRGHQLVLGYYYYNGLDVWGQGTTVTVSS
A78T-V88A-
V97A)
Light Chain 98 GATATTGTGaTGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAG
DNA (variable AGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCTTGTATAGTA
domain 3.1.1L- ATGGATACAACTTTTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTC
L4M-L83V) CACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGA
CAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAG
CAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCT
ACAAACTCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA
Light Chain 99 DIVMT QSPLSLPVTPGEPASIS CRS S QSLLYSNGYNFLDWYLQKPGQSPQL
Protein (variable LIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPR
domain 3.1.1L- TFGQGTKVEIK
L4M-L83V)
Heavy Chain 100 ATGGAGTTTGGGCTGAGCTGGGTTTTCCTCGTTGCTCTTTTAAGAGGTG
DNA Sequence TCCAGTGTCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGC
for Antibody CTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCA
7.1.2 GTAGCTATGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTG
GAGTGGGTGGCAGTTATATCAAATGATGGAGATAATAAATACCATGCA
GACTCCGTGTGGGGCCGATTCACCATCTCCAGAGACAATTCCAGGAGC
ACGCTTTATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTA
TATTACTGTGCGAGAAGAGGCATGGGGTCTAGTGGGAGCCGTGGGGA
TTACTACTACTACTACGGTTTGGACGTCTGGGGCCAAGGGACCACGGT
CACCGTCTCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGC
GCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCGGCCCTGGGCTGCCT
GGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGG
CGCTCTGACCAGCGGCGTGCACACCTTCCCAGCTGTCCTACAGTCCTC
AGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTT
CGGCACCCAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACA
CCAAGGTGGACAAGACAGTTGAGCGCAAATGTTGTGTCGAGTGCCCA
CCGTGCCCAGCACCACCTGTGGCAGGACCGTCAGTCTTCCTCTTCCCCC
CAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGT
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GCGTGGTGGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAAC
TGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCACG
GGAGGAGCAGTTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGT
TGTGCACCAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCT
CCAACAAAGGCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAACC
AAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCG
GGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAG
GCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAG
CCGGAGAACAACTACAAGACCACACCTCCCATGCTGGACTCCGACGG
CTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCA
GCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAA
CCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA
Heavy Chain 101 MEFGLSWVFLVALLRGVQCQVQLVESGGGVVQPGRSLRLSCAASGFTFS
Protein SYGMHWVRQAPGKGLEWVAVISNDGDNKYHADSVWGRFTISRDNSRST
Sequence for LYLQMNSLRAEDTAVYYCARRGMGSSGSRGDYYYYYGLDVWGQGTTV
Antibody 7.1.2 TVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALT
SGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKT
VERKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHE
DPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGK
EYKCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCL
VKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHNHYTQKSLSLSPGK
Light Chain 102 ATGAGGCTCCCTGCTCAGCTCCTGGGGCTGCTAATGCTCTGGGTCTCTG
DNA Sequence GATCCAGTGGGGATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGT
for Antibody CACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCT
7.1.2 CTTGTATAGTAATGGATACAACTTTTTGGATTGGTACCTGCAGAAGCC
AGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCC
GGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACA
CTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGC
ATGCAAGCTCTACAAACTCCTCGGACGTTCGGCCAAGGGACCAAGGTG
GAAATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCA
TCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGA
ATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAAC
GCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAG
CAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAG
CAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAG
GGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTA
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G
Light Chain 103 MRLPAQLLGLLMLWVS GS S GDIVMT QSPLSLPVTPGEPASIS CRS S QSLLY
Protein SNGYNFLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTKISRV
Sequence for EAEDVGVYYCMQALQTPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSG
Antibody 7.1.2 TASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSS
TLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
Mature Variable 104 CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAG
Domain of GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTAT
Heavy Chain GGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGT
DNA Sequence GGCAGTTATATCAAATGATGGAGATAATAAATACCATGCAGACTCCGT
of Antibody GTGGGGCCGATTCACCATCTCCAGAGACAATTCCAGGAGCACGCTTTA
7.1.2 TCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTATATTACTG
TGCGAGAAGAGGCATGGGGTCTAGTGGGAGCCGTGGGGATTACTACT
ACTACTACGGTTTGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCT
CCTCA
Mature Variable 105 QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYGMHWVRQAPGKGLEWV
Domain of AVISNDGDNKYHADSVWGRFTISRDNSRSTLYLQMNSLRAEDTAVYYCA
Heavy Chain RRGMGSSGSRGDYYYYYGLDVWGQGTTVTVSS
Protein
Sequence of
Antibody 7.1.2
Mature Variable 106 GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAG
Domain of Light AGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCTTGTATAGTA
Chain DNA ATGGATACAACTTTTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTC
Sequence of CACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGA
Antibody 7.1.2 CAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAG
CAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCT
ACAAACTCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAA
Mature Variable 107 DIVMT QSPLSLPVTPGEPASIS CRS S QSLLYSNGYNFLDWYLQKPGQSPQL
Domain of Light LIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPR
Chain Protein TFGQGTKVEIK
Sequence of
Antibody 7.1.2
Heavy Chain 108 ATGAAACACCTGTGGTTCTTCCTCCTGCTGGTGGCAGCTCCCAGATGG
DNA Sequence GTCCTGTCCCAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAA
for Antibody GCCTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATC
10.8.3 AGTAGTTACTACTGGATCTGGATCCGGCAGCCCGCCGGGAAGGGACTG
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GAATGGATTGGGCGTGTCTATACCAGTGGGAGCACCAACTACAACCCC
TCCCTCAAGAGTCGAGTCACCATGTCAGTAGACACGTCCAAGAACCAG
TTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCCGTGTAT
TACTGTGCGAGAGATGGTCTTTACAGGGGGTACGGTATGGACGTCTGG
GGCCAAGGGACCACGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCA
TCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACA
GCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACG
GTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTTCCCA
GCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACC
GTGCCCTCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAGAT
CACAAGCCCAGCAACACCAAGGTGGACAAGACAGTTGAGCGCAAATG
TTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGACCGTC
AGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCG
GACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGACC
CCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATG
CCAAGACAAAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGTGTG
GTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAACGGCAAGGAG
TACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAGCCCCCATCGAGAA
AACCATCTCCAAAACCAAAGGGCAGCCCCGAGAACCACAGGTGTACA
CCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTG
ACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGG
GAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACACCTCCCAT
GCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGA
CAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCA
TGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCC
GGGTAAATGA
Heavy Chain 109 MKHLWFFLLLVAAPRWVLS QVQLQESGPGLVKPSETLSLTCTVSGGSISS
Protein YYWIWIRQPAGKGLEWIGRVYTSGSTNYNPSLKSRVTMSVDTSKNQFSL
Sequence for KLS S VTAADT AVYYCARDGLYRGYGMD VWGQGTTVTVS S AS T KGPS VF
Antibody 10.8.3 PLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS
GLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCP
APPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDG
VEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLP
APIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH
EALHNHYTQKSLSLSPGK
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Light Chain 110 ATGAGGCTCCCTGCTCAGCTCCTGGGGCTCCTGCTGCTCTGGTTCCCAG
DNA Sequence GTTCCAGATGCGACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGC
for Antibody ATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGCCTAT
10.8.3 TAGCAGCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTA
AACTCCTGATTTATTCTGCCTCCGGTTTGCAAAGTGGGGTCCCATCAAG
GTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAG
CCTGCAGCCTGAAGATTTTGCAACTTACTATTGTCAACAGACTGACAG
TTTCCCGCTCACTTTCGGCGGCGGGACCAAGGTGGAGATCAAACGAAC
TGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTG
AAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCA
GAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGT
AACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTA
CAGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAAC
ACAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCG
TCACAAAGAGCTTCAACAGGGGAGAGTGTTAG
Light Chain 111 MRLPAQLLGLLLLWFPGSRCDIQMT QSPS S VS AS VGDRVTITCRAS QPIS S
Protein WLAWYQQKPGKAPKLLIYSASGLQSGVPSRFSGSGSGTDFTLTISSLQPED
Sequence for FATYYCQQTDSFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVV
Antibody 10.8.3 CLLNNFYPREAKVQWKVDNAL QS GNS QES VTEQD S KD STYSLS STLTLS
K
ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
Mature Variable 112 CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGA
Domain of GACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGTAGTTAC
Heavy Chain TACTGGATCTGGATCCGGCAGCCCGCCGGGAAGGGACTGGAATGGAT
DNA Sequence TGGGCGTGTCTATACCAGTGGGAGCACCAACTACAACCCCTCCCTCAA
for Antibody GAGTCGAGTCACCATGTCAGTAGACACGTCCAAGAACCAGTTCTCCCT
10.8.3 GAAGCTGAGCTCTGTGACCGCCGCGGACACGGCCGTGTATTACTGTGC
GAGAGATGGTCTTTACAGGGGGTACGGTATGGACGTCTGGGGCCAAG
GGACCACGGTCACCGTCTCCTCA
Mature Variable 113 QVQLQESGPGLVKPSETLSLTCTVSGGSISSYYWIWIRQPAGKGLEWIGRV
Domain of YTSGSTNYNPSLKSRVTMSVDTSKNQFSLKLSSVTAADTAVYYCARDGL
Heavy Chain YRGYGMDVWGQGTTVTVSS
Protein
Sequence for
Antibody 10.8.3
Mature Variable 114 GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGA
Domain of Light GACAGAGTCACCATCACTTGTCGGGCGAGTCAGCCTATTAGCAGCTGG
Chain DNA TTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAACTCCTGATT
-66-
CA 03007311 2018-06-01
WO 2017/100305 PCT/US2016/065353
Sequence for TATTCTGCCTCCGGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGC
Antibody 10.8.3 AGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAGCCT
GAAGATTTTGCAACTTACTATTGTCAACAGACTGACAGTTTCCCGCTCA
CTTTCGGCGGCGGGACCAAGGTGGAGATCAAA
Mature Variable 115 DIQMTQSPSSVSASVGDRVTITCRASQPISSWLAWYQQKPGKAPKLLIYSA
Domain of Light SGLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQTDSFPLTFGGGTK
Chain Protein VEIK
Sequence for
Antibody 10.8.3
Heavy Chain 116 ATGAAACATCTGTGGTTCTTCCTTCTCCTGGTGGCAGCTCCCAGATGGG
DNA Sequence TCCTGTCCCAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGC
for Antibody CTTCGGAGACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAG
15.1.1 AAGTTACTACTGGACCTGGATCCGGCAGCCCCCAGGGAAGGGACTGG
AGTGGATTGGATATATCTATTACAGTGGGAGCACCAACTACAATCCCT
CCCTCAAGAGTCGAGTCACCATATCAGTAGACATGTCCAAGAACCAGT
TCTCCCTGAAGCTGAGTTCTGTGACCGCTGCGGACACGGCCGTTTATT
ACTGTGCGAGAAAGGGTGACTACGGTGGTAATTTTAACTACTTTCACC
AGTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGG
GCCCATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGA
GCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGG
TGACGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCT
TCCCAGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGT
GACCGTGCCCTCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGT
AGATCACAAGCCCAGCAACACCAAGGTGGACAAGACAGTTGAGCGCA
AATGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGAC
CGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTC
CCGGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGTCACGAAG
ACCCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATA
ATGCCAAGACAAAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGT
GTGGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAACGGCAAG
GAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAGCCCCCATCGA
GAAAACCATCTCCAAAACCAAAGGGCAGCCCCGAGAACCACAGGTGT
ACACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGC
CTGACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAG
TGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACACCTCC
CATGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTG
GACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGAT
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CA 03007311 2018-06-01
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GCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTC
TCCGGGTAAATGA
Heavy Chain 117 MKHLWFFLLLVAAPRWVLS QVQLQESGPGLVKPSETLSLTCTVSGGSIRS
Protein YYWTWIRQPPGKGLEWIGYIYYSGSTNYNPSLKSRVTISVDMSKNQFSLK
Sequence for LS S VTAADTAVYYC ARKGD YGGNFNYFHQWGQGTLVTVS S AS T KGPS V
Antibody 15.1.1 FPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQS
SGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPC
PAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVD
GVEVHNAKTKPREEQFNSTFRVVS VLTVVHQDWLNGKEYKCKVSNKGL
PAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMH
EALHNHYTQKSLSLSPGK
Light Chain 118 ATGAGGCTCCCTGCTCAGCTCCTGGGGCTGCTAATGCTCTGGGTCTCTG
DNA Sequence GATCCAGTGGGGATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGT
for Antibody CACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCT
15.1.1 CCTACATACTAATGGATACAACTATTTCGATTGGTACCTGCAGAAGCC
AGGGCAGTCTCCACAACTCCTGATCTATTTGGGTTCTAATCGGGCCTCC
GGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACA
CTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGC
ATGCAAGCTCTACAAACTCCGTACAGTTTTGGCCAGGGGACCAAGCTG
GAGATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCA
TCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGA
ATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAAC
GCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAG
CAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAG
CAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAG
GGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTA
G
Light Chain 119 MRLPAQLLGLLMLWVS GS S GDIVMT QSPLSLPVTPGEPASIS CRS S QSLLH
Protein TNGYNYFDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKIS
Sequence for RVEAEDVGVYYCMQALQTPYSFGQGTKLEIKRTVAAPSVFIFPPSDEQLK
Antibody 15.1.1 SGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL
SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
Mature Variable 120 CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGA
Domain of GACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGAAGTTAC
Heavy Chain TACTGGACCTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGAT
DNA Sequence TGGATATATCTATTACAGTGGGAGCACCAACTACAATCCCTCCCTCAA
-68-
CA 03007311 2018-06-01
WO 2017/100305 PCT/US2016/065353
for Antibody GAGTCGAGTCACCATATCAGTAGACATGTCCAAGAACCAGTTCTCCCT
15.1.1 GAAGCTGAGTTCTGTGACCGCTGCGGACACGGCCGTTTATTACTGTGC
GAGAAAGGGTGACTACGGTGGTAATTTTAACTACTTTCACCAGTGGGG
CCAGGGAACCCTGGTCACCGTCTCCTCA
Mature Variable 121 QVQLQESGPGLVKPSETLSLTCTVSGGSIRSYYWTWIRQPPGKGLEWIGYI
Domain of YYSGSTNYNPSLKSRVTISVDMSKNQFSLKLSSVTAADTAVYYCARKGD
Heavy Chain YGGNFNYFHQWGQGTLVTVSS
Protein
Sequence for
Antibody 15.1.1
Mature Variable 122 GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAG
Domain of Light AGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTACATACTA
Chain DNA ATGGATACAACTATTTCGATTGGTACCTGCAGAAGCCAGGGCAGTCTC
Sequence for CACAACTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGA
Antibody 15.1.1 CAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAG
CAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCT
ACAAACTCCGTACAGTTTTGGCCAGGGGACCAAGCTGGAGATCAAA
Mature Variable 123 DIVMT QSPLSLPVTPGEPASIS CRS S QSLLHTNGYNYFDWYLQKPGQSPQL
Domain of Light LIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPY
Chain Protein SFGQGTKLEIK
Sequence for
Antibody 15.1.1
Heavy Chain 124 ATGGACTGGACCTGGAGGATCCTCTTCTTGGTGGCAGCAGCCACAGGA
DNA Sequence GCCCACTCCCAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAA
for Antibody GCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACACCTT
21.4.1 CACCGGCTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCT
TGAGTGGATGGGATGGATCAACCCTGACAGTGGTGGCACAAACTATGC
ACAGAAGTTTCAGGGCAGGGTCACCATGACCAGGGACACGTCCATCA
GCACAGCCTACATGGAGCTGAACAGGCTGAGATCTGACGACACGGCC
GTGTATTACTGTGCGAGAGATCAGCCCCTAGGATATTGTACTAATGGT
GTATGCTCCTACTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTC
TCCTCAGCCTCCACCAAGGGCCCATCGGTCTTCCCCCTGGCGCCCTGCT
CCAGGAGCACCTCCGAGAGCACAGCGGCCCTGGGCTGCCTGGTCAAG
GACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCTCTG
ACCAGCGGCGTGCACACCTTCCCAGCTGTCCTACAGTCCTCAGGACTC
TACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTTCGGCACC
CAGACCTACACCTGCAACGTAGATCACAAGCCCAGCAACACCAAGGT
-69-
CA 03007311 2018-06-01
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GGACAAGACAGTTGAGCGCAAATGTTGTGTCGAGTGCCCACCGTGCCC
AGCACCACCTGTGGCAGGACCGTCAGTCTTCCTCTTCCCCCCAAAACC
CAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACGTGCGTGGT
GGTGGACGTGAGCCACGAAGACCCCGAGGTCCAGTTCAACTGGTACG
TGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCACGGGAGGAG
CAGTTCAACAGCACGTTCCGTGTGGTCAGCGTCCTCACCGTTGTGCAC
CAGGACTGGCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAA
AGGCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAACCAAAGGGC
AGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGAGGAG
ATGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTAC
CCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAA
CAACTACAAGACCACACCTCCCATGCTGGACTCCGACGGCTCCTTCTT
CCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGA
ACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACA
CGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATGA
Heavy Chain 125 MDWTWRILFLVAAATGAHS QVQLVQS GAEVKKPGAS VKVS C KAS GYTF
Protein TGYYMHWVRQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSI
Sequence for STAYMELNRLRSDDTAVYYCARDQPLGYCTNGVCSYFDYWGQGTLVTV
Antibody 21.4.1 S S AS T KGPS VFPLAPCSRST S ESTAALGCLVKD YFPEPVTVSWNS GALT
S G
VHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVE
RKCCVECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP
EVQFNWYVDGVEVHNAKTKPREEQFNSTFRVVS VLTVVHQDWLNGKEY
KCKVSNKGLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVK
GFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQ
GNVFSCSVMHEALHNHYTQKSLSLSPGK
Light Chain 126 ATGAGGCTCCCTGCTCAGCTCCTGGGGCTCCTGCTGCTCTGGTTCCCAG
DNA Sequence GTTCCAGATGCGACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGC
for Antibody ATCTGTAGGAGACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTAT
21.4.1 TTACAGCTGGTTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAA
CCTCCTGATCTATACTGCATCCACTTTACAAAGTGGGGTCCCATCAAG
GTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAG
CCTGCAACCTGAAGATTTTGCAACTTACTATTGTCAACAGGCTAACATT
TTCCCGCTCACTTTCGGCGGAGGGACCAAGGTGGAGATCAAACGAACT
GTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGA
AATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCCCAG
AGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTA
ACTCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTAC
-70-
CA 03007311 2018-06-01
WO 2017/100305 PCT/US2016/065353
AGCCTCAGCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACA
CAAAGTCTACGCCTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGT
CACAAAGAGCTTCAACAGGGGAGAGTGTTAG
Light Chain 127 MRLPAQLLGLLLLWFPGSRCDIQMT QSPS S VS AS VGDRVTITCRAS QGIYS
Protein WLAWYQQKPGKAPNLLIYTAS TLQS GVPSRFS GS GS GTDFTLTIS SLQPED
Sequence for FATYYCQQANIFPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVV
Antibody 21.4.1 CLLNNFYPREAKVQWKVDNAL QS GNS QES VTEQD S KD STYSLS STLTLS K
AD YEKHKVYACEVTHQGLS SPVT KSFNRGEC
Mature Variable 128 AGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCC
Domain of TCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACACCTTCACCGGCTAC
Heavy Chain TATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATG
DNA Sequence GGATGGATCAACCCTGACAGTGGTGGCACAAACTATGCACAGAAGTTT
of Antibody CAGGGCAGGGTCACCATGACCAGGGACACGTCCATCAGCACAGCCTA
21.4.1 CATGGAGCTGAACAGGCTGAGATCTGACGACACGGCCGTGTATTACTG
TGCGAGAGATCAGCCCCTAGGATATTGTACTAATGGTGTATGCTCCTA
CTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA
Mature Variable 129 QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEW
Domain of MGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVYY
Heavy Chain CARD QPLGYCTNGVC S YFD YWGQGTLVTVS S
Protein
Sequence of
Antibody 21.4.1
Mature Variable 130 GACATCCAGATGACCCAGTCTCCATCTTCCGTGTCTGCATCTGTAGGA
Domain of Light GACAGAGTCACCATCACTTGTCGGGCGAGTCAGGGTATTTACAGCTGG
Chain DNA TTAGCCTGGTATCAGCAGAAACCAGGGAAAGCCCCTAACCTCCTGATC
Sequence of TATACTGCATCCACTTTACAAAGTGGGGTCCCATCAAGGTTCAGCGGC
Antibody 21.4.1 AGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAACCT
GAAGATTTTGCAACTTACTATTGTCAACAGGCTAACATTTTCCCGCTCA
CTTTCGGCGGAGGGACCAAGGTGGAGATCAAA
Mature Variable 131 DIQMT QSPS S VS AS VGDRVTITCRAS QGIYSWLAWYQ QKPGKAPNLLIYT
Domain of Light AS TLQS GVPSRFS GS GS GTDFTLTIS SLQPEDFATYYC QQANIFPLTFGGGT
Chain Protein KVEIK
Sequence of
Antibody 21.4.1
Heavy Chain 132 CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAG
DNA Sequence GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTAT
for Antibody GTCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGT
-71-
CA 03007311 2018-06-01
WO 2017/100305 PCT/US2016/065353
21.2.1
GGCAGTTATGTCATATGATGGAAGTAGTAAATACTATGCAAACTCCGT
GAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTA
TCTGCAAATAAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTG
TGCGAGAGATGGGGGTAAAGCAGTGCCTGGTCCTGACTACTGGGGCC
AGGGAATCCTGGTCACCGTCTCCTCAG
Heavy Chain
133 QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYVMHWVRQAPGKGLEWV
Protein
AVMS YDGSSKYYANS VKGRFTISRDNS KNTLYLQINSLRAEDTAVYYC A
Sequence for RDGGKAVPGPDYWGQGILVTVSS
Antibody 21.2.1
Light Chain
134 GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAG
DNA Sequence
AGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGTGTTCTGTATAGTA
for Antibody
ATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTC
21.2.1
CACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGA
CAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAG
CAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGTTTT
ACAAACTCCATTCACTTTCGGCCCTGGGACCAAAGTGGATATCAAAC
Light Chain
135 DIVMT QSPLSLPVTPGEPASIS CRS S QS VLYSNGYNYLDWYLQKPGQSPQL
Protein
LIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQVLQTPF
Sequence for TFGPGTKVDIK
Antibody 21.2.1
Heavy Chain
136 CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAG
DNA Sequence
GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTCGCTAT
for Antibody
GGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGT
22.1.1
GGCAGTTATATCATCTGATGGAGGTAATAAATACTATGCAGACTCCGT
GAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTA
TCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTG
TACGAGAAGAGGGACTGGAAAGACTTACTACCACTACTGTGGTATGG
ACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAG
Heavy Chain
137 QVQLVESGGGVVQPGRSLRLSCAASGFTFSRYGMHWVRQAPGKGLEWV
Protein
AVIS SDGGNKYYAD S VKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCT
Sequence for RRGTGKTYYHYCGMDVWGQGTTVTVSS
Antibody 22.1.1
Light Chain
138 GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAG
DNA Sequence
AGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGTATAGTA
for Antibody
ATGGATATAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTC
22.1.1
CACACCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGA
CAGGTTCAGTGGCAGTGGTTCAGGCACTGATTTTACACTGAAAATCAG
-72-
CA 03007311 2018-06-01
WO 2017/100305 PCT/US2016/065353
CAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCT
ACAAACTCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAAC
Light Chain
139 DIVMT QSPLS LPVTPGEPASIS CRS S QS LLYSNGYNYLDWYLQKPGQSPHL
Protein
LIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPR
Sequence for TFGQGTKVEIK
Antibody 22.1.1
Heavy Chain
140 CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTCCAGCCTGGGAGGTC
DNA Sequence
CCTGAGACTCTCCTGTGTAGCCTCTGGATTCACCTTCAGTAACTATGGC
for Antibody
ATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGC
23.5.1
AATTATATCATATGATGGAAGTAATAAATACTATGCAGACTCCGTGAA
GGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATGT
GCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACTGTGC
GAGACGCGGTCACTACGGGAGGGATTACTACTCCTACTACGGTTTGGA
CGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAG
Heavy Chain
141 QVQLVESGGGVVQPGRSLRLSCVASGFTFSNYGMHWVRQAPGKGLEWV
Protein
AIISYDGSNKYYADS VKGRFTISRDNS KNTLYVQMNS LRAEDTAVYYC AR
Sequence for RGHYGRDYYSYYGLDVWGQGTTVTVSS
Antibody 23.5.1
Light Chain
142 GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAG
DNA Sequence
AGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCCTGGTA
for Antibody
ATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTC
23.5.1
CACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGA
CAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACACTGAAAATCAG
CAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGCATGCAAGCTCT
ACAAACTCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAAC
Light Chain
143 DIVMT QSPLS LPVTPGEPASIS CRS S QS LLPGNGYNYLDWYLQKPGQSPQL
Protein
LIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCMQALQTPR
Sequence for TFGQGTKVEIK
Antibody 23.5.1
Heavy Chain
144 CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGA
DNA Sequence
CACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGAGGTTAC
for Antibody
TACTGGAGCTGGATCCGGCAGCCCCCTGGGAAGGGACTGGAGTGGAT
23.28.1
TGGGTATATCTATTACAGTGGGAGCACCAACTACAACCCCTCCCTCAA
GAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCT
GAAGCTGAACTCTGTGACCGCTGCGGACACGGCCGTGTATTATTGTGC
GAGAAAGGGGGGCCTCTACGGTGACTACGGCTGGTTCGCCCCCTGGG
GCCAGGGAACCCTGGTCACCGTCTCCTCAG
-73-
CA 03007311 2018-06-01
WO 2017/100305 PCT/US2016/065353
Heavy Chain 145 QVQLQESGPGLVKPSDTLSLTCTVSGGSIRGYYWSWIIRQPPGKGLEWIGY
Protein IYYSGSTNYNPSLKSRVTISVDTSKNQFSLKLNSVTAADTAVYYCARKGG
Sequence for LYGDYGWFAPWGQGTLVTVSS
Antibody
23.28.1
Light Chain 146 GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGG
DNA Sequence GAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCAG
for Antibody CGACTTAGCCTGGCACCAGCAGAAACCTGGCCAGGCTCCCAGACTCCT
23.28.1 CATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAG
TGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGA
GCCTGAAGATTTTGCAGTGTATTACTGTCAGCACTGTCGTAGCTTATTC
ACTTTCGGCCCTGGGACCAAAGTGGATATCAAAC
Light Chain 147 EIVLT QS PGTLS LS PGERATLS CRAS QS VS S S
DLAWHQQKPGQAPRLLIYG
Protein ASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQHCRSLFTFGPGTK
Sequence for VDIK
Antibody
23.28.1
Heavy Chain 148 CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGA
DNA Sequence GACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGAGGTTAC
(variable domain TACTGGAGCTGGATCCGGCAGCCCCCTGGGAAGGGACTGGAGTGGAT
23.28.1H-D16E) TGGGTATATCTATTACAGTGGGAGCACCAACTACAACCCCTCCCTCAA
GAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACOAGTTCTCCCT
GAAGCTGAACTCTGTGACCGCTGCGGACACGGCCGTGTATTATTGTGC
GAGAAAGGGGGGCCTCTACGGTGACTACGGCTGGTTCGCCCCCTGGG
GCCAGGGAACCCTGGTCACCGTCTCCTCAG
Heavy Chain 149 QVQLQESGPGLVKPSLTLSLTCTVSGGSIRGYYWSWIRQPPGKGLEWIGYI
Protein YYSGSTNYNPSLKSRVTISVDTSKNQFSLKLNSVTAADTAVYYCARKGGL
Sequence YGDYGWFAPWGQGTLVTVSS
(variable domain
23.28.1H-D16E)
Heavy Chain 150 CAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGCCTGGGAG
DNA Sequence GTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCAGTAGCTAT
of Antibody GCCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGT
23.29.1 GGCAGTTATATCATATGATGGAAGTAATAAATACTATGCAGACTCCGT
GAAGGGCCGATTCACCATCTACAGAGACAATTCCAAGAACACGCTGT
ATCTGCAAATGAACAGCCTGAGAGCTGAGGACACGGCTGTGTATTACT
GTGCGAGACGCGGTCACTACGGGAATAATTACTACTCCTATTACGGTT
-74-
CA 03007311 2018-06-01
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TGGACGTCTGGGGCCAAGGGACCACGGTCACCGTCTCCTCAG
Heavy Chain 151 QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWV
Protein AVIS YDGSNKYYADS VKGRFTIYRDNS KNTLYLQMNS LRAEDTAVYYC A
Sequence for RRGHYGNNYYSYYGLDVWGQGTTVTVSS
Antibody
23.29.1
Light Chain 152 GATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGTCACCCCTGGAG
DNA Sequence AGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGCCTCCTGCCTGGTA
for Antibody ATGGATACAACTATTTGGATTGGTACCTGCAGAAGCCAGGGCAGTCTC
23.29.1 CACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCCGGGGTCCCTGC
AGGTTCAGTGGCAGTGGCTCAGGCACAGATTTTACACTGAAAATCAGC
AGAGTGGAGGCTGAGGATGTTGGGATTTATTACTGCATGCAAGCTCTA
CAAACTCCTCGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAAC
Light Chain 153 DIVMT QSPLS LPVTPGEPASIS CRS S QS LLPGNGYNYLDWYLQKPGQSPQL
Protein LIYLGSNRASGVPDRFSGSGSGTDFTLKISRVEAEDVGIYYCMQALQTPRT
Sequence for FGQGTKVEIK
Antibody
23.29.1
Heavy Chain 154 CAGGTGCAGCTGCAGGAGTCGGGCCCAGGACTGGTGAAGCCTTCGGA
DNA Sequence GACCCTGTCCCTCACCTGCACTGTCTCTGGTGGCTCCATCAGAGGTTAC
for Antibody TACTGGAGCTGGATCCGGCAGCCCCCAGGGAAGGGACTGGAGTGGAT
24.2.1 TGGGTATATCTATTACAGTGGGAGCACCAACTACAACCCCTCCCTCAA
GAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCT
GAAGCTGAGTTCTGTGACCGCTGCGGACACGGCCGTGTATTACTGTGC
GAGAAGGGGGGGCCTCTACGGTGACTACGGCTGGTTCGCCCCCTGGG
GCCAGGGAACCCTGGTCACCGTCTCCTCAG
Heavy Chain 155 QVQLQESGPGLVKPSETLSLTCTVSGGSIRGYYWSWIRQPPGKGLEWIGYI
Protein YYSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARRGGL
Sequence for YGDYGWFAPWGQGTLVTVSS
Antibody 24.2.1
Light Chain 156 GAAATTGTGTTGACGCAGTCTCCAGGCACCCTGTCTTTGTCTCCAGGG
DNA Sequence GAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAGCACC
for Antibody TACTTAGCCTGGTACCAGCAGAAACCTGGCCAGGCTCCCAGGCTCCTC
24.2.1 ATCTATGGTGCATCCAGCAGGGCCACTGGCATCCCAGACAGGTTCAGT
GGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGACTGGAG
CCTGAAGATTTTGCAGTGTATTACTGTCAGCAGTATAGTAGCTTATTCA
CTTTCGGCCCTGGGACCAAAGTGGATATCAAAC
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Light Chain 157 ETVLTQSPGTLSLSPGERATLSCRASQSVSSTYLAWYQQKPGQAPRLLIYG
Protein ASSRATGIIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYSSLFTFGPGTK
Sequence for VDIK
Antibody 24.2.1
Heavy Chain 158 ATGGAGTTTGGGCTGAGCTGGGTTTTCCTCGTTGCTCTTTTAAGAGGTG
DNA Sequence TCCAGTGTCAGGTGCAGCTGGTGGAGTCTGGGGGAGGCGTGGTCCAGC
for Antibody CTGGGAGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTCA
21.2.1 GTAGCTATGTCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGG
AGTGGGTGGCAGTTATGTCATATGATGGAAGTAGTAAATACTATGCAA
ACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAATTCCAAGAACA
CGCTGTATCTGCAAATAAACAGCCTGAGAGCTGAGGACACGGCTGTGT
ATTACTGTGCGAGAGATGGGGGTAAAGCAGTGCCTGGTCCTGACTACT
GGGGCCAGGGAATCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCC
CATCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCA
CAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGA
CGGTGTCGTGGAACTCAGGCGCTCTGACCAGCGGCGTGCACACCTTCC
CAGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGA
CCGTGCCCTCCAGCAACTTCGGCACCCAGACCTACACCTGCAACGTAG
ATCACAAGCCCAGCAACACCAAGGTGGACAAGACAGTTGAGCGCAAA
TGTTGTGTCGAGTGCCCACCGTGCCCAGCACCACCTGTGGCAGGACCG
TCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCC
GGACCCCTGAGGTCACGTGCGTGGTGGTGGACGTGAGCCACGAAGAC
CCCGAGGTCCAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAAT
GCCAAGACAAAGCCACGGGAGGAGCAGTTCAACAGCACGTTCCGTGT
GGTCAGCGTCCTCACCGTTGTGCACCAGGACTGGCTGAACGGCAAGGA
GTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCAGCCCCCATCGAGA
AAACCATCTCCAAAACCAAAGGGCAGCCCCGAGAACCACAGGTGTAC
ACCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCT
GACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTG
GGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACACCTCCCA
TGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGG
ACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGC
ATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTC
CGGGTAAATGA
Heavy Chain 159 MEFGLSWVFLVALLRGVOCQVQLVESGGGVVQPGRSLRLSCAASGFTFS
Protein SYVMHWVRQAPGKGLEWVAVMSYDGSSKYYANSVKGRIFTISRDNSKN
Sequence for TLYLQINSLRAEDTAVYYCARDGGKAVPGPDYWGQGILVTVSSASTKGP
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Antibody 21.2.1 SVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVL
QSSGLYSLSSVVTVPSSNTGTQTYTCNVDHKPSNTKVDKTVERKCCVECP
PCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWY
VDGVEVHNAKTKPREEQFNSTFRVVS VLTVVHQDWLNGKEYKCKVSNK
GLPAPIEKTISKTKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIA
VEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFSCSV
MHEALHNHYTQKSLSLSPGK
Light Chain 160 ATGAGGCTCCCTGCTCAGCTCCTGGGGCTGCTAATGCTCTGGGTCTCTG
DNA Sequence GATCCAGTGGGGATATTGTGATGACTCAGTCTCCACTCTCCCTGCCCGT
for Antibody CACCCCTGGAGAGCCGGCCTCCATCTCCTGCAGGTCTAGTCAGAGTGT
21.2.1 TCTGTATAGTAATGGATACAACTATTTGGATTGGTACCTGCAGAAGCC
AGGGCAGTCTCCACAGCTCCTGATCTATTTGGGTTCTAATCGGGCCTCC
GGGGTCCCTGACAGGTTCAGTGGCAGTGGATCAGGCACAGATTTTACA
CTGAAAATCAGCAGAGTGGAGGCTGAGGATGTTGGGGTTTATTACTGC
ATGCAAGTTTTACAAACTCCATTCACTTTCGGCCCTGGGACCAAAGTG
GATATCAAACGAACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCAT
CTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGA
ATAACTTCTATCCCAGAGAGGCCAAAGTACAGTGGAAGGTGGATAAC
GCCCTCCAATCGGGTAACTCCCAGGAGAGTGTCACAGAGCAGGACAG
CAAGGACAGCACCTACAGCCTCAGCAGCACCCTGACGCTGAGCAAAG
CAGACTACGAGAAACACAAAGTCTACGCCTGCGAAGTCACCCATCAG
GGCCTGAGCTCGCCCGTCACAAAGAGCTTCAACAGGGGAGAGTGTTA
Light Chain 161 MRLPAQLLGLLMLWVS GS S GDIVMT QSPLSLPVTPGEPASIS CRSS QS VLY
Protein SNGYNYLDWYLQKPGQSPQLLIYLGSNRASGVPDRFSGSGSGTDFTLKIS
Sequence for RVEAEDVGVYYCMQVLQTPFTFGPGTKVDWRTVAAPSVFIFPPSDEQLKS
Antibody 21.2.1 GTASVVCLLNNFYPREAKVQWKVDNALQSGNS QESVTEQDSKDSTYSLS
STLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
[0208] Additional anti-CD40 antibody sequences that can be used in the
antibody construct can
comprise a sequence with greater than 70%, greater than 75%, greater than 80%,
greater than
85%, greater than 90%, greater than 95% or greater than 99% homology to any
sequence in
TABLE 1.
[0209] Antibody constructs disclosed herein can be non-natural, designed,
and/or engineered.
Antibody constructs disclosed herein can be non-natural, designed, and/or
engineered scaffolds
comprising an antigen binding domain. Antibody constructs disclosed herein can
be non-natural,
designed, and/or engineered antibodies. Antibody constructs can be monoclonal
antibodies.
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Antibody constructs can be human antibodies. Antibody constructs can be
humanized antibodies.
Antibody constructs can be monoclonal humanized antibodies. Antibody
constructs can be
recombinant antibodies.
[0210] An antigen binding domain of an antibody construct can be selected in
order to recognize
an antigen. For example, an antigen can be a cell surface marker on a target
cell associated with a
disease or condition. An antigen can be expressed on an immune cell. An
antigen can be a
peptide or fragment thereof. An antigen can be expressed on an antigen-
presenting cell. An
antigen can be expressed on a dendritic cell, a macrophage, or a B cell. An
antigen can be a
peptide presented in a major histocompatibility complex by cell. As another
example, a cell
surface marker recognized by the antigen binding domain can include
macromolecules associated
with viral and bacterial diseases or infections, autoimmune diseases and
cancerous diseases. An
antigen can be CD40 and an antigen binding domain can recognize a CD40
antigen. An antigen
can be a tumor antigen or fragment thereof. A tumor antigen can be any antigen
listed on tumor
antigen databases, such as TANTIGEN, or peptide databases for T cell-defined
tumor antigens,
such as the Cancer Immunity Peptide database. A tumor antigen can also be any
antigen listed in
the review by Chen (Chen, Cancer Immun 2004 [updated 2004 Mar 10; cited 2004
Apr 1]). Note
that the 'antibody' can recognize the 'tumor antigen' or a peptide derived
thereof, bound to an
MHC molecule. An antigen can have at least 80% homology to or can be CD5,
CD19, CD20,
CD25, CD37, CD30, CD33, CD45, CAMPATH-1, BCMA, CS-1, PD-L1, B7-H3, B7-DC,
BCMA, CS-1, PD-L1, B7-H3, B7-DC, HLD-DR, carcinoembryonic antigen, TAG-72,
EpCAM,
MUC1, folate-binding protein, A33, G250, prostate-specific membrane antigen,
ferritin, GD2,
GD3, GM2, Leg, CA-125, CA19-9, epidermal growth factor, p185HER2, IL-2
receptor, de2-7
EGFR, fibroblast activation protein, tenascin, metalloproteinases, endosialin,
vascular endothelial
growth factor, avB3, WT1, LMP2, HPV E6 E7, EGFRvIII, Her-2/neu, idiotype, MAGE
A3, p53
nonmutant, NY-ES0-1, PMSA, GD2, CEA, MelanA/MART1, Ras mutant, gp100, p53
mutant,
PR1, bcr-abl, tyronsinase, survivin, PSA, hTERT, Sarcoma translocation
breakpoints, EphA2,
PAP, ML-IAP, AFP, ERG, NA17, PAX3, ALK, androgen receptor, cyclin Bl,
polysialic acid,
MYCN, RhoC, TRP-2, fucosyl GM1, mesothelin, PSCA, MAGE Al, sLe(animal),
CYP1B1,
PLAV1, GM3, BORIS, Tn, GloboH, ETV6-AML, NY-BR-1, RGS5, SART3, STn, Carbonic
anhydrase IX, PAX5, 0Y-TES1, Sperm protein 17, LCK, HMWMAA, AKAP-4, 55X2, XAGE
1, B7H3, Legumain, Tie 3, Page4, VEGFR2, MAD-CT-1, PDGFR-B, MAD-CT-2, ROR2,
TRAILl, MUC16, MAGE A4, MAGE C2, GAGE, or Fos-related antigen 1. An antigen
binding
domain can be capable of recognizing a single antigen. An antigen binding
domain can be
capable of recognizing two or more different antigens.
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[0211] An antibody construct can comprise an antibody heavy chain. A heavy
chain can be a
heavy chain of an anti-HER2 monoclonal antibody which can bind a HER2 antigen.
A heavy
chain of an anti-HER2 antibody can be an IgG1 isotype. A heavy chain of an
anti-HER2
antibody can be SBT-050 VH-hIgG1 wt (pertuzumab). SBT-050 VH-hIgG1 wt can
comprise an
amino acid sequence
EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGG
SIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGQGTLV
TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV
LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE
LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR
EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTL
PPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT
VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 30). SBT-050 VH-
hIgG1 wt can comprise an amino acid sequence with greater than 70%, greater
than 75%, greater
than 80%, greater than 85%, greater than 90%, greater than 95% or greater than
99% homology
to SEQ ID NO: 30. A heavy chain of an anti-HER2 antibody can comprise a CDR. A
heavy
chain of an anti-HER2 antibody can comprise a CDR with an amino acid sequence
GFTFTDYT
(SEQ ID NO: 31). A heavy chain of an anti-HER2 antibody can comprise a CDR
with an amino
acid sequence VNPNSGGS (SEQ ID NO: 32). A heavy chain of an anti-HER2 antibody
can
comprise a CDR with an amino acid sequence ARNLGPSFYFDY (SEQ ID NO: 33). A
heavy
chain CDR of an anti-HER2 antibody can comprise an amino acid sequence with
greater than
70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%,
greater than 95%
or greater than 99% homology to SEQ ID NO: 31. A heavy chain CDR of an anti-
HER2 antibody
can comprise an amino acid sequence with greater than 70%, greater than 75%,
greater than 80%,
greater than 85%, greater than 90%, greater than 95% or greater than 99%
homology to SEQ ID
NO: 32. A heavy chain CDR of an anti-HER2 antibody can comprise an amino acid
sequence
with greater than 70%, greater than 75%, greater than 80%, greater than 85%,
greater than 90%,
greater than 95% or greater than 99% homology to SEQ ID NO: 33.
[0212] An antibody construct can comprise an antibody light chain. A light
chain can be a light
chain of a HER2 monoclonal antibody which can bind a HER2 antigen. A light
chain of an anti-
HER2 antibody can be SBT-050 VL-Ck (pertuzumab). SBT-050 VL-Ck can comprise an
amino
acid sequence
DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIYSASYRYTGVPS
RFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIKRTVAAPSVFIFPPSD
EQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTL
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SKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 34). SBT-050 VL-Ck can
comprise an amino acid sequence with greater than 70%, greater than 75%,
greater than 80%,
greater than 85%, greater than 90%, greater than 95% or greater than 99%
homology to SEQ ID
NO: 34. A light chain of an anti-HER2 antibody can comprise a CDR. A light
chain of an anti-
HER2 antibody can comprise a CDR with an amino acid sequence QDVSIG (SEQ ID
NO: 35).
A light chain of an anti-HER2 antibody can comprise a CDR with an amino acid
sequence SAS
(SEQ ID NO: 36). A light chain of an anti-HER2 antibody can comprise a CDR
with an amino
acid sequence QQYYIYPYT (SEQ ID NO: 37). A light chain CDR of an anti-HER2
antibody
can comprise an amino acid sequence with greater than 70%, greater than 75%,
greater than 80%,
greater than 85%, greater than 90%, greater than 95% or greater than 99%
homology to SEQ ID
NO: 35. A light chain CDR of an anti-HER2 antibody can comprise an amino acid
sequence with
greater than 70%, greater than 75%, greater than 80%, greater than 85%,
greater than 90%,
greater than 95% or greater than 99% homology to SEQ ID NO: 36. A light chain
CDR of an
anti-HER2 antibody can comprise an amino acid sequence with greater than 70%,
greater than
75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or
greater than
99% homology to SEQ ID NO: 37.
[0213] An antibody construct can comprise an Fc region with an Fc domain. An
Fc domain is a
structure that can bind to Fc receptors (FcRs). An antibody construct can
comprise an Fc domain.
Fc domains can be bound by FcRs. An Fc domain can be from an antibody. An Fc
domain can be
at least 80% homologous to an Fc domain from an antibody. An Fc region can be
in a scaffold.
An Fc region with an Fc domain can be in an antibody scaffold. An Fc region
with an Fc domain
can be in a non-antibody scaffold. An antibody construct can comprise an Fc
region with an Fc
domain in an antibody scaffold. An antibody construct can comprise an Fc
region with an Fc
domain in a non-antibody scaffold. An Fc domain can be in a scaffold. An Fc
domain can be in
an antibody scaffold. An Fc domain can be in a non-antibody scaffold. An
antibody construct can
comprise an Fc domain in an antibody scaffold. An antibody construct can
comprise an Fc
domain in a non-antibody scaffold. Fc domains of antibodies, including those
of the present
disclosure, can be bound by FcRs. Fc domains can be a portion of the Fc region
of an antibody.
FcRs can bind to an Fc domain of an antibody. FcRs can bind to an Fc domain of
an antibody
bound to an antigen. FcRs can be organized into classes (e.g., gamma (y),
alpha (a) and epsilon
(6)) based on the class of antibody that the FcR recognizes. The FcaR class
can bind to IgA and
includes several isoforms, FcaRI (CD89) and Fca R. The FcyR class can bind to
IgG and
includes several isoforms, FcyRI (CD64), FcyRIIA (CD32a), FcyRIIB (CD32b),
FcyRIIIA
(CD16a), and FcyRIIIB (CD16b). An FcyRIIIA (CD16a) can be an FcyRIIIA (CD16a)
F158
variant. An FcyRIIIA (CD16a) can be an FcyRIIIA (CD16a) V158 variant. Each
FcyR isoform
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can differ in affinity to the Fc region of the IgG antibody. For example,
FcyRI can bind to IgG
with greater affinity than FcyRII or FcyRIII. The affinity of a particular
FcyR isoform to IgG can
be controlled, in part, by a glycan (e.g., oligosaccharide) at position CH2
84.4 of the IgG
antibody. For example, fucose containing CH2 84.4 glycans can reduce IgG
affinity for FcyRIIIA.
In addition, GO glucans can have increased affinity for FcyRIIIA due to the
lack of galactose and
terminal GlcNAc moiety.
[0214] Binding of an Fc domain to an FcR can enhance an immune response. FcR-
mediated
signaling that can result from an Fc region binding to an FcR can lead to the
maturation of
immune cells. FcR-mediated signaling that can result from an Fc domain binding
to an FcR can
lead to the maturation of dendritic cells. FcR-mediated signaling that can
result from an Fc
domain binding to an FcR can lead to more efficient immune cell antigen uptake
and processing.
FcR-mediated signaling that can result from an Fc region binding to an FcR can
lead to more
efficient dendritic cell antigen uptake and processing. FcR-mediated signaling
that can result
from an Fc region binding to an FcR can increase antigen presentation. FcR-
mediated signaling
that can result from an Fc region binding to an FcR can increase antigen
presentation by immune
cells. FcR-mediated signaling that can result from an Fc region binding to an
FcR can increase
antigen presentation by antigen presenting cells. FcR-mediated signaling that
can result from an
Fc domain binding to an FcR can increase antigen presentation by dendritic
cells. FcR-mediated
signaling that can result from an Fc domain binding to an FcR can promote the
expansion and
activation of T cells. FcR-mediated signaling that can result from an Fc
domain binding to an
FcR can promote the expansion and activation of CD8+ T cells. FcR-mediated
signaling that can
result from an Fc domain binding to an FcR can influence immune cell
regulation of T cell
responses. FcR-mediated signaling that can result from an Fc domain binding to
an FcR can
influence immune cell regulation of T cell responses. FcR-mediated signaling
that can result
from an Fc domain binding to an FcR can influence dendritic cell regulation of
T cell responses.
FcR-mediated signaling that can result from an Fc domain binding to an FcR can
influence
functional polarization of T cells (e.g., polarization can be toward a TH1
cell response).
[0215] The profile of FcRs on a DC can impact the ability of the DC to respond
upon
stimulation. For example, most DC can express both CD32a and CD32b, which can
have
opposing effects on IgG-mediated maturation and function of DCs: binding of
IgG to CD32a can
mature and activate DCs in contrast with CD32b, which can mediate inhibition
due to
phosphorylation of immunoreceptor tyrosine-based inhibition motif (ITIM),
after CD32b binding
of IgG. Therefore, the activity of these two receptors can establish a
threshold of DC activation.
Furthermore, difference in functional avidity of these receptors for IgG can
shift their functional
balance. Hence, altering the Fc domain binding to FcRs can also shift their
functional balance,
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allowing for manipulation (either enhanced activity or enhanced inhibition) of
the DC immune
response.
A modification in the amino acid sequence of the antibody construct can alter
the recognition and
binding of an FcR for the Fc domain. For example, a modification of the amino
acid sequence of
the Fc domain in an antibody construct can increase the binding affinity
and/or avidity of the Fc
domain for FcRs. This increase in binding affinity and/or avidity can specific
for a type of FcR.
However, such modifications can still allow for FcR-mediated signaling. A
modification can be a
substitution of an amino acid at a residue (e.g., wildtype) for a different
amino acid at that
residue. For example, a wildtype Fc domain can comprise
ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSS
GLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGG
PSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQY
NSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSR
EEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDK
SRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 162), and a modified Fc
domain can comprise a substitution of an amino acid in comparison with SEQ ID
NO: 162. A
modification can permit binding of an FcR to a site on the Fc region of an
antibody construct that
the FcR may not otherwise bind to. A modification can increase binding
affinity of an FcR to the
Fc domain of an antibody construct that the FcR may have reduced binding
affinity for. A
modification can decrease binding affinity of an FcR to a site on the Fc
domain of an antibody
construct that the FcR may have increased binding affinity for. A modification
can increase the
subsequent FcR-mediated signaling after Fc binding to an FcR.
[0216] An antibody construct can comprise an Fc region with at least one amino
acid change as
compared to the sequence of the wild-type Fc region. A wild-type Fc region can
comprise SEQ
ID NO: 162. An antibody construct can comprise an Fc domain with at least one
amino acid
change as compared to the sequence of the wild-type Fc domain. A wild-type Fc
domain can
comprise SEQ ID NO: 162. For example, an antibody construct can comprise an Fc
domain with
at least one amino acid change as compared to the sequence of the wild-type Fc
domain
comprising SEQ ID NO: 162. An amino acid change in an Fc region of an antibody
construct can
allow the antibody construct to bind to at least one Fc receptor with greater
affinity compared to
a wild-type Fc region. An amino acid change in an Fc domain of an antibody
construct can allow
the antibody to bind to at least one Fc receptor with greater affinity
compared to a wild-type Fc
domain. An Fc region can comprise an amino acid sequence having at least one,
two, three, four,
five, six, seven, eight, nine or ten modifications but not more than 40, 35,
30, 25, 20, 15 or 10
modifications of the amino acid sequence relative to the natural or original
amino acid sequence.
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An Fc domain can comprise an amino acid sequence having at least one, two,
three, four, five,
six, seven, eight, nine or ten modifications but not more than 40, 35, 30, 25,
20, 15 or 10
modifications of the amino acid sequence relative to the natural or original
amino acid sequence.
An Fc region can be an Fc region of an anti-CD40 antibody. An Fc domain can be
an Fc domain
of an anti-CD40 antibody. An Fc region can contain an Fc domain. An Fc region
can be an Fc
domain.
[0217] An antibody construct can be an antibody comprising a sequence of the
IgG1 isoform that
has been modified from the wild type IgG1 sequence. A wild type IgG1 sequence
can comprise
SEQ ID NO: 162. A modification can comprise a substitution at more than one
amino acid
residue such as at 5 different amino acid residues including
L235V/F243L/R292P/Y300L/P396L
(G1VLPLL). The numbering of amino acids residues described herein can be
according to the
EU index as in Kabat. The 5 amino acid residues can be located in a portion of
an antibody
sequence which can encode an Fc region of the antibody and in particular, can
be located in
portions of the Fc region that can bind to Fc receptors (i.e., the Fc domain).
A modification can
comprise a substitution at more than one amino acid residue such as at 2
different amino acid
residues including 5239D/I332E (GIDE). The 2 amino acid residues can be
located in a portion
of an antibody sequence which encodes an Fc region of the antibody and in
particular, are located
in portions of the Fc region that can bind to Fc receptors (i.e., the Fc
domain). A modification can
comprise a substitution at more than one amino acid residue such as at 3
different amino acid
residues including 5298A/E333A/K334A (GlAAA). The 3 amino acid residues can be
located in
a portion of an antibody sequence which can encode an Fc region of the
antibody and in
particular, can be located in portions of the Fc region that can bind Fc
receptors (i.e., the Fc
domain).
[0218] An antibody construct can be a monoclonal anti-CD40 human antibody
comprising a
sequence of the IgG1 isoform that has been modified from the wildtype IgG1
sequence. A
wildtype IgG1 sequence can comprise SEQ ID NO: 15. A modification can comprise
a
substitution at more than one amino acid residue such as at 5 different amino
acid residues
including L235V/F243L/R292P/Y300L/P396L (SBT-040-G1VLPLL). The numbering of
amino
acids residues described herein can be according to the EU index as in Kabat.
The 5 amino acid
residues can be located in a portion of an antibody sequence which can encode
an Fc region of
the antibody and in particular, can be located in portions of the Fc region
that can bind to Fc
receptors (i.e., the Fc domain). A modification can comprise a substitution at
more than one
amino acid residue such as at 2 different amino acid residues including
5239D/I332E (SBT-040-
GIDE). The 2 amino acid residues can be located in a portion of an antibody
sequence which
encodes an Fc region of the antibody and in particular, are located in
portions of the Fc region
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that can bind to Fc receptors (i.e., the Fc domain). A modification can
comprise a substitution at
more than one amino acid residue such as at 3 different amino acid residues
including
S298A/E333A/K334A (SBT-040-G1AAA). The 3 amino acid residues can be located in
a
portion of an antibody sequence which can encode an Fc region of the antibody
and in particular,
can be located in portions of the Fc region that can bind Fc receptors (i.e.,
the Fc domain).
[0219] Binding of Fc receptors to an Fc region can be affected by amino acid
substitutions. For
example, FIGURE 4C illustrates SBT-040-G1VLPLL, which is an antibody with an
amino acid
sequence (SEQ ID NO: 16) of a heavy chain of human anti-CD40 monoclonal
antibody with
modifications to a wild-type IgG1 Fc domain (L235V/F243L/R292P/Y300L/P396L).
Binding of
some Fc receptors to the Fc region of SBT-040-G1VLPLL can be enhanced compared
to wild-
type by as result of the L235V/F243L/R292P/Y300L/P396L amino acid
modifications. However,
binding of other Fc receptors to the Fc region of SBT-040-G1VLPLL can be
reduced compared
to wild-type by the L235V/F243L/R292P/Y300L/P396L amino acid modifications.
For example,
the binding affinities of SBT-040-G1VLPLL to FcyRIIIA and to FcyRIIA can be
enhanced
compared to wild-type whereas the binding affinity of SBT-040-G1VLPLLto
FcyRIIB can be
reduced compared to wild-type. FIGURE 4D illustrates an SBT-040-G1DE antibody,
which is
an antibody with an amino acid sequence (SEQ ID NO: 17) of a heavy chain of
human anti-
CD40 monoclonal antibody with modifications to a wild-type IgG1 Fc domain
(5239D/I332E).
Binding of Fc receptors to the Fc region of SBT-040-DE can be enhanced
compared to wild-type
as a result of the 5239D/I332E amino acid modification. However, binding of
some Fc receptors
to the Fc region of SBT-040-G1DE can be reduced compared to wild-type by
5239D/I332E
amino acid modification. For example, the binding affinities of SBT-040-G1DE
to FcyRIIIA and
to FcyRIIB can be enhanced compared to wild-type. Binding of Fc receptors to
an Fc region of
are affected by amino acid substitutions. FIGURE 4E illustrates an SBT-040-
G1AAA antibody,
which is an antibody with an amino acid sequence (SEQ ID NO: 18) of a heavy
chain of a human
anti-CD40 monoclonal antibody with modifications to a wild-type IgG1 Fc domain
(5298A/E333A/K334A). Binding of Fc receptors to an Fc region of SBT-040-G1AAA
can be
enhanced compared to wild-type as a result of the 5298A/E333A/K334A amino acid
modification. However, binding of some Fc receptors to the Fc region of SBT-
040-G1AAA can
be reduced compared to wild-type by 5298A/E333A/K334A amino acid modification.
Binding
affinities of SBT-040-G1AAA to FcyRIIIA can be enhanced compared to wild-type
whereas the
binding affinity of SBT-040-G1AAA to FcyRIIB can be reduced compared to
wildtype.
[0220] In some embodiments, the heavy chain of a human IgG2 antibody can be
mutated at
cysteines as positions 127, 232, or 233. In some embodiments, the light chain
of a human IgG2
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antibody can be mutated at a cysteine at position 214. The mutations in the
heavy and light
chains of the human IgG2 antibody can be from a cysteine residue to a serine
residue.
[0221] An antibody construct can be a heavy chain of an anti-CD40 antibody. A
heavy chain of
an anti-CD40 antibody can be SBT-040-G1VLPLL. SBT-040-G1VLPLL be expressed
from a
DNA sequence comprising
ATGGACTGGACCTGGAGGATCCTCTTCTTGGTGGCAGCAGCCACAGGAGCCCACTCC
CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAA
GGTCTCCTGCAAGGCTTCTGGATACACCTTCACCGGCTACTATATGCACTGGGTGCG
ACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAACCCTGACAGTGGTG
GCACAAACTATGCACAGAAGTTTCAGGGCAGGGTCACCATGACCAGGGACACGTCC
ATCAGCACAGCCTACATGGAGCTGAACAGGCTGAGATCTGACGACACGGCCGTGTA
TTACTGTGCGAGAGATCAGCCCCTAGGATATTGTACTAATGGTGTATGCTCCTACTTT
GACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCA
TCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCGGCCCTG
GGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGC
GCTCTGACCAGCGGCGTGCACACCTTCCCAGCTGTCCTACAGTCCTCAGGACTCTAC
TCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTTCGGCACCCAGACCTACACC
TGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGACAGTTGAGCCCAA
ATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCGTGGGGGG
ACCGTCAGTCTTCCTCCTGCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGAC
CCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGT
TCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCCTGAG
GAGCAGTACAACAGCACGCTGCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGAC
TGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCC
CATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACA
CCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTG
GTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCC
GGAGAACAACTACAAGACCACGCCTCTGGTGCTGGACTCCGACGGCTCCTTCTTCCT
CTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCAT
GCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGT
CCCCGGGTAAATGA (SEQ ID NO: 9) wherein the DNA sequence comprises DNA
nucleotide
modifications that correspond to L235V, F243L, R292P, Y300L and P396L amino
acid residue
modifications compared to a wild-type DNA sequence. SBT-040-G1VLPLL can be
expressed
from a DNA sequence comprising greater than 70%, greater than 75%, greater
than 80%, greater
than 85%, greater than 90%, greater than 95% or greater than 99% homology to
SEQ ID NO: 9.
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A variable region of SBT-040-G1VLPLL can be expressed from a DNA sequence
comprising
SEQ ID NO: 13. A variable region of SBT-040-G1VLPLL can be expressed from a
DNA
sequence comprising greater than 70%, greater than 75%, greater than 80%,
greater than 85%,
greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO:
13.
Additionally, anti-CD40 antibodies comprising SBT-040-G1VLPLL expressed from
SEQ ID
NO: 9, or expressed from a DNA sequence comprising greater than 70% homology
to SEQ ID
NO: 9 can have a dissociation constant (Kd) for CD40 that is less than 10 nM.
Anti-CD40
antibodies comprising SBT-040-G1VLPLL expressed from DNA sequence comprising
SEQ ID
NO: 9, or comprising greater than 70% homology to SEQ ID NO: 9 can have a
dissociation
constant (Kd) for CD40 that is less than 1 nM, less than 100 pM, less than 10
pM, less than 1 pM,
or less than 0.1 pM. SBT-040-G1VLPLL can be expressed with any anti-CD40 light
chain or
fragment thereof. SBT-040-G1VLPLL can also be expressed with any anti-CD40
light chain or
fragment thereof to form an anti-CD40 antibody or fragment thereof. The anti-
CD40 antibody or
fragment thereof can be purified, and can be combined with a pharmaceutically
acceptable
carrier. The anti-CD40 antibody can be an antibody construct. Additionally,
one skilled in the art
would recognize that these same concepts could apply to antibody constructs
comprising anti-
CD40 antibodies created for use in the veterinary sciences and/or in
laboratory animals.
[0222] SBT-040-G1VLPLL can comprise an amino acid sequence
MDWTWRILFLVAAATGAHSQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWV
RQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVY
YCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGC
LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVD
HKPSNTKVDKTVEPKSCDKTHTCPPCPAPELVGGPSVFLLPPKPKDTLMISRTPEVTCVV
VDVSHEDPEVKFNWYVDGVEVHNAKTKPPEEQYNSTLRVVSVLTVLHQDWLNGKEYK
CKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPLVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ
KSLSLSPGK (SEQ ID NO: 16) wherein the amino acid sequence comprises L235V,
F243L,
R292P, Y300L, and P396L amino acid residue modifications compared to a wild-
type amino
acid sequence. SBT-040-G1VLPLL can comprise an amino acid sequence with
greater than 70%,
greater than 75%, greater than 80%, greater than 85%, greater than 90%,
greater than 95% or
greater than 99% homology to SEQ ID NO: 16. SBT-040-G1VLPLL can comprise an
amino acid
sequence SEQ ID NO: 20. A variable region of SBT-040-G1VLPLL can comprise an
amino acid
sequence with greater than 70%, greater than 75%, greater than 80%, greater
than 85%, greater
than 90%, greater than 95% or greater than 99% homology to SEQ ID NO: 20.
Additionally,
anti-CD40 antibodies comprising SBT-040-G1VLPLL with SEQ ID NO: 16 or with an
amino
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acid sequence with greater than 70% homology to SEQ ID NO: 16 can have a
dissociation
constant (Kd) for CD40 that is less than 10 nM. Anti-CD40 antibodies
comprising SBT-040-
G1VLPLL with SEQ ID NO: 16 or with an amino acid sequence with greater than
70%
homology to SEQ ID NO: 16 can have a dissociation constant (Kd) for CD40 that
is less than 1
nM, less than 100 pM, less than 10 pM, less than 1 pM, or less than 0.1 pM.
SBT-040-G1VLPLL
can be purified. SBT-040-G1VLPLL can be combined with any anti-CD40 light
chain or
fragment thereof to form an anti-CD40 antibody or fragment thereof. The anti-
CD40 antibody or
fragment thereof can be purified, and can be combined with a pharmaceutically
acceptable
carrier. The anti-CD40 antibody can be an antibody construct. Additionally,
one skilled in the art
would recognize that these same concepts could apply to antibody constructs
comprising anti-
CD40 antibodies created for use in the veterinary sciences and/or in
laboratory animals.
[0223] A heavy chain of an anti-CD40 antibody can be SBT-040-G1DE. SBT-040-
G1DE be
expressed from a DNA sequence comprising
ATGGACTGGACCTGGAGGATCCTCTTCTTGGTGGCAGCAGCCACAGGAGCCCACTCC
CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAA
GGTCTCCTGCAAGGCTTCTGGATACACCTTCACCGGCTACTATATGCACTGGGTGCG
ACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAACCCTGACAGTGGTG
GCACAAACTATGCACAGAAGTTTCAGGGCAGGGTCACCATGACCAGGGACACGTCC
ATCAGCACAGCCTACATGGAGCTGAACAGGCTGAGATCTGACGACACGGCCGTGTA
TTACTGTGCGAGAGATCAGCCCCTAGGATATTGTACTAATGGTGTATGCTCCTACTTT
GACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCA
TCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCGGCCCTG
GGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGC
GCTCTGACCAGCGGCGTGCACACCTTCCCAGCTGTCCTACAGTCCTCAGGACTCTAC
TCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTTCGGCACCCAGACCTACACC
TGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGACAGTTGAGCCCAA
ATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGG
ACCGGATGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGAC
CCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGT
TCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAG
GAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGAC
TGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCC
CGAGGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACA
CCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTG
GTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCC
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GGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCT
CTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCAT
GCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGT
CCCCGGGTAAATGA (SEQ ID NO: 10) wherein the DNA sequence comprises DNA
nucleotide modifications that correspond to 5239D and 1332E amino acid residue
modifications
compared to a wild-type DNA sequence. SBT-040-G1DE can be expressed from a DNA
sequence comprising greater than 70%, greater than 75%, greater than 80%,
greater than 85%,
greater than 90%, greater than 95% or greater than 99% homology to SEQ ID NO:
10. A variable
region of SBT-040-G1DE can be expressed from a DNA sequence comprising SEQ ID
NO: 13.
A variable region of SBT-040-G1DE can be expressed from a DNA sequence
comprising greater
than 70%, greater than 75%, greater than 80%, greater than 85%, greater than
90%, greater than
95% or greater than 99% homology to SEQ ID NO: 13. Additionally, anti-CD40
antibodies
comprising SBT-040-G1DE expressed from SEQ ID NO: 10, or expressed from a DNA
sequence
comprising greater than 70% homology to SEQ ID NO: 10 can have a dissociation
constant (Kd)
for CD40 that is less than 10 nM. Anti-CD40 antibodies comprising SBT-040-G1DE
expressed
from DNA sequence comprising SEQ ID NO: 10, or comprising greater than 70%
homology to
SEQ ID NO: 10 can have a dissociation constant (Kd) for CD40 that is less than
1 nM, less than
100 pM, less than 10 pM, less than 1 pM, or less than 0.1 pM. SBT-040-G1DE can
be expressed
with any anti-CD40 light chain or fragment thereof. SBT-040-G1DE can also be
expressed with
any anti-CD40 light chain or fragment thereof to form an anti-CD40 antibody or
fragment
thereof. The anti-CD40 antibody or fragment thereof can be purified, and can
be combined with a
pharmaceutically acceptable carrier. The anti-CD40 antibody can be an antibody
construct.
Additionally, one skilled in the art would recognize that these same concepts
could apply to
antibody constructs comprising anti-CD40 antibodies created for use in the
veterinary sciences
and/or in laboratory animals.
[0224] SBT-040-G1DE can comprise an amino acid sequence
MDWTWRILFLVAAATGAHSQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWV
RQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVY
YCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGC
LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVD
HKPSNTKVDKTVEPKSCDKTHTCPPCPAPELLGGPDVFLFPPKPKDTLMISRTPEVTCVV
VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPEEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV
EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT
QKSLSLSPGK (SEQ ID NO: 17) wherein the amino acid sequence comprises 5239D and
1332E
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amino acid residue modifications compared to a wild-type amino acid sequence.
SBT-040-G1DE
can comprise an amino acid sequence with greater than 70%, greater than 75%,
greater than 80%,
greater than 85%, greater than 90%, greater than 95% or greater than 99%
homology to SEQ ID
NO: 17. SBT-040-G1DE can comprise an amino acid sequence SEQ ID NO: 20. A
variable
region of SBT-040-G1DE can comprise an amino acid sequence with greater than
70%, greater
than 75%, greater than 80%, greater than 85%, greater than 90%, greater than
95% or greater
than 99% homology to SEQ ID NO: 20. Additionally, anti-CD40 antibodies
comprising SBT-
040-G1DE with SEQ ID NO: 17 or with an amino acid sequence with greater than
70%
homology to SEQ ID NO: 17 can have a dissociation constant (Kd) for CD40 that
is less than 10
nM. Anti-CD40 antibodies comprising SBT-040-G1DE with SEQ ID NO: 17 or with an
amino
acid sequence with greater than 70% homology to SEQ ID NO: 17 can have a
dissociation
constant (Kd) for CD40 that is less than 1 nM, less than 100 pM, less than 10
pM, less than 1 pM,
or less than 0.1 pM. SBT-040-G1DE can be purified. SBT-040-G1DE can be
combined with any
anti-CD40 light chain or fragment thereof to form an anti-CD40 antibody or
fragment thereof.
The anti-CD40 antibody or fragment thereof can be purified, and can be
combined with a
pharmaceutically acceptable carrier. The anti-CD40 antibody can be an antibody
construct.
Additionally, one skilled in the art would recognize that these same concepts
could apply to
antibody constructs comprising anti-CD40 antibodies created for use in the
veterinary sciences
and/or in laboratory animals.
[0225] A heavy chain of an anti-CD40 antibody can be SBT-040-G1AAA. SBT-040-
G1AAA be
expressed from a DNA sequence comprising
ATGGACTGGACCTGGAGGATCCTCTTCTTGGTGGCAGCAGCCACAGGAGCCCACTCC
CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAA
GGTCTCCTGCAAGGCTTCTGGATACACCTTCACCGGCTACTATATGCACTGGGTGCG
ACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAACCCTGACAGTGGTG
GCACAAACTATGCACAGAAGTTTCAGGGCAGGGTCACCATGACCAGGGACACGTCC
ATCAGCACAGCCTACATGGAGCTGAACAGGCTGAGATCTGACGACACGGCCGTGTA
TTACTGTGCGAGAGATCAGCCCCTAGGATATTGTACTAATGGTGTATGCTCCTACTTT
GACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCCTCCACCAAGGGCCCA
TCGGTCTTCCCCCTGGCGCCCTGCTCCAGGAGCACCTCCGAGAGCACAGCGGCCCTG
GGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGC
GCTCTGACCAGCGGCGTGCACACCTTCCCAGCTGTCCTACAGTCCTCAGGACTCTAC
TCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAACTTCGGCACCCAGACCTACACC
TGCAACGTAGATCACAAGCCCAGCAACACCAAGGTGGACAAGACAGTTGAGCCCAA
ATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGG
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ACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGAC
CCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGT
TCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAG
GAGCAGTACAACGCCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGAC
TGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCC
CATCGCCGCTACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACA
CCCTGCCCCCATCCCGGGAGGAGATGACCAAGAACCAGGTCAGCCTGACCTGCCTG
GTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCC
GGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCT
CTATAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCAT
GCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGT
CCCCGGGTAAATGA (SEQ ID NO: 11) wherein the DNA sequence comprises DNA
nucleotide modifications that correspond to 5298A, E333A, and K334A amino acid
residue
modifications compared to a wild-type DNA sequence. SBT-040-G1AAA can be
expressed from
a DNA sequence comprising greater than 70%, greater than 75%, greater than
80%, greater than
85%, greater than 90%, greater than 95% or greater than 99% homology to SEQ ID
NO: 11. A
variable region of SBT-040-G1AAA can be expressed from a DNA sequence
comprising SEQ
ID NO: 13. A variable region of SBT-040-G1AAA can be expressed from a DNA
sequence
comprising greater than 70%, greater than 75%, greater than 80%, greater than
85%, greater than
90%, greater than 95% or greater than 99% homology to SEQ ID NO: 13.
Additionally, anti-
CD40 antibodies comprising SBT-040-G1AAA expressed from SEQ ID NO: 11, or
expressed
from a DNA sequence comprising greater than 70% homology to SEQ ID NO: 11 can
have a
dissociation constant (Kd) for CD40 that is less than 10 nM. Anti-CD40
antibodies comprising
SBT-040-G1AAA expressed from DNA sequence comprising SEQ ID NO: 11, or
comprising
greater than 70% homology to SEQ ID NO: 11 can have a dissociation constant
(Kd) for CD40
that is less than 1 nM, less than 100 pM, less than 10 pM, less than 1 pM, or
less than 0.1 pM.
SBT-040-G1AAA can be expressed with any anti-CD40 light chain or fragment
thereof. SBT-
040-G1AAA can also be expressed with any anti-CD40 light chain or fragment
thereof to form
an anti-CD40 antibody or fragment thereof. The anti-CD40 antibody or fragment
thereof can be
purified, and can be combined with a pharmaceutically acceptable carrier. The
anti-CD40
antibody can be an antibody construct. Additionally, one skilled in the art
would recognize that
these same concepts could apply to antibody constructs comprising anti-CD40
antibodies created
for use in the veterinary sciences and/or in laboratory animals.
SBT-040-G1AAA can comprise an amino acid sequence
MDWTWRILFLVAAATGAHSQVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWV
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RQAPGQGLEWMGWINPDSGGTNYAQKFQGRVTMTRDTSISTAYMELNRLRSDDTAVY
YCARDQPLGYCTNGVCSYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGC
LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSNFGTQTYTCNVD
HKPSNTKVDKTVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVV
VDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNATYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIAATISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAV
EWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYT
QKSLSLSPGK (SEQ ID NO: 18) wherein the amino acid sequence comprises 5298A,
E333A,
and K334A amino acid residue modifications compared to a wild-type amino acid
sequence.
SBT-040-G1AAA can comprise an amino acid sequence with greater than 70%,
greater than
75%, greater than 80%, greater than 85%, greater than 90%, greater than 95% or
greater than
99% homology to SEQ ID NO: 18. SBT-040-G1AAA can comprise an amino acid
sequence
SEQ ID NO: 20. A variable region of SBT-040-G1AAA can comprise an amino acid
sequence
with greater than 70%, greater than 75%, greater than 80%, greater than 85%,
greater than 90%,
greater than 95% or greater than 99% homology to SEQ ID NO: 20. Additionally,
anti- CD40
antibodies comprising SBT-040-G1AAA with SEQ ID NO: 18 or with an amino acid
sequence
with greater than 70% homology to SEQ ID NO: 18 can have a dissociation
constant (Kd) for
CD40 that is less than 10 nM. Anti-CD40 antibodies comprising SBT-040-G1AAA
with SEQ ID
NO: 18 or with an amino acid sequence with greater than 70% homology to SEQ ID
NO: 18 can
have a dissociation constant (Kd) for CD40 that is less than 1 nM, less than
100 pM, less than 10
pM, less than 1 pM, or less than 0.1 pM. SBT-040-G1AAA can be purified. SBT-
040-G1AAA
can be combined with any anti-CD40 light chain or fragment thereof to form an
anti-CD40
antibody or fragment thereof. The anti-CD40 antibody or fragment thereof can
be purified, and
can be combined with a pharmaceutically acceptable carrier. The anti-CD40
antibody can be an
antibody construct. Additionally, one skilled in the art would recognize that
these same concepts
could apply to anti-CD40 antibodies created for use in the veterinary sciences
and/or in
laboratory animals.
[0226] While an antibody construct of the present disclosure can comprise an
anti-CD40
antibody with wild-type or modified amino acid sequences encoding the Fc
region or Fc domain,
the modifications of the Fc region or the Fc domain from the wild-type
sequence may not
significantly alter binding and/or affinity of the anti-CD40 antibody for
CD40. For example,
binding and/or affinity of SBT-040-G1WT, SBT-040-G1VLPLL, SBT-040-G1DE, and
SBT-
040-G1AAA may not be significantly altered by modification of an Fc region or
Fc domain
amino acid sequence compared to a wild-type sequence. Modifications of an Fc
region or Fc
domain from a wild-type sequence may not alter binding and/or affinity of
antibodies that bind to
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CD40 in an antibody construct. Additionally, the binding and/or affinity of
the antibodies
described herein that bind to CD40 and are antibody constructs, for example
SBT-040-G1WT,
SBT-040-G1VLPLL, SBT-040-G1DE, and SBT-040-G1AAA, may be comparable to the
binding
and/or affinity of wild-type antibodies that can bind to CD40.
[0227] Sequences that can be used to produce antibodies for antibody
constructs can include
leader sequences. Leader sequences can be signal sequences. Leader sequences
useful with the
compositions and methods described herein can include, but are not limited to,
a DNA sequence
comprising
ATGAGGCTCCCTGCTCAGCTCCTGGGGCTCCTGCTGCTCTGGTTCCCAGGTTCCAGAT
GC(SEQ ID NO: 2) or
ATGGACTGGACCTGGAGGATCCTCTTCTTGGTGGCAGCAGCCACAGGAGCCCACTCC
(SEQ ID NO: 12), or an amino acid sequence comprising MRLPAQLLGLLLLWFPGSRC
(SEQ
ID NO: 5) and MDWTWRILFLVAAATGAHS (SEQ ID NO: 19). Leader sequence can
comprise a DNA sequence with greater than 70%, greater than 75%, greater than
80%, greater
than 85%, greater than 90%, greater than 95% or greater than 99% homology to
SEQ ID NO: 2
or SEQ ID NO: 12. Leader sequence can comprise an amino acid sequence with
greater than
70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%,
greater than 95%
or greater than 99% homology to SEQ ID NO: 5 or SEQ ID NO: 19. Any of the
sequences
described herein can be used with or without a leader sequence. Additionally,
one skilled in the
art would recognize that these same concepts can apply to antibody constructs
comprising anti-
CD40 antibodies created for use in the veterinary sciences and/or in
laboratory animals.
Targeting Binding Domain
[0228] An antibody construct can further comprise a targeting binding domain.
A targeting
domain can comprise a domain that binds to a target. A target can be an
antigen. A targeting
domain can comprise an antigen binding domain. A targeting domain can be a
domain that can
specifically bind to an antigen A targeting domain can be an antigen-binding
portion of an
antibody or an antibody fragment. A targeting domain can be one or more
fragments of an
antibody that can retain the ability to specifically bind to an antigen. A
targeting domain can be
any antigen binding fragment. A targeting domain can be in a scaffold, in
which a scaffold is a
supporting framework for the antigen binding domain. A targeting domain can
comprise an
antigen binding domain in a scaffold.
[0229] A targeting domain can comprise an antigen binding domain which can
refer to a portion
of an antibody comprising the antigen recognition portion, i.e., an antigenic
determining variable
region of an antibody sufficient to confer recognition and binding of the
antigen recognition
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portion to a target, such as an antigen, i.e., the epitope. Examples of a
targeting domain can
include, but are not limited to, Fab, single chain variable fragment (scFv),
variable Fv fragment
and other fragments, combinations of fragments or types of fragments known or
knowable to one
of ordinary skill in the art. A targeting domain can comprise an antigen
binding domain which
can refer to a portion of an antibody comprising the antigen recognition
portion, i.e., an antigenic
determining variable region of an antibody sufficient to confer recognition
and binding of the
antigen recognition portion to a target, such as an antigen, i.e., the
epitope. Examples of a
targeting domain can include, but are not limited to, Fab, single chain
variable fragment (scFv),
variable Fv fragment and other fragments, combinations of fragments or types
of fragments
known or knowable to one of ordinary skill in the art.
[0230] A targeting domain can comprise an antigen binding domain of an
antibody. An antigen
binding domain of an antibody can comprise one or more light chain (LC) CDRs
and one or
more heavy chain (HC) CDRs. For example, an antibody binding domain of an
antibody can
comprise one or more of the following: a light chain complementary determining
region 1 (LC
CDR1), a light chain complementary determining region 2 (LC CDR2), or a light
chain
complementary determining region 3 (LC CDR3). For another example, an antibody
binding
domain can comprise one or more of the following: a heavy chain complementary
determining
region 1 (HC CDR1), a heavy chain complementary determining region 2 (HC
CDR2), or a
heavy chain complementary determining region 3 (HC CDR3).
[0231] An antibody construct can comprise an antibody fragment. An antibody
fragment can
include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL
and CHi domains;
(ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments
linked by a disulfide
bridge at the hinge region; and (iii) a Fv fragment consisting of the VL and
VH domains of a
single arm of an antibody. Although the two domains of the Fv fragment, VL and
VH, can be
coded for by separate genes, they can be linked by a synthetic linker to be
made as a single
protein chain in which the VL and VH regions pair to form monovalent
molecules.
[0232] F(ab')2 and Fab' moieties can be produced by treating immunoglobulin
(monoclonal
antibody) with a protease such as pepsin and papain, and can include an
antibody fragment
generated by digesting immunoglobulin near the disulfide bonds existing
between the hinge
regions in each of the two H chains.
[0233] An Fv can be the minimum antibody fragment which contains a complete
antigen-
recognition and antigen-binding site. This region can consist of a dimer of
one heavy chain and
one light chain variable domain in tight, non-covalent association. In this
configuration the three
hypervariable regions of each variable domain can interact to define an
antigen-binding site on
the surface of the VH-VL dimer. A single variable domain (or half of an Fv
comprising only three
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hypervariable regions specific for an antigen) can recognize and bind antigen,
although at a lower
affinity than the entire binding site.
[0234] A targeting domain can be at least 80% homologous to an antigen binding
domain
selected from, but not limited to, a monoclonal antibody, a polyclonal
antibody, a recombinant
antibody, or a functional fragment thereof, for example, a heavy chain
variable domain (VH) and
a light chain variable domain (VL), a DARPin, an affimer, an avimer, a
knottin, a monobody, an
affinity clamp, an ectodomain, a receptor ectodomain, a receptor, a cytokine,
a ligand, an
immunocytokine, a T cell receptor, or a recombinant T cell receptor.
[0235] A targeting domain can comprise an antigen binding domain comprising a
light chain and
a heavy chain from a monoclonal antibody. In one aspect, a targeting domain
binds to CD40 and
comprises the light chain of an anti-CD40 antibody and the heavy chain of an
anti-CD40
antibody, which bind a CD40 antigen. In another aspect, the targeting domain
binds to a tumor
antigen comprises the light chain of a tumor antigen antibody and the heavy
chain of a tumor
antigen antibody, which bind the tumor antigen.
[0236] A targeting domain can bet attached to an antibody construct. For
example, an antibody
construct can be fused with a targeting binding domain to create an antibody
construct targeting
binding domain fusion. The antibody construct- targeting binding domain fusion
can be the result
of the nucleic acid sequence of the targeting binding domain being expressed
in frame with the
nucleic acid sequence of the antibody construct. The antibody construct-
targeting binding domain
fusion can be the result of an in-frame genetic nucleotide sequence or a
contiguous peptide
sequence encoding the antibody construct with the targeting binding domain. As
another
example, a targeting binding domain can be linked to an antibody construct. A
targeting binding
domain can be linked to an antibody construct by a chemical conjugation. The
targeting binding
domain can direct the antibody construct to, for example, a particular cell or
cell type. A
targeting binding domain of an antibody construct can be selected in order to
recognize an
antigen. For example, an antigen can be expressed on an immune cell. An
antigen can be a
peptide or fragment thereof. An antigen can be expressed on an antigen-
presenting cell. An
antigen can be expressed on a dendritic cell, a macrophage, or a B cell. An
antigen can be CD40
and a targeting binding domain can recognize a CD40 antigen. A targeting
binding domain can
be a CD40 agonist. A targeting domain can recognize CD40 on, for example, an
antigen-
presenting cell. As another example, an antigen can be a tumor antigen. The
tumor antigen can be
any tumor antigen described herein.
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Immune-Stimulatory Compounds
[0237] Pattern recognition receptors (PRRs) can recognize pathogen-associated
molecular
patterns (PAMPs) and damage-associated molecular patterns (DAMPs). A PRR can
be
membrane bound. A PRR can be cytosolic. A PRR can be a toll-like receptor
(TLR). A PRR can
be RIG-I-like receptor. A PRR can be a receptor kinase. A PRR can be a C-type
lectin receptor.
A PRR can be a NOD-like receptor. A PRR can be TLR1, TLR2, TLR3, TLR4, TLR5,
TLR6,
TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 or TLR13.
[0238] A PRR agonist can be pathogen-associated molecular pattern (PAMP)
molecule. A
PAMP molecule can be a toll-like receptor agonist. A PRR agonist can be a toll-
like receptor
agonist. A toll-like receptor agonist can be any molecule that acts as an
agonist to at least one
toll-like receptor. A toll-like receptor agonist can be bacterial lipoprotein.
A toll-like receptor
agonist can be bacterial peptidoglycans. A toll-like receptor agonist can be
double stranded RNA.
A toll-like receptor agonist can be lipopolysaccharides. A toll-like receptor
agonist can be
bacterial flagella. A toll-like receptor agonist can be single stranded RNA. A
toll-like receptor
can be CpG DNA. A toll-like receptor agonist can be imiquimod. A toll-like
receptor agonist can
be CL307. A toll-like receptor agonist can be S-27609. A toll-like receptor
agonist can be
resiquimod. A toll-like receptor agonist can be UC-IV150. A toll-like receptor
agonist can be
gardiquimod. A toll-like receptor agonist can be motolimod. A toll-like
receptor agonist can be
VTX-1463. A toll-like receptor agonist can be GS-9620. A toll-like receptor
agonist can be
GSK2245035. A toll-like receptor agonist can be TMX-101. A toll-like receptor
agonist can be
TMX-201. A toll-like receptor agonist can be TMX-202. A toll-like receptor
agonist can be
isatoribine. A toll-like receptor agonist can be AZD8848. A toll-like receptor
agonist can be
MEDI9197. A toll-like receptor agonist can be 3M-051. A toll-like receptor
agonist can be 3M-
852. A toll-like receptor agonist can be 3M-052. A toll-like receptor agonist
can be 3M-854A. A
toll-like receptor agonist can be S-34240. A toll-like receptor agonist can be
CL663. A RIG-I
agonist can be KIN1148. A RIG-I agonist can be SB-9200. A RIG-I agonist can be
KIN700,
KIN600, KIN500, KIN100, KIN101, KIN400, or KIN2000. A toll-like receptor
agonist can be
KU34B.
[0239] A PRR agonist can be a damage-associated molecular pattern (DAMP)
molecule. A
DAMP molecule can be an intracellular protein. A DAMP molecule can be a heat-
shock protein.
A DAMP molecule can be an HMGB1 protein. A DAMP molecule can be a protein
derived from
the extracellular matrix that is generated after tissue injury. A DAMP
molecule can be a
hyaluronan fragment. A DAMP molecule can be DNA. A DAMP molecule can be RNA. A
DAMP molecule can be an S100 molecule. A DAMP molecule can be nucleotides. A
DAMP
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molecule can be an ATP. A DAMP molecule can be nucleosides. A DAMP molecule
can be an
adenosine. A DAMP molecule can be uric acid.
[0240] Additionally, stimulator of interferon genes (STING) can act as a
cytosolic DNA sensor
wherein cytosolic DNA and unique bacterial nucleic acids called cyclic
dinucleotides are
recognized by STING, and therefore STING agonists. Interferon Regulatory
Factor (IRF) agonist
can be KIN-100. Non-limiting examples of STING agonists include:
,0
0
Xs3 _______
0
.....?..,..
X6
0X.......,_
¨ F 3 . ..0 0
ii \ _
0 X2
, wherein in some embodiments, Xi=X2=0;
X3=G; X4=G; X5=CO(CH2)12CH3; X6=2 TEAH; in some embodiments, X1=X2=S [Rp,Rp];
X3=G;
X4=A; X5=H; X6=2 TEAH; in some embodiments, X1=X2=S [Rp,Rp] ; X3=A; X4=A;
X5=H; X6=2
Na; in some embodiments, X1=X2=S [Rp,Rp] ; X3=A; X4=A; X5=H; X6=2 NH4; and in
some
embodiments, Xi=X2=0 ; X3=G; X4=A; X5=H; X6=2 TEAH,
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1
¨ 0......,,,,
/
0
sr =1 :.= , </ X i IT I #1
?%$ ,, ,,, -..0 N ,'. st"-.
k'\''' '''''N----µi' i \ '¨' ",..k's<, sk.--'<i
?4, = . _.;), P' --ANkiN
a ,,,A,,, = (¨Y
1 : z
-ss......p....... s
S-
Ns.4
Its...4(
Ons
\,..,....4
4 4
0 S.
¨d
)00$
4 '44NM 2
0 %
14: 0 0
N
<is sliLT Ni ti i V
"I\ \- . = \ µ7
s,=....c. -6,...,,,,,,,
0
0
4, ..- ,..c.,.
%,..el\s....i NisOf V''',P t
,.,....
N--.,"¨N.,1
. - '1'
c". .1_,...1
* No. = 4$ ''. -
0 0 R
, wherein
Ri=R2=H; Ri=propargyl, R2=H; Ri=H, R2=propargyl; Ri=allyl, R2=H; Ri=H,
R2=ally1;
Ri=methyl, R2=H; Ri=H, R2=methyl; Ri=ethyl, R2=H; Ri=H, R2=ethyl; Ri=propyl,
R2=H; Ri=H,
R2=propyl; Ri=benzyl, R2=H; Ri=H, R2=benzyl; Ri=myristoyl, R2=H; Ri=H,
R2=myristoyl;
Ri=R2=heptanoyl; Ri=R2=hexanoyl; or Ri=R2=pentanoyl,
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i=3;34-.,--t\ .... 1 1 /
C.3 ________________________________________ i l'E
i¨c ' 3.. ......, ,vµ:` ' a '..., ,,t'"=(,
'''''N , -1, *3%.., /1 ''''===(" '''''''A ', El
N =,....,z,õ,,,t,i,
' ..\Se."' Y 1 . e.3õ,...2..,, ,0=E
i
N.)
i3 3...q.
= =====..Ø..- . ,,, F3 ,. c,
., = ;
41, \ & ..,,
..S...
EE -....., E=E .- .,
*y.;======(/ El itEl
\_,,, _ 4:-; 1 P93.--kst (
i µ S ,...., ,:" '''sve/".t.; 1¨
.)' 1(s'P - õ ' -7N> J
- --3õ---
\''''''
u z
`µy, ON: ,,, :. ),,y," = ==:=)p..z
('''..' '' <, 6R =I=,., tt
õ
= ...we,. .= t .3
.....,,f,,,m3) =01.,
IV `= ... it \ ..
0 S c3. S
,
wherein Ri=R2=H; Ri=propargyl, R2=H; Ri=H, R2=propargyl; Ri=allyl, R2=H; Ri=H,
R2=ally1;
Ri=methyl, R2=H; Ri=H, R2=methyl; Ri=ethyl, R2=H; Ri=H, R2=ethyl; Ri=propyl,
R2=H; Ri=H,
R2=propyl; Ri=benzyl, R2=H; Ri=H, R2=benzyl; Ri=myristoyl, R2=H; Ri=H,
R2=myristoyl;
Ri=R2=heptanoyl; Ri=R2=hexanoyl; or Ri=R2=pentanoyl,
N ;$
S...4
.. .w. / \ .. f=ti=i ,,...r.,..<3 \ ti
rt .t.',..=/ \ 3.3 0342
,..E.E...õ...\ ...1
==z S.. et
i 3.4' = o 41. 1 .s 3., t .3 N> ' =It = '3 , . , , , r.3
_õfrk.......,tki f`=.i 'Sy, 4P '"'= =-*".L. N
t ,
Nr..i, <ty
',
.õ/õ.
. -(Ns. N,..-
SoR2 µ....../
1-0õ,..6 6,-4 1---:,..,= ".......i.; i* o ,. -I
6k
.......õ..... .4
4% 0,
0 W ....) .1.;.- o
E,i=
..,":.. N ri.k
= Wt.?
,=( \
3p.k.
3e 'IN, k'
.,. F ..:.,,.,;
,, \%=.11 . ., ...4.(1
1 I 344,m, ll
,...c
0..0 <1. ) N ., .,3 3....,3-3."=3) 3
ii =
\'''-e \-=-..' .3 .^ = ,:="
\,.µ, ''......c .....ak..,,,,,,,,)
. NI"
''' = µ. R0.......1
6k*,.....p.....- , :
. .
,
wherein Ri=R2=H; Ri=propargyl, R2=H; Ri=H, R2=propargyl; Ri=allyl, R2=H; Ri=H,
R2=ally1;
Ri=methyl, R2=H; Ri=H, R2=methyl; Ri=ethyl, R2=H; Ri=H, R2=ethyl; Ri=propyl,
R2=H; Ri=H,
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R2=propyl; Ri=benzyl, R2=H; Ri=H, R2=benzyl; Ri=myristoyl, R2=H; Ri=H,
R2=myristoyl;
Ri=R2=heptanoyl; Ri=R2=hexanoyl; or Ri=R2=pentanoyl,
RI
P ¨0 0
0
r.
R4 0
¨PE ¨011,
R2
, wherein each X is independently 0 or S, and R3
and R4 are each independently H or an optionally substituted straight chain
alkyl of from 1 to 18
carbons and from 0 to 3 heteroatoms, an optionally substituted alkenyl of from
1-9 carbons, an
optionally substituted alkynyl of from 1-9 carbons, or an optionally
substituted aryl, wherein
substitution(s), when present, may be independently selected from the group
consisting of C1,6
alkyl straight or branched chain, benzyl, halogen, trihalomethyl, Ci_6 alkoxy,
¨NO2, ¨NH2, ¨
OH, =0, ¨COOR' where R' is H or lower alkyl, ¨CH2OH, and ¨CONH2, wherein R3
and R4
are not both H,
0
I
P- B2
,O,
0
HO
iNNINN"\\
B!
, wherein Xi=X2=0; Xi=X2=S; or X1=0 and X2=S,
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1
:i\L-
--...
o <,-/
H --0----- , 11
N--- 7, --
1 \ , 0y / '6'
\
S'......"
o
Ho
\ 1
\
o \
o
/
I
r'''': ' t === t N \ -.------N, `" (-..3.-----Pz----,,
!*== 1 .õ,.,
NN2
,
0
N. sit,
.,,,. NH --.....,
0
i
-----, -- -,
Nr....-'
1
HO i
0
i
\
2 / \
i
/ \
0 QH
1......1\ /
. \
1 . 6
NH
ri N
1 t
0 ,
NH.,
0
I
1 RP \
k
o
H9
i
\ i
CI
i \
?
1 1
o o H
,...,-... \...,, ....n-
" 1 '--= 0
I 1
-S
N -N_
"== N ..,'"'''', -<./
N...,....-- ===-,t,
N N.2
,
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0
,N,-., --------....
c
i
\
..$ 1-AP-- ¨ N-- N'N''.'7'.\'`
..,..,, /
......." ,,,,,e Nh 2
1
0
HO i
2 h\p
/ s
i \
1
n 011
tip ,ts; , '''.0---A Bp i
`N...õ......," 'N.,õN
I 1
0 ,
19 H2
I
...:-.... ,.,..õ..,...,' N".....,-:::õN
0
....-' "..,,. i
0
Hq
/
1 k
o /OH
l I
õ.N r N or" \&p/
e.'...2. .....--=.ii'' ......'Ø...--'4''....... C.--
!
ji
i
1
N Hz
,
0
I
N"..
===.t.4 ti
0
St/ i 1
\
\ / i
0',..õ
0
HO i
\
¨\
i
\111"1"1"41
1 /
o
, 0 H
H >14 N S p i
- -=.õ.....5. ,..õ k.....
-"--s-
H \' I
0
i
i
0
,
-10 1-
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.õØ.õ
Sp1,--Q"Nj N NH?
0
1 ==1.3p
it tt
0
'N
0 ,and
NOt,
Sp NN.
c,
0
in/
rNN.
0 s.SPI
-tsi =
\ i/
o
N N
µN,N,
Nfi2
[0241] An immune-stimulatory compound can be a PRR agonist. An immune-
stimulatory
compound can be a PAMP. An immune-stimulatory compound can be a DAMP. An
immune-
stimulatory compound can be a TLR agonist. An immune-stimulatory compound can
be a
STING agonist. An immune-stimulatory compound can be a cyclic dinucleotide.
[0242] An immune-stimulatory compound can be a drug.
[0243] The specificity of the antigen-binding domain to an antigen of an
antibody construct in an
antibody construct immune-stimulatory compound conjugate as disclosed herein
can be
influenced by the presence of an immune-stimulatory compound. The antigen-
binding domain of
the antibody construct in an antibody construct immune-stimulatory compound
conjugate can
bind to an antigen with at least about 10%, about 20%, about 30%, about 40%,
about 50%, about
60%, about 70%, about 80%, about 85%, about 90%, about 95%, or about 100% of a
specificity
of the antigen-binding domain to the antigen in the absence of the immune-
stimulatory
compound.
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[0244] The specificity of the Fc domain to an Fc receptor of an antibody
construct in an antibody
construct immune-stimulatory compound conjugate as disclosed herein can be
influenced by the
presence of an immune-stimulatory compound. The Fc domain of the antibody
construct in an
antibody construct immune-stimulatory compound conjugate can bind to an Fc
receptor with at
least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about
70%, about
80%, about 85%, about 90%, about 95%, or about 100% of a specificity of the Fc
domain to the
Fc receptor in the absence of the immune-stimulatory compound.
[0245] The affinity of the antigen-binding domain to an antigen of an antibody
construct in an
antibody construct immune-stimulatory compound conjugate as disclosed herein
can be
influenced by the presence of an immune-stimulatory compound. The antigen-
binding domain of
the antibody construct in an antibody construct immune-stimulatory compound
conjugate can
bind to an antigen with at least about 1%, about 5%, about 10%, about 20%,
about 30%, about
40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, about
95%, or
about 100% of an affinity of the antigen-binding domain to the antigen in the
absence of the
immune-stimulatory compound.
[0246] The affinity of the Fc domain to an Fc receptor of an antibody
construct in an antibody
construct immune-stimulatory compound conjugate as disclosed herein can be
influenced by the
presence of an immune-stimulatory compound. The Fc domain of the antibody
construct in an
antibody construct immune-stimulatory compound conjugate can bind to an Fc
receptor with at
least about 1%, about 5%, about 10%, about 20%, about 30%, about 40%, about
50%, about
60%, about 70%, about 80%, about 85%, about 90%, about 95%, or about 100% of
an affinity of
the Fc domain to the Fc receptor in the absence of the immune-stimulatory
compound.
[0247] The Kd for binding of an antigen-binding domain of an antibody
construct immune-
stimulatory compound conjugate to an antigen in the presence of an immune-
stimulatory
compound can be about 2 times, about 3 times, about 4 times, about 5 times,
about 6 times, about
7 times, about 8 times, about 9 times, about 10 times, about 15 times, about
20 times, about 25
times, about 30 times, about 35 times, about 40 times, about 45 times, about
50 times, about 60
times, about 70 times, about 80 times, about 90 times, about 100 times, about
110 times, or about
120 times greater than the Kd for binding of the antigen binding domain to the
antigen of an
antibody construct in the absence of the immune-stimulatory compound. The Kd
for binding of
an antigen-binding domain of an antibody construct immune-stimulatory compound
conjugate to
an antigen in the presence of the immune-stimulatory compound can be less than
10 nM. The Kd
for binding of an antigen-binding domain of an antibody construct immune-
stimulatory
compound conjugate to an antigen in the presence of the immune-stimulatory
compound can be
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less than 100 nM, less than 50 nM, less than 20 nM, less than 5 nM, less than
1 nM, or less than
0.1 nM.
[0248] The Kd for binding of an Fc domain of an antibody construct immune-
stimulatory
compound conjugate to a Fc receptor in the presence of the immune-stimulatory
compound can
be about 2 times, about 3 times, about 4 times, about 5 times, about 6 times,
about 7 times, about
8 times, about 9 times, about 10 times, about 15 times, about 20 times, about
25 times, about 30
times, about 35 times, about 40 times, about 45 times, about 50 times, about
60 times, about 70
times, about 80 times, about 90 times, about 100 times, about 110 times, or
about 120 times
greater than the Kd for binding of the Fc domain to the Fc receptor in the
absence of the immune-
stimulatory compound. The Kd for binding of an Fc domain of an antibody
construct immune-
stimulatory compound conjugate to an Fc receptor in the presence of the immune-
stimulatory
compound can be less than 10 nM. The Kd for binding of an Fc domain of an
antibody construct
immune-stimulatory compound conjugate to an Fc receptor in the presence of the
immune-
stimulatory compound can be less than 10 M, less than 1 M, less than 100 nM,
less than 50
nM, less than 20 nM, less than 5 nM, less than 1 nM, or less than 0.1 nM.
[0249] Affinity can be the strength of the sum total of noncovalent
interactions between a single
binding site of a molecule, for example, an antibody, and the binding partner
of the molecule, for
example, an antigen. The affinity can also measure the strength of an
interaction between an Fc
portion of an antibody and the Fc receptor. Unless indicated otherwise, as
used herein, "binding
affinity" can refer to intrinsic binding affinity which reflects a 1:1
interaction between members
of a binding pair (e.g., antibody and antigen or Fc domain and Fc receptor).
The affinity of a
molecule X for its partner Y can generally be represented by the dissociation
constant (Kd).
Affinity can be measured by common methods known in the art, including those
described
herein. Specific illustrative and exemplary embodiments for measuring binding
affinity are
described in the following.
[0250] In some embodiments, an antibody provided herein can have a
dissociation constant (Kd)
of about 1 M, about 100 nM, about 10 nM, about 5 nM, about 2 nM, about 1 nM,
about 0.5 nM,
about 0.1 nM, about 0.05 nM, about 0.01 nM, or about 0.001 nM or less (e.g.,
10-8 M or less, e.g.,
from 10-8M to 10-13 M, e.g., from 10 M to 10-13 M). An affinity matured
antibody can be an
antibody with one or more alterations in one or more complementarity
determining regions
(CDRs), compared to a parent antibody, which may not possess such alterations,
such alterations
resulting in an improvement in the affinity of the antibody for antigen. These
antibodies can bind
to their antigen with a Kd of about 5x10-9 M, about 2x10-9 M, about 1x10-9 M,
about 5x10-1 M,
-. .-,
about 2x10-9 M, about 1x10-10 m about 5x10- 1 1 M, about 1x10-" M, about 5x10-
12 M, about
1x10-12 M, or less. In some embodiments, the antibody construct can have an
increased affinity
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of at least 1.5-fold, 2-fold, 2.5-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-
fold, or greater as
compared to an antibody construct without alterations in one or more
complementarity
determining regions.
[0251] Kd can be measured by any suitable assay. For example, Kd can be
measured by a
radiolabeled antigen binding assay (RIA). For example, Kd can be measured
using surface
plasmon resonance assays (e.g., using a BIACOREC)-2000 or a BIACOREC)-3000).
[0252] Agonism can be described as the binding of a chemical to a receptor to
induce a
biological response. A chemical can be, for example, a small molecule, a
compound, or a protein.
An agonist causes a response, an antagonist can block the action of an
agonist, and an inverse
agonist can cause a response that is opposite to that of the agonist. A
receptor can be activated by
either endogenous or exogenous agonists.
[0253] The molar ratio of an antibody construct immune-stimulatory compound
conjugate can
refer to the average number of immune-stimulatory compounds conjugated to the
antibody
construct in a preparation of an antibody construct immune-stimulatory
compound conjugate.
The molar ratio can be determined, for example, by Liquid Chromatography/Mass
Spectrometry
(LC/MS), in which the number of immune-stimulatory compounds conjugated to the
antibody
construct can be directly determined. Additionally, as non-limiting examples,
the molar ratio can
be determined based on hydrophobic interaction chromatography (HIC) peak area,
by liquid
chromatography coupled to electrospray ionization mass spectrometry (LC-ESI-
MS), by UV/Vis
spectroscopy, by reversed-phase-HPLC (RP-HPLC), or by matrix-assisted laser
desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS).
[0254] In some embodiments, the molar ratio of immune-stimulatory compound to
antibody can
be less than 8. In other embodiments, the molar ratio of immune-stimulatory
compound to
antibody can be 8, 7, 6, 5, 4, 3, 2, or 1.
[0255] In some aspects, the present disclosure provides a compound represented
by the structure
of Formula (I):
0 X1
p¨l-J
/ B1
iy\k
0
0
oi \NN/(R1)2
B2 /
0-1"
/ 0
X2
(I)
or a pharmaceutically acceptable salt thereof, wherein:
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Xl is selected from ¨0R2 and ¨SR2;
X2 is selected from ¨0R3 and ¨SR3;
B1 and B2 are independently selected from optionally substituted nitrogenous
bases;
Y is selected from ¨0124, ¨NR4R4, and halogen;
121, R2, R3 and R4 are independently selected at each occurrence from
hydrogen, -
c(=o)Rioo, _
C(=0)0Rim and -C(=0)NRim; Ci_10 alkyl, C2-10 alkenyl, C2-10 alkynyl, each of
which is independently optionally substituted at each occurrence with one or
more substituents
selected from halogen, -ORm , -se , _Nei:52, _s(0)R100, _s(0)2R100, _c(0)eo, _
C(0)0Rim,
-0C(0)Rim, -NO2, =0, =s, =Nem) , _
P(0)(0R1m)2, -0P(0)(0Rim)2, -CN, C3_10 carbocycle and
3- to 10-membered heterocycle; and C3-10 carbocycle and 3- to 10-membered
heterocycle,
wherein each C3-10 carbocycle and 3- to 10-membered heterocycle in 121, R2, R3
and R4 is
independently optionally substituted with one or more substituents selected
from halogen, -
()R' , -SR100, _N(R100)2, _s(o)R100, _s(0)2R100 _c(o)R100, _
C(0)ORM, -0C(0)R' , -NO2, =0,
=s, =NR100), _
P(0)(0R1m)2, -0P(0)(0Rim)2, -CN, C1_6 alkyl, C2_6 alkenyl, and C2_6 alkynyl;
and
Rm at each occurrence is independently selected from hydrogen; and C1_10
alkyl, C2-10
alkenyl, C2_10 alkynyl, C3-10 carbocycle, and 3- to 10-membered heterocycle
each of which is
independently optionally substituted at each occurrence with one or more
substituents selected
from halogen, -CN, -NO2, =0, =S, and haloalkyl.
[0256] In some embodiments, the compound of Formula (I) is represented by
Formula (IA):
xi
0 1
¨0
ZP
\
Y 0 B1
0
B2 0 N(R1)2
0¨p
/ 0
X2
(IA) ,
or pharmaceutically acceptable salts thereof.
[0257] In an alternative embodiment, the compound of Formula (I) is
represented by Formula
(TB):
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X1
0 I
P-0
B1
2
N (R1)2C0
/P
x2 0 (16),
or a pharmaceutically acceptable salt thereof.
[0258] In various embodiments, B1 and B2 are independently selected from
optionally substituted
I
-r-N
purines. In certain embodiments, B1 and B2 are i L1\
ndependently selected from: H .
In
certain embodiments, B1 and B2 are independently selected from optionally
substituted
pyrimidines.
[0259] In some embodiments, optionally substituted purines may include
optionally substituted
adenine, optionally substituted guanine, optionally substituted xanthine,
optionally substituted
hypoxaanthine, optionally substituted theobromine, optionally substituted
caffeine, optionally
substituted uric acid, and optionally substituted isoguanine. In certain
embodiments, B1 and B2
are independently selected from:
o CH3 o CH3
NH2 0 0 0
HN \
Fil\aEN 1\1"---).(NH
CI H3
NH2 ON N
CH3
0 NH2
o yN
NNH
H H , and H , optionally substituted by one or more
additional
substituents.
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NH2
N.--)N
I
N"---N-
[0260] In certain embodiments, B1 and B2 are independently selected from: -1-
,
0 0 0 0 NH2
NDNHi I Hy o,\1 --IIIN N----)i . 1 ) NH 11 r N-
---NH
N, N NH2 0'...*'N N N----.N N, ---NO \N- ".--
NiL,0
H ,L , 1- Au. H , and 4-- ,
wherein the
,
point of connectivity of B1 to the remainder of the compound is represented by
1.
[0261] In a preferred embodiment, B1 and B2 are independently selected from
optionally
substituted adenine and optionally substituted guanine. In certain
embodiments, B1 and B2 are
NH2 0
N-.....)
N N-.....A
I ) I X-I
--"N N N NH2
independently selected from: H , and I-1 ---
N , optionally further
substituted by
one or more substituents. In certain embodiments, B1 and B2 are independently
selected from:
NH2 0
N --...)
N N--ANH
pi ---- N' N---N NH2
/
and 'II- .
[0262] In some embodiments, B1 and B2 are independently optionally substituted
with one or
more substituents, wherein the optional substituents on B1 and B2 are
independently selected at
each occurrence from halogen, =0, =S, -ORM, -SR100, _N(R100)2, _s(0
)R100, _s(0
)2R100, _
C(0)R' ,
_
C(0)0R100, -0C(0)Rioo, _
NO2,.2 _
-P(0)(0R1oo), OP(0)(0Rin2 and -CN; C1_10 alkyl,
C2_10 alkenyl, C2-10 alkynyl, each of which is independently optionally
substituted at each
occurrence with one or more substituents selected from halogen, -0R100, -
SR100, _N(R100)2, _
S(0)R' ,
_s(o)2R100, _c(o)R100, _
C(0)0R100, -0C(0)R' , -NO2, =0, =S,
=Nen,
-P(0)(0R1m2, -0P(0)(0R1
) )2, -C N, C3-10 carbocycle and 3- to 10-
membered
heterocycle; and C3-10 carbocycle and 3- to 10-membered heterocycle, wherein
each C3-10
carbocycle and 3- to 10-membered heterocycle is independently optionally
substituted with one
or more substituents selected from halogen, -0R100, -SR100, _N(R100)2,
_s(o)R100, _s(0)2R100 _
C(0)R' ,
_
C(0)0R100, -0C(0)R' , -NO2, =0, =S,
=Nen,
-P(0)(0R1m2, -0P(0)(0R1
) )2, -C N, C1_6 alkyl, C2lk
_6 aenyl, and C2_6 alkynyl.
[0263] In certain embodiments, B1 and B2 are independently optionally
substituted with one or
more substituents, wherein the optional substituents on B1 and B2 are
independently selected at
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each occurrence from halogen, =0, =S, -0Rioo, _N(Rioo)2, _s(0)Rioo,
_s(0)2Rioo, _
c(0)Rioo, _
C(0)0Rioo, -0C(0)R'
,
NO2,oos2 _
-P(0)(0R1 ), OP(0)(0R1 )2, ¨CN and C1_10 alkyl.
[0264] In some embodiments, B1 is an optionally substituted guanine. In
certain embodiments,
O 0
Xi Dae
N N NH2
B1 is 11 = NH2 ,
. In certain embodiments, B1 is Ar. ,
wherein the point of
connectivity of B' tothe remainder of the compound is represented by . In
some
NH2
NN
II
NN
embodiments, B1 is an optionally substituted adenine. In certain embodiments,
B1 is H
NH2
NN
In certain embodiments, B1 is -1- ,
wherein the point of connectivity of B1 to the
remainder of the compound is represented by .
[0265] In some embodiments, B2 is an optionally substituted guanine. In
certain, embodiments,
O 0
Xi Dae
N N NH2
2 = NH2 ,
B is H . In certain embodiments, B2 is Ar. ,
wherein the point of
connectivity on B2 isrepresented by . In some embodiments, B2 is an
optionally substituted
NH NH2
N NN
I I
adenine. In certain embodiments, B2 is H . In certain embodiments, B2 is
'4"
wherein the point of connectivity on B2 isrepresented by .
[0266] In some embodiments, B1 is an optionally substituted guanine and B2 is
an optionally
substituted guanine. In some embodiments, B1 is an optionally substituted
adenine and B2 is an
optionally substituted guanine.
[0267] In various embodiments, Xl is selected from ¨OH and ¨SH. For example,
Xl may be ¨
OH. In various embodiments, X2 is selected from ¨OH and ¨SH. For example, X2
may be ¨OH.
In some embodiments, Xl is ¨OH and X2 is ¨OH. In some embodiments, Xl is ¨SH
and X2 is ¨
SH.
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[0268] In various embodiments, Y is selected from ¨OH, ¨0-C1_10 alkyl,
¨NH(C1_10 alkyl), and ¨
NH2. For example, Y may be ¨OH.
[0269] In various embodiments, R1- is independently selected at each
occurrence from hydrogen
and C1_10 alkyl optionally substituted at each occurrence with one or more
substituents selected
from halogen, -CN, -NO2, =0, and =S.
[0270] In various embodiments, the compound of Formula (I) is represented by
Formula (IC):
PH
0
N
P--- 1.3:11H
Z 0,
H 0
II N NI N NH2
r01
H2N )\1 N (:), NH2
H N jN 0-1D
/ 0 (IC)
HO
0 ,
or a pharmaceutically acceptable salt thereof. In some embodiments, the
compound of Formula
(IC) is represented by Formula (ID):
0.9H N Aid
PO
HO 0) N N NH2
c
0/
0 \ ________________________________________
H2N1\1 N ,0 NH2
I j
HN / 0 (ID)
N
HO
0 ,
or a pharmaceutically acceptable salt thereof.
[0271] In various embodiments, the compound is a pharmaceutically acceptable
salt. In some
embodiments, the compound or salt is a modulator of a stimulator of interferon
genes (STING).
The compound or salt may agonize a stimulator of interferon genes (STING). In
certain
embodiments, the compound or salt may cause STING to coordinate multiple
immune responses
to infection, including the induction of interferons and STAT6-dependent
response and selective
autophagy response. In certain embodiments, the compound or salt may cause
STING to mediate
type I interferon production.
[0272] In some aspects, the present disclosure provides an antibody drug
conjugate, comprising a
compound or salt previously described, an antibody, and a linker group,
wherein the compound
or salt is linked, e.g., covalently bound, to the antibody through the linker
group. The linker
group may be selected from a cleavable or non-cleavable linker. In some
embodiments, the linker
group is cleavable. In alternative embodiments, the linker group is non-
cleavable. Linkers are
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further described in the present application in the subsequent section, any
one of which may be
used to connect an antibody to a compound described herein.
[0273] In some aspects, the present disclosure provides a compound represented
by the structure
of Formula (II):
0 X1
/ ,
p-u
/ B1
Y 0
k 0
0 Z
B2 /
0-p
/ 0
X2
(II)
or a pharmaceutically acceptable salt thereof, wherein:
Xl is selected from -0R2 and -SR2;
X2 is selected from -0R3 and -SR3;
B1 and B2 are independently selected from optionally substituted nitrogenous
bases,
wherein each optional substituent is independently selected from halogen, -
OW00, -SR100, _
N(Rioo)2; _s(0)Rioo; _s(0)2Rioo; _c(0)Rioo; _
C(0)0Rioo; -0C(0)Rim, -NO2, =0, =S,
=N(Rioo); _cN; R6; and _)(3;
Y is selected from -0124, -S124, -NR4R4, and halogen;
Z is selected from -0R5' -SR5, and -NR5R5;
Rl; R2; R3; -4,
K and R5 are independently selected from a -X3; hydrogen, -C(=o)Rioo; _
C(=0)0Rim and -C(=0)NRin C1_10 alkyl, C2-10 alkenyl, C2-10 alkynyl, each of
which is
independently optionally substituted at each occurrence with one or more
substituents selected
from halogen, -0R100, -SR100, _N(R100)2, _s(0)R100, _s(0)2R100, _c(0)R100, _
C(0)0R1M, -
OC(0)R100, -NO2, =0, =s, =N(R100),
-P(0)(0R1w)2, -0 P(0)(0Rin2, -CN, C3-10 carbocycle and
3- to 10-membered heterocycle; and C3-10 carbocycle and 3- to 10-membered
heterocycle,
wherein each C3_10 carbocycle and 3- to 10-membered heterocycle in 121, R2,
R3, R4, and R5 is
optionally substituted with one or more substituents selected from halogen, -
OW00, -SR100, _
N(Rioo)2; _s(0)Rioo; _s(0)2Rioo _c(0)Rioo; _
C(0)0Rioo; -0C(0)Rim, -NO2, =0, =S,
=N(Rioo);
-P(0)(0R1w)2, -0 P(0)(0R1m)2, -CN, C1_6 alkyl, C2-6 alkenyl, C2_6 alkynyl;
R6 isindependently selected from -C(=o)Rioo; _
C(=0)0Rim and -C(=0)NRin Cl-lo
alkyl, C2-10 alkenyl, C2-10 alkynyl, each of which is independently optionally
substituted at each
occurrence with one or more substituents selected from halogen, -0R100, -
SR100, _N(R100)2, _
S(0)R' ,
_s(0)2R100, _c(0)R100, _
C(0)0R100, -0C(0)R' , -NO2, =0, =S,
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=N(R),
-P(0)(0R1 ), -0P(0)(ORM)2, -C N, C3-10 carbocycle and 3- to 10-membered
heterocycle; and C3_10 carbocycle and 3- to 10-membered heterocycle, wherein
each C3_10
carbocycle and 3- to 10-membered heterocycle in R6 is optionally substituted
with one or more
substituents selected from halogen, -0Rioo, _sRioo, _N(Rioo)2, _s(0)Rioo,
_s(0)2Rioo _
C(0)R' , _
C(0)0Rioo, -0C(0)R1 , -NO2, =0, =S,
=N(Rioo),
-P(0)(0R1 ), -0P(0)(ORM)2, -C N, C1_6 alkyl, C2lk
-6 aenyl, C2_6 alkynyl;
Rm at each occurrence is independently selected from hydrogen; and C1_10
alkyl, C2-10
alkenyl, C2_10 alkynyl, C3-10 carbocycle, and 3- to 10-membered heterocycle
each of which is
independently optionally substituted at each occurrence with one or more
substituents selected
from halogen, -CN, -NO2, =0, =S, and haloalkyl; and
X3 is a linker moiety, wherein at least one of Ri, R2, R3, R4, Rs, xl, )(2, a
bi-1
substituent
and a B2 substituent is -X3.
[0274] In various embodiments, the compound of Formula (II) is represented by
a structure of
Formula (IA):
xi
0 1
¨0
ZP
\
Y 0 B1
0
B2 /0 N(R1)2
0-P
/ 0
X2
(IA) ,
or pharmaceutically acceptable salts thereof.
[0275] In various embodiments, the compound of Formula (II) is represented by
a structure of
Formula (JIB):
X1
0 I
P-0
7
B1
0
NH(R1)2 0
0----
/P
X2 0 (IIB),
or a pharmaceutically acceptable salt thereof.
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[0276] In various embodiments, B1 and B2 are independently selected from
optionally substituted
purines. B1 and B2 may be each, independently selected from one another,
adenine, guanine, and
derivatives thereof. B1 and B2 may be independently selected from optionally
substituted
adenine, optionally substituted guanine, optionally substituted xanthine,
optionally substituted
hypoxanthine, optionally substituted theobromine, optionally substituted
caffeine, optionally
substituted uric acid, and optionally substituted isoguanine. In a preferred
embodiment, B1 and B2
are independently selected from optionally substituted adenine and optionally
substituted
guanine.
[0277] In various embodiments, B1 is substituted by X3 and optionally one or
more additional
substituents independently selected from halogen, -0R100, -SR100, _N(R100)2, _
S(0)R1 , -
S(0)2R100, _c(0)R100, _
C(0)0R100, -0C(0)R100, -NO2, =0, =s, =N(R100), _
CN, and R6. For
--X3
HN
1\1)\1
N N
example, B1 may be represented by: =,-1, , and wherein B1 is optionally
further substituted
by one or more substituents.
[0278] In various embodiments, B2 is substituted by X3 and optionally one or
more additional
substituents independently selected from halogen, -0R100, -SR100, _N(R100)2, _
S(0)R1 , -
S(0)2R100, _c(0)R100, _
C(0)0R100, -0C(0)R100, -NO2, =0, =s, =N(R100), _
CN, and R6. For
--X3
HN
1\136\1
N N
I
example, B2 may be represented by: ¨ , and wherein B2 is optionally further
substituted
by one or more substituents.
--X3
HN
1\1)\1
N N
[0279] In some embodiments, B1 is represented by =,-1, and B2 is
represented by
,X3
NH2 HN
361 N
, b
N N N N
-I- . In some embodiments, B1 is represented by =,-1, and
B2 is represented by
0
N D.NH
; I
N N NH2
-1,-. .
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[0280] In various embodiments, Xl is selected from -0- X3 and-S-X3. In some
embodiments, Xl
is selected from ¨OH and ¨SH. In some embodiments, Xl is -SH.
[0281] In various embodiments, X2 is selected from -0- X3 and-S-X3. In some
embodiments, X2
is selected from ¨OH and ¨SH. In some embodiments, X2 is -S-X3.
[0282] In some embodiments, Xl is ¨SH and X2 is -S-X3.
[0283] In certain embodiments, Y is selected from -NR4X3, -S-X3, and -0- X3.
In some
embodiments, Y is selected from ¨OH, -SH, ¨0-Ci_io alkyl, ¨NH(Ci_io alkyl),
and ¨NH2. In a
preferred embodiment, Y is selected from ¨OH.
[0284] In various embodiments, Z is selected from -NR4X3, -S-X3, and -0- X3.
In some
embodiments, Z is selected from ¨OH, -SH, ¨0-Ci_io alkyl, ¨NH(Ci_io alkyl),
and ¨NH2.
[0285] In various embodiments, ¨X3 is represented by the formula:
0 j(
N peptide
[0286] In some embodiments, ¨X3 is represented by the formula:
0 i(
N peptide¨RX
, wherein RX comprises a reactive moiety, such a maleimide.
[0287] In some embodiments, ¨X3 is represented by the formula:
0
N peptide¨RX*¨Antibody
*
, wherein RX is a reactive moiety that has reacted
with a moiety on an antibody to form an antibody-drug conjugate.
[0288] In some embodiments, ¨X3 is represented by the formula:
RX
1-4 H , wherein RX is
a reactive moiety, such as
a maleimide.
[0289] In some embodiments, ¨X3 is represented by the formula:
0 0
RX* Antibody
0 0
/1-4 H , wherein RX* is a reactive
moiety that has reacted with a moiety on an antibody to form an antibody drug
conjugate.
[0290] In some embodiments, the compound is represented by the formula:
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o
NI-J1'NH
I *I,
XP N N NH2
P-0
HO ,
4_0 c 0
_k y ? _N0
0 H
0 0
0-P,
H2 N y,;(1 1 N
0 0
0 X
0
HN I
0 ,
or a pharmaceutically acceptable salt thereof. The compound may be represented
by the formula:
N2- N H
I *1,
HOP N N NH
, P-0
F___. c_0_? H
H2N ,N
00H 0 0
EI)N7j1\ 0
N
O , or a pharmaceutically
acceptable salt thereof. The compound may be represented by the formula:
</N X N1...H
HS Q N N NH2
P-
H
HN--r",,,,..-0,õõõo.-"--õ,õ--0.f.o.-----..õ- N (Dr?
H2N ,N 07 0
-Ek
0 SH 0
E 1);j I \ 0
N
O , or a pharmaceutically
acceptable salt thereof.
[0291] In some embodiments, the compound is represented by the formula:
o
Ni'll'NH
x1P N N NH2
FLO
HO 7
H 91-13 0 H 0
4.--0-* 0 Ny:,N)5c,irwNji
01,, (2 H N Niro ipi 0 H
H2Ny,;(1.N d x o
o,...1
HN I N/> 0
O , or a pharmaceutically acceptable salt
thereof. The compound may be represented by the formula:
<J\1115,1H
HOP N N NH2
r P-0
H1,0_4 Lo_? H g H3y
=H2 N-ri-N
N \ HN
y:rr11N 0 0 H 0
0 OH
HN I /> 0 0
N
O , or a pharmaceutically acceptable salt
thereof. The compound may be represented by the formula:
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<P1/12::õNLH
HS. P N N NH2
.../ P-0
HITL_21 'V,L?:) HcH3y
HN 40 NY'N Y.'....3
N
H2NNrIN 0-ffl y) 0 H 0 /
o SH
HN I 0 0
N
O , or a pharmaceutically acceptable salt
thereof.
[0292] In some embodiments, the compound is represented by the formula:
o
<PIT'IIIIH
xP N N NH2
FLO
OI-141_0 Zk' 0--,2
F
0--r
H2N -Ss ,
,cN, x2 0 0 F F
H1) N I NJ'
F
O , or a pharmaceutically acceptable
salt thereof. The compound may be represented by the formula:
<,N1 2:õ...NLEI
HOP N N NH2
P-0
OH
F
0-7
1473(0-cr '-" F
r`m-',31:1-'0 -r 0
H2N N N.. OH
NV /> 0 F F
H N
F
O , or a pharmaceutically acceptable
salt thereof. The compound may be represented by the formula:
HS. P N N NH2
P-0
OH
F
0-7
H2N N N1473(0-oRZ FIN--tr`A)-'0"0 -r 0 F
..
NV /> SH 0 F F
H N
F
O , or a pharmaceutically acceptable
salt thereof.
[0293] In some embodiments, the compound is represented by the formula:
o
.NH Fi2Nyo
x1P N N NH2 1-IN
FLO
HO -r
j__,0 IcLCL? HgC
0H 0
(---Cr-ko_Kro HN,yo 41110 Nr,\,,-N.,_, ...
H2N N 0 /
FX):NNS d x 0 0
O , or a pharmaceutically acceptable salt
thereof. The compound may be represented by the formula:
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H 2N ,to
HOP N N NH2 HN
P-0
Fi!: 'VI IL:) H\ H
'
H lb Nr
'i ...,"
oHN"11^-------)L3
H2NN 0 irN crsA N 0 0
i 1 0 OH
0 0
HNt
N
O , or a pharmaceutically acceptable salt
thereof. The compound may be represented by the formula:
1\11-12:,,NLF 1 H2N Nr.o
HS. P N N NH2 HN
H/ PO HO., IcILD
N,tr,.." N.....,NHIr.,..,.....õ...,........).13
(ye r ill 0 H
H2N.,.;;N HN....o
rIN 0
I 1 0 SH
0 0
HN
N
O , or a pharmaceutically acceptable salt
thereof.
[0294] In some embodiments, the compound is represented by the formula:
o
xP N N, NH2
P-0
OI-417
0
0--r
H2 N Nd x2 o 0
HV NI 0
N
O , or a pharmaceutically acceptable
salt thereof.
[0295] The compound is represented by the formula:
HOP N N NH2
, P-0
OF2_41r IciL?D
H2N N
NV N
00H 0 0 0
H )
O , or a pharmaceutically acceptable
salt thereof.
[0296] The compound may be represented by the formula:
HS. P N N NH2
, P-0
OH ' IciL?D
I4-.Ao_s
H2N N
NV N
0 SH 0 0 0
H )
O , or a pharmaceutically acceptable
salt thereof.
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[0297] In some embodiments, the compound is represented by the formula:
o
o o
HN=CDCDN).1\1-jc
H ...,
NI'L,Ki 0
' I
)( P N N
, FLO
1-1C4LAi
0 OH
H2N);NrIN 0 /
.---FLX2
HN I
O , or a pharmaceutically
acceptable salt thereof. The compound may be represented by the formula:
HN N
H
q,N1.-"L. N
1 0
Hog N N
7_.._.
0 p OH
H2N N N 0-, /
Nv \ S- OH
H IT
O , or a pharmaceutically
acceptable salt thereof. The compound may be represented by the formula:
H1\10 0 . NH Ij..3
/
Nf,'KI
' I 0
HS. P N N
P- 0
H. IcLO_
o1 P S H
H2N
NV /> lx- OH
H N
O , or a pharmaceutically
acceptable salt thereof.
[0298] In some embodiments, the compound is represented by the formula:
o
o o
FINjOC)0 N).L.N-jc
NI/IN 0
</ 1
X19 N N
P-0
1-1(4Z:kr
cLO_
OH/0
H2Ny:NirlIN o"----, /
HN I N/> cilD'X2
O , or a pharmaceutically
acceptable salt thereof. The compound may be represented by the formula:
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11...3N /
I 0
HOP N N
P-0
7_._. 1cLO_
O OH ,0
H2N..rN)x
li..N
HN I 1\1 P'
0 OH
O , or a pharmaceutically
actable salt thereof. The compound may be represented by the formula:
11...3N /
I 1 0
HS. P N N
P- 0
H. 1cLO_
o1 oH,0
H2N NN
HN1.IT
); \
S'SH
O , or a pharmaceutically
acceptable salt thereof.
[0299] In some embodiments, the compound is represented by the formula:
o
-...,
N
H 0
X1P N N
FLO
o-p,----"
H2NX) N N d x2
F :N
o , or a pharmaceutically acceptable
salt thereof. The compound may be represented by the formula:
-õ,
HNJLO 110
N Lc Nis.... ENi ,..ss..........Z
NI/IN
Ir'H
I H 0 0
HOP N N
HNY7_,sr( P-hc0
O OH
0_ Ft------ 0
H2NN N 6 OH
T
N
O , or a pharmaceutically acceptable
salt thereof. The compound may be represented by the formula:
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0 0
--,..-
HN---k0
H 1rN
H 0
HS, P N N
IHC4L0
077
0-R----"
H2N.T.:Nirt.N
d SH
HN )N'>
NI>
O , or a pharmaceutically acceptable
salt thereof.
In some embodiments, the compound is represented by the formula:
o o
--,--
HN----11- N0 iii EN,
\'D'I -..r---
H I.:El 0
X1P N N
HN
1-1C4L0Z:k'
H2N0
07-7
o-p,----"
H2Ny.,NrIN d x2
HNI I NP
O , or a pharmaceutically acceptable
salt thereof. The compound may be represented by the formula:
0
...,--
H NriL 0 io N H 1
1....õ......,,,,.......... ....p
NX..11 NY'N
I H 0 H 0
HOP N N
P-0 HN
H2N 0
0R__------- L.
H2 N ,N
0 OH
El);j I
N
O , or a pharmaceutically acceptable
salt thereof. The compound may be represented by the formula:
0
...,--
HNIO io N H 1
1....õ......,,,,.......... ....p
NX..11 N
NY'
I H 0H 0
HS. P N N
P-0 HN
H2N 0
0 OH
H2N
crik-----
,N1
0 SH
E;(1j 1\
N
O , or a pharmaceutically acceptable
salt thereof.
Linkers
[0300] The compositions and methods described herein can comprise a linker,
e.g., a peptide
linker. Linkers of the compositions and methods described herein may not
affect the binding of
active portions of a conjugate (e.g., active portions include antigen binding
domains, Fc domains,
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targeting binding domains, antibodies, agonists or the like) to a target,
which can be a cognate
binding partner such as an antigen. A linker sequence can form a linkage
between different parts
of a composition. A composition can be a conjugate. A conjugate can comprise
multiple linkers.
These linkers can be the same linkers or different linkers.
[0301] Attachment via a linker can involve incorporation of a linker between
parts of a
composition or conjugate. A linker can be short, flexible, rigid, cleavable,
non-cleavable,
hydrophilic, or hydrophobic. A linker can contain segments that have different
characteristics,
such as segments of flexibility or segments of rigidity. The linker can be
chemically stable to
extracellular environments, for example, chemically stable in the blood
stream, or may include
linkages that are not stable. The linker can include linkages that are
designed to cleave and/or
immolate or otherwise breakdown specifically or non- specifically inside
cells. A cleavable
linker can be sensitive to enzymes. A cleavable linker can be cleaved by
enzymes such as
proteases. A cleavable linker can be a valine-citrulline linker or a valine-
alanine linker. A valine-
citrulline or valine-alanine linker can contain a pentafluorophenyl group. A
valine-citrulline or
valine-alanine linker can contain a succimide group. A valine-citrulline or
valine-alanine linker
can contain a para aminobenzoic acid (PABA) group. A valine-citrulline or
valine-alanine linker
can contain a PABA group and a pentafluorophenyl group. A valine-citrulline or
valine-alanine
linker can contain a PABA group and a succinimide group. A non-cleavable
linker can be
protease insensitive. A non-cleavable linker can be maleimidocaproyl linker. A
maleimidocaproyl linker can comprise N-maleimidomethylcyclohexane-l-
carboxylate. A
maleimidocaproyl linker can contain a succinimide group. A maleimidocaproyl
linker can
contain pentafluorophenyl group. A linker can be a combination of a
maleimidocaproyl group
and one or more polyethylene glycol molecules. A linker can be a maleimide-
PEG4 linker. A
linker can be a combination of a maleimidocaproyl linker containing a
succinimide group and
one or more polyethylene glycol molecules. A linker can be a combination of a
maleimidocaproyl linker containing a pentafluorophenyl group and one or more
polyethylene
glycol molecules. A linker can contain maleimides linked to polyethylene
glycol molecules in
which the polyethylene glycol can allow for more linker flexibility or can be
used lengthen the
linker. A linker can be a (maleimidocaproy1)-(valine-citrulline)-(para-
aminobenzyloxycarbonly)-
(NH2) linker. A linker can be a THIOMAB linker. A THIOMAB linker can be a
(maleimidocaproy1)-(valine-citrulline)-(para-aminobenzyloxycarbonly)-(NH2)
linker. A linker
can also be an alkylene, alkenylene, alkynylene, polyether, polyester,
polyamide, polyamino
acids, polypeptides, cleavable peptides, or aminobenzylcarbamates. A linker
can contain a
maleimide at one end and an N-hydroxysuccinimidyl ester at the other end. A
linker can contain
a lysine with an N-terminal amine acetylated, and a valine-citrulline cleavage
site. A linker can
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be a link created by a microbial transglutaminase, wherein the link can be
created between an
amine-containing moiety and a moiety engineered to contain glutamine as a
result of the enzyme
catalyzing a bond formation between the acyl group of a glutamine side chain
and the primary
amine of a lysine chain. A linker can contain a reactive primary amine. A
linker can be a Sortase
A linker. A Sortase A linker can be created by a Sortase A enzyme fusing an
LXPTG recognition
motif (SEQ ID NO: 21) to an N-terminal GGG motif to regenerate a native amide
bond. The
linker created can therefore link a moiety attached to the LXPTG recognition
motif (SEQ ID NO:
21) with a moiety attached to the N-terminal GGG motif. A linker can be a link
created between
an unnatural amino acid on one moiety reacting with oxime bond that was formed
by modifying
a ketone group with an alkoxyamine on another moiety. A moiety can be an
antibody construct.
A moiety can be an antibody. A moiety can be an immune-stimulatory compound. A
moiety can
be a targeting binding domain. A linker can be a portion of a linker can be
unsubstituted or
substituted, for example, with a substituent. A substituent can include, for
example, hydroxyl
groups, amino groups, nitro groups, cyano groups, azido groups, carboxyl
groups,
carboxaldehyde groups, imine groups, alkyl groups, alkenyl groups, alkynyl
groups, alkoxy
groups, acyl groups, acyloxy groups, amide groups, and ester groups.
[0302] In the antibody construct immune-stimulatory compound conjugate
described herein, the
immune-stimulatory compound is linked to the antibody construct by way of
linkers. The linker
linking an immune-stimulatory compound to the antibody of an antibody
construct immune-
stimulatory compound conjugate can be short, long, hydrophobic, hydrophilic,
flexible or rigid,
or may be composed of segments that each independently have one or more of the
above-
mentioned properties such that the linker may include segments having
different properties. The
linkers can be polyvalent such that they covalently link more than one immune-
stimulatory
compound to a single site on the antibody construct, or monovalent such that
covalently they link
a single immune-stimulatory compound to a single site on the antibody
construct.
[0303] As will be appreciated by skilled artisans, the linkers link the immune-
stimulatory
compound to the antibody by forming a covalent linkage to the immune-
stimulatory compound at
one location and a covalent linkage to the antibody construct at another. The
covalent linkages
are formed by reaction between functional groups on the linker and functional
groups on the
inhibitors and antibody construct. As used herein, the expression "linker" is
intended to include (i)
unconjugated forms of the linker that include a functional group capable of
covalently linking the
linker to an immune-stimulatory compound and a functional group capable of
covalently linking
the linker to an antibody construct; (ii) partially conjugated forms of the
linker that include a
functional group capable of covalently linking the linker to an antibody
construct and that is
covalently linked to an immune-stimulatory compound, or vice versa; and (iii)
fully conjugated
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forms of the linker that is covalently linked to both an immune-stimulatory
compound and an
antibody construct. In some specific embodiments of intermediate synthons and
antibody
construct immune-stimulatory compound conjugates described herein, moieties
comprising the
functional groups on the linker and covalent linkages formed between the
linker and antibody
construct are specifically illustrated as Rx and LK, respectively. One
embodiment pertains to an
antibody construct immune-stimulatory compound conjugate formed by contacting
an antibody
construct that binds a cell surface receptor or tumor associated antigen
expressed on a tumor cell
with a synthon described herein under conditions in which the synthon
covalently links to the
antibody construct. One embodiment pertains to a method of making an antibody
construct
immune-stimulatory compound conjugate formed by contacting a synthon described
herein under
conditions in which the synthon covalently links to the antibody construct.
One embodiment
pertains to a method of stimulating immune activity in a cell that expresses
CD40, comprising
contacting the cell with an antibody construct immune-stimulatory compound
conjugate
described herein that is capable of binding the cell, under conditions in
which the antibody
construct immune-stimulatory compound conjugate binds the cell.
[0304] Exemplary polyvalent linkers that may be used to link many immune-
stimulatory
compounds to an antibody construct are described. For example, Fleximer
linker technology
has the potential to enable high-DAR antibody construct immune-stimulatory
compound
conjugate with good physicochemical properties. As shown below, the Fleximer
linker
technology is based on incorporating drug molecules into a solubilizing poly-
acetal backbone via
a sequence of ester bonds. The methodology renders highly-loaded antibody
construct immune-
stimulatory compound conjugates (DAR up to 20) whilst maintaining good
physicochemical
properties. This methodology could be utilized with immune-stimulatory
compound as shown in
the Scheme below.
X1
X' 01
01
p-o
Z Y 0 B1
0
_,....
2
, o
0
B2 0 HN )
/ 0-
0-p HN I:)
/ C)
1% )(2 0
x2 OH
01_
NH2
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0 - =
yL'77N),,L3 ^c.,,yrOya Thr6yeThe,0
g
Ho OH OH n
Ho
ado. F.'eximer 4nker
INF'
HN HN
0 01117K
b¨Drug" o¨Dwg' 0¨Drug'
[0305] To utilize the Fleximer linker technology depicted in the scheme
above, an aliphatic
alcohol can be present or introduced into the immune-stimulatory compound. The
alcohol moiety
is then conjugated to an alanine moiety, which is then synthetically
incorporated into the
Fleximer linker. Liposomal processing of the antibody construct immune-
stimulatory
compound conjugate in vitro releases the parent alcohol-containing drug.
[0306] By way of example and not limitation, some cleavable and noncleavable
linkers that may
be included in the antibody construct immune-stimulatory compound conjugates
described herein
are described below.
[0307] Cleavable linkers can be cleavable in vitro and in vivo. Cleavable
linkers can include
chemically or enzymatically unstable or degradable linkages. Cleavable linkers
can rely on
processes inside the cell to liberate an immune-stimulatory compound, such as
reduction in the
cytoplasm, exposure to acidic conditions in the lysosome, or cleavage by
specific proteases or
other enzymes within the cell. Cleavable linkers can incorporate one or more
chemical bonds that
are either chemically or enzymatically cleavable while the remainder of the
linker can be non-
cleavable.
[0308] A linker can contain a chemically labile group such as hydrazone and/or
disulfide groups.
Linkers comprising chemically labile groups can exploit differential
properties between the
plasma and some cytoplasmic compartments. The intracellular conditions that
can facilitate
immune-stimulatory compound release for hydrazone containing linkers can be
the acidic
environment of endosomes and lysosomes, while the disulfide containing linkers
can be reduced
in the cytosol, which can contain high thiol concentrations, e.g.,
glutathione. The plasma stability
of a linker containing a chemically labile group can be increased by
introducing steric hindrance
using substituents near the chemically labile group.
[0309] Acid-labile groups, such as hydrazone, can remain intact during
systemic circulation in
the blood's neutral pH environment (pH 7.3-7.5) and can undergo hydrolysis and
can release the
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immune-stimulatory compound once the antibody construct immune-stimulatory
compound
conjugate is internalized into mildly acidic endosomal (pH 5.0-6.5) and
lysosomal (pH 4.5-5.0)
compartments of the cell. This pH dependent release mechanism can be
associated with
nonspecific release of the drug. To increase the stability of the hydrazone
group of the linker, the
linker can be varied by chemical modification, e.g., substitution, allowing
tuning to achieve more
efficient release in the lysosome with a minimized loss in circulation.
[0310] Hydrazone-containing linkers can contain additional cleavage sites,
such as additional
acid-labile cleavage sites and/or enzymatically labile cleavage sites.
Antibody construct immune-
stimulatory compound conjugates including exemplary hydrazone-containing
linkers can include,
for example, the following structures:
0 -
H
,N
N S Ab
g U 8
_ n
'0
hAb
J 6
dSI
--41 n
N' N
I
(If) H3C
H 1
--Ab
8 n
wherein D and Ab represent the immune-stimulatory compound and antibody
construct,
respectively, and n represents the number of immune-stimulatory compound -
linkers linked to
the antibody construct. In certain linkers such as linker (Ig), the linker can
comprise two
cleavable groups¨ a disulfide and a hydrazone moiety. For such linkers,
effective release of the
unmodified free immune-stimulatory compound can require acidic pH or disulfide
reduction and
acidic pH. Linkers such as (Ih) and (Ii) can be effective with a single
hydrazone cleavage site.
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[0311] Other acid-labile groups that can be included in linkers include cis-
aconityl-containing
linkers. cis-Aconityl chemistry can use a carboxylic acid juxtaposed to an
amide bond to
accelerate amide hydrolysis under acidic conditions.
[0312] Cleavable linkers can also include a disulfide group. Disulfides can be
thermodynamically stable at physiological pH and can be designed to release
the immune-
stimulatory compound upon internalization inside cells, wherein the cytosol
can provide a
significantly more reducing environment compared to the extracellular
environment. Scission of
disulfide bonds can require the presence of a cytoplasmic thiol cofactor, such
as (reduced)
glutathione (GSH), such that disulfide-containing linkers can be reasonably
stable in circulation,
selectively releasing the immune-stimulatory compound in the cytosol. The
intracellular enzyme
protein disulfide isomerase, or similar enzymes capable of cleaving disulfide
bonds, can also
contribute to the preferential cleavage of disulfide bonds inside cells. GSH
can be present in cells
in the concentration range of 0.5-10 mM compared with a significantly lower
concentration of
GSH or cysteine, the most abundant low-molecular weight thiol, in circulation
at approximately
M. Tumor cells, where irregular blood flow can lead to a hypoxic state, can
result in enhanced
activity of reductive enzymes and therefore even higher glutathione
concentrations. The in vivo
stability of a disulfide-containing linker can be enhanced by chemical
modification of the linker,
e.g., use of steric hindrance adjacent to the disulfide bond.
[0313] Antibody construct immune-stimulatory compound conjugates including
exemplary
disulfide-containing linkers can include the following structures:
R R
(ti)
(1k) D S Ab
R R
(11)
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wherein D and Ab represent the immune-stimulatory compound and antibody
construct,
respectively, n represents the number of immune-stimulatory compound -linkers
linked to the
antibody construct and R is independently selected at each occurrence from
hydrogen or alkyl,
for example. Increasing steric hindrance adjacent to the disulfide bond can
increase the stability
of the linker. Structures such as (Ij) and (I1) can show increased in vivo
stability when one or
more R groups is selected from a lower alkyl such as methyl.
[0314] Another type of linker that can be used is a linker that is
specifically cleaved by an
enzyme. For example, the linker can be cleaved by a lysosomal enzyme. Such
linkers can be
peptide-based or can include peptidic regions that can act as substrates for
enzymes. Peptide
based linkers can be more stable in plasma and extracellular milieu than
chemically labile linkers.
[0315] Peptide bonds can have good serum stability, as lysosomal proteolytic
enzymes can have
very low activity in blood due to endogenous inhibitors and the unfavorably
high pH value of
blood compared to lysosomes. Release of an immune-stimulatory compound from an
antibody
construct can occur due to the action of lysosomal proteases, e.g., cathepsin
and plasmin. These
proteases can be present at elevated levels in certain tumor tissues. The
linker can be cleavable
by a lysosomal enzyme. The lysosomal enzyme can be, for example, cathepsin B,
f3-
glucuronidase, or P-galactosidase.
[0316] The cleavable peptide can be selected from tetrapeptides such as Gly-
Phe-Leu-Gly, Ala-
Leu-Ala-Leu or dipeptides such as Val-Cit, Val-Ala, and Phe-Lys. Dipeptides
can have lower
hydrophobicity compared to longer peptides.
[0317] A variety of dipeptide-based cleavable linkers can be used in the
antibody constructs
immune-stimulatory compound conjugates described herein.
[0318] Enzymatically cleavable linkers can include a self-immolative spacer to
spatially separate
the immune-stimulatory compound from the site of enzymatic cleavage. The
direct attachment of
an immune-stimulatory compound to a peptide linker can result in proteolytic
release of an amino
acid adduct of the immune-stimulatory compound, thereby impairing its
activity. The use of a
self-immolative spacer can allow for the elimination of the fully active,
chemically unmodified
immune-stimulatory compound upon amide bond hydrolysis.
[0319] One self-immolative spacer can be a bifunctional para-aminobenzyl
alcohol group, which
can link to the peptide through the amino group, forming an amide bond, while
amine containing
immune-stimulatory compounds can be attached through carbamate functionalities
to the
benzylic hydroxyl group of the linker (to give a p-amidobenzylcarbamate,
PABC). The resulting
pro- immune-stimulatory compound can be activated upon protease- mediated
cleavage, leading
to a 1,6-elimination reaction releasing the unmodified immune-stimulatory
compound, carbon
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dioxide, and remnants of the linker group. The following scheme depicts the
fragmentation of p-
amidobenzyl carbamate and release of the immune-stimulatory compound:
0 ,
0 ,
---'.-- ""'\-0' X¨D -smi =
-NT ';'" 11 o'
µ..4-:o 1,z,linittltiTL, +co2
.p....,pticte" N'''N.. ''' -=-= --,..'4",/
HA µN\
HN':::>.c.\'=I'''
..
H ¨ X ¨0
wherein X-D represents the unmodified immune-stimulatory compound.
Heterocyclic variants of this self-immolative group have also been described.
[0320] The enzymatically cleavable linker can be a B-glucuronic acid-based
linker. Facile release
of the immune-stimulatory compound can be realized through cleavage of the B-
glucuronide
glycosidic bond by the lysosomal enzyme B-glucuronidase. This enzyme can be
abundantly
present within lysosomes and can be overexpressed in some tumor types, while
the enzyme
activity outside cells can be low. 13- Glucuronic acid-based linkers can be
used to circumvent the
tendency of an antibody construct immune-stimulatory compound conjugate to
undergo
aggregation due to the hydrophilic nature of B-glucuronides. In certain
embodiments, 13-
glucuronic acid-based linkers can link an antibody construct to a hydrophobic
immune-
stimulatory compound. The following scheme depicts the release of an immune-
stimulatory
compound (D) from an antibody construct (Ab) immune-stimulatory compound
conjugate
containing a B-glucuronic acid-based linker:
HO
HO ¨,
D 11-qh.teurorsidas,e. ---;.' I I' 0--
-' Q 1,6-Mi.miriatioll ¨
HO
z...\\0
----7:71'
D 0
o o õ
OH
[0321] A variety of cleavable P-glucuronic acid-based linkers useful for
linking drugs such as
auristatins, camptothecin and doxorubicin analogues, CBI minor-groove binders,
and psymberin
to antibodies have been described. All of these P-glucuronic acid-based
linkers may be used in
the ADCs described herein. In certain embodiments, the enzymatically cleavable
linker is a f3-
galactoside-based linker. P-Galactoside is present abundantly within
lysosomes, while the
enzyme activity outside cells is low.
[0322] Additionally, immune-stimulatory compounds containing a phenol group
can be
covalently bonded to a linker through the phenolic oxygen. One such linker
relies on a
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methodology in which a diamino-ethane "Space Link" is used in conjunction with
traditional
"PABO" -based self-immolative groups to deliver phenols.
[0323] Cleavable linkers can include non-cleavable portions or segments,
and/or cleavable
segments or portions can be included in an otherwise non-cleavable linker to
render it cleavable.
By way of example only, polyethylene glycol (PEG) and related polymers can
include cleavable
groups in the polymer backbone. For example, a polyethylene glycol or polymer
linker can
include one or more cleavable groups such as a disulfide, a hydrazone or a
dipeptide.
[0324] Other degradable linkages that can be included in linkers can include
ester linkages
formed by the reaction of PEG carboxylic acids or activated PEG carboxylic
acids with alcohol
groups on an immune-stimulatory compound, wherein such ester groups can
hydrolyze under
physiological conditions to release the immune-stimulatory compound.
Hydrolytically
degradable linkages can include, but are not limited to, carbonate linkages;
imine linkages
resulting from reaction of an amine and an aldehyde; phosphate ester linkages
formed by reacting
an alcohol with a phosphate group; acetal linkages that are the reaction
product of an aldehyde
and an alcohol; orthoester linkages that are the reaction product of a formate
and an alcohol; and
oligonucleotide linkages formed by a phosphoramidite group, including but not
limited to, at the
end of a polymer, and a 5' hydroxyl group of an oligonucleotide.
[0325] A linker can contain an enzymatically cleavable peptide moiety, for
example, a linker
comprising structural formula (IVa), (IVb), (IVc), or (IVd):
RY 0
R' H 010 =tz 0 tv"---
IVO
-N ; T peptde
H
RY
'
(Ivo
*N\----hir-c-i-'-'--perfticte¨N
RY 0
0
(Ivo
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9
c?
1lVd)
*". 'Veptide¨N'
or a salt thereof, wherein: peptide represents a peptide (illustrated N¨>C,
wherein peptide
includes the amino and carboxy "termini") a cleavable by a lysosomal enzyme; T
represents a
polymer comprising one or more ethylene glycol units or an alkylene chain, or
combinations
thereof; Ra is selected from hydrogen, alkyl, sulfonate and methyl sulfonate;
RY is hydrogen or
Ci_4 alkyl-(0),-(C14 a1kylene)s-G1 or Ci_4 alkyl-(N)-[(C14 alkylene)-G1]2; Rz
is C1-4 alkyl-(0)r-
(C14 a1kylene)s-G2; Gl is SO3H, CO2H, PEG 4-32, or sugar moiety; G2 is SO3H,
CO2H, or PEG
4-32 moiety; r is 0 or 1; s is 0 or 1; p is an integer ranging from 0 to 5; q
is 0 or 1; xis 0 or 1; y is
0 or 1; represents the point of attachment of the linker to the immune-
stimulatory compound;
and * represents the point of attachment to the remainder of the linker.
[0326] In certain embodiments, the peptide can be selected from a tripeptide
or a dipeptide. In
particular embodiments, the dipeptide can be selected from: Val-Cit; Cit-Val;
Ala-Ala; Ala-Cit;
Cit-Ala; Asn-Cit; Cit-Asn; Cit-Cit; Val-Glu; Glu-Val; Ser-Cit; Cit-Ser; Lys-
Cit; Cit-Lys; Asp-Cit;
Cit-Asp; Ala-Val; Val-Ala; Phe-Lys; Lys-Phe; Val-Lys; Lys-Val; Ala-Lys; Lys-
Ala; Phe-Cit;
Cit-Phe; Leu- Cit; Cit-Leu; Ile-Cit; Cit-Ile; Phe-Arg; Arg-Phe; Cit-Trp; and
Trp-Cit, or salts
thereof.
[0327] Exemplary embodiments of linkers according to structural formula (IVa)
that can be
included in the antibody construct immune-stimulatory compound conjugates
described herein
can include the linkers illustrated below (as illustrated, the linkers include
a group suitable for
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covalently linking the linker to an antibody construct):
1,13
-....
0 ...., *-,`-,----"---
iy-'---,
, 3 I H 2 1:01
N 1 m M k Ltn4
....,,,I
ht;;)
....A.,-,
H21',4 '0
3 .
0 -serk
(ilia 2)
9
---,... ....kti
CI 0 ' ' i_. 0
0
50.0i
0
0,jc4
IQ H 3 ''N.
(IVa.4) fal -11,
sk,,,,. N'''',----'''=-=,.--- -I - -
H H
0 L'=
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õIL
(iVa.5) '
0
NH-2
0 ID-
(iVa.6)
b
0
0
,1
(fli 'Ti
"ee
7Ft.
H
(EVa 9\\ L.
0
0
[0328] Exemplary embodiments of linkers according to structural formula (IVb),
(IVc), or (IVd)
that can be included in the antibody construct immune-stimulatory compound
conjugates
described herein can include the linkers illustrated below (as illustrated,
the linkers can include a
group suitable for covalently linking the linker to an antibody construct):
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9
it) 0 1
f",-.N 0,,,,,,a1^,.. =. a."'1/4,. a Na..,,,,,,ala , ..,--kN,,,,
0 A
NH
O'L'5LNH2
9
0 ". '-`1, ''''''..--".4"-- 0- v===
'=;:, fl , k= rz g t
(1Vb :2)
0 =
H2 r4 '1\)='
0
trfP
'2 ---- 0 (-:;---e---0-11-1--
,q =k,.-- -'14 -. =,''
0 ' .
Ci
(tVb .4)
0
1
-r
1 1
0
(Rib 5)
Ills:1
Le NH 2
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0 =
0\1).6) it 1
y-
H
'0
Viy0
3-04
011
(1\lb.7.)
0
Q
OjNIkklti
-
Y
,eo
(V1),$) -
(13 ki A g H
OH
0
*,1!
\tro.N.
01,1b .9.) 6 H
04k Ni-I2
N*6
- 0
Wit$,IA)
,
0
Cr*:LNK-,
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0
( Mb . 1 1 ) te- = - N .."'
1 14 i H
HO-437,0 0 7
6
NH
OANIZ-42
0
e 9.
4..
;:....?
cl,
JA
i'. .: H
Fi0-6=0 0
6
t,....,
ON
NH.,
ON 0
0
ri: 0 ti 0 4SyN0)11A
/ 4 A u Ne-*Nj''= N --'S''',',):
(i lib . 1 :3) o4 4"iH
. NLI
0 Ni-i.,
0
0
p4,..õ..õ......A, Kr?
(IVb . 1 4) 0
0 ....,...,
H>:N A0
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5)
===:µ) '-'sag-i NI,,,
= MI
t=ye"
0"4-1,Thik
0
(. IV b , I 6)
A---/--
0
x),_0,0,------e= k _ ., ..):11 ...i F.
HO 'la
--,
k.,..;µ,...õ....%,
(I Nib . r)
-I.
)µ1
-.- H
MO
$,0
0
(1Vb. 13)
,N---4
'µ,..õõe"-"r'seP'-'"µ 0
0 = * 11,-.1(.''--N
H
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crrzk
OH
f
,
(1Vb .. 19) H
H kH
at
() U
0
0 ?gY
r'r H
.1\c )
14
(IVC,2) jrIYH H P
N
04111
() 0
(11k,3) 7 r] t
-
. H = g,
1...Li 4ity-
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_ 0
r--1/4\õAõ._.õ( it \
\---\ p-o-- 1 inl"-----
l' 4
( 1Vc,4) , 0
_.r.
0--
.,,
rl = ====-- N..- '' ,---s, -,'''''''re's
(tVe.5) ,
HO
Q õ1 ,OH
-....;
(1V 6)o
'-'- c"
U
,, m=õ,
0 HN
NH
0
0
(IVO) 0
0 4i E H
= N.,
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)
(
6.1 IV& I ) ,
4,,
r i 0 .kAk2,
l f 11 =$-i 1---"
b
',,.e`Ck.,-="-`40,'.
'k4P
(1.1
( I \M. 2.)
.z. iij H 1 cz,...õ.õ...,0,,
'C413e7411"k1
(1Vd .3) A
fyirIT"\---14',...---Nses.....Ø6,-"Kr"Nes0-1
. s H
1..,51õ,õ.- ..
= ....=H
C'tft---NH2
I-g',..1)
-)
A 4-1 R
6 0
k.
i
066*0
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[0329] The linker can contain an enzymatically cleavable sugar moiety, for
example, a linker
comprising structural formula (Va), (Vb), (Vc), (Vd), or (Ve):
0 *
\. :, t 1
, ==: : Z' ;
(Va) H .
,OH
Q 1
0....00,4...... OH
.
il
OH OH
OH OH
..-1,.. .0H
,1
0
OH
I '
(Vb)
Q 0
xl Air
0
.,
,
(VC) u
LOH
ID)0eL
CY
' . OH
-
t
OH OH
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OH OH
A 1,, .01-i
6 k,
oH
6
(vd.)
=
9
111,xl
(vo 0
H 4
OH
'**
1
011
or a salt thereof, wherein: q is 0 or 1; r is 0 or 1; X is CH2, 0 or NH;
/represents the point of
attachment of the linker to the immune-stimulatory compound; and * represents
the point of
attachment to the remainder of the linker.
[0330] Exemplary embodiments of linkers according to structural formula (Va)
that can be
included in the antibody construct immune-stimulatory compound conjugates
described herein
can include the linkers illustrated below (as illustrated, the linkers include
a group suitable for
covalently linking the linker to an antibody construct):
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tlkIN
16
,l,k,,,,, 0
0
9
w'ANY'''''''' 0.3,õ......0
:
KY'y'tm
Olk
J
r
( Val)
0 0
q -
1 0
)10''' = '''''''OH
\\L
OIN
v
IL, 0 0 NH
tif,,,lAstO1P
4 .1,,,,,.,, 3,,/,,,,,,,,,,=:
.-.
H. H
wrANIe. Nbee
iid µIbti
to,
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yy0
i.,
1
!,.
Z,,,.., , 21.1õ, ......essµ AN.1 ,,,,,,,,,,,z,,,,,..".õ.....,õ,..a.,,,,,-
.4.,NeNN.eaks,""NtiAN.,-= iki
(Vas5)
. = . ,,
,..,,,,., ,,,,,,
..,
HoeYkkm
,itt.0õ.,
(
, ..;.=
( Va, 6) o 14
KI,Aksts"'"
!,,..
6i
0
Ill )
u fr'tN P Ci 0
\a.7) o \I
ti 4
,
t H
\N \
ey
=:! a 9, ,, 0
H
H t4
0
W 1
e=K) CI
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ltiol
I ?
, ?
---I0 N--N------N-"'N,-----N,,",,,,
NO i
04yX
I ,
(Va: 1 0)
0
HOe \\I".*D-i
Ok
AO
6
sopH o
i N
0 '==,.,'-''''''' N ' N.0 (r*
1 i
ics.,,,0
......,,,
=
L 0,1...õ..,.õ,z,
\\ TDI
=$,,,,, _ ..,..,,,,,,,mi
(Va. 12)
d õL õIL., 0
=\\.,,,,,o- N ., N ,0 `-
0
H H
Hos,..A4,õ..e.¨........õ*N.,
k 1
Ni
[0331] Exemplary embodiments of linkers according to structural formula (Vb)
that may be
included in the antibody construct immune-stimulatory compound conjugates
described herein
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include the linkers illustrated below (as illustrated, the linkers include a
group suitable for
covalently linking the linker to an antibody construct):
s'st..sk,
Or.4\
0 0
q
t
(Vb., I)
N 0.
e,..,.
jr0v 'j
4 ,
Ho ko4
01-tc
0
144-,43,
(vb,2)h" A)
es ,õ,.<4 _
c. H ,.......õe
...., _
0 N-,
0¨c
k--\*..õ
0
HOCK,.,440H =w''''"
4 Nt=¨=
HO t4I
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4-P
r \,,,,
,!,,,.,.(,-...k ,,,,
',,-,. =
8 Li: 501 Q
i
0.0,..N.,-,0,õ..",õ0 = Ki
N
o PI
: k
bii 15 i
\.,,,, = ,,..w
.4=µ"=-µ,'
:I. r*
); ====="'N'=\=,---"N,,,(' sOM
k
.>
0 HN ,001 ("NY
..,,,
(VM) " ==''',AN,'"Ng';''''. \ NkS"V
Ck N
H
0,4111\A'ON
ho
. N
0. ThLfP
"4.1 HO
1, PH
(Vb3) ==%. 1,-K=),,, ..., "s\ OH
2
o
.74)
t gm
)
0
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HO 0
DH
(Vb.:7) 0\
`k
r"%t=01
OH
HO 0
b,8)
(-Nod
0
0
071c,
/N*-
>4.00
911
'\sis
(Vbs9)
i)=0
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0 '0
9
(Vb.10)
1.1=H
(t4*(IFN`NeZ
1-1('
OH o
[0332] Exemplary embodiments of linkers according to structural formula (Vc)
that may be
included in the antibody construct immune-stimulatory compound conjugates
described herein
include the linkers illustrated below (as illustrated, the linkers include a
group suitable for
covalently linking the linker to an antibody construct):
41'0. " 2
(VC. )
PH
NO
Nokk,..et
0
0001
0
4)\'`µ(( 0
,
(Vc,2)
XL'o
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,..i2
,i(lke$kNõ,k4S1
Z 1
Ofk \\O'''ANCOP
k .
(ile. $) C " 0
0
:
N4\40
NO
1., pH
,..,
HO,,,s -i,
0 0 H \ -V
( V0,4)
1 / o
H .
2, 0
RNN''''c
= .4
,
,s .......õ..
.2:, ..
(VC:
....7
4-,
.z,
Ho-' : -4"-te*'''
,,1-,\\,,,,,
He -it-.
OH
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Qt,
o -rsk
d i
(Vc.6) i k
= .10
0
110'''''''' \=r**6
z
CAI
9 o,..õ.µ
HI -11,
,:4õ.2...;\II 0....,...õ.õ.., a
0 ,
õ.... u,õõõ,,,\\
(VC:1)
...e.P
0 1
j
Hoe' \\Ir *OH
OH
HO
..-IP4
0 A i
....,
(Ve:8)
..eNi
0
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a.
0
(VC,9)
110se.\\T"OH
OH
(VC:
= 401 Ls, 0,
cr's 1"-A
0
N
Agl
HO
LJ
=
H00 k
0 0
(VC.1 0"L s(
) 0
C's
e \OH
X's-v
[0333] Exemplary embodiments of linkers according to structural formula (Vd)
that may be
included in the antibody construct immune-stimulatory compound conjugates
described herein
include the linkers illustrated below (as illustrated, the linkers include a
group suitable for
covalently linking the linker to an antibody construct):
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) = =
0= QJ
0
H
. 4k
4
)ft
X 0
CNN OH
(Vd2) = ,k
04-4õ
-"N
(Vd3)
s'N
=-=*õ/
j
oros
9
(Vd54)
Oro
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AP
P.
6, =
CL?
fili=¨=\ ,,,
(Vd.3) No--
-\\
I
\_s
tid
Cir.. Is
9
11
=,:d's
(\d 6) ia,,,,,. . L., .9
.r. , \1/41,, 94
043 \\OPI
[0334] Exemplary embodiments of linkers according to structural formula (Ve)
that may be
included in the antibody construct immune-stimulatory compound conjugates
described herein
include the linkers illustrated below (as illustrated, the linkers include a
group suitable for
covalently linking the linker to an antibody construct):
.,0
0
(Ve, I)
- .voies
u i
\
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,----'
o õ
(Vo. 2)
\
H ht5
40N r
[0335] Although cleavable linkers can provide certain advantages, the linkers
comprising the
conjugate described herein need not be cleavable. For non-cleavable linkers,
the immune-
stimulatory compound release may not depend on the differential properties
between the plasma
and some cytoplasmic compartments. The release of the immune-stimulatory
compound can
occur after internalization of the antibody construct immune-stimulatory
compound conjugate via
antigen-mediated endocytosis and delivery to lysosomal compartment, where the
antibody
construct can be degraded to the level of amino acids through intracellular
proteolytic
degradation. This process can release a immune-stimulatory compound
derivative, which is
formed by the immune-stimulatory compound, the linker, and the amino acid
residue to which
the linker was covalently attached. The immune-stimulatory compound derivative
from antibody
construct immune-stimulatory compound conjugates with non-cleavable linkers
can be more
hydrophilic and less membrane permeable, which can lead to less bystander
effects and less
nonspecific toxicities compared to antibody construct immune-stimulatory
compound conjugates
with a cleavable linker. Antibody construct immune-stimulatory compound
conjugates with non-
cleavable linkers can have greater stability in circulation than antibody
construct immune-
stimulatory compound conjugates with cleavable linkers. Non-cleavable linkers
can be alkylene
chains, or can be polymeric, such as, for example, based upon polyalkylene
glycol polymers,
amide polymers, or can include segments of alkylene chains, polyalkylene
glycols and/or amide
polymers. The linker can contain a polyethylene glycol segment having from 1
to 6 ethylene
glycol units.
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[0336] The linker can be non-cleavable in vivo, for example, a linker
according to the
formulations below:
0 0
0
(Via)
0 N
6'4
0
(Wb)
o-to
0
(Vic)
N =
H = C-9
0
(Vid)
Fe 04
or salts thereof, wherein: Ra is selected from hydrogen, alkyl, sulfonate and
methyl sulfonate; 12'
is a moiety including a functional group capable of covalently linking the
linker to an antibody
construct; and 1 represents the point of attachment of the linker to the
immune-stimulatory
compound.
[0337] Exemplary embodiments of linkers according to structural formula (VIa)-
(VId) that may
be included in the conjugates described herein include the linkers illustrated
below (as illustrated,
the linkers include a group suitable for covalently linking the linker to an
antibody construct, and
represents the point of attachment to an immune-stimulatory compound):
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9 a
\ 0 0
(Va. =1 )
,
0".
9
(vic.t) \s"
0
0
(V10.1)
.0,Lx
(vitt
0
0
0
(V id.2)
SOH 0
0 0
0
0
,
(V Id. 4)
S 03H 0
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[0338] Attachment groups that are used to attach the linkers to an antibody
can be electrophilic
in nature and include, for example, maleimide groups, activated disulfides,
active esters such as
NHS esters and HOBt esters, haloformates, acid halides, alkyl, and benzyl
halides such as
haloacetamides. There are also emerging technologies related to "self-
stabilizing" maleimides
and "bridging disulfides" that can be used in accordance with the disclosure.
[0339] One example of a "self-stabilizing" maleimide group that hydrolyzes
spontaneously under
antibody construct conjugation conditions to give an antibody construct immune-
stimulatory
compound conjugate species with improved stability is depicted in the
schematic below. Thus,
the maleimide attachment group is reacted with a sulfhydryl of an antibody
construct to give an
intermediate succinimide ring. The hydrolyzed form of the attachment group is
resistant to
deconjugation in the presence of plasma proteins.
itigirrW .5sTerT3 :
-41 mit \
nft A it*
9b . AP
1
nip\ 0
6 tASrpla Q
0 -,....
'-''''4=,--1( 1-1. fwile 0 ,y,.., pi,a,.k'41 .#
..,' 'N.¨, ..., ,semmmw, Y=NN __ ..,..
A 0
1-11
0
0 /
'-.-5---\ r
1 N--
S,,,,i L-M'
)c.).
.0
0
&SSW'S t. 'DAR kase owe .11m.
Siettf=Oeb4iz:.,-k; attacrneRt
6 0 "k= mickb k
A 'Lt=iN nAH
ii
0 H2 N.' 0 B26/ OH
=tains ma:4.11M =Wm svcd9Irri::Nje -
Mg rsttg h>t
Kz.wod 'f.Jr,-=!& of sixeinft:62 rk,-.q
hy4r0.1 torrowstro stliAt in pslavrt,1
[0340] A method for bridging a pair of sulfhydryl groups derived from
reduction of a native
hinge disulfide bond has been disclosed and is depicted in the schematic
below. An advantage of
this methodology is the ability to synthesize homogenous DAR4 antibody
construct immune-
stimulatory compound conjugates by full reduction of IgGs (to give 4 pairs of
sulfhydryls)
followed by reaction with 4 equivalents of the alkylating agent. Antibody
construct immune-
stimulatory compound conjugates containing "bridged disulfides" are also
claimed to have
increased stability.
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,.............,_.N,
d------s---.0
r
.TWkir.:e a.lulfblf:,
',. ,-' ., ."'" ' .0
.=
0¨:...
/ ',,-.1
.,'fflr ,.....;.
n
,.1.-
--,"--,, &la Glirespatcq
1
(As.71..,'%; . Ac.f.:;1.....$,,.....,µ ,, = ,b, ..
9,
6
.....,)ogvki
[0341] Similarly, as depicted below, a maleimide derivative that is capable of
bridging a pair of
sulfhydryl groups has been developed.
.1 N
N,..i,,,,,,,=,.,
S 0
.,)
___________________________ =. :
, S
0
.--L, ,cir.,.->= - N ,s
N
[0342] The attachment moiety can contain the following structural formulas
(VIIa), (VIIb), or
(VIIc):
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õP
er¨f 0
-*
(vw 0
0, 0
k'm
-stµ
e e kX
$,
(Vlib 0 6
'y
G'
(V11c)
N
Ltr
or salts thereof, wherein: Rq is H or-0-(CH2CH20)11-CH3; x is 0 or 1; y is 0
or 1; G2 is-
CH2CH2CH2S03H or-CH2CH20-(CH2CH20)ii-CH3; is-O-CH2CH2S03H or-NH(C0)-
CH2CH20-(CH2CH20)12-CH3; and * represents the point of attachment to the
remainder of the
linker.
[0343] Exemplary embodiments of linkers according to structural formula (VIIa)
and (VIIb) that
can be included in the antibody construct immune-stimulatory compound
conjugates described
herein can include the linkers illustrated below (as illustrated, the linkers
can include a group
suitable for covalently linking the linker to an antibody construct):
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'...... K
&
=-4( $
\
1
tg
foftle:L.s. 0.4
ss).....t
k t,,,,,;k,(
e'
(4,...,
)3.?=,,."'s\µµ k Isi
/ .."
es--1
õ.....
(V 142)
r'"=-='
0.....,
J r-No
I rm J
r I r
r ..,x;
r'
I
r
õj
i....,,,
,..---
,r,.. j
.r.
= ,,,,----3,-"Nrs'''''
( VI lav3)
.i
¨160¨
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ets
I-4
ir
r
f
e)
r4
_...1,
( V I fa . 4) ,..)
.Ã
I
ili,=i
o 824'ik's\'' k
(VIM. fl :1)
r
)
f V3
0
'-'tire'N'Ne*C1/41."'"Ncye-\ "k.,04) \ .,=-='''\ N'o='"NN.0=4)')
='' ,===
'=A ,=-k. r"..ty"...,,A:X,Nee-,...,0õ0"Nts,,,..,,,ONses,"...õ0õ...,N,w,,,C)
=-.
0 11204,1s 14,14¨
Viib.2) :21,=
LIe'NH 0 0
) 1
'`µ,,=-= . .."' e\ 1`" .,----3
H
..,
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0
7 9
Nõ..-Ascm
(Yitb,3) 0A
ovztc
64 6H
,
\
H
N
(Vilbs,t) ''\11 =! === N.,,e
0 '
=-= G-1-1c#)
k.OH
k
OH OH:
WN4
pslcr
0
(Vilb,6) Li
-kze,
' 0
,
,
oti e5H
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\\ci,...--=,,,õ,,O.N.,,,,s-Nr..."...40,..0,..\,,,,,,..0,,,-\\,..,,O,,,k
õA
.).....N i:
(Vitt). 7) H
lir"''''Iws's
.."-
o
6i1 6,4
, ek
H lz Y H 0
f" ),'=
(\lib 8) 4-N
:IN, ,AGH
"N.,,"'%\,..s,",o4"Nve =N.-''''''''to-'"N=eA,Ne"No
"el\'`=,-"INOH
.&,i 6sd
[0344] Exemplary embodiments of linkers according to structural formula (VIIc)
that can be
included in the antibody construct immune-stimulatory compound conjugates
described herein
can include the linkers illustrated below (as illustrated, the linkers can
include a group suitable
for covalently linking the linker to an antibody construct):
0
0
¨ 0
V.!
'''' \ tole\ r...3' 0
,..,.,
11
.k
(VIIC, 1) CI se0
OH 0 )
(:sr.' '44 +=,,,.
: ,t''''`k
O
0 H
Ck'''rl '''''N \ r OH
OH OH
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= '= e
( ile.2)
f"e
A
N.=61t4
/===
rks
%
ON,
(VIle:3)
r
T
õ.te 0,e õ...= z4
Nõ,re
kAZ ======2,
%
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HA 100
141k1.
(Vile 4)
n k s ,,,....-
''' \' r-- g
...
(0 0
RV'
Os-bt
ge0 0
K1 ,
HO Zo
IL Pi
HOw
0
g.'
k, ''''. ,,,,"*.N)1.\-=.'14 '''*N''
/
v
0
0 H , .õ.,
h
loNytN \võ,-,.',,,N, ,,,õ NNe ".-N' `TAN \,,,,,,
, k H .. ,-,, ,u
.....õ . , 0 1,01-. 0
(Vikv6) 0 Os- NH
(.) I
r\ Z.47'-tir
OH
Conjugates
[0345] A composition as described herein can be a conjugate. A conjugate can
comprise an
antibody construct, an immune-stimulatory compound, and a linker. A conjugate
can comprise an
antibody construct, a pattern recognition receptor (PRR) agonist, and a
linker. A conjugate can
comprise an antibody construct, a pattern-associated molecular pattern (PAMP)
molecule, and a
linker. A conjugate can comprise an antibody construct, a damage-associated
molecular pattern
(DAMP) molecule, and a linker. A conjugate can comprise an antibody construct,
a STING
agonist, and a linker. A conjugate can comprise an antibody construct, a toll-
like receptor agonist
molecule, and a linker. A conjugate can comprise an antibody construct,
imiquimod, and a linker.
A conjugate can comprise an antibody construct, S-27609, and a linker. A
conjugate can
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comprise an antibody construct, CL307, and a linker. A conjugate can comprise
an antibody
construct, resiquimod, and a linker. A conjugate can comprise an antibody
construct,
gardiquimod, and a linker. A conjugate can comprise an antibody construct, UC-
IV150, and a
linker. A conjugate can comprise an antibody construct, KU34B, and a linker. A
conjugate can
comprise an antibody construct, motolimod, and a linker. A conjugate can
comprise an antibody
construct, VTX-1463, and a linker. A conjugate can comprise an antibody
construct, GS-9620,
and a linker. A conjugate can comprise an antibody construct, GSK2245035, and
a linker. A
conjugate can comprise an antibody construct, TMX-101, and a linker. A
conjugate can comprise
an antibody construct, TMX-201, and a linker. A conjugate can comprise an
antibody construct,
TMX-202, and a linker. A conjugate can comprise an antibody construct,
isatoribine, and a
linker. A conjugate can comprise an antibody construct, AZD8848, and a linker.
A conjugate can
comprise an antibody construct, MEDI9197, and a linker. A conjugate can
comprise an antibody
construct, 3M-051, and a linker. A conjugate can comprise an antibody
construct, 3M-852, and a
linker. A conjugate can comprise an antibody construct, 3M-052, and a linker.
A conjugate can
comprise an antibody construct, 3M-854A, and a linker. A conjugate can
comprise an antibody
construct, S-34240, and a linker. A conjugate can comprise an antibody
construct, CL663, and a
linker. A conjugate can comprise an antibody construct, KIN1148, and a linker.
A conjugate can
comprise an antibody construct, SB-9200, and a linker. A conjugate can
comprise an antibody
construct, KIN-100, and a linker. A conjugate can comprise an antibody
construct, ADU-S100,
and a linker. A conjugate can comprise an antibody construct, KU34B, and a
linker. An antibody
construct of any of the conjugates described herein can have a modified Fc
domain of the
antibody construct. The modified Fc domain can comprise a substitution at more
than one amino
acid residue such as at 5 different amino acid residues including
L235V/F243L/R292P/Y300L/P396L, as at 2 different amino acid residues including
S239D/I332E, or as at 3 different amino acid residues including
S298A/E333A/K334A. The
numbering of amino acids residues described herein can be according to the EU
index as in
Kabat.
[0346] A conjugate can comprise an antibody construct, a targeting binding
domain, an immune-
stimulatory compound, and a linker. A conjugate can comprise an antibody
construct, a targeting
binding domain, a pattern recognition receptor (PRR) agonist, and a linker. A
conjugate can
comprise an antibody construct, a targeting binding domain, a pattern-
associated molecular
pattern (PAMP) molecule, and a linker. A conjugate can comprise an antibody
construct, a
targeting binding domain, a damage-associated molecular pattern (DAMP)
molecule, and a
linker. A conjugate can comprise an antibody construct, a targeting binding
domain, a STING
agonist, and a linker. A conjugate can comprise an antibody construct, a
targeting binding
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domain, a toll-like receptor agonist molecule, and a linker. A conjugate can
comprise an antibody
construct, a targeting binding domain, imiquimod, and a linker. A conjugate
can comprise an
antibody construct, a targeting binding domain, S-27609, and a linker. A
conjugate can comprise
an antibody construct, a targeting binding domain, CL307, and a linker. A
conjugate can
comprise an antibody construct, a targeting binding domain, resiquimod, and a
linker. A
conjugate can comprise an antibody construct, a targeting binding domain,
gardiquimod, and a
linker. A conjugate can comprise an antibody construct, a targeting binding
domain, UC-IV150,
and a linker. A conjugate can comprise an antibody construct, a targeting
binding domain,
motolimod, and a linker. A conjugate can comprise an antibody construct, a
targeting binding
domain, VTX-1463, and a linker. A conjugate can comprise an antibody
construct, a targeting
binding domain, GS-9620, and a linker. A conjugate can comprise an antibody
construct, a
targeting binding domain, GSK2245035, and a linker. A conjugate can comprise
an antibody
construct, a targeting binding domain, TMX-101, and a linker. A conjugate can
comprise an
antibody construct, a targeting binding domain, TMX-201, and a linker. A
conjugate can
comprise an antibody construct, a targeting binding domain, TMX-202, and a
linker. A conjugate
can comprise an antibody construct, a targeting binding domain, isatoribine,
and a linker. A
conjugate can comprise an antibody construct, a targeting binding domain,
AZD8848, and a
linker. A conjugate can comprise an antibody construct, a targeting binding
domain, MEDI9197,
and a linker. A conjugate can comprise an antibody construct, a targeting
binding domain, 3M-
051, and a linker. A conjugate can comprise an antibody construct, a targeting
binding domain,
3M-852, and a linker. A conjugate can comprise an antibody construct, a
targeting binding
domain, 3M-052, and a linker. A conjugate can comprise an antibody construct,
a targeting
binding domain, 3M-854A, and a linker. A conjugate can comprise an antibody
construct, a
targeting binding domain, S-34240, and a linker. A conjugate can comprise an
antibody
construct, a targeting binding domain, CL663, and a linker. A conjugate can
comprise an
antibody construct, a targeting binding domain, KIN1148, and a linker. A
conjugate can
comprise an antibody construct, a targeting binding domain, SB-9200, and a
linker. A conjugate
can comprise an antibody construct, a targeting binding domain, KIN-100, and a
linker. A
conjugate can comprise an antibody construct, a targeting binding domain, ADU-
S100, and a
linker. A conjugate can comprise an antibody construct, a targeting binding
domain, KU34B, and
a linker. An antibody construct of any of the conjugates described herein can
have a modified Fc
domain of the antibody construct. The modified Fc domain can comprise a
substitution at more
than one amino acid residue such as at 5 different amino acid residues
including
L235V/F243L/R292P/Y300L/P396L, as at 2 different amino acid residues including
S239D/I332E, or as at 3 different amino acid residues including
S298A/E333A/K334A.
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[0347] The linker can be a linker as described herein. A linker can be
cleavable, non-cleavable,
hydrophilic, or hydrophobic. A cleavable linker can be sensitive to enzymes. A
cleavable linker
can be cleaved by enzymes such as proteases. A cleavable linker can be a
valine-citrulline or a
valine-alanine linker. A valine-citrulline or valine-alanine linker can
contain a pentafluorophenyl
group. A valine-citrulline or valine-alanine linker can contain a succimide
group. A valine-
citrulline or valine-alanine linker can contain a PABA group. A valine-
citrulline or valine-alanine
linker can contain a PABA group and a pentafluorophenyl group. A valine-
citrulline or valine-
alanine linker can contain a PABA group and a succinimide group. A non-
cleavable linker can be
protease insensitive. A non-cleavable linker can be maleimidocaproyl linker. A
maleimidocaproyl linker can comprise N-maleimidomethylcyclohexane-l-
carboxylate. A
maleimidocaproyl linker can contain a succinimide group. A maleimidocaproyl
linker can
contain pentafluorophenyl group. A linker can be a combination of a
maleimidocaproyl group
and one or more polyethylene glycol molecules. A linker can be a maleimide-
PEG4 linker. A
linker can be a combination of a maleimidocaproyl linker containing a
succinimide group and
one or more polyethylene glycol molecules. A linker can be a combination of a
maleimidocaproyl linker containing a pentafluorophenyl group and one or more
polyethylene
glycol molecules. A linker can contain maleimides linked to polyethylene
glycol molecules in
which the polyethylene glycol can allow for more linker flexibility or can be
used lengthen the
linker. A linker can be a (maleimidocaproy1)-(valine-citrulline)-(para-
aminobenzyloxycarbonly)-
(NH2) linker. A linker can be a THIOMAB linker. A THIOMAB linker can be a
(maleimidocaproy1)-(valine-citrulline)-(para-aminobenzyloxycarbonly)-(NH2)
linker. A linker
can also be an alkylene, alkenylene, alkynylene, polyether, polyester,
polyamide, polyamino
acids, polypeptides, cleavable peptides, or aminobenzylcarbamates. A linker
can contain a
maleimide at one end and an N-hydroxysuccinimidyl ester at the other end. A
linker can contain
a lysine with an N-terminal amine acetylated, and a valine-citrulline cleavage
site. A linker can
be a link created by a microbial transglutaminase, wherein the link is created
between an amine-
containing moiety and a moiety engineered to contain glutamine as a result of
the enzyme
catalyzing a bond formation between the acyl group of a glutamine side chain
and the primary
amine of a lysine chain. A linker can contain a reactive primary amine. A
linker can be a Sortase
A linker. A Sortase A linker can be created by a Sortase A enzyme fusing an
LXPTG (SEQ ID
NO: 21) recognition motif to an N-terminal GGG motif to regenerate a native
amide bond. The
linker created can therefore link a moiety attached to the LXPTG (SEQ ID NO:
21) recognition
motif with a moiety attached to the N-terminal GGG motif. A linker can be a
link created
between an unnatural amino acid on one moiety reacting with oxime bond that
was formed by
modifying a ketone group with an alkoxyamine on another moiety. A moiety can
be an antibody
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construct. A moiety can be a targeting binding domain. A moiety can be an
antibody. A moiety
can be an immune-stimulatory compound.
[0348] The antibody construct can be as described herein. The antibody
construct can be an anti-
tumor antigen antibody construct. The antibody construct can be an anti-tumor
antigen antibody.
An antigen recognized by the antibody construct can be CD5, CD19, CD20, CD25,
CD37, CD30,
CD33, CD45, CAMPATH-1, BCMA, CS-1, PD-L1, B7-H3, B7-DC,HLD-DR,
carcinoembryonic
antigen, TAG-72, EpCAM, MUC1, folate-binding protein, A33, G250, prostate-
specific
membrane antigen, ferritin, GD2, GD3, GM2, Leg, CA-125, CA19-9, epidermal
growth factor,
p185HER2, IL-2 receptor, de2-7 EGFR, fibroblast activation protein, tenascin,
metalloproteinases, endosialin, vascular endothelial growth factor, avB3, WT1,
LMP2, HPV E6
E7, EGFRvIII, Her-2/neu, idiotype, MAGE A3, p53 nonmutant, NY-ES0-1, PMSA,
GD2, CEA,
MelanA/MART1, Ras mutant, gp100, p53 mutant, PR1, bcr-abl, tyronsinase,
survivin, PSA,
hTERT, Sarcoma translocation breakpoints, EphA2, PAP, ML-IAP, AFP, ERG, NA17,
PAX3,
ALK, androgen receptor, cyclin Bl, polysialic acid, MYCN, RhoC, TRP-2, fucosyl
GM1,
mesothelin, PSCA, MAGE Al, sLe(animal), CYP1B1, PLAV1, GM3, BORIS, Tn, GloboH,
ETV6-AML, NY-BR-1, RGS5, SART3, STn, Carbonic anhydrase IX, PAX5, 0Y-TES1,
Sperm
protein 17, LCK, HMWMAA, AKAP-4, 55X2, XAGE 1, B7H3, Legumain, Tie 3, Page4,
VEGFR2, MAD-CT-1, PDGFR-B, MAD-CT-2, ROR2, TRAILl, MUC16, MAGE A4, MAGE
C2, GAGE, or Fos-related antigen 1. The antibody construct can recognize an
antigen that can be
expressed on a cell. The antibody construct can recognize an antigen that can
be expressed by a
cell. The antibody construct can recognize an antigen that can be expressed in
the context of a
Major Histocompatibility Complex. The antibody construct can recognize an
antigen that can
stimulate activity of a cell. The antibody construct can recognize an antigen
that can stimulate an
immune response. The antibody construct can recognize an antigen that can
reduce an immune
response. The antibody construct can recognize an antigen can reduce activity
of a cell. The
antibody construct can recognize an antigen that can be expressed on an immune
cell. The
antibody construct can recognize an antigen that can be expressed by an immune
cell. The
antibody construct can recognize an antigen that can be in the context of a
Major
Histocompatibility Complex. The antibody construct can recognize an antigen on
a cell wherein
the antigen can be involved in stimulating activity of a cell. The antibody
construct can recognize
an antigen on an immune cell that can be involved in the costimulation of an
immune cell. The
antibody construct can recognize an antigen on an immune cell that can be
involved in the
costimulation of an immune cell during an immune response. The antibody
construct can
recognize a receptor. The antibody construct can recognize a receptor on a
cell. The antibody
construct can recognize a receptor ligand. The antibody construct can
recognize a receptor on a
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cell wherein the receptor can be involved in stimulating activity of a cell.
The antibody construct
can recognize a receptor on an immune cell. The antibody construct can
recognize a receptor on
an immune cell that can be involved in stimulating activity of an immune cell.
The antibody
construct can recognize a receptor on an immune cell that can be involved in
the costimulation of
an immune cell. The antibody construct can recognize a receptor on an immune
cell that can be
involved in the costimulation of an immune cell during an immune response. The
antibody
construct can recognize an antigen that can be expressed on an immune cell and
that can
stimulate activity of an immune cell. The antibody construct can recognize an
antigen that can be
expressed on an immune that can reduce activity of an immune cell. The
antibody construct can
be an anti-CD40 antibody. The antibody construct can comprise a light chain of
an SBT-040
antibody. The antibody construct can comprise an SBT-040-G1WT heavy chain. The
antibody
construct can comprise an SBT-040-G1VLPLL heavy chain. The antibody construct
can
comprise an SBT-040-G1DE heavy chain. The antibody construct can comprise an
SBT-040-
G1AAA heavy chain. The antibody construct can comprise an SBT-040-CDR
sequence. The
antibody construct can be capable of recognizing a single antigen. The
antibody construct can be
capable of recognizing two or more antigens. The Kd for binding of an antigen-
binding domain of
an antibody construct immune-stimulatory compound conjugate to an antigen in
the presence of
an immune-stimulatory compound can be about 2 times, about 3 times, about 4
times, about 5
times, about 6 times, about 7 times, about 8 times, about 9 times, about 10
times, about 15 times,
about 20 times, about 25 times, about 30 times, about 35 times, about 40
times, about 45 times,
about 50 times, about 60 times, about 70 times, about 80 times, about 90
times, about 100 times,
about 110 times, or about 120 times greater than the Kd for binding of the
antigen binding
domain to the antigen of an antibody construct in the absence of the immune-
stimulatory
compound. The Kd for binding of an antigen-binding domain of an antibody
construct immune-
stimulatory compound conjugate to an antigen in the presence of the immune-
stimulatory
compound can be less than 10 nM. The Kd for binding of an antigen-binding
domain of an
antibody construct immune-stimulatory compound conjugate to an antigen in the
presence of the
immune-stimulatory compound can be less than 100 nM, less than 50 nM, less
than 20 nM, less
than 5 nM, less than 1 nM, or less than 0.1 nM.
[0349] An antibody construct can further comprise a targeting binding domain.
A targeting
binding domain of an antibody construct can recognize an antigen. For example,
an antigen can
be expressed on an immune cell. As another example, an antigen can be
expressed by a tumor or
cancer cell. An antigen can be a peptide or fragment thereof. An antigen can
be expressed on an
antigen-presenting cell. An antigen can be expressed on a dendritic cell, a
macrophage, or a B
cell. An antigen can be CD40 and a targeting binding domain can recognize a
CD40 antigen. An
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antigen can be a tumor antigen and a targeting binding domain can recognize a
tumor antigen. A
targeting binding domain of an antibody construct can be a CD40 agonist. A
targeting binding
domain of an antibody construct can bind to a tumor antigen.
[0350] The antibody construct can have an Fc domain that can bind to an FcR
when linked to an
immune-stimulatory compound. The antibody construct can have an Fc domain that
can bind to
an FcR to initiate FcR-mediated signaling when linked to an immune stimulatory
compound. The
antibody construct can bind to its antigen when linked to an immune-
stimulatory compound. The
antibody construct can bind to its antigen when linked to an immune-
stimulatory compound and
the Fc domain of the antibody construct can bind to an FcR when linked to an
immune-
stimulatory compound. The antibody construct can bind to its antigen when
linked to an immune-
stimulatory compound and the Fc domain of the antibody can bind to an FcR to
initiate FcR-
mediated signaling when linked to an immune stimulatory compound. The Fc
domain linked to
an immune-stimulatory compound can be a modified Fc domain. The modified Fc
domain can
comprise a substitution at more than one amino acid residue such as at 5
different amino acid
residues including L235V/F243L/R292P/Y300L/P396L, as at 2 different amino acid
residues
including S239D/I332E, or as at 3 different amino acid residues including
S298A/E333A/K334A. The Kd for binding of an Fc domain to a Fc receptor when
the Fc domain
is linked to an immune-stimulatory compound can be about 2 times, about 3
times, about 4 times,
about 5 times, about 6 times, about 7 times, about 8 times, about 9 times,
about 10 times, about
15 times, about 20 times, about 25 times, about 30 times, about 35 times,
about 40 times, about
45 times, about 50 times, about 60 times, about 70 times, about 80 times,
about 90 times, about
100 times, about 110 times, or about 120 times greater than the Kd for binding
of the Fc domain
to the Fc receptor in the absence of the immune-stimulatory compound. The Kd
for binding of an
Fc domain to an Fc receptor when linked to an immune-stimulatory compound can
be less than
nM. The Kd for binding of an Fc domain to an Fc receptor when linked to an
immune-
stimulatory compound can be less than 100 nM, less than 50 nM, less than 20
nM, less than 5
nM, less than 1 nM, or less than 0.1 nM.
[0351] The PRR agonist can be a toll-like receptor agonist. The toll-like
receptor agonist can be a
TLR1 agonist, a TLR2 agonist, a TLR3 agonist, a TLR4 agonist, a TLR5 agonist,
a TLR6
agonist, a TLR7 agonist, a TLR8 agonist, a TLR9 agonist, a TLR10 agonist, a
TLR11 agonist, a
TLR12 agonist or a TLR13 agonist. The toll-like receptor agonist can activate
two or more
TLRs. The PAMP molecule can be a RIG-I agonist.
[0352] A conjugate can be formed by a linker that can connect an antibody
construct to a PRR. A
conjugate can be formed by a linker that can connect an antibody construct to
a PAMP molecule.
A conjugate can be formed by a linker that can connect an antibody construct
and a DAMP
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molecule. A conjugate can be formed by a linker that can connect an antibody
construct to a
PRR, and a linker that can connect an antibody construct and a targeting
binding domain. A
conjugate can be formed by a linker that can connect an antibody construct to
a PAMP molecule,
and a linker that can connect an antibody construct and a targeting binding
domain. A conjugate
can be formed by a linker that can connect an antibody construct and a DAMP
molecule, and a
linker that can connect an antibody construct and a targeting binding domain.
[0353] A linker can be connected to an antibody construct by a direct linkage
between the
antibody construct and the linker. A linker can be connected to an anti-CD40
antibody construct
by a direct linkage between the anti-CD40 antibody construct and the linker. A
linker can be
connected to an anti-CD40 antibody by a direct linkage between the anti-CD40
antibody and the
linker. A linker can be connected to an anti-tumor antigen antibody construct
by a direct linkage
between the anti-tumor antigen antibody construct and the linker. A linker can
be connected to an
anti-tumor antigen antibody by a direct linkage between the anti-tumor antigen
antibody and the
linker. A direct linkage can be a covalent bond. For example, a linker can be
attached to a
terminus of an amino acid sequence of an antibody construct, or could be
attached to a side chain
modification to the antibody construct, such as the side chain of a lysine,
serine, threonine,
cysteine, tyrosine, aspartic acid, a non-natural amino acid residue, or
glutamic acid residue. An
attachment can be via any of a number of bonds, for example but not limited
to, an amide bond,
an ester bond, an ether bond, a carbon-nitrogen bond, a carbon-carbon single
double or triple
bond, a disulfide bond, or a thioether bond. A linker can have at least one
functional group,
which can be linked to the antibody. Non-limiting examples of the functional
groups can include
those which form an amide bond, an ester bond, an ether bond, a carbonate
bond, a carbamate
bond, or a thioether bond, such functional groups can be, for example, amino
groups; carboxyl
groups; aldehyde groups; azide groups; alkyne and alkene groups; ketones;
carbonates; carbonyl
functionalities bonded to leaving groups such as cyano and succinimidyl and
hydroxyl groups. A
linker can be connected to an antibody construct at a hinge cysteine. A linker
can be connected to
an antibody construct at a light chain constant domain lysine. A linker can be
connected to an
antibody construct at an engineered cysteine in the light chain. A linker can
be connected to an
antibody construct at an engineered light chain glutamine. A linker can be
connected to an
antibody construct at an unnatural amino acid engineered into the light chain.
A linker can be
connected to antibody construct at an unnatural amino acid engineered into the
heavy chain.
Amino acids can be engineered into an amino acid sequence of a composition as
described
herein, for example, a linker of a conjugate. Engineered amino acids can be
added to a sequence
of existing amino acids. Engineered amino acids can be substituted for one or
more existing
amino acids of a sequence of amino acids. A linker can be conjugated to
antibody construct via a
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sulfhydryl group. A linker can be conjugated to an antibody construct via a
primary amine. A
linker can be a link created between an unnatural amino acid on an antibody
construct reacting
with oxime bond that was formed by modifying a ketone group with an
alkoxyamine on an
immune-stimulatory compound. When a linker is connected to an antibody
construct at the sites
described herein, an Fc domain of the antibody construct can bind to Fc
receptors. When a linker
is connected to an antibody construct at the sites described herein, the
antigen binding domain of
the antibody construct can bind its antigen. When a linker is connected to an
antibody construct
at the sites described herein, a targeting binding domain of said antibody
construct can bind its
antigen.
[0354] An antibody with engineered reactive cysteine residues (THIOMAB) can be
used to link
a targeting binding domain to the antibody. A linker can connect an antibody
construct to a
targeting binding domain via Sortase A linker. A Sortase A linker can be
created by a Sortase A
enzyme fusing an LXPTG (SEQ ID NO: 21) recognition motif to an N-terminal GGG
motif to
regenerate a native amide bond. The linker created can therefore link an
antibody construct
attached to the LXPTG (SEQ ID NO: 21) recognition motif with a targeting
binding domain
attached to the N-terminal GGG motif. A targeting binding domain can be
connected to a linker
by a direct linkage. A direct linkage can be a covalent bond. For example, a
linker can be
attached to a terminus of an amino acid sequence of a targeting binding
domain, or could be
attached to a side chain modification to the targeting binding domain, such as
the side chain of a
lysine, serine, threonine, cysteine, tyrosine, aspartic acid, a non-natural
amino acid residue, or
glutamic acid residue. An attachment can be via any of a number of bonds, for
example but not
limited to, an amide bond, an ester bond, an ether bond, a carbon-nitrogen
bond, a carbon-carbon
single double or triple bond, a disulfide bond, or a thioether bond. A linker
can have at least one
functional group, which can be linked to the targeting binding domain. Non-
limiting examples of
the functional groups can include those which form an amide bond, an ester
bond, an ether bond,
a carbonate bond, a carbamate bond, or a thioether bond, such functional
groups can be, for
example, amino groups; carboxyl groups; aldehyde groups; azide groups; alkyne
and alkene
groups; ketones; carbonates; carbonyl functionalities bonded to leaving groups
such as cyano and
succinimidyl and hydroxyl groups. Amino acids can be engineered into an amino
acid sequence
of the targeting binding domain. Engineered amino acids can be added to a
sequence of existing
amino acids. Engineered amino acids can be substituted for one or more
existing amino acids of a
sequence of amino acids. A linker can be conjugated to a targeting binding
domain via a
sulfhydryl group. A linker can be conjugated to a targeting binding domain via
a primary amine.
A targeting binding domain can be conjugated to the C-terminal of an Fc domain
of an antibody
construct.
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[0355] An antibody with engineered reactive cysteine residues (THIOMAB) can be
used to link
an immune-stimulatory compound to the antibody. A linker can connect an
antibody construct to
an immune-stimulatory compound via THIOMAB linker. A linker can connect an
antibody
construct to an immune-stimulatory compound via Sortase A linker. A Sortase A
linker can be
created by a Sortase A enzyme fusing an LXPTG (SEQ ID NO: 21) recognition
motifto an N-
terminal GGG motif to regenerate a native amide bond. The linker created can
therefore link an
antibody construct attached the LXPTG (SEQ ID NO: 21) recognition motif with
an immune-
stimulatory compound attached to the N-terminal GGG motif. A linker can be a
link created
between an unnatural amino acid on an antibody construct reacting with oxime
bond that was
formed by modifying a ketone group with an alkoxyamine on an immune-
stimulatory compound.
The immune-stimulatory compound can comprise one or more rings selected from
carbocyclic
and heterocyclic rings. The immune-stimulatory compound can be covalently
bound to a linker
by a bond to an exocyclic carbon or nitrogen atom on said immune-stimulatory
compound. A
linker can be conjugated to an immune-stimulatory compound via an exocyclic
nitrogen or
carbon atom of an immune-stimulatory compound. A linker can be connected to a
STING
agonist, for example:
õ-,
=
,,N õ.õõ
4:" 8
HO H H
0H 0"
/*--\
-------
WWinslitio-WI-Akt-PAE,/A¨GAMP
H4,4 .t4
"r
0
N
I
N
/es=
HOõ 9
OH 0'
()'
M3:::0:8.6004>ECAMP
4) 0 t'41
HN, =
0
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0
= NH
J1,..
HO. p
N. -NH2
OH
H 0 CY .
H
= =,,,,ve = N
4¨Y4m\ Hk j A ¨ H /I
¨ 0
HN
1 riss'
gi
d
N
N
0
Miklimkto-Vat-AWPABA,..cs=G(2's=NH)p4P
or
9,
il*s-tv." 'NH
< I
HO... P N' 'j"
\.)
HN
efr"'".-4S, = .:?-
~-s>.
HN,
0 0
o
wIwtnitio-PEG.4,<:45(2`44H2V.GP
[0356] A linker agonist complex can dissociate under physiological conditions
to yield an active
agonist.
[0357] A linker can be connected to a PRR agonist by a direct linkage between
the PRR agonist
and the linker. A linker can be connected to a PAMP molecule by a direct
linkage between the
PAMP molecule and the linker. A linker can be connected to a toll-like
receptor agonist by a
direct linkage between the toll-like receptor agonist and the linker.
[0358] Examples of toll-like receptor agonists connected to a linker in a
manner able to release
an active toll-like receptor agonist under physiologic condition can include:
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o
o 0
H
.
110
0 0 H 0 * OAN
H
\ H cHr
o INN NJ(
- N
0 NH2 \ H i H
1 , N
0 0 =
NH2
Maleincle-PEG4-KU34b Maleincle-
Val-Ala-PABA-KU34b
r,e0 0
0
H
0 0 0 I 0AN
0 4 0 IN N H
.,....µ1,0)(N,N)(ir Nj(N H
N H = H 1
, N
H2
0 0 =
0
NHS-PEG5-KU34b NHS-Val-Ala-PABA-KU34b
NH2
[0359] Examples of RIG-I agonists connected to a linker in a manner able to
release an active
toll-like receptor agonist under physiologic conditions can include:
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0
H
CI
H
0 .'" N
S
OC H3
Maleimide-PEG4-KIN700
0
CI 0 ...... N ,A......../ \., 0./
,.... ......)1\0.11,R
H
-5 0
0 ---= N
140
OC H3
NHS-PEG5-KIN700
S 0
CI A 0
0 N 0 0 0 H 0
0 ---= N H
H H
SO0H30 0
OC H3
Maleimide-Val-Ala-PABC-KIN700
S 0
CI A
N 0 0 0 HI..0 0 0
H
*ON ---=
H H
SO 0H30 00
OC H3
NHS-Val-Ala-PABC-KIN700
[0360] A linker can be connected to a DAMP molecule by a direct linkage
between the DAMP
molecule and the linker. A direct linkage can be a covalent bond. For example,
a linker can be
attached to a terminus of an amino acid sequence of an antibody, or could be
attached to a side
chain modification to the antibody, such as the side chain of a lysine,
serine, threonine, cysteine,
tyrosine, aspartic acid, a non-natural amino acid residue, or glutamic acid
residue. An attachment
can be via any of a number of bonds, for example but not limited to, an amide
bond, an ester
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bond, an ether bond, a carbon-nitrogen bond, a carbon-carbon single double or
triple bond, a
disulfide bond, or a thioether bond. A linker can have at least one functional
group, which can be
linked to the antibody construct. Non-limiting examples of the functional
groups can include
those which form an amide bond, an ester bond, an ether bond, a carbonate
bond, a carbamate
bond, or a thioether bond, such functional groups can be, for example, amino
groups; carboxyl
groups; aldehyde groups; azide groups; alkyne and alkene groups; ketones;
carbonates; carbonyl
functionalities bonded to leaving groups such as cyano and succinimidyl and
hydroxyl groups.
[0361] An ATAC can be formed by conjugating a noncleavable maleimide-PEG4
linker
containing a succinimide group with an immune-stimulatory compound. For
example, an ATAC
can be N-((4-amino-1-(2-hydroxy-2-methylpropy1)-1H-imidazo[4,5-c]quinolin-2-
yl)methyl)-1-
(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-N-ethyl-3,6,9,12-
tetraoxapentadecan-
15-amide (ATAC11); N-(5-(2-amino-3-pentylquinolin-5-yl)penty1)-1-(3-(2,5-dioxo-
2,5-dihydro-
1H-pyrrol-1-yl)propanamido)-3,6,9,12-tetraoxapentadecan-15-amide (ATAC12); 1-
(3-(2,5-
dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-N-(3-pentylquinolin-2-y1)-
3,6,9,12-
tetraoxapentadecan-15-amide (ATAC13); 1-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-
yl)propanamido)-N-(1-isobuty1-1H-imidazo[4,5-c]quinolin-4-y1)-3,6,9,12-
tetraoxapentadecan-
15-amide (ATAC14); 1-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-N-
methyl-N-(2-
(3-(7-methylbenzo[1,2-d:3,4-0bis(thiazole)-2-yl)ureido)ethyl)-3,6,9,12-
tetraoxapentadecan-15-
amide (ATAC15); (S)-1-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-N-
(1-((7-
methylbenzo[1,2-d:3,4-0bis(thiazole)-2-yOamino)-1-oxo-3-phenylpropan-2-y1)-
3,6,9,12-
tetraoxapentadecan-15-amide (ATAC16); N-(benzo[d]thiazol-2-y1)-1-(3-(2,5-dioxo-
2,5-dihydro-
1H-pyrrol-1-yl)propanamido)-N-((8-hydroxyquinolin-7-y1)(4-
(trifluoromethoxy)phenyOmethyl)-
3,6,9,12-tetrao xapentadecan- 15-amide (ATAC17); N-((2R,3R,3 aS ,7
aR,9R,10R,10aS ,14aR)-2,9-
bis(2-amino-6-oxo-1H-purin-9(6H)-y1)-5,10,12-trihydroxy-5,12-
dioxidodecahydrodifuro[3,2-
d:3',2'-j][1,3,7,9,2,8]tetra-oxadiphosphacyclododecin-3-y1)-1-(3-(2,5-dioxo-
2,5-dihydro-1H-
pyrrol-1-yl)propanamido)-3,6,9,12-tetraoxapentadecan-15-amide (ATAC18); N-
((2R,3R,3 aS,7 aR,9R,10R,10aS ,14aR)-2,9-bis(2-amino-6-oxo-1H-purin-9(6H)-y1)-
10-hydroxy-
5,12-dimercapto-5,12-dioxidodecahydrodifuro[3,2-d:3',2'-
j][1,3,7,9,2,8]tetraoxadiphosphacyclododecin-3-y1)-1-(3-(2,5-dioxo-2,5-dihydro-
1H-pyrrol-1-
yl)propanamido)-3,6,9,12-tetraoxapentadecan-15-amide (ATAC19); N-(9-
((2R,3R,3 aS,7aR,9R,10R,10aS ,14aR)-9-(2-amino-6-oxo- 1H-purin-9(6H)-y1)-3
,5,10,12-
tetrahydroxy-5,12-dioxidodecahydrodifuro[3,2-d:3',2'-j][1,3,7,9,2,8]tetra-
oxadiphosphacyclododecin-2-y1)-9H-purin-6-y1)-1-(3-(2,5-dioxo-2,5-dihydro-1H-
pyrrol-1-
yl)propanamido)-3,6,9,12-tetraoxapentadecan-15-amide (ATAC20); or
((2R,3R,3 aS,7aR,9R,10R,10aS ,14aR)-9-(2-amino-6-oxo- 1H-purin-9(6H)-y1)-3
,5,10,12-
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tetrahydroxy-5,12-dioxidodecahydrodifuro[3,2-d:3',2'-
j][1,3,7,9,2,8]tetraoxadiphosphacyclododecin-2-y1)-9H-purin-6-y1)-1-(3-(2,5-
dioxo-2,5-dihydro-
1H-pyrrol-1-yl)propanamido)-3,6,9,12-tetraoxapentadecan-15-amide (ATAC21).
[0362] An ATAC can be formed by conjugating a cleavable valine-alanine or
valine-citrulline
linker containing a PABA group and a succinimide group with an immune-
stimulatory
compound. For example, an ATAC can be 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-
dihydro-1H-pyrrol-
1-yl)hexanamido)-3-methylbutanamido)propanamido)benzyl ((4-amino-1-(2-hydroxy-
2-methyl-
propy1)-1H-imidazo[4,5-c]quinolin-2-yl)methyl)(ethyl)carbamate (ATAC22); 4-
((S)-2-((S)-2-(6-
(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methyl-
butanamido)propanamido)benzyl
(5-(2-amino-3-pentylquinolin-5-yl)penty1)-carbamate (ATAC23); 4-((S)-2-((S)-2-
(6-(2,5-dioxo-
2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutan-amido)-5-
ureidopentanamido)benzyl-
(5-(2-amino-3-pentylquinolin-5-yl)penty1)-carbamate (ATAC24); 4-((S)-2-((S)-2-
(6-(2,5-dioxo-
2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methylbutanamido)-5-
ureidopentanamido)benzyl((4-
amino-1-(2-hydroxy-2-methylpropy1)- 1H-imidazo[4,5-c]quinolin-2-
yl)methyl)(ethyl)carbamate
TFA salt (ATAC25); 2-(3-124N-Methyle[p-RS)-2-1(S)-246-(2,5-dioxo-1H-pyrrol-1-
yl)hexanoylamino]-3-methylbutyrylamino}-5-
ureidovalerylamino]phenyl}methoxycarbonyl)aminolethyl}ureido)-7-methy1-1,6-
dithia-3,8-
diaza-as-indacene (ATAC26); 2-1 [(8-Hydroxy-7-quinoly1)(p-
trifluoromethoxyphenyOmethyl] (1 p- RS )-2-{ (S)-2-[6-(2,5-dioxo-1H-pyrrol-1-
yOhexanoylamino]-
3-methylbutyrylamino}-5-ureidovalerylamino]phenyl}methoxycarbonyl)amino}-1,3-
benzothiazole (ATAC27); (1R,6R,8R,95,10S,15R,17R,18S)-18-(1p-RS)-2-1(S)-246-
(2,5-Dioxo-
1H-pyrrol-1-y1)hexanoylamino]-3-methylbutyrylamino}-5-
ureidovalerylamino]phenyl}methoxycarbonylamino)-8,17-bis(2-amino-6-oxo-1,9-
dihydropurin-
9-y1)-3,12-dihydroxy-9-hydroxy-2.4.7.11.13.16-hexaoxa-3k5.1215-
diphosphatricyclo[13.3Ø06,10]octadecane-3,12-dione (ATAC28);
(1R,6R,8R,95,10S,15R,17R,18S)-18-(1p-RS)-2-1(S)-246-(2,5-Dioxo-1H-pyrrol-1-
yl)hexanoylamino]-3-
methylbutyrylamino}propionylamino]phenyl}methoxycarbonylamino)-
8,17-bis(2-amino-6-oxo-1,9-dihydropurin-9-y1)-3,12-dihydroxy-9-hydroxy-
2.4.7.11.13.16-
hexaoxa-3k5.1215-diphosphatricyclo[13.3Ø06,10]octadecane-3,12-dione
(ATAC29);
(1R,6R,8R,95,10S,15R,17R,18S)-18-(1p-RS)-2-1(S)-246-(2,5-Dioxo-1H-pyrrol-1-
yl)hexanoylamino]-3-methylbutyrylamino}-5-
ureidovalerylamino]phenyl}methoxycarbonylamino)-8,17-bis(2-amino-6-oxo-1,9-
dihydropurin-
9-y1)-9-hydroxy-3,12-dimercapto-2.4.7.11.13.16-hexaoxa-3k5.1215-
diphosphatricyclo [13 .3 Ø06,10]octadecane-3,12-dione (ATAC30); lp- RS )-2-{
(S)-246-(2,5-
Dio xo-1H-pyrrol-1-yOhexanoylamino]-3-methylbutyrylamino }-5-
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ureidovalerylamino]phenyl} methyl 9-1 (1S ,6R,8R,9S ,10S ,15R,17R,18S )-8-(2-
amino-6-oxo- 1,9-
dihydropurin-9-y1)-3,12-dihydroxy-9,18-dihydroxy-3,12-dioxo-2.4.7.11.13.16-
hexaoxa-
3k5 .1215-dipho sphatric yclo [13 .2.1.06,10] o ctadec-17- yl} -9a-adenineec
arbo xylate (ATAC31; 1-
I 6-RI 7-Amino-3-(2-hydroxy-2-methylpropy1)-3.5.8-triazatricyclo [7
.4Ø02,6]tridec a-
1(9),2(6),4,7,10,12-hexaen-4- yl} methyl)-N-ethylamino] -6-o xohexyl } -1H-
pyrrole-2,5-dione
(ATAC32); 1- I [441 6- R I 7-Amino-3-(2-hydroxy-2-methylpropy1)-3 .5.8-
triazatric yclo [7 .4Ø02,6] tridec a- 1(9),2(6),4,7,10,12-hexaen-4- yl}
methyl)-N-ethylamino] -6-
oxohexylamino }carbonyl)cyclohexyl]methy1}-1H-pyrrole-2,5-dione (ATAC33); or
14(4- I R I 7-
Amino-3-(2-hydroxy-2-methylpropy1)-3.5.8-triazatricyclo[7.4Ø02,6]trideca-
1(9),2(6),4,7,10,12-
hexaen-4- yl} methyl)-N-ethylamino] -carbonyl } c yclo hexyl)methyl] - 1H-p
yrro le-2,5-dione
(ATAC34).
[0363] An ATAC can be formed by conjugating a noncleavable maleimide-PEG4
linker
containing an activated ester such as a pentafluorophenyl group or an N-
hydroxysuccinimide
group with an immune-stimulatory compound. For example, an ATAC can be
pentafluorophenyl
25-(2-amino-3-pentylquinolin-5-y1)-19-oxo-4,7,10,13,16-pentaoxa-20-
azapentacosanoate
(ATAC1); perfluorophenyl 3-((4-amino-1-(2-hydroxy-2-methylpropy1)-1H-imidazo
[4,5-
c]quinolin-2-yl)methyl)-4-oxo-7,10,13,16,19-pentaoxa-3-azadocosan-22-oate
(ATAC2);
pentafluorophenyl 25-(2-amino-3-pentylquinolin-5-y1)-19-oxo-4,7,10,13,16-
pentaoxa-20-
azapentacosanoate (ATAC3); or 2,5-Dioxopyrrolidin-1-y134(4-amino-1-(2-hydroxy-
2-
methylpropy1)-1H-imidazo-[4,5-c]quinolin-2-y1)methyl)-4-oxo-7,10,13,16,19-
pentaoxa-3-
azadocosan-22-oate (ATAC4).
[0364] An ATAC can be formed by conjugating a cleavable valine-alanine or
valine-citrulline
linker containing a PABA group and an activated ester such as a
pentafluorophenyl group or an
N-hydroxysuccinimde group to an immune-stimulatory compo pound. For example,
an ATAC
can be 2,5-dioxopyrrolidin-l-y16-(((S)-1-(((S)-1-((4-((((5-(2-amino-3-
pentylquinolin-5-
yl)pentyl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3 -methyl-
l-oxobutan-
2-yl)amino)-6-oxohexanoate (ATAC5); 2,5-dioxopyrrolidin-1-y17-(((S)-1-(((S)-1-
((4-(((((4-
amino-1-(2-hydroxy-2-methylpropy1)-1H-imidazo [4,5-c] quinolin-2-
yl)methyl)(ethyl)c arbamo yl)oxy)methyl)phenyl)amino)-1-oxopropan-2-y1) amino)-
3 -methyl-1-
oxobutan-2-yl)amino)-7-oxoheptanoate (ATAC6); 2,5-dioxopyrrolidin-1-y17-(((S)-
1-(((S)-1-((4-
(((((4-amino-1-(2-hydroxy-2-methylpropy1)-1H-imidazo [4,5-c] quino lin-2-
yl)methyl)(ethyl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-
yl)amino)-3-
methyl-l-oxobutan-2-yl)amino)-7-oxoheptanoate (ATAC7); perfluorophenyl 6-(((S)-
1-(((S)-1-
((4-((((5-(2-amino-3-pentylquinolin-5-
yl)pentyl)carbamoyl)oxy)methyl)phenyl)amino)-1-
oxopropan-2-yl)amino)-3-methyl-l-oxobutan-2-yl)amino)-6-oxohexanoate (ATAC8);
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perfluorophenyl 7-(((S)-1-(((S)-1-((4-(((((4-amino-1-(2-hydroxy-2-
methylpropy1)-1H-
imidazo[4,5-c]quinolin-2-yl)methyl)(ethyl)carbamoyl)oxy)methyl)phenyl)amino)-1-
oxopropan-
2-yl)amino)-3-methyl-1-oxobutan-2-y1)amino)-7-oxoheptanoate (ATAC9); or
perfluorophenyl 7-
(((S)-1-(((S )-1-((4-(((((4-amino-1-(2-hydroxy-2-methylpropy1)-1H-imidazo [4,5-
c] quino lin-2-
yl)methyl)(ethyl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-
yl)amino)-3-
methyl-l-oxobutan-2-yl)amino)-7-oxoheptanoate (ATAC10).
[0365] An antibody construct can comprise an anti-CD40 antibody. An anti-CD40
antibody can
comprise two SBT-040-G1WT heavy chains and two light chain from a SBT-040
antibody,
which can be referred to as SBT-040-WT or as SBT-040-Gl. An anti-CD40 antibody
can
comprise two SBT-040-G1VLPLL heavy chains and two light chains from a SBT-040
antibody,
which can be referred to as SBT-040-VLPLL. An anti-CD40 antibody can comprise
two SBT-
040-G1DE heavy chains and two light chains from a SBT-040 antibody, which can
be referred to
as SBT-040-DE. An anti-CD40 antibody can comprise two SBT-040-G1AAA heavy
chains and
two light chains from a SBT-040 antibody, which can be referred to as SBT-040-
AAA. An anti-
CD40 antibody can comprise two IgG2 heavy chains and two light chain from a
SBT-040
antibody, which can be referred to as SBT-040-G2.
[0366] A conjugate can comprise SBT-040-WT-ATAC1. A conjugate can comprise SBT-
040-
WT-ATAC2. A conjugate can comprise SBT-040-WT-ATAC3. A conjugate can comprise
SBT-
040-WT-ATAC4. A conjugate can comprise SBT-040-WT-ATAC5. A conjugate can
comprise
SBT-040-WT-ATAC6. A conjugate can comprise SBT-040-WT-ATAC7. A conjugate can
comprise SBT-040-WT-ATAC8. A conjugate can comprise SBT-040-WT-ATAC9. A
conjugate
can comprise SBT-040-WT-ATAC10. A conjugate can comprise SBT-040-WT-ATAC11. A
conjugate can comprise SBT-040-WT-ATAC12. A conjugate can comprise SBT-040-WT-
ATAC13. A conjugate can comprise SBT-040-WT-ATAC14. A conjugate can comprise
SBT-
040-WT-ATAC15. A conjugate can comprise SBT-040-WT-ATAC16. A conjugate can
comprise SBT-040-WT-ATAC17. A conjugate can comprise SBT-040-WT-ATAC18. A
conjugate can comprise SBT-040-WT-ATAC19. A conjugate can comprise SBT-040-WT-
ATAC20. A conjugate can comprise SBT-040-WT-ATAC21. A conjugate can comprise
SBT-
040-WT-ATAC22. A conjugate can comprise SBT-040-WT-ATAC23. A conjugate can
comprise SBT-040-WT-ATAC24. A conjugate can comprise SBT-040-WT-ATAC25. A
conjugate can comprise SBT-040-WT-ATAC26. A conjugate can comprise SBT-040-WT-
ATAC27. A conjugate can comprise SBT-040-WT-ATAC28. A conjugate can comprise
SBT-
040-WT-ATAC29. A conjugate can comprise SBT-040-WT-ATAC30. A conjugate can
comprise SBT-040-WT-ATAC31. A conjugate can comprise SBT-040-WT-ATAC32. A
conjugate can comprise SBT-040-WT-ATAC33. A conjugate can comprise SBT-040-WT-
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ATAC34. A conjugate can comprise SBT-040-WT-ATAC43. A conjugate can comprise
SBT-
040-VLPLL-ATAC1. A conjugate can comprise SBT-040-VLPLL-ATAC2. A conjugate can
comprise SBT-040-VLPLL-ATAC3. A conjugate can comprise SBT-040-VLPLL-ATAC4. A
conjugate can comprise SBT-040-VLPLL-ATAC5. A conjugate can comprise SBT-040-
VLPLL-
ATAC6. A conjugate can comprise SBT-040-VLPLL-ATAC7. A conjugate can comprise
SBT-
040-VLPLL-ATAC8. A conjugate can comprise SBT-040-VLPLL-ATAC9. A conjugate can
comprise SBT-040-VLPLL-ATAC10. A conjugate can comprise SBT-040-VLPLL-ATAC11.
A
conjugate can comprise SBT-040-VLPLL-ATAC12. A conjugate can comprise SBT-040-
VLPLL-ATAC13. A conjugate can comprise SBT-040-VLPLL-ATAC14. A conjugate can
comprise SBT-040-VLPLL-ATAC15. A conjugate can comprise SBT-040-VLPLL-ATAC16.
A
conjugate can comprise SBT-040-VLPLL-ATAC17. A conjugate can comprise SBT-040-
VLPLL-ATAC18. A conjugate can comprise SBT-040-VLPLL-ATAC19. A conjugate can
comprise SBT-040-VLPLL-ATAC20. A conjugate can comprise SBT-040-VLPLL-ATAC21.
A
conjugate can comprise SBT-040-VLPLL-ATAC22. A conjugate can comprise SBT-040-
VLPLL-ATAC23. A conjugate can comprise SBT-040-VLPLL-ATAC24. A conjugate can
comprise SBT-040-VLPLL-ATAC25. A conjugate can comprise SBT-040-VLPLL-ATAC26.
A
conjugate can comprise SBT-040-VLPLL-ATAC27. A conjugate can comprise SBT-040-
VLPLL-ATAC28. A conjugate can comprise SBT-040-VLPLL-ATAC29. A conjugate can
comprise SBT-040-VLPLL-ATAC30. A conjugate can comprise SBT-040-VLPLL-ATAC31.
A
conjugate can comprise SBT-040-VLPLL-ATAC32. A conjugate can comprise SBT-040-
VLPLL-ATAC33. A conjugate can comprise SBT-040-VLPLL-ATAC34. A conjugate can
comprise SBT-040-VLPLL-ATAC43.A conjugate can comprise SBT-040-DE-ATAC1. A
conjugate can comprise SBT-040-DE-ATAC2. A conjugate can comprise SBT-040-DE-
ATAC3.
A conjugate can comprise SBT-040-DE-ATAC4. A conjugate can comprise SBT-040-DE-
ATAC5. A conjugate can comprise SBT-040-DE-ATAC6. A conjugate can comprise SBT-
040-
DE-ATAC7. A conjugate can comprise SBT-040-DE-ATAC8. A conjugate can comprise
SBT-
040-DE-ATAC9. A conjugate can comprise SBT-040-DE-ATAC10. A conjugate can
comprise
SBT-040-DE-ATAC11. A conjugate can comprise SBT-040-DE-ATAC12. A conjugate can
comprise SBT-040-DE-ATAC13. A conjugate can comprise SBT-040-DE-ATAC14. A
conjugate can comprise SBT-040-DE-ATAC15. A conjugate can comprise SBT-040-DE-
ATAC16. A conjugate can comprise SBT-040-DE-ATAC17. A conjugate can comprise
SBT-
040-DE-ATAC18. A conjugate can comprise SBT-040-DE-ATAC19. A conjugate can
comprise
SBT-040-DE-ATAC20. A conjugate can comprise SBT-040-DE-ATAC21. A conjugate can
comprise SBT-040-DE-ATAC22. A conjugate can comprise SBT-040-DE-ATAC23. A
conjugate can comprise SBT-040-DE-ATAC24. A conjugate can comprise SBT-040-DE-
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ATAC25. A conjugate can comprise SBT-040-DE-ATAC26. A conjugate can comprise
SBT-
040-DE-ATAC27. A conjugate can comprise SBT-040-DE-ATAC28. A conjugate can
comprise
SBT-040-DE-ATAC29. A conjugate can comprise SBT-040-DE-ATAC30. A conjugate can
comprise SBT-040-DE-ATAC31. A conjugate can comprise SBT-040-DE-ATAC32. A
conjugate can comprise SBT-040-DE-ATAC33. A conjugate can comprise SBT-040-DE-
ATAC34. A conjugate can comprise SBT-040-DE-ATAC43. A conjugate can comprise
SBT-
040-AAA-ATAC1. A conjugate can comprise SBT-040-AAA-ATAC2. A conjugate can
comprise SBT-040-AAA-ATAC3. A conjugate can comprise SBT-040-AAA-ATAC4. A
conjugate can comprise SBT-040-AAA-ATAC5. A conjugate can comprise SBT-040-AAA-
ATAC6. A conjugate can comprise SBT-040-AAA-ATAC7. A conjugate can comprise
SBT-
040-AAA-ATAC8. A conjugate can comprise SBT-040-AAA-ATAC9. A conjugate can
comprise SBT-040-AAA-ATAC10. A conjugate can comprise SBT-040-AAA-ATAC11. A
conjugate can comprise SBT-040-AAA-ATAC12. A conjugate can comprise SBT-040-
AAA-
ATAC13. A conjugate can comprise SBT-040-AAA-ATAC14. A conjugate can comprise
SBT-
040-AAA-ATAC15. A conjugate can comprise SBT-040-AAA-ATAC16. A conjugate can
comprise SBT-040- AAA -ATAC17. A conjugate can comprise SBT-040-AAA-ATAC18. A
conjugate can comprise SBT-040-AAA-ATAC19. A conjugate can comprise SBT-040-
AAA-
ATAC20. A conjugate can comprise SBT-040-AAA-ATAC21. A conjugate can comprise
SBT-
040-AAA-ATAC22. A conjugate can comprise SBT-040-AAA-ATAC23. A conjugate can
comprise SBT-040-AAA-ATAC24. A conjugate can comprise SBT-040-AAA-ATAC25. A
conjugate can comprise SBT-040-AAA-ATAC26. A conjugate can comprise SBT-040-
AAA-
ATAC27. A conjugate can comprise SBT-040-AAA-ATAC28. A conjugate can comprise
SBT-
040-AAA-ATAC29. A conjugate can comprise SBT-040-AAA-ATAC30. A conjugate can
comprise SBT-040-AAA-ATAC31. A conjugate can comprise SBT-040-AAA-ATAC32. A
conjugate can comprise SBT-040-AAA-ATAC33. A conjugate can comprise SBT-040-
AAA-
ATAC34. A conjugate can comprise SBT-040-AAA-ATAC33. A conjugate can comprise
SBT-
040-AAA-ATAC43. The Kd for binding of the CD40 binding domain of any of these
conjugates
to CD40 can be about 2 times, about 3 times, about 4 times, about 5 times,
about 6 times, about 7
times, about 8 times, about 9 times, about 10 times, about 15 times, about 20
times, about 25
times, about 30 times, about 35 times, about 40 times, about 45 times, about
50 times, about 60
times, about 70 times, about 80 times, about 90 times, about 100 times, about
110 times, or about
120 times greater than the Kd for binding of the CD40 binding domain to CD40
in the absence of
the immune-stimulatory compound or ATAC. The Kd for binding of the CD40
binding domain of
any of these conjugates to CD40 can be less than 10 nM. The Kd for binding of
the CD40 binding
domain of any of the conjugates to CD40 can be less than 100 nM, less than 50
nM, less than 20
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nM, less than 5 nM, less than 1 nM, or less than 0.1 nM. The Kd for binding of
the Fc domain of
any of the conjugates to an Fc receptor can be about 2 times, about 3 times,
about 4 times, about
times, about 6 times, about 7 times, about 8 times, about 9 times, about 10
times, about 15
times, about 20 times, about 25 times, about 30 times, about 35 times, about
40 times, about 45
times, about 50 times, about 60 times, about 70 times, about 80 times, about
90 times, about 100
times, about 110 times, or about 120 times greater than the Kd for binding of
the Fc domain to the
Fc receptor in the absence of the immune-stimulatory compound or ATAC. The Kd
for binding of
the Fc domain of any of the conjugates to an Fc receptor of an can be less
than 10 nM. The Kd for
binding of the Fc domain of any of the conjugates to an Fc receptor can be
less than 100 nM, less
than 50 nM, less than 20 nM, less than 5 nM, less than 1 nM, or less than 0.1
nM.
[0367] In a conjugate, an antibody can be linked to an immune-stimulatory
compound in such a
way that the antibody can still bind to an antigen and the Fc domain of the
antibody can still bind
to an FcR. In a conjugate, an antibody construct is linked to an immune-
stimulatory compound in
such a way that the linking does not interfere with ability of the antigen
binding domain of the
antibody construct to bind to antigen, the ability of the Fc domain of the
antibody construct to
bind to an FcR, or FcR-mediated signaling resulting from the Fc domain of the
antibody
construct from binding to an FcR. In a conjugate, an immune-stimulatory
compound can be
linked to an antibody construct in such a way the linking does not interfere
with the ability of the
immune-stimulatory compound to bind to its receptor. A conjugate can produce
stronger immune
stimulation and a greater therapeutic window than components of the conjugate
alone. In an anti-
CD40 antibody linked to a TLR agonist conjugate, the combination of CD40
agonism, TLR
agonism, and an accessible Fc domain of the anti-CD40 antibody to allow FcR-
mediated
signaling can produce stronger immune stimulation and a greater therapeutic
window than the
CD40 agonism, TLR agonism, or the FcR-mediated signaling alone.
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Methods of Synthesis of Antibody Construct Immune-Stimulatory Compound
Conjugate
Components
Synthesis of Immune-Stimulatory Compounds
[0368] An immune stimulatory compound can be synthesized as shown in Scheme
Al.
Scheme Al:
O 1. Tf20 o o
2. NaNO2 / H20 N-...)LN-H 1. DMTrCI N-..)LN-H
N,AN-H
--1 3. Tf20 2. H2 1
, ,
' N N NHIBu __________ . N"---N HIBu
N N NHIBu
4. NaN3
3. CbzCI
A OH 5. TBAF N3 4. CEM
DMTr0)0.....NHCbz
Rsi,o,sbi / HO OH
0-p
JD
NCr---/ \N__..(
.1
i iii
1. TFA/pyr
o
o 2. deprotect
N-HN-...,AN-H 3.
deprotect
NH
--1 o
N N NHIBu 1 ell/A
N---eiNHiBu N N4'NH Su 0
J...... DMTr0...,OTBS
OH NHCbz CN .),_.).... N,AN-1-1
1. oxidize NHCbz
o=P\ a\ ,OH 2. deprotect L ,=0
P : NC 11.-(
--\ N---N NHIBu
'---C2 3. cyclize
\-- CIPO2H
,P\ . ______________ Q. . ______
.)0.....,,,NHCbz
0 \c)
TBSO
TBSO HO
cr '''
crODMTr
OPO2H
IBuHN N N
.T, .1õ IBuHN N N
NI--
H-N1CN viv iv
H-N1CN
o
o
1. hydrogenate
2. deprotect
o o
N,AN-H kl-...,AN-H
--t---
N N Nh12 N N Nh12
J..., J.....
OH ' "NH2 OH
i 0
---. i ,,,=0
01 b\ pH (:)=P\ o\ pH
'--Q. deprotect L-C2
cr0 =0 cr0 \c)
TBSO''' HO'',
H21\1 N N H21\1 N N
v
1 ii viii
H-N1CN H-N1CN
o o
[0369] Synthesis of the C-2' amino cyclic dinucleotide (viii) can be
accomplished using a
multistep synthesis as outlined in scheme Al above and described below in
EXAMPLE 3.
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Synthesis of ATAC Compounds
[0370] An ATAC compound can be synthesized by various methods. For example,
ATAC
compounds, such as ATAC1 ¨ ATAC4, can be synthesized as shown in Scheme Bl.
Scheme Bl:
1.
0 00 0
H2N
R0
.."<j0 N
2. DIC/ROH
R = NHS, pentafluorophenyl
ISC: immune-stimulatory compound
[0371] A PEGylated carboxylic acid (i) that has been activated for amide bond
formation can be
reacted with an appropriately substituted amine containing immune-stimulatory
compound to
afford an intermediate amide. Formation of an activated ester (ii) can be
achieved by reaction the
intermediate amide-containing carboxylic using a reagent such as N-
hydroxysuccinimide or
pentafluorophenol in the presence of a coupling agent such as
diisopropylcarbodiimide (DIC) to
provide compounds (ii).
[0372] An ATAC compound can be synthesized by various methods. For example,
ATAC
compounds, such as ATAC5 ¨ ATAC10, can be synthesized as shown in Scheme B2.
Scheme B2:
1=
0 IR H 0 0 0 H 2 N
R 3 0 N))iNN
40R30re 0 Ri H 0 N
H H ,1N.,\ANAliNy& 0N
0 u R2
0 0 R2
NO2
deprotect
)0
0 Ri H 0 is 0, N i
couple 0 Ri H 0 0 N
R40=Te\e"\AN). yN yjk.N
0 0 R2
0 0 R2
Iv
R4 = NHS, Perfluorofenyl
ISC: immune-stimulatory compound
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[0373] An activated carbonate such as (i) can be reacted with an appropriately
substituted amine
containing immune-stimulatory compound to afford carbamates (ii) which can be
deprotected
using standard methods based on the nature of the R3 ester group. The
resulting carboxylic acid
(iii) can then by coupled with an activating agent such as N-
hydroxysuccinimide or
pentafluorophenol to provide compounds (iv).
[0374] An ATAC compound can be synthesized by various methods. For example,
ATAC
compounds, such as ATAC11 ¨ ATAC21, can be synthesized as shown in Scheme B3.
Scheme B3:
0 0
0
HN
)()K
0 0 0 0
i-a; X = NHS ii
i-b; X = H
ISO: immune-stimulatory compound
[0375] An activated carboxylic ester such as (i-a) can be reacted with an
appropriately
substituted amine containing immune-stimulatory compound to afford amides
(ii). Alternatively,
carboxylic acids of type (i-b) can be coupled to an appropriately substituted
amine containing
immune-stimulatory compound in the presence of an amide bond forming agent
such as
dicyclohexycarbodiimde (DCC) to provide the desired ATAC compounds.
[0376] An ATAC compound can be synthesized by various methods. For example,
ATAC
compounds, such as ATAC22 ¨ ATAC31, can be synthesized as shown in Scheme B4.
Scheme B4:
41)
cr)., ri 0 0 H2N
0 R2
NO2
O Jo(
Fr\i40 N
N'Thr
0 R2
ISO: immune-stimulatory compound
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[0377] An activated carbonate such as (i) can be reacted with an appropriately
substituted amine
containing immune-stimulatory compound to afford carbamates (ii) as the target
ATAC
compounds.
[0378] An ATAC compound can be synthesized by various methods. For example,
ATAC
compounds, such as ATAC32 ¨ ATAC34, can be synthesized as shown in Scheme B5.
Scheme B5:
H2N
N
0 H
0
i-a ii-a
0
CD 0
......4.,----ciy,õ........õ.....õ).õ
H2N
0 0 N
0 H
0
i-b ii-b
0 H2N CD
ce. jciti3O x . a 0
c0,xD)LN
N H
N
0 0
i-c ii-c
ISC: immune-stimulatory compound
[0379] An activated carboxylic acid such as (i-a, i-b, i-c) can be reacted
with an appropriately
substituted amine containing immune-stimulatory compound to afford amides (ii-
a, ii-b, ii-c) as
the target ATAC compounds.
[0380] These antibody construct immune-stimulatory conjugates can be made by
various
methods. It is understood that one skilled in the art may be able to make
these compounds by
similar methods or by combining other methods known to one skilled in the art.
It is also
understood that one skilled in the art would be able to make, in a similar
manner as described
herein by using the appropriate starting materials and modifying the synthetic
route as needed.
Starting materials and reagents can be obtained from commercial vendors or
synthesized
according to sources known to those skilled in the art or prepared as
described herein.
Pharmaceutical Formulations
[0381] The compositions and methods described herein can be considered useful
as
pharmaceutical compositions for administration to a subject in need thereof.
Pharmaceutical
compositions can comprise at least the compositions described herein and one
or more
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pharmaceutically acceptable carriers, diluents, excipients, stabilizers,
dispersing agents,
suspending agents, and/or thickening agents. The composition can comprise the
conjugate having
an antibody construct and an agonist. The composition can comprise the
conjugate having an
antibody construct, a targeting binding domain, and an agonist. The
composition can comprise
any conjugate described herein. Often, the antibody construct is an anti-CD40
antibody. A
conjugate can comprise an anti-CD40 antibody and a PAMP molecule. A conjugate
can comprise
an anti-CD40 antibody and a DAMP molecule. A pharmaceutical composition can
further
comprise buffers, antibiotics, steroids, carbohydrates, drugs (e.g.,
chemotherapy drugs),
radiation, polypeptides, chelators, adjuvants and/or preservatives.
[0382] Pharmaceutical compositions can be formulated using one or more
physiologically-
acceptable carriers comprising excipients and auxiliaries. Formulation can be
modified
depending upon the route of administration chosen. Pharmaceutical compositions
comprising a
composition as described herein can be manufactured, for example, by
lyophilizing the
conjugate, mixing, dissolving, emulsifying, encapsulating or entrapping the
conjugate. The
pharmaceutical compositions can also include the compositions described herein
in a free-base
form or pharmaceutically-acceptable salt form.
[0383] Methods for formulation of the conjugates described herein can include
formulating any
of the conjugates described herein with one or more inert, pharmaceutically-
acceptable excipients
or carriers to form a solid, semi-solid, or liquid composition. Solid
compositions can include, for
example, powders, tablets, dispersible granules and capsules, and in some
aspects, the solid
compositions further contain nontoxic, auxiliary substances, for example
wetting or emulsifying
agents, pH buffering agents, and other pharmaceutically-acceptable additives.
Alternatively, the
compositions described herein can be lyophilized or in powder form for re-
constitution with a
suitable vehicle, e.g., sterile pyrogen-free water, before use
[0384] Pharmaceutical compositions of the conjugates described herein can
comprise at least an
active ingredient. The active ingredients can be entrapped in microcapsules
prepared, for
example, by coacervation techniques or by interfacial polymerization (e.g.,
hydroxymethylcellulose or gelatin microcapsules and poly-(methylmethacylate)
microcapsules,
respectively), in colloidal drug-delivery systems (e.g., liposomes, albumin
microspheres,
microemulsions, nano-particles and nanocapsules) or in macroemulsions.
[0385] Pharmaceutical compositions as described herein often further can
comprise more than
one active compound as necessary for the particular indication being treated.
The active
compounds can have complementary activities that do not adversely affect each
other. For
example, the composition can comprise a chemotherapeutic agent, cytotoxic
agent, cytokine,
growth-inhibitory agent, anti-hormonal agent, anti- angiogenic agent, and/or
cardioprotectant.
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Such molecules can be present in combination in amounts that are effective for
the purpose
intended.
[0386] The compositions and formulations can be sterilized. Sterilization can
be accomplished
by filtration through sterile filtration.
[0387] The compositions described herein can be formulated for administration
as an injection.
Non-limiting examples of formulations for injection can include a sterile
suspension, solution or
emulsion in oily or aqueous vehicles. Suitable oily vehicles can include, but
are not limited to,
lipophilic solvents or vehicles such as fatty oils or synthetic fatty acid
esters, or liposomes.
Aqueous injection suspensions can contain substances which increase the
viscosity of the
suspension. The suspension can also contain suitable stabilizers. Injections
can be formulated for
bolus injection or continuous infusion. Alternatively, the compositions
described herein can be
lyophilized or in powder form for reconstitution with a suitable vehicle,
e.g., sterile pyrogen-free
water, before use.
[0388] For parenteral administration the conjugates can be formulated in a
unit dosage injectable
form (e.g., use letter solution, suspension, emulsion) in association with a
pharmaceutically
acceptable parenteral vehicle. Such vehicles can be inherently nontoxic, and
non-therapeutic. A
vehicles can be water, saline, Ringer's solution, dextrose solution, and 5%
human serum
albumin. Nonaqueous vehicles such as fixed oils and ethyl oleate can also be
used. Liposomes
can be used as carriers. The vehicle can contain minor amounts of additives
such as substances
that enhance isotonicity and chemical stability (e.g., buffers and
preservatives).
[0389] Sustained-release preparations can be also be prepared. Examples of
sustained-release
preparations can include semipermeable matrices of solid hydrophobic polymers
that can contain
the antibody, and these matrices can be in the form of shaped articles (e.g.,
films or
microcapsules). Examples of sustained-release matrices can include polyesters,
hydrogels (e.g.,
poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides,
copolymers of L-
glutamic acid and y ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,
degradable lactic
acid-glycolic acid copolymers such as the LUPRON DEPOTM (i.e., injectable
microspheres
composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and
poly-D-( ¨ )-3-
hydroxybutyric acid.
[0390] Pharmaceutical formulations of the compositions described herein can be
prepared for
storage by mixing a conjugate with a pharmaceutically acceptable carrier,
excipient, and/or a
stabilizer. This formulation can be a lyophilized formulation or an aqueous
solution. Acceptable
carriers, excipients, and/or stabilizers can be nontoxic to recipients at the
dosages and
concentrations used. Acceptable carriers, excipients, and/or stabilizers can
include buffers such
as phosphate, citrate, and other organic acids; antioxidants including
ascorbic acid and
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methionine; preservatives, polypeptides; proteins, such as serum albumin or
gelatin; hydrophilic
polymers; amino acids; monosaccharides, disaccharides, and other carbohydrates
including
glucose, mannose, or dextrins; chelating agents such as EDTA; sugars such as
sucrose, mannitol,
trehalose or sorbitol; salt-forming counter-ions such as sodium; metal
complexes; and/or non-
ionic surfactants or polyethylene glycol.
Therapeutic Applications
[0391] The compositions and methods of the present disclosure can be useful
for a plurality of
different subjects including, but are not limited to, a mammal, human, non-
human mammal, a
domesticated animal (e.g., laboratory animals, household pets, or livestock),
non-domesticated
animal (e.g., wildlife), dog, cat, rodent, mouse, hamster, cow, bird, chicken,
fish, pig, horse, goat,
sheep, rabbit, and any combination thereof.
[0392] The compositions and methods described herein can be useful as a
therapeutic, for
example a treatment that can be administered to a subject in need thereof. A
therapeutic effect of
the present disclosure can be obtained in a subject by reduction, suppression,
remission, or
eradication of a disease state, including, but not limited to, a symptom
thereof. A therapeutic
effect in a subject having a disease or condition, or pre-disposed to have or
is beginning to have
the disease or condition, can be obtained by a reduction, a suppression, a
prevention, a remission,
or an eradication of the condition or disease, or pre-condition or pre-disease
state.
[0393] In practicing the methods described herein, therapeutically-effective
amounts of the
compositions described herein can be administered to a subject in need
thereof, often for treating
and/or preventing a condition or progression thereof. A pharmaceutical
composition can affect
the physiology of the subject, such as the immune system, inflammatory
response, or other
physiologic affect. A therapeutically-effective amount can vary widely
depending on the severity
of the disease, the age and relative health of the subject, the potency of the
compounds used, and
other factors.
[0394] Treat and/or treating can refer to any indicia of success in the
treatment or amelioration of
the disease or condition. Treating can include, for example, reducing,
delaying or alleviating the
severity of one or more symptoms of the disease or condition, or it can
include reducing the
frequency with which symptoms of a disease, defect, disorder, or adverse
condition, and the like,
are experienced by a patient. Treat can be used herein to refer to a method
that results in some
level of treatment or amelioration of the disease or condition, and can
contemplate a range of
results directed to that end, including but not restricted to prevention of
the condition entirely.
[0395] Prevent, preventing and the like can refer to the prevention of the
disease or condition,
e.g., tumor formation, in the patient. For example, if an individual at risk
of developing a tumor
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or other form of cancer is treated with the methods of the present disclosure
and does not later
develop the tumor or other form of cancer, then the disease has been
prevented, at least over a
period of time, in that individual.
[0396] A therapeutically effective amount can be the amount of a composition
or an active
component thereof sufficient to provide a beneficial effect or to otherwise
reduce a detrimental
non-beneficial event to the individual to whom the composition is
administered. A
therapeutically effective dose can be a dose that produces one or more desired
or desirable (e.g.,
beneficial) effects for which it is administered, such administration
occurring one or more times
over a given period of time. An exact dose can depend on the purpose of the
treatment, and can
be ascertainable by one skilled in the art using known techniques.
[0397] The conjugates described herein that can be used in therapy can be
formulated and
dosages established in a fashion consistent with good medical practice taking
into account the
disorder to be treated, the condition of the individual patient, the site of
delivery of the
composition, the method of administration and other factors known to
practitioners. The
conjugates described herein can be prepared according to the description of
preparation described
herein.
[0398] Pharmaceutical compositions can be considered useful with the
compositions and
methods described herein can be administered to a subject in need thereof
using a technique
known to one of ordinary skill in the art which can be suitable as a therapy
for the disease or
condition affecting the subject. One of ordinary skill in the art would
understand that the amount,
duration and frequency of administration of a pharmaceutical composition
described herein to a
subject in need thereof depends on several factors including, for example but
not limited to, the
health of the subject, the specific disease or condition of the patient, the
grade or level of a
specific disease or condition of the patient, the additional therapeutics the
subject is being or has
been administered, and the like.
[0399] The methods and compositions described herein can be for administration
to a subject in
need thereof. Often, administration of the compositions described herein can
include routes of
administration, non-limiting examples of administration routes include
intravenous, intraarterial,
subcutaneous, subdural, intramuscular, intracranial, intrasternal,
intratumoral, or
intraperitoneally. Additionally, a pharmaceutical composition can be
administered to a subject by
additional routes of administration, for example, by inhalation, oral, dermal,
intranasal, or
intrathecal administration.
[0400] Compositions of the present disclosure can be administered to a subject
in need thereof in
a first administration, and in one or more additional administrations. The one
or more additional
administrations can be administered to the subject in need thereof minutes,
hours, days, weeks or
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months following the first administration. Any one of the additional
administrations can be
administered to the subject in need thereof less than 21 days, or less than 14
days, less than 10
days, less than 7 days, less than 4 days or less than 1 day after the first
administration. The one or
more administrations can occur more than once per day, more than once per week
or more than
once per month.
Diseases, Conditions and the Like
[0401] The compositions and methods provided herein can be useful for the
treatment of a
plurality of diseases, conditions, preventing a disease or a condition in a
subject or other
therapeutic applications for subjects in need thereof. Often the compositions
and methods
provided herein can be useful for treatment of hyperplastic conditions,
including but not limited
to, neoplasms, cancers, tumors and the like. A condition, such as a cancer,
can be associated with
expression of a molecule on the cancer cells. Often, the molecule expressed by
the cancer cells
can comprise an extracellular portion capable of recognition by the antibody
portion of the
conjugate. A molecule expressed by the cancer cells can be a tumor antigen. An
antibody portion
of the conjugate can recognize a tumor antigen. A tumor antigen can include
CD5, CD19, CD20,
CD25, CD37, CD30, CD33, CD45, CAMPATH-1, BCMA, CS-1, PD-L1, B7-H3, B7-DC, HLD-
DR, carcinoembryonic antigen, TAG-72, EpCAM, MUC1, folate-binding protein,
A33, G250,
prostate-specific membrane antigen, ferritin, GD2, GD3, GM2, Leg, CA-125, CA19-
9, epidermal
growth factor, p185HER2, IL-2 receptor, de2-7 EGFR, fibroblast activation
protein, tenascin,
metalloproteinases, endosialin, vascular endothelial growth factor, avB3, WT1,
LMP2, HPV E6
E7, EGFRvIII, Her-2/neu, idiotype, MAGE A3, p53 nonmutant, NY-ES0-1, PMSA,
GD2, CEA,
MelanA/MART1, Ras mutant, gp100, p53 mutant, PR1, bcr-abl, tyronsinase,
survivin, PSA,
hTERT, Sarcoma translocation breakpoints, EphA2, PAP, ML-IAP, AFP, ERG, NA17,
PAX3,
ALK, androgen receptor, cyclin Bl, polysialic acid, MYCN, RhoC, TRP-2, fucosyl
GM1,
mesothelin, PSCA, MAGE Al, sLe(animal), CYP1B1, PLAV1, GM3, BORIS, Tn, GloboH,
ETV6-AML, NY-BR-1, RGS5, SART3, STn, Carbonic anhydrase IX, PAX5, 0Y-TES1,
Sperm
protein 17, LCK, HMWMAA, AKAP-4, 55X2, XAGE 1, B7H3, Legumain, Tie 3, Page4,
VEGFR2, MAD-CT-1, PDGFR-B, MAD-CT-2, ROR2, TRAILl, MUC16, MAGE A4, MAGE
C2, GAGE, or Fos-related antigen 1.
[0402] As described herein, an antigen binding domain portion of the
conjugate, can be
configured to recognize a molecule expressed by a cancer cell, such as for
example, a disease
antigen, tumor antigen or a cancer antigen. Often such antigens are known to
those of ordinary
skill in the art, or newly found to be associated with such a condition, to be
commonly associated
with, and/or, specific to, such conditions. For example, a disease antigen,
tumor antigen or a
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cancer antigen is, but is not limited to, CD5, CD19, CD20, CD25, CD37, CD30,
CD33, CD45,
CAMPATH-1, BCMA, CS-1, PD-L1, B7-H3, B7-DC, HLD-DR, carcinoembryonic antigen,
TAG-72, EpCAM, MUC1, folate-binding protein, A33, G250, prostate-specific
membrane
antigen, ferritin, GD2, GD3, GM2, Leg, CA-125, CA19-9, epidermal growth
factor, p185HER2,
IL-2 receptor, de2-7 EGFR, fibroblast activation protein, tenascin,
metalloproteinases,
endosialin, vascular endothelial growth factor, avB3, WT1, LMP2, HPV E6 E7,
EGFRvIII, Her-
2/neu, idiotype, MAGE A3, p53 nonmutant, NY-ES0-1, PMSA, GD2, CEA,
MelanA/MART1,
Ras mutant, gp100, p53 mutant, PR1, bcr-abl, tyronsinase, survivin, PSA,
hTERT, Sarcoma
translocation breakpoints, EphA2, PAP, ML-IAP, AFP, ERG, NA17, PAX3, ALK,
androgen
receptor, cyclin Bl, polysialic acid, MYCN, RhoC, TRP-2, fucosyl GM1,
mesothelin, PSCA,
MAGE Al, sLe(animal), CYP1B1, PLAV1, GM3, BORIS, Tn, GloboH, ETV6-AML, NY-BR-
1,
RGS5, SART3, STn, Carbonic anhydrase IX, PAX5, 0Y-TES1, Sperm protein 17, LCK,
HMWMAA, AKAP-4, 55X2, XAGE 1, B7H3, Legumain, Tie 3, Page4, VEGFR2, MAD-CT-1,
PDGFR-B, MAD-CT-2, ROR2, TRAILl, MUC16, MAGE A4, MAGE C2, GAGE, EGFR,
CMET, HER3, MUC1, MUC15, MSLN, CA6, NAPI2B, TROP2, CLDN18.2, RON, LY6E, FRA,
DLL3, PTK7, LIV1, ROR1, MAGE-A3, or Fos-related antigen 1. Additionally, such
tumor
antigens can be derived from the following specific conditions and/or families
of conditions,
including but not limited to, cancers such as brain cancers, skin cancers,
lymphomas, sarcomas,
lung cancer, liver cancer, leukemias, uterine cancer, breast cancer, ovarian
cancer, cervical
cancer, bladder cancer, kidney cancer, hemangio sarcomas, bone cancers, blood
cancers, testicular
cancer, prostate cancer, stomach cancer, intestinal cancers, pancreatic
cancer, and other types of
cancers as well as pre-cancerous conditions such as hyperplasia or the like.
[0403] Non-limiting examples of cancers can include Acute lymphoblastic
leukemia (ALL);
Acute myeloid leukemia; Adrenocortical carcinoma; Astrocytoma, childhood
cerebellar or
cerebral; Basal-cell carcinoma; Bladder cancer; Bone tumor,
osteosarcoma/malignant fibrous
histiocytoma; Brain cancer; Brain tumors, such as, cerebellar astrocytoma,
malignant glioma,
ependymoma, medulloblastoma, visual pathway and hypothalamic glioma; Brainstem
glioma;
Breast cancer; Bronchial adenomas/carcinoids; Burkitt's lymphoma; Cerebellar
astrocytoma;
Cervical cancer; Cholangiocarcinoma; Chondrosarcoma; Chronic lymphocytic
leukemia;
Chronic myelogenous leukemia; Chronic myeloproliferative disorders; Colon
cancer; Cutaneous
T-cell lymphoma; Endometrial cancer; Ependymoma; Esophageal cancer; Eye
cancers, such as,
intraocular melanoma and retinoblastoma; Gallbladder cancer; Glioma; Hairy
cell leukemia;
Head and neck cancer; Heart cancer; Hepatocellular (liver) cancer; Hodgkin
lymphoma;
Hypopharyngeal cancer; Islet cell carcinoma (endocrine pancreas); Kaposi
sarcoma; Kidney
cancer (renal cell cancer); Laryngeal cancer; Leukaemia, such as, acute
lymphoblastic, acute
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myeloid, chronic lymphocytic, chronic myelogenous and, hairy cell; Lip and
oral cavity cancer;
Liposarcoma; Lung cancer, such as, non-small cell and small cell; Lymphoma,
such as, AIDS-
related, Burkitt; Lymphoma, cutaneous T-Cell, Hodgkin and Non-Hodgkin,
Macroglobulinemia,
Malignant fibrous histiocytoma of bone/osteosarcoma; Melanoma; Merkel cell
cancer;
Mesothelioma; Multiple myeloma/plasma cell neoplasm; Mycosis fungoides;
Myelodysplastic
syndromes; Myelodysplastic/myeloproliferative diseases; Myeloproliferative
disorders, chronic;
Nasal cavity and paranasal sinus cancer; Nasopharyngeal carcinoma;
Neuroblastoma;
Oligodendroglioma; Oropharyngeal cancer; Osteosarcoma/malignant fibrous
histiocytoma of
bone; Ovarian cancer; Pancreatic cancer; Parathyroid cancer; Pharyngeal
cancer;
Pheochromocytoma; Pituitary adenoma; Plasma cell neoplasia; Pleuropulmonary
blastoma;
Prostate cancer; Rectal cancer; Renal cell carcinoma (kidney cancer); Renal
pelvis and ureter,
transitional cell cancer; Rhabdomyosarcoma; Salivary gland cancer; Sarcoma,
Ewing family of
tumors; Sarcoma, Kaposi; Sarcoma, soft tissue; Sarcoma, uterine; Sezary
syndrome; Skin cancer
(non-melanoma); Skin carcinoma; Small intestine cancer; Soft tissue sarcoma;
Squamous cell
carcinoma; Squamous neck cancer with occult primary, metastatic; Stomach
cancer; Testicular
cancer; Throat cancer; Thymoma and thymic carcinoma; Thymoma,; Thyroid cancer;
Thyroid
cancer, childhood; Uterine cancer; Vaginal cancer; Waldenstrom
macroglobulinemia; Wilms
tumor and any combination thereof.
EXAMPLE 1
Fc Receptor Binding to anti-CD40 Antibodies
[0404] An anti-CD40 antibody is comprised of two SBT-040-G1WT heavy chains and
two light
chain from a SBT-040 antibody, which is referred to as a SBT-040-WT antibody.
An anti-CD40
antibody is comprised of two SBT-040-G1VLPLL heavy chains and two light chains
from a
SBT-040 antibody, which is referred to as a SBT-040-VLPLL antibody. An anti-
CD40 antibody
is comprised two SBT-040-G1DE heavy chains and two light chains from a SBT-040
antibody,
which is referred to as a SBT-040-DE antibody. An anti-CD40 antibody is
comprised of two
SBT-040-G1AAA heavy chains and two light chains from a SBT-040 antibody, which
is referred
to as a SBT-040-AAA antibody.
[0405] SBT-040-WT antibody, SBT-040-VLPLL antibody, SBT-040-DE antibody, and
SBT-
040-AAA antibody are produced by standard methods for producing antibodies.
These antibodies
are purified, and each antibody's affinity for soluble glycosylated
ectodomains from all human
Fey receptors (FcyRs) is measured. These affinities are measured by
experiments using surface
plasmon resonance. In these experiments, biotinylated soluble glycosylated
FcyR ectodomains
from all human FcyRs are immobilized on a streptavidin-coated surface. The
ability of each
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antibody to bind to soluble glycosylated FcyR ectodomains from all human FcyRs
is then
measured by surface plasmon resonance using a Biacore instrument. The data
from this
experiment shows that the Fc domain of a SBT-040-WT antibody, the Fc domain of
a SBT-040-
VLPLL antibody, the Fc domain of a SBT-040-DE antibody, and the Fc domain of a
SBT-040-
AAA antibody are each bound to soluble glycosylated FcyR ectodomains from all
human FcyRs.
Therefore, the surface plasmon resonance experiments show that the Fc domain
of the SBT-040-
G1WT antibody and variants of the Fc domain of a SBT-040-G1WT antibody (i.e.,
the Fc
domain of a SBT-040-G1VLPLL antibody, the Fc domain of a SBT-040-DE antibody
and the Fc
domain of a SBT-040-AAA antibody) are each bound to all human FcyRs. The
affinity of each
antibody for each human FcyRs is also shown by these experiments.
EXAMPLE 2
Synthesis of Linkers with Immune-Stimulatory Compounds
[0406] A linker is linked with an immune-stimulatory compound. A linker linked
to an immune-
stimulatory compound is formed to make a linker-immune stimulatory compound
conjugate
(ATAC). Subsequently, an ATAC is conjugated to an antibody, in which the ATAC
is any one of
ATAC1 ¨ ATAC34 or ATAC 43 (each of which is described in the below EXAMPLES).
[0407] A linker is linked with an antibody, in which the linker is a pegylated
linker, a valine-
alanine linker, a valine-citrulline linker, or an N-Maleimidomethylcyclohexane-
l-carboxylate
(MCC) linker. Subsequently, an immune-stimulatory compound is conjugated to
the linker linked
with the antibody, in which the immune-stimulatory compound is a TLR ligand, a
Nod-like
receptor ligand, a RIG-Like receptor ligand, a CLR ligand, a CDS ligand, or an
inflammasome
inducer.
[0408] A linker is linked with an antibody, in which the linker is a pegylated
linker, a valine-
alanine linker, a valine-citrulline linker, or an N-Maleimidomethylcyclohexane-
l-carboxylate
(MCC) linker. Subsequently, an immune-stimulatory compound is conjugated to
the linker linked
with the antibody, in which the immune-stimulatory compound is gardiquimod or
an analog of a
cyclic dinucleotide.
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EXAMPLE 3
Synthesis of (1R,6R,8R,9R,10S,15R,17R,18R)-9-Amino-8,17-bis(2-amino-6-oxo-1,9-
dihydropurin-9-y1)-3,12-dihydroxy-18-hydroxy-2.4.7.11.13.16-hexaoxa-313.1215-
diphosphatricyclo [13.3Ø06,10]octadecane-3,12-dione (Compound 21)
[0409] This example shows the synthesis of (1R,6R,8R,9R,10S,15R,17R,18R)-9-
Amino-8,17-
bis(2-amino-6-oxo-1,9-dihydropurin-9-y1)-3,12-dihydroxy-18-hydroxy-
2.4.7.11.13.16-hexaoxa-
3k5.1215-diphosphatricyclo [13.3Ø06,10]octadecane-3,12-dione (Compound 21).
0
N1)LNH
0
1 NNH2
OHO
oA I) 111-12
H2N NN 0-0-0H
XI?: Nj> 0
0
Compound 21
Step A: Preparation of Int 2.13-1
0 0
N
NIalH .L
0
HO )N NNH2 1. TBSCI, Py, 25 C, 3h Ho N NZ N1___
\.-0-,)
2. isobutyric anhydride, 25 C, 16h ).- \-- -ti _____________ ) H
6H 6H 3. NH3H20, 25 C, 1h 6H 6H
Int 2.13-1
[0410] Guanosine (200 g, 706.71 mmol, 1.00 equiv) was suspended in dry
pyridine (4000 mL)
under a nitrogen atmosphere, and TB5C1 (572 g, 5.30 mol, 7.50 equiv) was added
dropwise at
0 C. The reaction was stirred at ambient temperature for 3h then cooled to 0 C
before adding
isobutyric anhydride (167 g, 1.06 mol, 1.5 equiv) dropwise over 20 min. The
solution was
allowed to warm to room temperature and stirred for 16h. The reaction solution
was cooled to
0 C and the reaction was quenched by the addition of water (500 mL). After
stirring for 20 min
at 0 C, 1000 mL of concentrated aqueous NH4OH was added dropwise at 0 C. After
stirring for
an additional lh at room temperature, the resulting mixture was concentrated
and the residue was
dissolved in 3000 mL of water and washed with 1500 mL of Et0Ac. The aqueous
phase was
concentrated to ¨1000 mL whereby the product precipitated from water. The
product was filtered
to afford 174 g of N-[9-[(2R,3R,4S,5R)-3,4-dihydroxy-5-(hydroxymethyl)oxolan-2-
y11-6-oxo-
6,9-dihydro-1H-purin-2-y11-2-methylpropanamide (Int 2.13-1) as a white solid.
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Step B. Preparation of Int 2.13-2
0
0
NH
I
fx_
N
0 v
HO N )1 o DMTrCI , Py DMTrO 0 N
25 C,16h
________________________________________________________ (
61-1 oH
OH OH
Int 2.13-1 Int 2.13-2
[0411] To a stirred suspension of Int 2.13-1 (200.0 g, 566.57 mmol, 1.00
equiv) in pyridine (3 L)
under a nitrogen atmosphere was added 4,4'-
(chloro(phenyl)methylene)bis(methoxybenzene)
(211 g, 623.23 mmol, 1.10 equiv). The resulting mixture was left to stir for
16 hat room
temperature. The reaction was quenched with methanol (100 mL) and the mixture
was
concentrated under vacuum. The residue was dissolved in 3000 mL of
dichloromethane, washed
with 2 x 1500 mL of saturated sodium bicarbonate solution and 1500 mL of
saturated sodium
chloride solution respectively. The organic phase was dried over anhydrous
sodium sulfate,
filtered and concentrated under vacuum. The crude product was applied onto a
silica gel column
with DCM/methanol (with 0.05% triethylamine) (50/1-20/1). This resulted in 278
g (75%) of N-
[9-[(2R,3R,4S,5R)-5-[[bis(4-methoxyphenyl)(phenyl)methoxylmethyl]-3,4-
dihydroxyoxolan-2-
y1]-6-oxo-6,9-dihydro-1H-purin-2-y1]-2-methylpropanamide (Int 2.13-2) as a
light yellow solid.
Step C: Preparation of Int 2.13-3a and Int 2.13-3b
0
r\IfNH
1\111-1 0 n111
DMTrO N
DMTrOKNN TBSCI, Py, IM N N VO,)
VO-.)
25 C, 16h TBSO OH
OH OH OH OTBS
Int 2.13-2 Int 2.13-3a Int 2.13-3b
[0412] Compound Int 2.13-2 (150 g, 229 mmol, 1.00 equiv) was dissolved in 1500
mL of
pyridine under a nitrogen atmosphere. 1H-imidazole (46.71 g, 687.02 mmol, 3.00
equiv) was
added, followed by addition of TBS-Cl (51.6 g, 343.51 mmol, 1.50 equiv) in
portions at 25 C.
The resulting solution was stirred for 16 h at 25 C then concentrated and
dissolved in 2000 mL
of dichloromethane. The organic extract was washed with 2 x 1000 mL of
saturated sodium
bicarbonate solution and 1000 mL of saturated sodium chloride solution,
respectively. The
organic phase was dried over anhydrous sodium sulfate, filtered and
concentrated under vacuum.
The crude product was applied onto a silica gel column with ethyl
acetate/dichloromethane
(1/50-1/1) and then purified by flash with the following conditions: silica
gel column; ethyl
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acetate in dichloromethane with 0.05% triethylamine:15% up to 70% within 10
min and 70%
maintained 10 min; This resulted in 62 g (35%) of N49-[(2R,3R,4R,5R)-5-Dis(4-
methoxyphenyl)(phenyl)methoxy]methyl]-3-[(tert-butyldimethylsily1)oxy]-4-
hydroxyoxolan-2-
y1]-6-oxo-6,9-dihydro-1H-purin-2-y1]-2-methylpropanamide (Int 2.13-3a) as a
yellow solid and
44 g (25%) of N49-[(2R,3R,4S,5R)-5-[[bis(4-
methoxyphenyl)(phenyl)methoxy]methyl]-4-[(tert-
butyldimethylsilyl)oxy]-3-hydroxyoxolan-2-y1]-6-oxo-6,9-dihydro-1H-purin-2-y1]-
2-
methylpropanamide (Int 2.13-3b).
Step D: Preparation of Int 2.13-4
0
0 NNH
1 *L 0
DMTrO N N Ni____
I\13(y H \ \
DMTrO N N
0
CEOP[NOPO2l2,
H DCI
N'I_____ --0-.4
DCM ___________________________________________ 1-
,6 o-ms
_
OH a-ms 25 C, lh NC--./-0-RN
¨r
Int 2.13-3a Int 2.13-4
[0413] Int 2.13-3a (28 g, 36.41 mmol, 1.00 equiv) was dissolved in 280 mL of
dichloromethane
under a nitrogen atmosphere. 1H-imidazole-4,5-dicarbonitrile (12.9 g, 109.23
mmol, 3.00 equiv)
and 3-(bis[bis(propan-2-yl)amino]phosphanyloxy)propanenitrile (43.84 g, 145.64
mmol, 4.00
equiv) were added in order. The resulting solution was stirred for 1 h at 25 C
and the resulting
solution was diluted with 500 mL of dichloromethane and washed with 4 x 400 mL
of saturated
sodium bicarbonate solution and 1x400 mL of saturated sodium chloride
solution, respectively.
The organic phase was dried over anhydrous sodium sulfate, filtered and
concentrated under
vacuum. The crude product was purified by flash chromatography (C18 silica
gel; mobile phase,
acetonitrile in water gradient: 40% up to 100% within 8 min and 100%
maintained 10 min) to
afford 25 g of Int 2.13-4 as a white solid.
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Step E. Preparation of Int 2.13-5
0
0 Ii
NI6F_I 0 hi 0
HO N NN
TPDSCI2, Py N
H 25 C, 16h
SI-0 OH
8H 8H 7A
Int 2.13-1 Int 2.13-5
[0414] To a solution of Int 2.13-1 (190 g, 538.24 mmol) in 3000 mL of pyridine
was added
1,1,3,3-tetraisopropy1-1,3-dichlorodisiloxane (152.6 g, 484.42 mmol, 0.9
equiv) dropwise at 0 C.
The resulting solution was stirred for 16 h at 25 C. The reaction was quenched
by the addition of
30 mL of methanol and the resulting solution was concentrated under vacuum.
The residue was
applied onto a silica gel column with dichloromethane/methanol (50/1-30/1) to
afford 189 g of
N49-[(6aR,8R,9R,9aS)-9-hydroxy-2,2,4,4-tetrakis(propan-2-y1)-hexahydro-2H-
furo[3,2-
f][1,3,5,2,4]trioxadisilocin-8-y1]-6-oxo-6,9-dihydro-1H-purin-2-y1]-2-
methylpropanamide (Int
2.13-5) as a white solid.
Step F: Preparation of Int 2.13-6
0
0
N1)1\1H 0
I 0 N N
P N N N Tf20, Py, DMAP
/
DCM, 0 C, 2h / 01. uTf
Si¨u OH Si¨u
Int 2.13-5 Int 2.13-6
[0415] Into a 250-mL 3-necked round-bottom flask purged and maintained with an
inert
atmosphere of nitrogen, was placed a solution of 195 g (327.73 mmol) of N49-
[(6aR,8R,9R,9aS)-9-hydroxy-2,2,4,4-tetrakis(propan-2-y1)-hexahydro-2H-furo[3,2-
f][1,3,5,2,4]trioxadisilocin-8-y1]-6-oxo-6,9-dihydro-1H-purin-2-y1]-2-
methylpropanamide (Int
2.13-5) in 4000 mL of dichloromethane, 129.5 g (5.00 equiv) of pyridine and 4-
dimethylaminopyridine (20 g, 163.86 mmol, 0.50 equiv). The solution was cooled
to 0 C and
treated with 184.8 g (655.5 mmol, 2.0 equiv) triflic anhydride dropwise. The
resulting solution
was stirred for 2 h at 0 C then quenched by the addition of 4000 mL of
water/ice. The resulting
solution was extracted with 3 x 4000 mL of dichloromethane and the organic
layers were
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combined. The organic extracts were washed with 2 x 4000 mL of water/ice and 1
x 4000 mL of
saturated sodium chloride solution, respectively. The organic phase was dried
over anhydrous
sodium sulfate, filtered and concentrated to provide 213 g (crude) of
(6aR,8R,9R,9aR)-842-(2-
methylpropanamido)-6-oxo-6,9-dihydro-1H-purin-9-y11-2,2,4,4-tetrakis(propan-2-
y1)-hexahydro-
2H-furo[3,2-f][1,3,5,2,4]trioxadisilocin-9-yltrifluoromethanesulfonate (Int
2.13-6) as a yellow
solid.
Step G. Preparation of Int 2.13-7
0
0
la\(1H 0
leLZI 0
N
NaNO2, DMF N N N
25 C, 16h u"--2OH
Si-6 6-rf / g, ___
7A =
7A
Int 2.13-6 Int 2.13-7
[0416] Int 2.13-6 (213 g, crude) was dissolved in 2100 mL of DMF and treated
with sodium
nitrite (158.3 g, 2.29 mol, 7.00 equiv). After stirring for16 h the solution
was filtered and
concentrated under vacuum. The resulting solution was diluted with 6000 mL of
DCM and
washed with 2 x 3000 mL of saturated sodium chloride solution, dried over
anhydrous sodium
sulfate, filtered and concentrated under vacuum. The residue was applied to a
silica gel column
with dichloromethane/methanol (50/1-30/1) and then purified using the
following conditions:
C18 silica gel, 50% Me0H/water to 100% water over 10 min then 100% water for
10 min to
provide 50 g (26% for 2 steps) of Int 2.13-7 as a white solid.
Step H: Preparation of Int 2.13-8
0 0
la\(1H
la\(1H 0 0
N N
N Tf20, DMAP, DCM SfçO
----r __ -20H
0 C, lh _________ -Zon
cc-6
7A
Int 2.13-7 Int 2.13-8
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[0417] N49-[(6aR,8R,9R,9aS)-9-hydroxy-2,2,4,4-tetrakis(propan-2-y1)-hexahydro-
2H-furo[3,2-
f][1,3,5,2,4]trioxadisilocin-8-y1]-6-oxo-6,9-dihydro-1H-purin-2-y1]-2-
methylpropanamide (Int
2.13-7) (50 g, 84.03 mmol) and DMAP (30.8 g, 252.10 mmol, 3.00 equiv) were
dissolved in 500
mL of DCM and the mixture was cooled to 0 C. Triflic anhydride (30.8 g, 109.24
mmol, 1.30
equiv) was then added dropwise with stirring at 0 C. The resulting solution
was stirred at this
temperature for 1 h. The reaction was then quenched by the addition of 500 mL
of ice/water then
extracted with 3 x 500 mL of dichloromethane and the organic layers were
combined. The
organic layer was washed with 2 x 100 mL of saturated sodium chloride
solution. The solution
was dried over anhydrous sodium sulfate, filtered and concentrated under
vacuum. The crude
product (Int 2.13-8) (55 g) was thus isolated and used directly in the next
step.
Step I: Preparation of Int 2.13-9
0 0
ItNX 0 0
P N N NaN3, DMF ,:S., N N N
/ 01.
O.
Si¨O Si¨O n3
7A 7A
Int 2.13-8 Int 2.13-9
[0418] Int 2.13-8 (55g, crude) was dissolved in N,N-dimethylformamide (500 mL)
then treated
with sodium azide (27.8 g, 427.73 mmol, 5.1 equiv). The resulting solution was
stirred for 16 h at
room temperature. The resulting mixture was filtered and concentrated under
vacuum. The
residue was applied onto a silica gel column with dichloromethane/methanol
(20/1) to afford 15.0
g of Int 2.13-9 as a yellow solid.
Step J: Preparation of Int 2.13-10
0 0
AH 0 0
N N HO N
TBAF, THF
25 C, 10min _-
/ 0. OH N3
Si¨O n3
7A
Int 2.13-9 Int 2.13-10
[0419] Int 2.13-9 (23 g, 1.00 equiv) in THF (230mL) was treated with
tetrabutylammonium
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fluoride (37 mL, 1.0 equiv). The resulting solution was stirred for 10 min at
room temperature
then concentrated under vacuum. The residue was applied directly to a silica
gel column with
dichloromethane/methanol (100/1-20/1). This resulted in 12.4 g (88%) of N49-
[(2R,3R,4S,5R)-
3-azido-4-hydroxy-5-(hydroxymethyl)oxolan-2-y1]-6-oxo-6,9-dihydro-1H-purin-2-
y1]-2-
methylpropanamide (Int 2.13-10) as a white solid.
Step K: Preparation of Int 2.13-11
0 0
N
11*-ILNH ,, 1\11?N1H
I u 0
\
HO 0N N NI____ DMTrCI, Py
....-0.....) H1-----
aH F13 25 C, 2h
OH F13
Int 2.13-10 Int 2.13-11
[0420] Into a 50-mL round-bottom flask, was placed a solution of 11 g of Int
2.13-10 in pyridine
(60 mL). 4,4'-(chloro(phenyl)methylene)bis(methoxybenzene) (14.75 g, 1.50
equiv) was added
and the resulting solution was stirred for 2 h at room temperature. The
reaction was quenched by
the addition of 20 mL of methanol and the resulting mixture was concentrated
under vacuum.
The residue was dissolved in 500 mL of dichloromethane, washed with 2 x 250 mL
of saturated
sodium bicarbonate solution and 1 x 250 mL of saturated sodium chloride
solution respectively.
The organic phase was dried over sodium sulfate, filtered and concentrated
under vacuum. The
residue was chromatographed with dichloromethane/methanol with 0.05%
triethylamine (100/1-
60/1) to afford 17.8 g of Int 2.13-11 as a white solid.
Step L: Preparation of Int 2.13-12
0 0
1\11.LNH I I.L _
u :1--1 N L1
0
\DMTrO N N NI____ H2, Pd/C DMTrO O N N N -
0,....) H V...) H
________________________________________________ ).-
aH F13 Me0H OH F1H2
25 C, lh
Int 2.13-11 Int 2.13-12
[0421] Into a 50-mL round-bottom flask was placed a solution of 10 g of Int
2.13-11 in methanol
(150 mL) and 10% anhydrous palladium on carbon (2g, w/w). The resulting
mixture was stirred
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under an atmosphere of hydrogen for lh at room temperature. The mixture was
filtered through
Celite and concentrated to provide 8.7 g of Int 2.13-12 as a yellow solid
which was used directly
without further purification.
Step M: Preparation of Int 2.13-13
0 0
NINHI *L 0 1\116H 0
DMTrO N N NI__ DMTrO N V NN-5____ O
H CbzCI, NaHCO3 VO H
_____________________________________________ ).-
OH 111-12 THF, H20 OH IIHCbz
25 C, 20min
Int 2.13-12 Int 2.13-13
[0422] To a solution of 16.8 g of N49-R2R,3R,4S,5R)-3-amino-5-Dis(4-
methoxyphenyl)(pheny1)-methoxylmethyl]-4-hydroxyoxolan-2-y1]-6-oxo-6,9-dihydro-
1H-purin-
2-y1]-2-methylpropanamide (Int 2.13-12) in 210 mL of THF/water (4/1) was added
sodium
bicarbonate (6.46g, 3.00 equiv) followed by the addition of Cbz-Cl (5.67g,
1.30 equiv). The
resulting solution was stirred for 20 min at room temperature. The reaction
was diluted with
saturated sodium carbonate solution (100 mL) and extracted with 3 x 200 mL of
dichloromethane.
The organic phase was dried over sodium sulfate, filtered and concentrated
under vacuum. The
residue was chromatographed with dichloromethane/methanol with 0.05%
triethylamine (100/1)
and then crystallized from dichloromethane (40 mL) to afford 14.8g (86% over
two steps) of Int
2.13-13 as a white solid.
Step N: Preparation of Int 2.13-14
0 0
N N
16H
la\LJH 0 0
DMTrO N N N¨ CEOP[NOPO2l2 DMTrO N N9'N1......_
8H Fl-HCbz 25 C, lh
Nõ8 11HICbz
C)CN
Int 2.13-13 Int 2.13-14
[0423] Int 2.13-13 (14.8 g, 18.78 mmol) was dissolved in 150 mL
dichloromethane under a
nitrogen atmosphere, 3-(bis[bis(propan-2-yl)amino]phosphanyloxy)propanenitrile
(22.6 g, 75.12
mmol, 4.00 equiv) and 1H-imidazole-4,5-dicarbonitrile (6.64 g, 56.34 mmol 3.00
equiv) were
added in order. The resulting solution was stirred for 1 h at 25 C and diluted
with 400 mL of
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dichloromethane. The solution was washed with 1 x 500 mL of saturated sodium
bicarbonate
solution and 1 x 500mL of saturated sodium chloride solution respectively. The
organic phase
was dried over anhydrous sodium sulfate, filtered and concentrated under
vacuum. The crude
product was purified by Flash with the following conditions: Column, C18
silica gel; mobile
phase, acetonitrile in water: 30% up to 80% within 8 min and 100% maintained
10 min to
provide 14.8 g (75%) of N49-[(2R,3R,4R,5R)-5-Dis(4-
methoxyphenyl)(phenyl)methoxy]methyl]-3-([[bis(propan-2-y1)amino](2-
cyanoethoxy)phosphanyl]oxy)-4-[(tert-butyldimethylsily1)oxy]oxolan-2-y1]-6-oxo-
6,9-dihydro-
1H-purin-2-y1]-2-methylpropanamide (Int 2.13-14) as a white solid.
Step O. Preparation of Int 2.13-15
0 0
1\1 H
0 0
DMTrO NDMTrO N
y ______________ Py TFA, H20
r1\1,1D,6 11HCbz HO _O F1HCbz
MeCN
C)CN 25 C, 10 min C)CN
Int 2.13-14 Int 2.13-15
[0424] To a solution of Int 2.13-14 (7.2 g, 7.20 mmol) in acetonitrile (600
mL) and water (260
mg) was added pyridinium triflate (1.66g, 8.38 mmol, 1.20 equiv). The
resulting solution was
stirred for 10 min at room temperature to provide a solution of Int 2.13-15
which was used
directly in the next step.
Step P: Preparation of Int 2.13-16
\i/NNH
DMTrOv N 0 1 0
N DMTrOv N-
0 0
E tBuN H2, H20
/ 111-1Cbz111-1Cbz
MeCN 0 H=P--
25 C, 30 min 0- tBuNH3+
ON
Int 2.13-16
Int 2.13-15
[0425] The solution containing Int 2.13-15 was treated with tert-butylamine
(36 mL) for 30 min
at room temperature. The mixture was then concentrated under vacuum to afford
7.5 g of Int
2.13-16 as a foam which was used directly at next step without further
purification.
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Step Q. Preparation of Int 2.13-17
i\i,,yFI
DMTrO _5__
vo)1 NN H0v0.4 NN
H H
Cl2CHCO2H, DCM a zi
)5 NHCbz25 C NHCbz
25 C, 10 min
0=P¨H
I 0=P¨H
1
a tBuNH3+
OH
Int 2.13-16 Int 2.13-17
[0426] Int 2.13-16 was dissolved in 75 mL of dichloromethane and the solution
was treated with
91.5 mL of 6% dichloroacetic acid in dichloromethane. Triethylsilane (150 ml)
was added after
minutes followed by pyridine (10 mL). The mixture was concentrated and the
residue was
dissolved in 50 mL of dry acetonitrile and concentrated. This process was
repeated twice. The
residue was finally dissolved in 20 mL of acetonitrile and used directly at
next step.
Step R: Preparation of Int 2.13-18
,Nir1Tr
DMTrO\õ.Ø4 N HN
1\11)1LXH iy
N&IBS H
DMTrOossy N N
NC ()
el6H i..... \--\
-1<
n-4 C 0 OTBS N NH
HO\___ )1 NN 0 It 2.13
---\ \---\
alD *L 0
0 H 0
:f _____________________________________________ ).- \---0--) N N¨
H
I NHCbz MeCN, 4A MS
C) ,0 NHCbz
0¨_/
25 C, 5min
1 /IDIH
OH HO
Int 2.13-17 Int 2.13-
18
[0427] To the above solution containing 3.0 g of Int 2.13-17 and 3.0 g of 4A
MS in 20 mL of
MeCN was added a solution of Int 2.13-4 (18 g, 18.56 mmol, 2.5 equiv) in 30 ml
of dry
acetonitrile. The reaction solution was stirred for 5 min at 25 C and used
directly in the next step.
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Step S. Preparation of Int 2.13-19
1\11JH j 1\11it.i&H
DMTrO DMTrO
09 N r !Lr 09 N Ill)Y
E. ________ E
NC\--\ 6 Om s e 1 NH
OTI0
Q oTBs e,IFI
0
V
4.0 eb TBHP a 0 Vo,...)
H
i ____________ E MeCN i E
(:) ,6 NHCbz25 C, ,6 F1HCbz
/1H 25 C, 30min
r-H
HO HO
Int 2.13-18 Int 2.13-19
[0428] To above reaction solution (Int 2.13-18) was added 8 mL of tert-butyl
hydroperoxide (5.5
M in decane). The resulting solution was stirred for 30 min at 25 C then
diluted with 250 mL of
ethyl acetate. The resulting solution was washed with 2 x 300 mL of water and
1 x 300 mL of
saturated sodium chloride solution. The mixture was dried over anhydrous
sodium sulfate,
filtered and concentrated under vacuum to afford 21 g of Int 2.13-19 which was
used directly in
the next step.
Step T: Preparation of Int 2.13-20
F\IIJH x eli).LNH jy
DMTrOl _10....._ IN N N HO N N N
,,,,, = E ,,,,, E
NC ck ams NC\____\ 0\ oTBSe . NH
\........\ N10 I 0 C12CHCO2H, DC 0
11 I 0
H
25 C, 10 min
z:. E
n / (: 6 F1HCbz 0 NHCbz
r
/ H
HO ,_, " HO
Int 2.13-19 Int 2.13-20
[0429] Int 2.13-19 (21 g, crude) was dissolved in methylene chloride (250 mL)
before adding
210 mL dichloroacetic acid (6% in methylene chloride) to the solution. After
stirring for 10 min,
420 mL of triethylsilane was added while stirring at room temperature. 50 mL
of pyridine was
then added and the mixture was concentrated under vacuum. The resulting
residue was purified
by flash column chromatography (C18 gel column, mobile phase,
acetonitrile/water with 0.04%
ammonium bicarbonate: 20% up to 80% within 10 min, then 100% for 5 min). This
resulted in
2.5 g (30% overall for 6 steps) of Int 2.13-20 as a white solid.
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Step U. Preparation of Int 2.13-21
I 1 x d jy
NC
N N N
HO\--0---) H
M i?
r\13X 0
0-p-O
NC P oTBSeN 1. DMOCP, Py, 4A MS, 25 C, 15min i 0)1 N III'
\--\ 1 *LNH _c
OTBSC1
0 Vo...) N 111 2.12, H20, 20 min
A 9
E
NHCbz
1 E HN N --p-
O ,0 11HCbz 11: 0 ii OH
0
PH
. 0 HN N
/
HO
0
Int 2.13-20 Int 2.13-21
[0430] Compound 2-chloro-5,5-dimethy1-1,3,2-dioxaphosphorinane-2-oxide (1.9 g,
10.39 mmol,
4.7 eq) was added to the solution of Int 2.13-20 (2.5 g, 2.21 mmol) in 50 mL
of pyridine and the
reaction solution was stirred for 15 min at 25 C. The reaction was quenched by
water (600 uL)
followed by iodine (0.84 g, 3.31 mmol, 1.5 equiv). After stirring for 20 min,
12.5 mL saturated
sodium thiosulfate solution was added. The mixture was concentrated to a foam
to provide 2.5 g
of Int 2.13-21 which was used directly in the next step.
Step V: Preparation of Int 2.13-22
o 0
NC
L-1 9 N
h-1 0
pe(Z1 0
0 N N N t-BuNH2, MeCN OTBSoõ) N riy
)...
O
25 C, 10min _ _
H C b z
1,N N rElsL,N N
0¨FLOH 0¨P-0-
11 di
IC di
0 HN N 0 HN N 2(t-BuNH3 )
0 0
Int 2.13-21 Int 2.13-22
[0431] To a solution of 2.5 g crude Int 2.13-21 in acetonitrile (12.5 mL) was
added tert-
butylamine (22.5 mL). After stirring for 10 min at 25 C, the reaction solution
was concentrated to
a yellow foam and the crude product was purified by flash chromatography (C18
gel column,
mobile phase, acetonitrile/water with 0.04% ammonium bicarbonate, gradient:
10% up to 50%
within 15 min, 100% for 5 min) to provide pure 1.2 g (50% over 2 steps) Int
2.13-22.
Step W: Preparation of Int 2.13-23
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N
ep ,-, q X
N
11 i......
, q ,X1 u- p¨o
N
H
Ni_... ()IBS Q N N
i ..\--.4).-..)
OTBS 1) H2 -
E . Me0H, PcVC ri-1 l'I (---- A
i ,, N H2
,,
H OA P 11 H Cbz
25 C, 1 h N 1 N1 0 0-1-0 -
/r N 1 \J 0-I--0- 0 HNII;,1 0
2(t-B u N H3 )
0
0 HN N 2(t -B u N H3 ) 0
0 Int 2.13-22 Int 2.13-23
[0432] A 50-mL round-bottom flask was purged with argon then charged with a
solution of Int
2.13-22 (500 mg, 0.46 mmol) in 25 mL of methanol. 10% anhydrous palladium
carbon (250 mg,
w/w) was then added and hydrogen was bubbled through the solution. The
reaction mixture was
stirred for 1 h at 25 C then filtered through Celite and concentrated to
provide 380 mg (87%) of
Int 2.13-23 as a white solid.
Step X: Preparation of Int 2.13-24
e 1X-1
-
N q
;LX1 _c_... 0-T -0-...k., õI N riF1
QTBSQ
-, q , 0
LP' rki.....k.... )1 N N OTBSO
0 H z __ r
(.......-0A - -
? NH2
(.......-0A ?
I NH2 2(t-BuNH3) Me NH2' Et OH H2N NV NJ
o-T-o-
o
H N N 2(MeNH3 )
R" VN NS 0-T-0-
0
0 HN N 0
0
Int 2.13-23 Int 2.13-24
[0433] Into a 100-mL round-bottom flask was placed Int 2.13-23 (380 mg, 0.40
mmol) and 25
mL of methylamine (33% in anhydrous ethanol). The resulting solution was
stirred for 16 h at
25 C then concentrated to provide Int 2.13-24 as a white foam which used
directly in the next
step.
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Step Y: Preparation of Compound 21
0
iNNIN NH2
I I
QTBSO
N N NH2 I 11E1
OA 9 R1112 Et3N , Et3N.3HF
QH Q Ck."1
- ________________________________________________________
H2N
(MeN H31 55 C, 1 h
H2N,
C) 1-12
.82 NH4
y=-=
0
HN 2 r\ji 0
0 0 0.18 + NH
Int 2.13-24 Compound 21
[0434] The above crude Int 2.13-24 was azeotroped with pyridine/triethylamine
(9mL/3mL)
three times then dissolved in 0.8 mL pyridine in a 100 mL round-bottom flask.
To this solution at
55 C was added 6 mL triethylamine and 4 mL triethylamine trihydrofluride
simultaneously.
After stirring lh, the bath was removed and 60 mL anhydrous acetone was added
immediately.
The mixture was stirred for 20 min and the white solid was collected by
filtration. The precipitate
was washed with 5 mL of anhydrous acetone. The product was purified by
preparative flash
chromatography (AQ-C18 silica gel; mobile phase = acetonitrile /water with
0.04% ammonium
bicarbonate; gradient 1% to 20% over 20 min, UV detector @ 210 nm). The
resulting solution
was lyophilized to provide 100 mg (36% for 2 steps) of Compound 21 as a white
solid. LC-MS-
SVT-001-1-24: (ESI, m/z): 690 [M+H] 1H NMR (D20): 6 7.86 (s, 1H); 7.83 (s,
1H); 5.80-5.92
(m, 2H); 4.96-5.09 (m, 1H); 4.84-4.88 (m, 1H); 4.69-4.83 (m, 1H); 4.32-4.39
(m, 1H); 4.11-4.39
(m, 4H); 3.91-4.02 (m, 2H); [0.18 equiv TEA: 2.98-3.12 (q, J=6.6, 1H); 1.05-
1.24(m, J=6.6,
1.6H)]. 31P NMR (D20) 6 -1.28, -1.39.
EXAMPLE 4
Synthesis of ATAC1 and ATAC2
[0435] This example shows the synthesis of Pentafluorophenyl 25-(2-amino-3-
pentylquinolin-5-
y1)-19-oxo-4,7,10,13,16-pentaoxa-20-azapentacosanoate (ATAC1) and
Perfluorophenyl 3-((4-
amino-1-(2-hydroxy-2-methylpropy1)-1H-imidazo [4,5-c] quinolin-2-yl)methyl)-4-
oxo-
7,10,13,16,19-pentaoxa-3-azadocosan-22-oate (ATAC2).
TFA
NH2
,
F 01..0c)000,iN
0 0
ATAC1
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Step A: Preparation of Int ATAC1-1
NH2
1 ''N
0 H2N 40
HO...rõ..no
_____________________________________________________ ,-
0 0
0
HCI
NH2
1 ''N
H
I'
0 0
ATAC1 -1
[0436] To a 0 C solution containing 271 mg (0.90 mmol) of 5-(5-aminopenty1)-3-
pentylquinolin-
2-amine in 4 mL of DCM was added 435 mg (1.00 mmol) of the NHS ester in 1 mL
of DCM
dropwise over 15 minutes. The reaction mixture was allowed to warm to ambient
temperature
over 19h before it was concentrated and purified by reverse phase
chromatography. Pure
fractions were lyophilized and dissolved in 3 mL of methanol then treated with
1 mL of 4N HC1
in dioxane. The solution was stirred for lh then concentrated to afford the
desired compound as
an HC1 salt which was used directly in the next step.
Step B: Preparation of ATAC1
H CI F
NH2 F Alt, OH
Ir
1 ''N F F
H
40 F
__________________________________________________________ ..
DIC / DM F
0 0
ATAC 1 -1
TFA
NH2
F
H
F
F 0 0F
0 0
F
ATAC 1
[0437] To a stirred solution of 25-(2-amino-3-pentylquinolin-5-y1)-19-oxo-
4,7,10,13,16-
pentaoxa-20 azapentacosanoic acid hydrochloride (130 mg, 0.198 mmol) and
pentafluorophenol
(146 mg, 0.792 mmol) in DMF (2.5 ml) at room temperature was added N,N'-
Diisopropylcarbodiimide (0.186 ml, 1.189 mmol) dropwise. The reaction was
stirred at room
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temperature for 18h then concentrated. The crude product was added to a 100g
C18 gold reverse
phase column and was eluted with water/acetonitrile (0.1% TFA) 10-100%. The
fractions were
combined and concentrated then freeze dried to give perfluorophenyl 25-(2-
amino-3-
pentylquinolin-5-y1)-19-oxo-4,7,10,13,16-pentaoxa-20-azapentacosanoate-2,2,2-
trifluoroacetate
(110 mg, 61.7 % yield) as a clear gum. 1H NMR (DMSO-d6) 8 13.7 (s, 1H), 8.37-
8.35 (m, 3H),
7.78 (t, J=5.5Hz, 1H), 7.63 (t, J=7.5Hz, 1H), 7.53 (d, J=8.5Hz, 1H), 7.31 (d,
J=7.0 Hz, 1H), 3.58
(t, J=6.0Hz, 2H), 3.63-3.43 (m, 20H), 3.04-2.96 (m, 6H), 2.73 (t, J=7.5Hz,
2H), 2.27 (t, J=7.5Hz,
2H), 1.60-1.55 (m, 4H), 1.44-1.33 (m, 9H), 0.88 (t, J=7.5Hz, 3H). LCMS [M-FH]
= 786.3.
[0438] The following compound in TABLE 2 can be prepared using a method
similar to that
described above for ATAC1.
TABLE 2
Compound Structure IUPAC M+1
ATAC2 H2N -N Perfluorophenyl 3-((4-amino-
800
F r- N \ AM 1-(2-hydroxy-2-
F 0 0...TorO......,,,0O,...,,,cOn2,N,..,,A.N /.
F F HO F methylpropy1)-1H-
imidazo[4,5-c]quinolin-2-
yl)methyl)-4-oxo-
7,10,13,16,19-pentaoxa-3-
azadocosan-22-oate
EXAMPLE 5
Synthesis of ATAC3 and ATAC4
[0439] This example shows the synthesis of pentafluorophenyl 25-(2-amino-3-
pentylquinolin-5-
y1)-19-oxo-4,7,10,13,16-pentaoxa-20-azapentacosanoate (ATAC3) and 2,5-
Dioxopyrrolidin-1-y1
3-((4-amino-1-(2-hydroxy-2-methylpropy1)-1H-imidazo-[4,5-c]quinolin-2-
y1)methyl)-4-oxo-
7,10,13,16,19-pentaoxa-3-azadocosan-22-oate (ATAC4).
H C I
NH2
1 ' N
I
0
el
H
0 0
0
ATAC3
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Step A. Preparation of ATAC3
[0440] To a stirred solution of Int ATAC1-1 (185 mg, 0.282 mmol) and N-
hydroxysuccinimide
(130 mg, 1.128 mmol) in DMF (3 ml) was added N,N'-diisopropylcarbodiimide
(0.221 ml, 1.411
mmol) dropwise and the reaction mixture was stirred at room temperature for 18
hours. The
reaction mixture was filtered and washed with acetonitrile and the filtrate
was evaporated. The
resulting residue was purified by silica gel Silica gel column chromatography
(DCM / Me0H /
HOAc) to give 65 mg of the desired product as the acetic acid salt which was
subsequently
dissolved in 2 mL of DCM and treated with 2M HC1 in diethyl ether. The
solution was stirred for
lh then concentrated and lyophilized to afford the desired compound as the HC1
salt. 1H NMR
(CDC13) 8 15.2 (s, 1H), 8.15 (d, J=7.8Hz, 1H), 7.68 (d, J=7.9Hz, 1H), 7.55 (t,
J=8.1Hz, 1H), 6.55
(bs, 1H), 3.98 (t, J=6.0Hz, 2H), 3.83-3.55 (m, 18H), 3.33-3.22 (m, 2H), 2.95-
2.56 (m, 11H), 2.27
(t, J=7.5Hz, 2H), 1.60-1.55 (m, 4H), 1.44-1.33 (m, 9H), 0.88 (t, J=7.5Hz, 3H).
LCMS [M+H] =
717.3.
[0441] The following 2,5-Dioxopyrrolidin-1-y13-((4-amino-1-(2-hydroxy-2-
methylpropy1)-1H-
imidazo-[4,5-c]quinolin-2-y1)methyl)-4-oxo-7,10,13,16,19-pentaoxa-3-azadocosan-
22-oate
(ATAC4) compound can be prepared using a method similar to that described
above for ATAC3.
H2N
_N
.......1.\µ1,01.(000(:)0.rN 1
'N
0 0
0
H-0-2
ATAC4
1H NMR (CDC13) 8 14.9 (s, 1H), 8.88 (bs, 1H), 8.15 (d, 1H), 7.85 (d, 1H), 7.61
(t, 1H), 7.45 (t,
1H), 4.72 (s, 2H), 3.83 (m, 4H), 3.65-3.45 (m, 18H), 2.90-2.71 (m, 9H), 1.43
(t, J=7.0Hz, 3H),
1.33 (s, 6H). LCMS [M+H] = 731.
EXAMPLE 6
Synthesis of ATAC5, ATAC6 and ATAC7
[0442] This example shows the synthesis of 2,5-dioxopyrrolidin-l-y16-(((S)-1-
(((S)-1-((4-((((5-
(2-amino-3-pentylquinolin-5-yl)pentyl)carbamoyl)oxy)methyl)phenyl)amino)-1-
oxopropan-2-
yl)amino)-3-methyl-l-oxobutan-2-yl)amino)-6-oxohexanoate (ATAC5), 2,5-
dioxopyrrolidin-1-y1
7-(((S)-1-(((S)-1-((4-(((((4-amino-1-(2-hydroxy-2-methylpropy1)-1H-imidazo
[4,5-c] quinolin-2-
yl)methyl)(ethyl)c arbamo yl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-
3-methy1-1-
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oxobutan-2-yl)amino)-7-oxoheptanoate (ATAC6), and 2,5-dioxopyrrolidin-l-y17-
(((S)-1-(((S)-
1-((4-(((((4-amino-1-(2-hydroxy-2-methylpropy1)-1H-imidazo[4,5-c]quinolin-2-
y1)methyl)(ethyl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-
yl)amino)-3-
methyl-1-oxobutan-2-y1)amino)-7-oxoheptanoate (ATAC7).
TFA
NH2
0 0 CH3
N ,?LN )H.r N
= H
0 0 0 10 0 EN1
0
0
ATAC5
Step A. Preparation of Int ATAC5-1
NH2
NO2 1
0 CH3 H2N
>0 NieyN
DIPEA / DCM
0 0 0 0y0
2 HCI
0
HCI
NH2
0H
HON
401 40
0 0 0
0
Int ATAC5-1
[0443] A solution of 5-(5-aminopenty1)-3-pentylquinolin-2-amine (300 mg, 1.00
mmol) in 5 mL
DCM was stirred at room temperature under nitrogen for 10 min before tert-
butyladipate-valine-
alanine-para-aminobenzy1-4-nitrophenylcarbonate (tBuAdip-va-PAB-OPNP, 656 mg,
1.00 mmol)
and DIPEA (0.26 nil, 1.5 mmol) in 3 mL of DCM were added and the mixture was
stirred at
room temperature overnight. The mixture was concentrated and purified by
column
chromatography. Clean fractions were combined and evaporated and the residue
was dissolved
in 2 mL of DCM and treated with 2M HC1 in diethyl ether. The solution was
stirred for lh then
concentrated and lyophilized to afford the desired compound Int ATAC5-1 as the
HC1 salt. MS
m/z 761 (M) .
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Step B. Preparation of ATAC5
HCI
NH2
EN1 ? ,i:rH I 1\1
HO N N dah H NHS/DIC ..
H
0 0 0 W ON 40
11
0
Int ATAC5-1
TFA
NH2
0 ENi 0 j;.-13..rH I
N
H H
0 0 0 W CD 40
0 11N
0
ATAC5
[0444] To a stirred solution of 6-(((S)-1-(((S)-1-((4-((((5-(2-amino-3-
pentylquinolin-5-
yl)pentyl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-
l-oxobutan-
2-yl)amino)-6-oxohexanoic acid hydrochloride (221 mg, 0.282 mmol) and N-
hydroxysuccinimide (130 mg, 1.12 mmol) in DMF (3 ml) was added N,N'-
diisopropylcarbodiimide (0.221 ml, 1.41 mmol) dropwise and the reaction
mixture was stirred at
room temperature for 5h. HPLC indicated some starting material remained so the
reaction was
stirred at ambient temperature overnight. The reaction mixture was filtered
and washed with
acetonitrile. The filtrate was evaporated and the residue was dissolved in
DMSO and purified by
reverse phase chromatography [water/acetonitrile (0.1% TFA)] from 10% followed
by a gradient
from 20 to 80%. Pure fractions were combined to give 2,5-dioxopyrrolidin-l-y16-
(((S)-1-(((S)-1-
((4-((((5-(2-amino-3-pentylquinolin-5-yl)penty1)-
carbamoyl)oxy)methyl)phenyl)amino)-1-
oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-y1)amino)-6-oxohexanoate 2,2,2-
trifluoroacetate
(109 mg, 40 % yield) as a white solid. 1H NMR (DMSO-d6) 8 13.6 (s, 1H), 9.92
(s, 1H), 8.32
(d, J=7.5Hz, 1H), 7.84 (d, J=8.5Hz,1H), 7.61-7.55 (m, 4H), 7.30-7.17 (m, 4H),
4.92 (s, 2H), 4.37
(t, J=7.0Hz, 1H), 4.18 (t, J=7.0Hz, 1H), 2.96 (m, 4H), 2.81-2.62 (m, 8H), 2.33-
2.11 (m, 2H), 1.95
(q, J=7.0Hz, 1H), 1.63-1.55 (m, 8H), 1.50-1.40 (m, 2H), 1.38-1.33 (m, 4H),
1.29 (d, J=7.0Hz,
3H), 0.83 (d, J=7.0Hz, 6H). LCMS [M+H] = 844.3.
[0445] The following ATAC6 compound and ATAC7 compound in TABLE 3 can be
prepared
using a method similar to that described above for ATAC5.
TABLE 3
Compound Structure Name M+ 1
ATAC6 0 N jt iH3 ENi H2N 2,5-
dioxopyrrolidin- 1 -y1 7- 872
-IµC),C(Cr (10r 0 NUI At
0 01( N (((S)-1-(((S)-1-((4-
(((((4-
H* amino- 1 -(2-hydroxy-2-
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methylpropy1)- 1H-
imidazo[4,5-c] quinolin-2-
yl)methyl)(ethyl)carbamoyl)o
xy)methyl)phenyl)amino)- 1 -
oxopropan-2-yl)amino)-3 -
methyl-1 -oxobutan-2-
yl)amino)-7-oxoheptanoate
ATAC7 H2N0 2,5-dioxopyrrolidin- 1 -y1 7-
958
A H2N (((S)-1-(((S)-1-((4-(((((4-
(N N
01r,cFNi,rH -N
amino-1 -(2-hydroxy-2-
0
HC>( methylpropy1)- 1H-
i
imidazo[4,5-c] quinolin-2-
yl)methyl)(ethyl)carbamoyl)o
xy)methyl)phenyl)amino)- 1 -
oxo-5-ureidopentan-2-
yl)amino)-3-methyl- 1 -
oxobutan-2-yl)amino)-7-
oxoheptanoate
EXAMPLE 7
Synthesis of ATAC8, ATAC9, and ATAC10
[0446] This example shows synthesis of Perfluorophenyl 6-(((S)-1-(((S)-1-((4-
((((5-(2-amino-3-
pentylquinolin-5-yl)pentyl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-
yl)amino)-3-
methyl-l-oxobutan-2-yl)amino)-6-oxohexanoate (ATAC8), perfluorophenyl 7-(((S)-
1-(((S)-1-
((4-(((((4-amino-1-(2-hydroxy-2-methylpropy1)-1H-imidazo[4,5-c]quinolin-2-
yl)methyl)(ethyl)carbamoyl)oxy)methyl)phenyl)amino)- 1 -o xoprop an-2- yl)
amino)-3 -methyl-1 -
oxobutan-2-yl)amino)-7-oxoheptanoate (ATAC9), and perfluorophenyl 7-(((S)-1-
(((S)-1-((4-
(((((4-amino- 1 -(2-hydro xy-2-methylprop y1)- 1H-imidazo [4,5-c] quino lin-2-
yl)methyl)(ethyl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxo-5-ureidopentan-2-
yl)amino)-3-
methyl-l-oxobutan-2-yl)amino)-7-oxoheptanoate (ATAC10).
TFA
NH 2
0 CH3 N
H J.L
F 01,..1,N N)Lir N
= H
0 ./rl
0 0 0
0
ATAC8
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Step A. Preparation of ATAC8
F
I-I CI NH2 F la OH
0 CH3 N
H I F F
HO El\llYN l F
0 0 0 ...
H H ei D I C
W ON
II
0
T FA
NH2
F 0 CH3 N
H I
F 10 0 Ell 1\IYN
W
H H
F F
0 0 0 OIIN
lei
F 0
ATAC8
[0447] To a stirred solution of 6-(((S)-1-(((S)-1-((4-((((5-(2-amino-3-
pentylquinolin-5-
yl)pentyl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-yl)amino)-3-methyl-
l-oxobutan-
2-yl)amino)-6-oxohexanoic acid hydrochloride (168 mg, 0.215 mmol) and
pentafluorophenol
(158 mg, 0.86 mmol) in DMF (3 ml) was added N,N'-diisopropylcarbodiimide
(0.166 ml, 1.07
mmol) dropwise and the reaction mixture was stirred at room temperature for
6h. The reaction
mixture was concentrated and the residue was dissolved in DMSO and purified by
reverse phase
chromatography [water/acetonitrile (0.1% TFA)] from 10% followed by a gradient
from 20 to
80%. Pure fractions were combined to give perfluorophenyl 6-(((S)-1-(((S)-1-
((4-((((5-(2-amino-
3-pentylquinolin-5-yl)pentyl)carbamoyl)oxy)methyl)phenyl)amino)-1-oxopropan-2-
yl)amino)-3-
methyl-l-oxobutan-2-yl)amino)-6-oxohexanoate 2,2,2-trifluoroacetate (122 mg)
as a white solid.
1H NMR (DMSO-d6) 8 13.5 (s, 1H), 9.92 (s, 1H), 8.35 (bs, 3H), 8.17 (d,
J=7.0Hz, 1H), 7.87 (d,
J=7.0Hz, 1H), 7.64-7.52 (m, 4H), 7.32-7.18 (m, 4H), 4.91 (s, 2H), 4.37 (t,
J=7.0Hz, 1H), 4.19 (t,
J=7.0Hz, 1H), 3.60-3.50 (m, 4H), 2.97 (m, 4H), 2.79 (t, J=7.0Hz, 2H), 2.74 (t,
J=7.0Hz, 2H),
2.31-2.22 (m, 2H), 1.96 (q, J=7.0Hz, 1H), 1.71-1.51 (m, 8H), 1.45-1.38 (m,
2H), 1.40-1.27 (m,
9H), 0.90-0.80 (m, 9H). LCMS [M+H] = 913.4.
[0448] The following compound in TABLE 4 can be prepared using a method
similar to that
described in above for ATAC8.
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TABLE 4
Compound Structure Name M+1
ATAC9FI,F1
F 0 -N perfluorophenyl 7-(((S)-1-(((S)-
941
up 0 0 - 0 411 0 FC,A \
F F F Y 1-((4-(((((4-amino-1- (2-
HO
hydroxy-2-methylpropy1)-1H-
imidazo [4,5-0 quinolin-2-
yl)methyl)(ethyl)carbamoyl)oxy
)methyl)phenyl)amino)-1-
oxopropan-2-yl)amino)-3 -
methyl-l-oxobutan-2-yl)amino)-
7-oxoheptanoate
ATAC10 Hplf
perfluorophenyl 7-(((S)-1-(((5)- 1027
1-((4-(((((4-amino-1- (2-
\
F oli¨õ¨õ¨I,1 0 N r. N
F F OTN IN hydroxy-2-methylpropy1)-1H-
F
F10->f) imidazo [4,5-0 quinolin-2-
yl)methyl)(ethyl)carbamoyl)oxy
)methyl)phenyl)amino)-1-oxo-5-
ureidopentan-2-yl)amino)-3-
methyl-l-oxobutan-2-yl)amino)-
7-oxoheptanoate
EXAMPLE 8
Synthesis of ATAC11
[0449] This example shows the synthesis of N-((4-amino-1-(2-hydroxy-2-
methylpropy1)-1H-
imidazo[4,5-c]quinolin-2-y1)methyl)-1-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-
yl)propanamido)-
N-ethyl-3,6,9,12-tetraoxapentadecan-15-amide (ATAC11).
0 0 0
C)OC)0)L N N NH2
H3C) N \ N
0
OH
ATAC11
Step A: Preparation of ATAC11
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0
V / NI-N
0 0 gardiquimod ...
DCC, DCM/ACN
0 0 0
0 0 0
NN NH2
0 0
H
) N
/\ N
0 H3C
/(.....
OH ilk
ATAC11
[0450] A solution of MAL-PEG4-acid (265.7 mg, 0.638 mmol) and N,N'-
dicyclohexylcarbodiimide (DCC, 144.8 mg, 0.702 mmol) in dry dichloromethane /
acetonitrile
(1:1,5 mL) was stirred at room temperature for lh, followed by addition of
compound 1 (100 mg,
0.319 mmol) in one portion. After 72h of stirring, volatile organics were
removed under vacuum.
The residue obtained was purified by flash column chromatography on silica
gel, eluting with
step gradients of methanol in dichloromethane at a ratio of v/v 1:20, 1:15,
and 1:9, to afford the
target product N-((4-amino-1-(2-hydroxy-2-methylpropy1)-1H-imidazo[4,5-
c[quinolin-2-
yl)methyl)-1-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-N-ethyl-
3,6,9,12-
tetraoxapentadecan-15-amide (80 mg, 35% yield) as white colored foamy solid
oil. 1H NMR
(300 MHz, CDC13) 8 8.40 ¨ 7.82 (br m, 1H), 7.74 (d, J=8.1 Hz, 1H), 7.44 (t,
J=7.5 Hz, 1H), 7.30
(t, J=7.4 Hz, 1H), 6.76 ¨ 6.28 (br m, 2H), 4.82 ¨ 4.32 (br m, 2H), 4.08 ¨ 3.64
(br m, 6H), 3.54 (br
s, 14H), 3.31 (br s, 3H), 2.63 (br s, 2H), 2.38 (t, J=6.9 Hz, 2H), 1.27 (br s,
4H), 1.20 ¨ 0.68 (br m,
5H). MS (ESI+) m/z 712 (M+1), 734 (M+Na).
EXAMPLE 9
Synthesis of ATAC12, ATAC13, ATAC14, ATAC15, ATAC16, ATAC17, ATAC18,
ATAC19, ATAC20, and ATAC21
[0451] This example shows the synthesis of N-(5-(2-amino-3-pentylquinolin-5-
yl)penty1)-1-(3-
(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-3,6,9,12-tetraoxapentadecan-
15-amide
(ATAC12), 1-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-N-(3-
pentylquinolin-2-
y1)-3,6,9,12-tetraoxapentadecan-15-amide (ATAC13), 1-(3-(2,5-dioxo-2,5-dihydro-
1H-pyrrol-1-
yl)propanamido)-N-(1-isobuty1-1H-imidazo[4,5-c[quinolin-4-y1)-3,6,9,12-
tetraoxapentadecan-
15-amide (ATAC14), 1-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-N-
methyl-N-(2-
(3-(7-methylbenzo[1,2-d:3,4-0bis(thiazole)-2-yl)ureido)ethyl)-3,6,9,12-
tetraoxapentadecan-15-
amide (ATAC15), (5)-1-(3-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamido)-N-
(1-((7-
methylbenzo[1,2-d:3,4-0bis(thiazole)-2-yflamino)-1-oxo-3-phenylpropan-2-y1)-
3,6,9,12-
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tetraoxapentadecan-15-amide (AT AC16), N-(benzo [d]thiazol-2-y1)-1-(3-(2,5-
dioxo-2,5-dihydro-
1H-pyrrol-1-y1)propanamido)-N-((8-hydroxyquinolin-7-y1)(4-
(trifluoromethoxy)phenyOmethyl)-
3,6,9,12-tetraoxapentadecan-15-amide (ATAC17), N-
((2R,3R,3aS,7aR,9R,10R,10aS,14aR)-2,9-
bis(2-amino-6-oxo-1H-purin-9(6H)-y1)-5,10,12-trihydroxy-5,12-
dioxidodecahydrodifuro [3,2-
d:3',2'-j] [1,3 ,7,9,2,8]tetra-oxadipho sphac yclododecin-3 -y1)-1-(3 -(2,5-
dioxo-2,5-dihydro-1H-
pyrrol-1-yl)propanamido)-3 ,6,9,12-tetraoxapentadecan-15-amide (AT AC18), N-
((2R,3R,3aS,7aR,9R,10R,10aS,14aR)-2,9-bis(2-amino-6-oxo-1H-purin-9(6H)-y1)-10-
hydroxy-
5,12-dimercapto-5,12-dioxidodecahydrodifuro [3 ,2-d:3',2'-
A [1,3 ,7,9,2,8]tetraoxadipho sphac yclododecin-3 -y1) -1-(3 -(2,5-dioxo-2,5-
dihydro-1H-pyrrol-1-
yl)propanamido)-3 ,6,9,12-tetraoxapentadecan-15-amide (AT AC19), N-(9-
((2R,3R,3 aS,7aR,9R,10R,10aS ,14aR)-9-(2-amino-6-oxo-1H-purin-9(6H)-y1)-3
,5,10,12-
tetrahydroxy-5,12-dioxidodecahydrodifuro [3,2-d:3',2'A [1,3,7,9,2,8]tetra-
oxadiphosphacyclododecin-2-y1)-9H-purin-6-y1)-1-(3-(2,5-dioxo-2,5-dihydro-1H-
pyrrol-1-
yl)propanamido)-3,6,9,12-tetraoxapentadecan-15-amide (AT AC20), and N-(9-
((2R,3R,3 aS,7aR,9R,10R,10aS ,14aR)-9-(2-amino-6-oxo-1H-purin-9(6H)-y1)-3
,5,10,12-
tetrahydroxy-5,12-dioxidodecahydrodifuro [3,2-d:3',2'-
A [1,3 ,7,9,2,8]tetraoxadipho sphac yclododecin-2-y1) -9H-purin-6-y1)-1-(3 -
(2,5-dioxo-2,5-dihydro-
1H-pyrrol-1-yl)propanamido)-3,6,9,12-tetraoxapentadecan-15-amide (AT AC21).
H3C
NH2
0 0 0 /
..._..C)L N..---.....,...C...,..,...."..0,...---...õ..õ.õ---...0,AN 0 IN
H H
0
ATAC 12
Step A: Preparation of ATAC12
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H3C
/ NH2 Mal-PEG4-NHS
N
_________________________________________ >
H2N
0 DCM
H3C
NH2
0 0 0 /
N
,..---..., ,..---...õ...Øõ.......õ.--, ,.....--...õ...0,..õ....õ.",õ
,..."...õ.õ...----,
\ N hl 0 0 hl
0
0
ATAC12
[0452] To a stirred solution containing 100 mg (0.33 mmol) of 5-(5-
aminopenty1)-3-
pentylquinolin-2-amine in 13 mL of CH2C12 under N2 was added a solution of MAL-
PEG4-NHS
[CAS No 756525-99-2] (171 mg, 0.33 mmol) in 3 mL of CH2C12 by syringe pump
over 90 mins.
The reaction mixture was stirred at room temperature for 16h then evaporated
to afford a residue
which was purified by silica gel chromatography (CombiFlash Gold (12g):
CH2C12/CH3OH/NH4OH) to afford a light yellow syrup which was dissolved in 5 mL
of CH3CN
and lyophilized to provide 164 mg of the desired compound. 1H NMR (CD30D) 8
7.95 (s, 1H),
7.38 (s, 1H), 7.37 (s, 1H), 7.07 (t, J=8.5Hz, 1H), 6.78 (s, 2H), 3.75 (t,
J=6.0Hz, 2H), 3.65 (t,
J=6.0Hz, 2H), 3.59-3.52 (m, 12H), 3.46 (t, J=5.5Hz, 2H), 3.28 (t, J=7.5Hz,
2H), 3.18 (t, J=7.5Hz,
2H), 2.98 (t, J=8.5Hz, 2H), 2.67 (t, J=7.5Hz, 2H), 2.44 (t, J=7.0 Hz, 2H),
2.40 (t, J=7.0 Hz, 2H),
1.76-1.68 (m, 4H), 1.58-1.52 (m, 2H), 1.46-1.40 (m, 6H), 0.94 (t, J=7.0Hz,
3H). (MS (ESI+) m/z
698 (M+1).
[0453] The following compounds in TABLE 5 can be prepared using a method
similar to that as
described above for ATAC12.
TABLE 5
Compound Structure Name M+1
ATAC13
, ,I. 1-(3-(2,5-dioxo-2,5-dihydro-1H- 613
I pyrrol-1-yl)propanamido)-N-(3-
0 "
HaC
pentylquinolin-2-y1)-3,6,9,12-
tetraoxapentadecan-15-amide
ATAC141-(3-(2,5-dioxo-2,5-dihydro-1H- 639
0 0n 0 N ' 1
4N-___ pyrrol-1-yl)propanamido)-N-(1-
0 "
isobuty1-1H-imidazo[4,5-
c]quinolin-4-y1)-3,6,9,12-
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tetraoxapentadecan-15-amide
ATAC15 )L 1-(3-(2,5-dioxo-2,5-dihydro-1H-
720
H &1,
0 pyrrol-1-yl)propanamido)-N-
methyl-N-(2-(3-(7-
methylbenzo[1,2-d:3,4-
dlbis(thiazole)-2-yOureido)ethyl)-
3,6,9,12-tetraoxapentadecan-15-
amide
ATAC16
(S)-1-(3-(2,5-dioxo-2,5-dihydro- 635
0 ,R
0 Fd 1H-pyrrol-1-y1)propanamido)-N-
0
N
yS (1-((7-methylbenzo[1,2-d:3,4-
CH' dlbis(thiazole)-2-yl)amino)-1-
oxo-3-phenylpropan-2-y1)-
3,6,9,12-tetraoxapentadecan-15-
amide
ATAC17
CI? N-(benzokflthiazol-2-y1)-1-(3-
734
S (2,5-dioxo-2,5-dihydro-1H-pyrrol-
o o
140
oH,-- 1-yl)propanamido)-N-((8-
o OH
0
hydroxyquinolin-7-y1)(4-
ocF3 (trifluoromethoxy)phenyl)methyl)-
3,6,9,12-tetraoxapentadecan-15-
amide
ATAC18N- 1088
HO, tNN eNIZNH 2
FIC/P-hcL49 0 ((2R,3R,3aS,7aR,9R,10R,10aS,14
2H7'1X1 0 aR)-2,9-bis(2-amino-6-oxo-1H-
0
purin-9(6H)-y1)-5,10,12-
trihydroxy-5,12-
dioxidodecahydrodifuro[3,2-
d:3',2'-j][1,3,7,9,2,8]tetra-
oxadiphosphacyclododecin-3-y1)-
1-(3-(2,5-dioxo-2,5-dihydro-1H-
pyrrol-1-yl)propanamido)-
3,6,9,12-tetraoxapentadecan-15-
amide
ATAC190
tNNH N- 1120
H").P-01c4 0 ((2R,3R,3aS,7aR,9R,10R,10aS,14
yf
nfl,r11:,N, SH 0 aR)-2,9-bis(2-amino-6-oxo-1H-
0
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purin-9(6H)-y1)-10-hydroxy-5,12-
dimercapto-5,12-
dioxidodecahydrodifuro[3,2-
d:3',2'-
j][1,3,7,9,2,8]tetraoxadiphosphacy
clododecin-3-y1)-1-(3-(2,5-dioxo-
2,5-dihydro-1H-pyrrol-1-
yl)propanamido)-3,6,9,12-
tetraoxapentadecan-15-amide
ATAC20 N-(9-1073
H
Ho 0
((2R,3R,3aS,7aR,9R,10R,10aS,14
0H)-hcj, N
C14-7\
H'N aR)-9-(2-amino-6-oxo-1H-purin-
H,Tp
O 9(6H)-y1)-3,5,10,12-tetrahydroxy-
5,12-dioxidodecahydrodifuro113,2-
d:3',2'-j]111,3,7,9,2,8]tetra-
oxadiphosphacyclododecin-2-y1)-
9H-purin-6-y1)-1-(3-(2,5-dioxo-
2,5-dihydro-1H-pyrrol-1-
yl)propanamido)-3,6,9,12-
tetraoxapentadecan-15-amide
ATAC21 N-(9-1073
µki1:61 H
Ho 0
((2R,3R,3aS,7aR,9R,10R,10aS,14
0H)-hcj, N
OH aR)-9-(2-amino-6-oxo-1H-purin-
Hp P H
9(6H)-y1)-3,5,10,12-tetrahydroxy-
5,12-dioxidodecahydrodifuro113,2-
d:3',2'-
j][1,3,7,9,2,8]tetraoxadiphosphacy
clododecin-2-y1)-9H-purin-6-y1)-
1-(3-(2,5-dioxo-2,5-dihydro-1H-
pyrrol-1-yl)propanamido)-
3,6,9,12-tetraoxapentadecan-15-
amide
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EXAMPLE 10
Synthesis of ATAC22, ATAC23, ATAC24, ATAC25, ATAC26, ATAC27, ATAC28,
ATAC29, ATAC30, and ATAC31
[0454] This example shows the synthesis of 4-((5)-2-((5)-2-(6-(2,5-dioxo-2,5-
dihydro-1H-
pyrrol-1-yl)hexanamido)-3-methylbutanamido)propanamido)benzyl ((4-amino-1-(2-
hydroxy-2-
methyl-propy1)-1H-imidazo[4,5-c]quinolin-2-yl)methyl)(ethyl)carbamate
(ATAC22), 4-((5)-2-
((5)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-methyl-
butanamido)propanamido)benzyl (5-(2-amino-3-pentylquinolin-5-yl)penty1)-
carbamate
(ATAC23), 4-((5)-2-((5)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-
3-
methylbutan-amido)-5-ureidopentanamido)benzyl-(5-(2-amino-3-pentylquinolin-5-
y1)penty1)-
carbamate (ATAC24), 4-((5)-2-((5)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-
yl)hexanamido)-3-
methylbutanamido)-5-ureidopentanamido)benzyl((4-amino-1-(2-hydroxy-2-
methylpropy1)- 1H-
imidazo[4,5-c]quinolin-2-yl)methyl)(ethyl)carbamate TFA salt (ATAC25), 2-(3-12-
11\1-
Methyl(1p-1(5)-2-1(S)-2-[6-(2,5-dioxo-1H-pyrrol-1-yOhexanoylamino]-3-
methylbutyrylamino}-
5-ureidovalerylamino]phenyl}methoxycarbonyl)amino]ethyl}ureido)-7-methyl-1,6-
dithia-3,8-
diaza-as-indacene (ATAC26), 2-11(8-Hydroxy-7-quinoly1)(p-
trifluoromethoxyphenyOmethyll(1p-1(5)-2-1(5)-2-[6-(2,5-dioxo-1H-pyrrol-1-
yOhexanoylamino]-
3-methylbutyrylamino }-5-ureidovalerylamino]phenyl}methoxycarbonyl)amino } -
1,3-
benzothiazole (ATAC27), (1R,6R,8R,95 ,105 ,15R,17R,185 )- 1841 p-RS )-2-1 (5 )-
2-[6-(2,5-Dioxo-
1H-pyrrol-1-yl)hexanoylamino]-3-methylbutyrylamino } -5-
ureidovalerylamino]phenyl} methoxycarbonylamino)-8,17-bis(2-amino-6-oxo-1,9-
dihydropurin-
9-y1)-3,12-dihydroxy-9-hydroxy-2.4.7.11.13.16-hexaoxa-3k5.1215-
diphosphatricyclo[13.3Ø06,10]octadecane-3,12-dione (ATAC28),
(1R,6R,8R,95,105,15R,17R,185)-18-(1p-1(5)-2-1(5)-2-[6-(2,5-Dioxo-1H-pyrrol-1-
yl)hexanoylamino]-3-
methylbutyrylamino}propionylamino]phenyl}methoxycarbonylamino)-
8,17-bis(2-amino-6-oxo-1,9-dihydropurin-9-y1)-3,12-dihydroxy-9-hydroxy-
2.4.7.11.13.16-
hexaoxa-3k5.1215-diphosphatricyclo[13.3Ø06,10]octadecane-3,12-dione
(ATAC29),
(1R,6R,8R,95,105,15R,17R,185)-18-(1p-1(5)-2-1(5)-2-[6-(2,5-Dioxo-1H-pyrrol-1-
yl)hexanoylamino]-3-methylbutyrylamino } -5-
ureidovalerylamino]phenyl} methoxycarbonylamino)-8,17-bis(2-amino-6-oxo-1,9-
dihydropurin-
9-y1)-9-hydroxy-3,12-dimercapto-2.4.7.11.13.16-hexaoxa-3k5.1215-
diphosphatricyclo[13.3Ø06,10]octadecane-3,12-dione (ATAC30), and 1p-1(5)-2-1
(5)-24642,5-
Dioxo-1H-pyrrol-1-yOhexanoylamino]-3-methylbutyrylamino } -5-
ureidovalerylamino]phenyl } methyl 9-1(15 ,6R,8R,95 ,105 ,15R,17R,185 )-8-(2-
amino-6-oxo- 1,9-
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dihydropurin-9- y1)-3,12-dihydro xy-9,18-dihydro xy-3,12-dio xo -2.4.7 .11.13
.16-hexao xa-
3k5 .1215-dipho sphatric yclo [13 .2.1.06,10]o ctadec-1'7- yl} -9a-adenineec
arbo xylate (ATAC31).
0
0 0c (:)N N NH2 if N XNEI
N H3C
) N / \ N
/(........._
H
0 H 0 CH3
OH lik
ATAC22
Step A: Preparation of ATAC22
c
0 0 NO2ifioo rEN,,). 0 A
Compound 1
0 0 0 ______________________ ..
DIPEA, DMF, r t 0).L0 ) N\_____N
N N
H H
/
0 0 CH3
MC-Val-Ala-PAB-PNP
cfctO i\).Lc) N 401
NH2
N \ N
H H
/
0 0 CH3
OH .
ATAC22
[0455] A solution of compound 1(150 mg, 0.479 mmol) and N,N'-
diisopropylethylamine (145.4
mg, 1.437 mmol) in dry DMF was stirred at room temperature for 5 min.,
followed by addition of
maleimidocaproyl-valine-alanine-p-aminobenzyl alcohol p-nitrophenyl-carbonate
(MC-Val-Ala-
PAB-PNP, 343.6 mg, 0.527 mmol). After stirring for 24 h, volatile organics
were removed under
vacuum. The residue obtained was triturated with dry acetonitrile. The
precipitated solid was
collected by filtration, washed with acetonitrile and dried under vacuum to
obtain unreacted MC-
Val-Ala-PAB-PNP (130 mg) as beige solid. The filtrate and washings were
combined and
concentrated under vacuum. The residue obtained was purified by flash column
chromatography
on silica gel, eluting with step gradients of Me0H in dichloromethane at a
ratio of v/v 1:20, 1:15,
and 1:10, to afford the target product mc-Val-Ala-PAB-GDQ (70 mg, 18% yield)
as beige
colored foamy solid. 1H NMR (DMSO-d6) 8 10.1 ¨ 9.75 (br m, 1H), 8.58 ¨ 8.24
(br m, 1H), 8.15
(d, J=6.6 Hz, 1H), 8.01 (br s, 1H), 7.81 (d, J=8.4 Hz, 1H), 7.71 (d, J=8.4 Hz,
1H), 7.65 ¨ 7.48 (m,
2H), 7.46 ¨7.34 (m, 2H), 7.29 (br s, 1H), 7.18 (br s, 1H), 6.99 (s, 2H), 5.03
(br s, 2H), 4.96 (br s,
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1H), 4.72 (br s, 1H), 4.48 ¨ 4.26 (m, 1H), 4.26 ¨ 4.04 (m, 1H), 2.22 ¨ 2.02
(m, 2H), 2.02 ¨ 1.80
(m, 1H), 1.58 ¨ 1.37 (m, 4H), 1.36 ¨0.92 (br m, 15H), 0.92 ¨0.53 ( br m, 7H).
MS (ESI+) m/z
826 (M+1).
[0456] The following ATAC30, ATAC31, ATAC32, ATAC33, ATAC34, ATAC35, ATAC36,
ATAC37, ATAC38, ATAC39, ATAC40, ATAC41, and ATAC42 can be prepared using a
method similar to that described above for ATAC29.
ATAC23: 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-
methyl-
butanamido)propanamido)benzyl (5-(2-amino-3-pentylquinolin-5-yl)penty1)-
carbamate
0
0
c 0 o oA f
N,LN)criRLAN
H H
0 0 CF-I3 N
H3C
NH2
ATAC23
[0457] 1H NMR (CD30D) 8 8.35 (s, 1H), 7.63 (t, J=8.5Hz, 1H), 7.55 (d, J=8.0Hz,
1H), 7.47 (d,
J=8.0Hz, 1H), 7.33 (d, J=8.0Hz, 1H), 7.27(d, J=8.0Hz, 1H), 6.78 (s, 2H), 5.00
(s, 2H), 4.46 (q,
J=7.0Hz, 2H), 4.13 (d, J=7.0Hz, 1H), 3.47-3.4 (m, 3H), 3.17 (t, J=7.0Hz, 2H),
3.05 (t, J=7.0Hz,
2H), 2.75 (t, J=7.5Hz, 2H), 2.27 (t, J=7.5Hz, 2H), 2.07 (q, J=7.0Hz, 1H), 1.72-
1.51 (m, 10H),
1.46-1.35 (m, 8H), 1.32-1.26 (m, 3H), 1.00-0.92 (m, 9H). LCMS [M+I-1] = 812.4.
ATAC24: 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-
yl)hexanamido)-3-
methylbutan-amido)-5-ureidopentanamido)benzyl-(5-(2-amino-3-pentylquinolin-5-
yl)penty1)-
carbamate
0
0
c---f 0 H 0 la 0 hi
H H
0 0
H3C N
NH NH2
hl2NO
ATAC24
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1H NMR (DMSO-d6) 8 13.5 (bs, 1H), 10.0 (s, 1H), 8.40 (m, 3H), 8.07 (d,
J=7.5Hz, 1H), 7.80 (d,
J=8.5 Hz, 1H), 7.6-7.5 (m, 4H), 7.35-7.25 (m, 2H), 6.01 (m, 1H), 5.42 (s, 1H),
4.89 (s, 2H), 4.41
(q, J=7.0Hz, 1H), 4.18 (t, J=7.0Hz, 1H), 3.10 ¨ 2.90 (m, 6H), 2.75 (t,
J=7.5Hz, 2H), 2.27 (t,
J=7.5Hz, 2H), 2.07 (q, J=7.0Hz, 1H), 1.72-1.51 (m, 10H), 1.46-1.35 (m, 8H),
1.32-1.26 (m, 3H),
1.00-0.92 (m, 9H). LCMS [M+H] = 898.
ATAC25: 4-((S)-2-((S)-2-(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamido)-3-
methylbutanamido)-5-ureidopentanamido)benzyl((4-amino-1-(2-hydroxy-2-
methylpropy1)- 1H-
imidazo[4,5-c]quinolin-2-yl)methyl)(ethyl)carbamate TFA salt
0
0 c NH2 f 0 0 1101 0 )11---N
NkN)c.r N N \ N
H H
0 0
OH
NH
H2NO TFA
ATAC25
1H NMR (DMSO-d6) 8 13.4 (bs, 1H), 9.99 ¨ 9.89 (br m, 1H), 9.09 ¨ 8.40 (m, 3H),
8.07 (d,
J=7.5Hz, 1H), 7.80 (d, J=8.5 Hz, 1H), 7.68 (t, J=8.0 Hz, 1H), 7.59 (bs, 1H),
7.51 (t, J=8.5Hz,
1H), 7.46 ¨ 7.14 (m, 2H), 7.00 (s, 1H), 5.99 (br s, 1H), 5.05 (br s, 1H), 4.95
(br s, 1H), 4.37 (q,
J=7.0Hz, 1H), 4.18 (t, J=7.0Hz, 1H), 3.37 (t, J=7.0Hz, 2H), 3.03 ¨ 2.93 (m,
2H), 2.22 ¨ 2.07 (m,
2H), 1.99 ¨ 1.92 (m, 1H), 1.75 ¨ 1.05 (br m, 20H), 0.85 (d, J=8.5Hz, 3H), 0.81
(d, J=8.5Hz, 3H).
MS (ESI+) m/z 912.5 (M+1).
TABLE 6
Compoun Structure Name
M+1
ATAC26 o
CH 2-(3-124N-Methyl({ p-(S)-2-{(S)-246-(2,5- 921
r'
0 dioxo-1H-pyrrol-1-yl)hexanoylamino]-3-
\H
methylbutyrylamino1-5-
ureidovalerylamino]phenyllmethoxycarbonyl
)amino]ethyllureido)-7-methy1-1,6-dithia-3,8-
diaza-as-indacene
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ATAC27 OCF, 2- R8-Hydroxy-7-quinoly1)(p-
1067
001
- OH trifluoromethoxyphenyl)methyll({ p-
RS)-2-
0
ji,Nxtro.õ 0 NIS 40
(S)-246-(2,5-dioxo-1H-pyrrol-1_
H 0 iF1
yl)hexanoylaminol-3-methylbutyrylaminol-5-
0NH2
ureidovalerylaminolphenyl I methoxycarbonyl
)amino I -1,3-benzothiazole
ATAC28 0
(1R,6R,8R,9S,10S,15R,17R,18S)-18-(lp-(S)- 1289
H0,9 H N NH 2 al
2-
* yl)hexanoylaminol-3-
methylbutyrylaminol-5-
H2NH,Nr 6 0H 'Icr:
0
O
ureidovalerylaminolphenyllmethoxycarbonyl
amino)-8,17-bis(2-amino-6-oxo-1,9-
dihydropurin-9-y1)-3,12-dihydroxy-9-
hydroxy-2.4.7.11.13.16-hexaoxa-35.1215-
diphosphatricyclol13.3Ø06,10loctadecane-
3,12-dione
ATAC29 0 (1R,6R,8R,9S,10S,15R,17R,18S)-18-
(lp4(S)- 1203
H0,2
HOL N N NH2
2- (S)-246-(2,5-Dioxo-1H-pyrrol-l-
4.-P Ic121_?
H CH,j.yfi 0
HN, * yl)hexanoylaminol -3-
H2NHIT,N* 6 0H
0
methylbutyrylaminolpropionylaminolphenyll
methoxycarbonylamino)-8,17-bis(2-amino-6-
oxo-1,9-dihydropurin-9-y1)-3,12-dihydroxy-9-
hydroxy-2.4.7.11.13.16-hexaoxa-35.1215-
diphosphatricyclol13.3Ø06,10loctadecane-
3,12-dione
ATAC30 N,INH (1R,6R,8R,95,105,15R,17R,185)-18-
(lp4(S)- 1321
H2N-.0
HS, P N
H0 N NH2 41
'-0
2- (S)-246-(2,5-Dioxo-1H-pyrrol-l-
--P -ic2L?
NH '
HN, = * yl)hexanoylaminol-3-
methylbutyrylaminol-5-
FoHt* sH
0
ureidovalerylaminolphenyllmethoxycarbonyl
amino)-8,17-bis(2-amino-6-oxo-1,9-
dihydropurin-9-y1)-9-hydroxy-3,12-
dimercapto-2.4.7.11.13.16-hexaoxa-35.1215-
diphosphatricyclol13.3Ø06,10loctadecane-
3,12-dione
ATAC31 .-10 ,Er { p-(S)-2-{ (S)-246-(2,5-Dioxo-1H-
pyrrol-1- 1274
HOo yl)hexanoylaminol -3-
methylbutyrylamino I -5-
HO cr.. Ic2_?
H2NHIO
0 ureidovalerylaminolphenyllmethyl 9-
H2NHTINTr 'OH
Nit
{(1S,6R,8R,9S,105,15R,17R,18S)-8-(2-
0
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amino-6-oxo-1,9-dihydropurin-9-y1)-3,12-
dihydroxy-9,18-dihydroxy-3,12-dioxo-
2.4.7.11.13.16-hexaoxa-35.1215-
diphosphatricyclo[13.2.1.06,10]octadec-17-
y11-9a-adenineecarboxylate
EXAMPLE 11
Synthesis of ATAC32
[0458] This example shows the synthesis of 1-16-1(17-Amino-3-(2-hydroxy-2-
methylpropy1)-
3.5.8-triazatricyclo[7.4Ø02,6}trideca-1(9),2(6),4,7,10,12-hexaen-4-y1}
methyl)-N-ethylamino} -6-
oxohexy1}-1H-pyrrole-2,5-dione (ATAC32).
0
0
IN N H2
0 ) N \
H3C N
OH
ATAC32
Step A: Preparation of ATAC32
0
0 0
0
cifILOH ___________________________________________________________________
cr(/\/\AN\c5,,N N H2
0 N N
0 H3C
/c;,
ATAC32
[0459] To an ice-cold solution of 6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-
yl)hexanoic acid (0.034
g, 0.16 mmol) in DCM (0.800 ml) was added 1-chloro-N,N,2-trimethylprop-1-en-1-
amine (0.021
mL, 0.160 mmol) dropwise. This was stirred at 0 C for lh then added to an ice-
cold mixture of
compound 1(50 mg, 0.160 mmol) and triethylamine (66.7 L, 0.479 mmol) in DCM
(800 L).
Overall molarity 0.1 M. The mixture was stirred to room temperature overnight
and then
chromatographed (DCM to 20% Me0H/DCM) without work-up. Fractions containing
product
were pooled and evaporated then dissolved in 1 mL of acetonitrile and treated
with 0.1 mL of
trifluoroacetic acid. The resulting material was evaporated to an oil then
redissolved in CH3CN
and lyophilized the sample to give ATAC32 (65 mg) as a white solid. 1H NMR
(400 MHz,
(DMSO-d6) 8 13.3 (s, 1H), 8.54 ¨ 8.50 (m, 3H), 7.81 (d, J=8.5 Hz, 1H), 7.76
(d, J=7.5 Hz, 1H),
7.51 (d, J=7.5 Hz, 1H), 6.99 (s, 1H), 6.95 (s, 1H), 3.51 (q, J=7.0 Hz, 2H),
3.43-3.31 (m, 3H),
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2.36-2.30 (m, 2H), 1.54-1.41 (m, 4H), 1.25-1.00 (m, 10H). 19F NMR (DMSO-d6) 8 -
74Ø LCMS
[A4 + H]' = 507.1.
EXAMPLE 12
Synthesis of ATAC33
[0460] This example shows the synthesis of 1-1 [4-(16-R17-Amino-3-(2-hydroxy-2-
methylpropy1)-3.5.8-triazatricyclo[7.4Ø02,6]trideca-1(9),2(6),4,7,10,12-
hexaen-4-y1}methyl)-N-
ethylamino}-6-oxohexylamino }carbonyl)cyclohexyl} methyl} -1H-pyrrole-2,5-
dione (ATAC33).
0
,0
N
0 NTh....õ-N NH2
H3C /c.
OH lik
ATAC33
Step A: Preparation of ATAC33
0
0
_.....N..rH.L
N
0 0
I
0 N _______________ ..-
0. rO compound 1
0
0
.rH).L
N
0 Ni="-N NI-12
H3C /c.......
OH ilk
ATAC33
[0461] To a stirred solution of 1-(4-amino-2-((ethylamino)methyl)-1H-
imidazo[4,5-c]quinolin-1-
y1)-2-methylpropan-2-ol (100 mg, 0.319 mmol) in DCM (10 mL) under nitrogen was
added via a
syringe pump a solution of 2,5-dioxopyrrolidin-1-y16-(44(2,5-dioxo-2,5-dihydro-
1H-pyrrol-1-
y1)methyl)cyclohexane-1-carboxamido)hexanoate (143 mg, 0.319 mmol) in DCM (5
mL) over a
period of 3.5 h. The reaction mixture was stirred at room temperature
overnight. The reaction
mixture was concentrated and the residue was purified by reverse phase column
chromatography.
Pure fractions identified by HPLC analysis were pooled and concentrated. The
residue was
lyophilized from CH3CN to provide a white solid (52.8, mg) as the TFA salt of
ATAC33 as a
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mixture of cis and trans isomers. 1H NMR (400 MHz, (CD30D) 8 8.54 and 8.48 (d,
J=8.3 Hz,
1H), 7.81-7.71 (m, 2H), 7.62-7.55 (m, 1H), 6.80 (s, 2H), 3.66 (q, J=7.0 Hz,
2H), 3.13 and 3.08 (t,
J=7.0 Hz, 2H), 2.45 and 2.38 (t, J=7.5 Hz, 2H), 2.1-2.0 (m, 1H), 1.8-1.47 (m,
10H), 1.46-1.15 (m,
16H), 1.54-1.41 (m, 4H), 1.25-1.00 (m, 10H). LCMS [M + H[ = 646.3.
EXAMPLE 13
Synthesis of ATAC34
[0462] This example shows the synthesis of 1-[(4-1[(17-Amino-3-(2-hydroxy-2-
methylpropy1)-
3.5.8-triazatricyclo [7 .4Ø02,6[tridec a-1(9),2(6),4,7,10,12-hexaen-4- yl}
methyl)-N-ethylaminol -
carbonyl } cyclohexyl)methyll- 1H-pyrrole-2,5-dione (ATAC34).
0 0
LII.7--1(NTh....,-N NH2
) N/ \ N
1---0 H3C /(.......
OH lik
ATAC34
Step A: Preparation of ATAC34
0
0
H3C
ANN NH2
/ \ N
corn 1 \ 5v........
0
0
OH .
ATAC34
[0463] To an ice-cold solution of (1r,40-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-
y1)methyl)cyclohexane-1-carboxylic acid (82 mg, 0.346 mmol) in DCM (1728 1)
was added 1-
chloro-N,N,2-trimethylprop-1-en-1-amine (50.3 L, 0.380 mmol) dropwise. This
was stirred at
0 C for lh then added to an ice-cold mixture of 1-(4-amino-2-
((ethylamino)methyl)-1H-
imidazo[4,5-c[quinolin-1-y1)-2-methylpropan-2-ol (100 mg, 0.319 mmol) and
triethylamine (133
[11, 0.957 mmol) in 1.6 mL of DCM. The mixture became a yellow solution as it
stirred overnight
to room temp. The reaction was concentrated to dryness, redissolved in
Me0H/CH2C12, silica
gel was added, then the solvents evaporated. Chromatography (12 g Gold silica,
DCM to 20%
Me0H/DCM, dry load) gave a solid which was dissolved in CH3CN, frozen and
lyophilized to
afford 170 mg of N-((4-((1-(dimethylamino)-2-methylprop-1-en-l-y1)amino)-1-(2-
((1-
(dimethylamino)-2-methyl-prop-1-en-1-y1)oxy)-2-methylpropyl)-1H-imidazo [4,5-
c] quino lin-2-
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yl)methyl)-44(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-y1)methyl)-N-ethylcyclohexane-
1-
carboxamide which was subsequently dissolved in 50% aqueous MeCN containing
0.1%TFA
and heated in a microwave reactor at 150 C for 60 min. The reaction mixture
was cooled and the
solvents were evaporated and chromatographed to give ATAC34 (72 mg) as a white
solid. 1H
NMR (400 MHz, (DMSO-d6) 8 13.3 (s, 1H), 8.70- 8.50 (m, 3H), 7.83-7.79 (m, 1H),
7.71-7.65
(m, 1H), 7.55-7.48 (m, 1H), 7.00 (s, 1H), 6.98 (s, 1H), 5.13 (bs, 1H), 4.83
(bs, 1H), 3.65 (q, J=7.0
Hz, 2H), 3.38 (m, 1H),3.25 and 3.18 (d, J=6.5 Hz, 2H), 1.69-1.52 (m, 5H), 1.45-
0.88 (m, 13H).
19F NMR (DMSO-d6) 8 -73.7. LCMS [M + H[ = 533.1.
EXAMPLE 14
Fc Receptor Binding to anti-CD40 Antibody Immune-Stimulatory Compound
Conjugates
[0464] An anti-CD40 antibody is comprised of two SBT-040-G1WT heavy chains and
two light
chains from a SBT-040 antibody, which is referred to as a SBT-040-WT antibody.
An anti-CD40
antibody is comprised of two SBT-040-G1VLPLL heavy chains and two light chains
from a
SBT-040 antibody, which is referred to as a SBT-040-VLPLL antibody. An anti-
CD40 antibody
is comprised of two SBT-040-G1DE heavy chains and two light chains from a SBT-
040
antibody, which is referred to as a SBT-040-DE antibody. An anti-CD40 antibody
is comprised
of two SBT-040-G1AAA heavy chains and two light chains from a SBT-040
antibody, which is
referred to as a SBT-040-AAA antibody.
[0465] Each antibody is purified and then each is conjugated to ATAC1, ATAC2,
ATAC3,
ATAC4, ATAC5, ATAC6, ATAC7, ATAC8. ATAC1, ATAC2, ATAC3, ATAC4, ATAC5,
ATAC6, ATAC7, ATAC8, ATAC9, ATAC10, ATAC11, ATAC12, ATAC13, ATAC14,
ATAC15, ATAC16, ATAC17, ATAC18, ATAC19, ATAC20. ATAC21, ATAC22, ATAC23,
ATAC24, ATAC25, ATAC26, ATAC27, ATAC28, ATAC29, ATAC30, ATAC31, ATAC32,
ATAC33, ATAC34 or ATAC43 as described in EXAMPLE 2. Each of these conjugates
is
characterized for the ability of their Fc domains to bind to and for their
affinity for soluble
glycosylated FcyR ectodomains from human FcyRs. This is shown by performing
surface
plasmon resonance experiments. In these experiments, biotinylated soluble
glycosylated FcyR
ectodomains from all human FcyRs are immobilized on a streptavidin-coated
surface. The ability
of each conjugate to bind to soluble glycosylated FcyR ectodomains from all
human FcyRs is
then measured by surface plasmon resonance using a Biacore instrument. The
data from these
experiments shows that the Fc domain of any one of the SBT-040-WT-ATAC1 - SBT-
040-WT-
ATAC34 or SBT-040-ATAC43 conjugates, the Fc domain of any one of the SBT-040-
VLPLL-
ATAC1 - SBT-040-VLPLL-ATAC34 or SBT-040-VLPLL-ATAC43 conjugates, the Fc domain
of any one of the SBT-040-DE-ATAC1 - SBT-040-DE-ATAC34 or SBT-040-DE-ATAC 43
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conjugates, the Fc domain of any one of the SBT-040-AAA-ATAC1 ¨ the SBT-040-
AAA-
ATAC34 or SBT-040-AAA-ATAC43 conjugates is bound to soluble glycosylated FcyR
ectodomains from human FcyRs. Therefore, the surface plasmon resonance
experiments show
that the ability of the Fc domain of the antibody component of the conjugate
to bind to human
FcyRs is not interfered with by the conjugation of the components of the
conjugate. The affinity
of each conjugate for each human FcyRs is also shown by the surface plasmon
resonance
experiments. These affinity measurements are compared with the affinity
measurements for each
antibody alone (as can be shown by EXAMPLE 1). The similarity in affinity of
each antibody
alone for soluble glycosylated FcyR ectodomains from all human FcyRs with the
affinity of each
corresponding conjugate for soluble glycosylated FcyR ectodomains from all
human FcyRs is
shown by this comparison.
EXAMPLE 15
Affinity of anti-CD40 Antibodies to CD40
[0466] An anti-CD40 antibody is comprised of two SBT-040-G1WT heavy chains and
two light
chain from a SBT-040 antibody, which are referred to as a SBT-040-WT antibody.
An anti-CD40
antibody is comprised of two SBT-040-G1VLPLL heavy chains and two light chains
from a
SBT-040 antibody, which are referred to as a SBT-040-VLPLL antibody. An anti-
CD40 antibody
is comprised of two SBT-040-G1DE heavy chains and two light chains from a SBT-
040
antibody, which is referred to as a SBT-040-DE antibody. An anti-CD40 antibody
is comprised
of two SBT-040-G1AAA heavy chains and two light chains from a SBT-040
antibody, which is
referred to as a SBT-040-AAA antibody.
[0467] SBT-040-WT antibody, SBT-040-VLPLL antibody, SBT-040-DE antibody, and
SBT-
040-AAA antibody are each produced by standard methods for producing
antibodies. Each
antibody is purified, and then is characterized for the ability to bind to
CD40. This
characterization is shown by experiments using flow cytometry. For these
experiments, the
human Burkitt's Lymphoma tumor cell lines Raji and Daudi, which are previously
shown to be
CD40-positive, and the human Chronic Myelogenous Leukemia tumor cell line
K562, which is
previously shown to be CD40-negative are first evaluated by flow cytometry to
assess their
relative expression levels of CD40. This is assessed by incubating each cell
line with a
commercially available CD40 antibody conjugated to a fluorochrome, and then
running samples
of the incubation on a flow cytometer. The relative fluorescent intensity
profiles for each cell line
is shown by this data, indicating the level of CD40 expression of each cell
line. The relative
fluorescent intensity profiles of human Burkitt's Lymphoma tumor cell lines
Raji and Daudi
show that CD40 is expressed in each of these cell lines, whereas the relative
fluorescent intensity
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profile of the human Chronic Myelogenous Leukemia tumor cell line K562 show
that CD40 is
not expressed in the cell line. Then, each cell line is separately incubated
with purified SBT-040-
WT antibody, SBT-040-VLPLL antibody, SBT-040G1DE antibody, SBT-040-AAA
antibody or
no antibody as a control. Each incubation is further incubated with a
secondary anti-human IgG1
antibody conjugated with FITC, which is then each assessed by flow cytometry
for the FITC
fluorescent intensity profile of each sample. The ability of each antibody to
detect CD40
expression on the cell lines is indicated by their FITC fluorescent intensity
profile. More
specifically, the similarity between the SBT-040-WT antibody fluorescent
intensity profile and
each antibody with an Fc-enhanced IgG1 isotype after incubation with each of
the cell lines is
shown by this data. Each Fc-enhanced IgG1 isotype is not altered by the
ability of the antibody to
bind to CD40-positive cells is also shown by this data.
EXAMPLE 16
Affinity of anti-CD40 Antibodies to CD40
[0468] An anti-CD40 antibody is comprised of two SBT-040-G1WT heavy chains and
two light
chain from a SBT-040 antibody, which is referred to as a SBT-040-WT antibody.
An anti-CD40
antibody is comprised of two SBT-040-G1VLPLL heavy chains and two light chains
from a
SBT-040 antibody, which is referred to as a SBT-040-VLPLL antibody. An anti-
CD40 antibody
is comprised two SBT-040-G1DE heavy chains and two light chains from a SBT-040
antibody,
which is referred to as a SBT-040-DE antibody. An anti-CD40 antibody is
comprised two SBT-
040-G1AAA heavy chains and two light chains from a SBT-040 antibody, which is
referred to as
a SBT-040-AAA antibody.
[0469] SBT-040-WT antibody, SBT-040-VLPLL antibody, SBT-040-DE antibody, and
SBT-
040-AAA antibody are each produced by standard methods for producing
antibodies. Each
antibody is purified, and each antibody's affinity for CD40 is measured. These
affinities are
measured by experiments using surface plasmon resonance. In these experiments,
biotinylated
recombinant CD40 is immobilized on a streptavidin-coated surface. The ability
of each antibody
to bind to recombinant CD40 is then measured by surface plasmon resonance
using a Biacore
instrument. The data from these experiments shows that SBT-040-WT antibody,
SBT-040-
VLPLL antibody, SBT-040-DE antibody, and SBT-040-AAA antibody are each bound
to
recombinant CD40. Therefore, each antibody's ability to bind to CD40 is not
interfered with by
the enhanced Fc-enhanced IgG1 isotypes is shown by the surface plasmon
resonance data.
[0470] Furthermore, surface plasmon resonance is used to show that CD4OL
binding to CD40 is
not blocked by these antibodies. In these experiments, biotinylated
recombinant CD40 is
immobilized on a streptavidin-coated surface. Surface plasmon resonance using
a Biacore
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instrument is then used to measure the binding affinity of CD4OL in the
presence of each
antibody or without any antibody as a control. The binding affinity of CD4OL
with recombinant
CD40 in presence of each antibody is shown to be the same as the binding
affinity of the CD4OL
with recombinant CD40 in the absence of any antibody. Therefore, CD40 and
CD4OL binding is
unaffected by the presence of SBT-040-WT antibody, SBT-040-G1VLPLL antibody,
SBT-040-
DE antibody, or SBT-040-AAA antibody.
EXAMPLE 17
Fc Receptor Binding to anti-CD40 Antibody Immune-Stimulatory Compound
Conjugates
[0471] An anti-CD40 antibody is comprised of two SBT-040-G1WT heavy chains and
two light
chain from a SBT-040 antibody, which is referred to as a SBT-040-WT antibody.
An anti-CD40
antibody is comprised of two SBT-040-G1VLPLL heavy chains and two light chains
from a
SBT-040 antibody, which is referred to as a SBT-040-VLPLL antibody. An anti-
CD40 antibody
is comprised of two SBT-040-G1DE heavy chains and two light chains from a SBT-
040
antibody, which is referred to as a SBT-040-DE antibody. An anti-CD40 antibody
is comprised
of two SBT-040-G1AAA heavy chains and two light chains from a SBT-040
antibody, which is
referred to as a SBT-040-AAA antibody.
[0472] SBT-040-WT antibody, SBT-040-VLPLL antibody, SBT-040-DE antibody, and
SBT-
040-AAA antibody are each made following standard methods for antibody
production. Each
antibody is purified and then each is conjugated to ATAC1, ATAC2, ATAC3,
ATAC4, ATAC5,
ATAC6, ATAC7, or ATAC8. ATAC1, ATAC2, ATAC3, ATAC4, ATAC5, ATAC6, ATAC7,
ATAC8, ATAC9, ATAC10, ATAC11, ATAC12, ATAC13, ATAC14, ATAC15, ATAC16,
ATAC17, ATAC18, ATAC19, ATAC20. ATAC21, ATAC22, ATAC23, ATAC24, ATAC25,
ATAC26, ATAC27, ATAC28, ATAC29, ATAC30, ATAC31, ATAC32, ATAC33, ATAC34 or
ATAC 43 are as described in EXAMPLE 2. The affinity of each conjugate for CD40
is then
measured by experiments using surface plasmon resonance. In these experiments,
biotinylated
recombinant CD40 is immobilized on a streptavidin-coated surface. The ability
of each conjugate
to bind to recombinant CD40 is then measured by surface plasmon resonance
using a Biacore
instrument. The data from these experiments shows that any one of the SBT-040-
WT-ATAC1 -
SBT-040-WT-ATAC34 or SBT-040-WT-ATAC43 conjugates, any one of the SBT-040-
VLPLL-
ATAC1 - SBT-040-VLPLL-ATAC34 or SBT-040-VLPLL-ATAC43 conjugates, any one of
the
SBT-040-DE-ATAC1 - SBT-040-DE-ATAC34 or SBT-040-DE-ATAC43 conjugates, or any
one of the SBT-040-AAA-ATAC1 - the SBT-040-AAA-ATAC34 or SBT-040-AAA-ATAC43
conjugates is bound to recombinant CD40. Therefore, the surface plasmon
resonance
experiments show that each component antibody's ability to bind to CD40 is not
interfered with
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by the enhanced Fc-enhanced IgG1 isotypes nor the antibody conjugation to
ATAC1, ATAC2,
ATAC3, ATAC4, ATAC5, ATAC6, ATAC7, or ATAC8. ATAC1, ATAC2, ATAC3, ATAC4,
ATAC5, ATAC6, ATAC7, ATAC8, ATAC9, ATAC10, ATAC11, ATAC12, ATAC13,
ATAC14, ATAC15, ATAC16, ATAC17, ATAC18, ATAC19, ATAC20. ATAC21, ATAC22,
ATAC23, ATAC24, ATAC25, ATAC26, ATAC27, ATAC28, ATAC29, ATAC30, ATAC31,
ATAC32, ATAC33, ATAC34 or ATAC43.
[0473] Furthermore, surface plasmon resonance is used to show that CD4OL
binding to CD40 is
not blocked in the presence of each conjugate. In these experiments,
biotinylated recombinant
CD40 is immobilized on a streptavidin-coated surface. Surface plasmon
resonance using a
Biacore instrument is then used to measure the binding affinity of CD4OL in
the presence of each
conjugate or without any conjugate as a control. The binding affinity of CD4OL
with
recombinant CD40 in presence of each conjugate is shown to be the same as the
binding affinity
of the CD4OL with recombinant CD40 in the absence of any conjugate by these
experiments.
Therefore, CD40 and CD4OL binding is unaffected by the presence of any one of
the SBT-040-
WT-ATAC1 - SBT-040-WT-ATAC34 or SBT-040-WT-ATAC43 conjugates, any one of the
SBT-040-VLPLL-ATAC1 - SBT-040-VLPLL-ATAC34 or SBT-040-VLPLL-ATAC43
conjugates, any one of the SBT-040-DE-ATAC1 - SBT-040-DE-ATAC34 or SBT-040-DE-
ATAC43conjugates, or any one of the SBT-040-AAA-ATAC1 - the SBT-040-AAA-ATAC34
or
SBT-040-AAA-ATAC43 conjugates.
EXAMPLE 18
Cytokine Production is Enhanced by anti-CD40 Antibody Immune-Stimulatory
Compound
Conjugates
[0474] This example shows that cytokine production by dendritic cells is
enhanced after
administration of antibody-immune stimulatory compound conjugates in culture.
In this
experiment, dendritic cells (DCs) are derived from peripheral blood
mononuclear cells (PBMCs).
DCs are obtained by putting human PBMCs into a culture dish. The resulting
adherent cells are
washed with RPMI containing 10% fetal calf serum, and then are incubated for 7
days in
complete medium containing 10 ng/mL IL-4 and 100 ng/mL GM-CSF. The non-
adherent cells
are isolated and are washed. These isolated cells are run by a flow cytometer
to ensure CD11c
expression, in which the DCs identity as DCs is confirmed by CD11c expression.
The DCs are
then incubated with either the antibodies as described in Example 1 or the
conjugates as
described in Example 3. More specifically, the DCs are incubated with any one
of SBT-040-WT
antibody, SBT-040-VLPLL antibody, SBT-040-DE antibody, SBT-040-AAA antibody,
the SBT-
040-WT-ATAC1 conjugate - SBT-040-WT-ATAC34 or SBT-040-WT-ATAC43 conjugate, the
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SBT-040-VLPLL-ATAC1 conjugate ¨ SBT-040-VLPLL-ATAC34 or SBT-040-VLPLL-
ATAC43conjugate, the SBT-040-DE-ATAC1 conjugate ¨ SBT-040-DE-ATAC34 or SBT-040-
DE-ATAC43 conjugate, the SBT-040-AAA-ATAC1 conjugate ¨ the SBT-040-AAA-ATAC34
or SBT-040-AAA-ATAC43 conjugate, or a non-binding isotype control antibody.
Each culture is
then incubated for 24 hours and the supernatant of each culture is analyzed
using a cytokine bead
array assay. Cytokine expression levels of IFNy, IL-8, IL-12 and IL-2 are
measured by the
cytokine bead array assay. The supernatant from the culture containing the non-
binding isotype
control shows the level of cytokine expression is decreased as compared to the
supernatant from
cultures containing SBT-040-WT, SBT-040-VLPLL, SBT-040-DE, or SBT-040-AAA.
Additionally, the level of cytokine expression in the supernatant from
cultures containing SBT-
040-WT, SBT-040-VLPLL, SBT-040-DE, or SBT-040-AAA is decreased as compared to
the
supernatant from cultures containing any one of the SBT-040-WT-ATAC1 conjugate
¨ SBT-
040-WT-ATAC34 or SBT-040-WT-ATAC43 conjugate, any one of the SBT-040-VLPLL-
ATAC1 conjugate ¨ SBT-040-VLPLL-ATAC34 or SBT-040-VLPLL ATAC43 conjugate, any
one of the SBT-040-DE-ATAC1 conjugate ¨SBT-040-DE-ATAC34 or SBT-040-DE-ATAC43
conjugate, or any one of the SBT-040-AAA-ATAC1 conjugate ¨SBT-040-AAA-ATAC34
or
SBT-040-AAA-ATAC43 conjugate.
EXAMPLE 19
Cytokine Production by Dendritic Cells from Multiple Donors Was Enhanced by
anti-
CD40 Antibody Immune-Stimulatory Compound Conjugates
[0475] Antibody-immune stimulatory compound conjugates enhanced
immunostimulatory
cytokines produced by human dendritic cells in a concentration dependent
manner when added to
and incubated with the cells. The human dendritic cells (DCs) from two donors
were derived
from CD14+ monocytes isolated from peripheral blood mononuclear cells (PBMCs)
by negative
selection using a commercially available kit. The monocytes were cultured in
RPMI containing
10% fetal calf serum for seven days in complete medium supplemented with
25ng/mL IL-4 and
lOng/mL GM-CSF. The media was replaced with fresh media plus cytokines on day
three. On
day six anti-CD40 antibody immune-stimulatory compound conjugates SBT-040-G1-
ATAC23
and SBT-040-G1-ATAC17 and control antibody were added to individual wells
containing the
dendritic cells. After 24 hours of further incubation, the supernatants were
collected and the
cytokines IL-6, TNFa, IL-12p70 and IL-10 produced by the dendritic cells were
quantitated by
electrochemiluminesence signal by multiplex ELISA using commercially available
reagents and
plate reader from Meso Scale Discovery. Results are shown for the immune
stimulatory
cytokines IL-12p70 and TNFa for dendritic cells derived from two donors.
FIGURE 31A shows
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the concentration of IL-12p70 produced by DCs from donor 358 after incubation
with SBT-040-
G1-ATAC23 or SBT-040-G1-ATAC17 as compared with SBT-050-WT. FIGURE 31B shows
the concentration of IL-12p70 produced by DCs from donor 363 after incubation
with SBT-040-
G1-ATAC23or SBT-040-G1-ATAC17 as compared with SBT-050-WT. FIGURE 31C shows
the concentration of TNFa produced by DCs from donor 358 after incubation with
SBT-040-G1-
ATAC23 or SBT-040-G1-ATAC17 as compared with an anti-HER2 antibody. FIGURE 31D
shows the concentration of TNFa produced by DCs from donor 363 after
incubation with SBT-
040-G1-ATAC23 or SBT-040-G1-ATAC17 as compared with an anti-HER2 antibody.
EXAMPLE 20
Immunostimulatory Cytokine Secretion Is Enhanced By Anti-CD40 Antibody Immune-
Stimulatory Compound Conjugates with Different Linkers and FcyR Binding
[0476] Antibody-immune stimulatory compound conjugates enhanced human
dendritic cells
cytokine production in a concentration dependent manner when added to and
incubated with the
cells. The human dendritic cells (DCs) were derived from CD14+ monocytes
isolated from
peripheral blood mononuclear cells (PBMCs) by negative selection using a
commercially
available kit. The monocytes were cultured in RPMI containing 10% fetal calf
serum for seven
days in complete medium supplemented with 25ng/mL IL-4 and lOng/mL GM-CSF. On
day six
anti-CD40 antibody immune-stimulatory compound conjugates and non-DC binding
control
antibody SBT-50 G1 and commercially available soluble CD4OL were added to
individual wells
containing the dendritic cells. More specifically, the DCs are incubated with
any one of SBT-
040-WT-ATAC4, SBT-040-WT-ATAC3, SBT-040-G2-ATAC4, SBT-040-G2-ATAC3, SBT-
040-AAA-ATAC29, SBT-040-VLPLL-ATAC29, SBT-040-WT-ATAC1, SBT-040-G2-ATAC1,
SBT-040-WT-ATAC12, SBT-040-G2-ATAC12, SBT-040-WT-ATAC30, SBT-040-AAA-
ATAC11, SBT-040-VLPLL-ATAC11, SBT-040-VLPLL-ATAC12, SBT-040-AAA-ATAC12,
SBT-040-VLPLL-ATAC30, and SBT-040-AAA-ATAC30. After 24 hours further
incubation
supernatants were collected and the cytokines IL-6, TNFa, IL-12p70 and IL-10
produced by the
dendritic cells were quantitated by electrochemiluminesence signal by
multiplex ELISA using
commercially available reagents and plate reader from Meso Scale Discovery.
FIGURE 32A
shows the concentration of IL-12p70 produced by DCs after incubation with SBT-
040-WT-
ATAC4, SBT-040-WT-ATAC3, SBT-040-G2-ATAC4, SBT-040-G2-ATAC3, SBT-040-AAA-
ATAC29, SBT-040-VLPLL-ATAC29, SBT-040-WT-ATAC1, SBT-040-G2-ATAC1, SBT-040-
WT-ATAC12, SBT-040-G2-ATAC12, SBT-040-WT-ATAC30, SBT-040-AAA-ATAC11, SBT-
040-VLPLL-ATAC11, SBT-040-VLPLL-ATAC12, SBT-040-AAA-ATAC12, SBT-040-
VLPLL-ATAC30, and SBT-040-AAA-ATAC30 as compared with SBT-050-G2 antibody or
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CD40 ligand. FIGURE 32B shows the concentration of IL-6 produced by DCs from
donor 2
after incubation with SBT-040-WT-ATAC4, SBT-040-WT-ATAC3, SBT-040-G2-ATAC4,
SBT-040-G2-ATAC3, SBT-040-AAA-ATAC29, SBT-040-VLPLL-ATAC29, SBT-040-WT-
ATAC1, SBT-040-G2-ATAC1, SBT-040-WT-ATAC12, SBT-040-G2-ATAC12, SBT-040-WT-
ATAC30, SBT-040-AAA-ATAC11, SBT-040-VLPLL-ATAC11, SBT-040-VLPLL-ATAC12,
SBT-040-AAA-ATAC12, SBT-040-VLPLL-ATAC30, and SBT-040-AAA-ATAC30 as
compared with SBT-050-G2 or CD40 ligand. Results are shown for the immune
stimulatory
cytokines IL-12p70 and IL-6. The treatment concentrations for each molecule,
depicted on the x-
axis from right to left, were 0.08 ug/mL, 0.310 ug/mL, 1.25 ug/mL and 5.00
ug/mL.
EXAMPLE 21
Anti-CD40 Antibody Immune-Stimulatory Compound Conjugates Increased Cell
Surface
Expression of Immune Activating Proteins
[0477] Human dendritic cells showed increased expression of CD83, CD86, and
MHC class II
cell surface proteins in after treatment with anti-CD40 antibody immune-
stimulatory compound
conjugates. The increased expression of these surface proteins was dose
dependent.
[0478] Human dendritic cells were derived from human PBMCs by isolation of
CD14+
monocytes followed by culture in RPMI containing 10% fetal calf serum for
seven days in
complete medium supplemented with lOng/mL IL-4 and 10Ong/mL GM-CSF. After
three days of
culture the media was removed and replaced with fresh media including cytokine
supplement. On
day six SBT-040-WT-ATAC30, SBT-040-WT-ATAC24, SBT-040-VLPLL-ATAC30, SBT-040-
AAA-ATAC30 or a control SBT-050-WT were added to separate aliquots of
dendritic cells.
After an additional 24 hour incubation the cells were collected and washed by
centrifugations
then stained for 30 minutes on ice using manufacturer's recommended
concentrations of
commercially available anti-CD83, anti-CD86 and anti-MHC class II monoclonal
antibodies
conjugated to laser sensitive fluors. A separate aliquot for each treatment
was stained with IgG
matched isotype control antibody conjugate for the anti-CD86 antibody, anti-
CD83 antibody, and
anti-MHC Class II antibody. After washing to remove unbound antibody-fluor
molecules, the
stained cells were subjected to FACS analysis using a Celesta flow cytometer
(BD Biosciences)
with gating on live cells. The output was analyzed by FlowJo v10.2 software
(FlowJo LLC) and
curve fit with Prism 7.01 software (GraphPad Software, Inc.). FIGURE 33A shows
a dose
dependent increase in CD86 expression on dendritic cells after treatment with
SBT-040-WT-
ATAC30, SBT-040-WT-ATAC24, SBT-040-VLPLL-ATAC30, SBT-040-AAA-ATAC30 as
compared to treatment a control SBT-050-WT or staining with an isotype
control. FIGURE 33B
shows a dose dependent increase in CD83 expression on dendritic cells after
treatment SBT-040-
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WT-ATAC30, SBT-040-WT-ATAC24, SBT-040-VLPLL-ATAC30, SBT-040-AAA-ATAC30
as compared to treatment a control SBT-050-WT or staining with an isotype
control. FIGURE
33C shows a dose dependent increase in MHC class II expression on dendritic
cells after
treatment with SBT-040-WT-ATAC30, SBT-040-WT-ATAC24, SBT-040-VLPLL-ATAC30,
SBT-040-AAA-ATAC30 as compared to treatment a control SBT-050-WT or staining
with an
isotype control. The graph shows plots of treatment protein concentration on
the x axis versus
mean fluorescence intensity for the cell surface protein on the y axis.
EXAMPLE 22
Treatment of Cancer By Administering a Conjugate
[0479] This example describes treatment of cancer with a conjugate. A human
patient is
diagnosed with a cancer. A conjugate as shown in the schematic of FIGURE 8 is
administered to
the patient with a pharmaceutically acceptable carrier. FIGURE 8 is a
conjugate comprising an
antibody construct and an immune stimulatory compound. The antibody construct
is an antibody,
which contains two heavy chains as shown in gray and two light chains as shown
in light gray.
The antibody comprises two antigen binding sites (810 and 815), and a portion
of the heavy
chains contain Fc domains (805 and 820). The immune-stimulatory compounds (830
and 840)
are conjugated to the antibody by linkers (860 and 870).
[0480] As another example, a human patient is diagnosed with a cancer. A
conjugate as shown in
the schematic of FIGURE 9 is administered to the patient with a
pharmaceutically acceptable
carrier. FIGURE 9 is a conjugate comprising an antibody construct, two
targeting binding
domains, and two immune stimulatory compounds. The antibody construct is an
antibody, which
contains two heavy chains as shown in gray and two light chains as shown in
light gray. The
antibody comprises two antigen binding sites (910 and 915), and a portion of
the heavy chains
contain Fc domains (905 and 920). The immune-stimulatory compounds (930 and
940) are
conjugated to the antibody by linkers (960 and 970). The targeting binding
domains are
conjugated to the antibody (980 and 985).
[0481] As an additional example, a human patient is diagnosed with a cancer. A
conjugate as
shown in the schematic of FIGURE 10 is administered to the patient with a
pharmaceutically
acceptable carrier. FIGURE 10 is a conjugate comprising an antibody construct
and two immune
stimulatory compounds. The antibody construct contains the Fc region of an
antibody with the
heavy chains shown in gray, and two scaffolds as shown in light gray. The
antibody construct
comprises two antigen binding sites (1010 and 1015) in the scaffolds, and a
portion of the heavy
chains contain Fc domains (1005 and 1020). The immune-stimulatory compounds
(1030 and
1040) are conjugated to the antibody construct by linkers (1060 and 1070).
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[0482] As another example, a human patient is diagnosed with a cancer. A
conjugate as shown in
the schematic of FIGURE 11 is administered to the patient with a
pharmaceutically acceptable
carrier. FIGURE 11 is a conjugate comprising an antibody construct, two
targeting domains, and
two immune stimulatory compounds. The antibody construct contains the Fc
region of an
antibody with the heavy chains shown in gray, and two scaffolds as shown in
light gray. The
antibody construct comprises two antigen binding sites (1110 and 1115) in the
scaffolds, and a
portion of the heavy chains contain Fc domains (1105 and 1120). The immune-
stimulatory
compounds (1130 and 1140) are conjugated to the antibody construct by linkers
(1160 and 1170).
The targeting binding domains are conjugated to the antibody construct (1180
and 1185).
[0483] As another example, a human patient is diagnosed with a cancer. A
conjugate as shown in
the schematic of FIGURE 12 is administered to the patient with a
pharmaceutically acceptable
carrier. FIGURE 12 is a conjugate comprising an antibody construct and two
immune
stimulatory compounds. The antibody construct contains the F(ab')2region of an
antibody with
heavy chains shown in gray and light chains shown in light gray, and two
scaffolds as shown in
dark gray. The antibody construct comprises two antigen binding sites (1210
and 1215), and a
portion of two scaffolds contain Fc domains (1220 and 1245). The immune-
stimulatory
compounds (1230 and 1240) are conjugated to the antibody construct by linkers
(1260 and 1270).
[0484] As another example, a human patient is diagnosed with a cancer. A
conjugate as shown in
the schematic of FIGURE 13 is administered to the patient with a
pharmaceutically acceptable
carrier. FIGURE 13 is a conjugate comprising an antibody construct, two
targeting binding
domains, and two immune stimulatory compounds. The antibody construct contains
the F(ab')2
region of an antibody with heavy chains shown in gray and light chains shown
in light gray, and
two scaffolds as shown in dark gray. The antibody construct comprises two
antigen binding sites
(1310 and 1315), and a portion of two scaffolds contain Fc domains (1320 and
1345). The
immune-stimulatory compounds (1330 and 1340) are conjugated to the antibody
construct by
linkers (1360 and 1370). The targeting binding domains are conjugated to the
antibody construct
(1380 and 1385).
[0485] As another example, a human patient is diagnosed with a cancer. A
conjugate as shown in
the schematic of FIGURE 14 is administered to the patient with a
pharmaceutically acceptable
carrier. FIGURE 14 is a conjugate comprising an antibody construct, and two
immune
stimulatory compounds. The antibody construct contains two scaffolds as shown
in light gray and
two scaffolds as shown in dark gray. The antibody construct comprises two
antigen binding sites
(1410 and 1415), and a portion of the two dark gray scaffolds contain Fc
domains (1420 and
1445). The immune-stimulatory compounds (1430 and 1440) are conjugated to the
antibody
construct by linkers (1460 and 1470).
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[0486] As another example, a human patient is diagnosed with a cancer. A
conjugate as shown in
the schematic of FIGURE 15 is administered to the patient with a
pharmaceutically acceptable
carrier. FIGURE 15 is a conjugate comprising an antibody construct, two
targeting binding
domains, and two immune stimulatory compounds. The antibody construct contains
two
scaffolds as shown in light gray and two scaffolds as shown in dark gray. The
antibody construct
comprises two antigen binding sites (1510 and 1515), and a portion of the two
dark gray
scaffolds contain Fc domains (1520 and 1545). The immune-stimulatory compounds
(1530 and
1540) are conjugated to the antibody construct by linkers (1560 and 1570). The
targeting binding
domains are conjugated to the antibody construct (1580 and 1585).
EXAMPLE 23
Determination of Ka Values
[0487] Kd is measured by a radiolabeled antigen binding assay (RIA) performed
with the Fab
version of an antibody of interest and its antigen as described by the
following assay.
[0488] Solution binding affinity of Fabs for antigen is measured by
equilibrating the Fab with a
minimal concentration of (125I)-labeled antigen in the presence of a titration
series of unlabeled
antigen, then capturing bound antigen with an anti-Fab antibody-coated plate
(See, e.g., Chen et
al., J. Mol. Biol. 293:865-881 (1999)). To establish conditions for the assay,
multi-well plates are
coated overnight with 5 g/mL of a capturing anti-Fab antibody (Cappel Labs)
in 50 mM sodium
carbonate (pH 9.6), and subsequently blocked with 2% (w/v) bovine serum
albumin in PBS for
two to five hours at room temperature (approximately 23 C). In a non-
adsorbent plate (Nunc
#269620), 100 pM or 26 pM [1251]-antigen are mixed with serial dilutions of a
Fab of interest
(e.g., consistent with assessment of the anti-VEGF antibody, Fab-12, in Presta
et al., Cancer Res.
57:4593-4599 (1997)). The Fab of interest is then incubated overnight;
however, the incubation
may continue for a longer period (e.g., about 65 hours) to ensure that
equilibrium is reached.
Thereafter, the mixtures are transferred to the capture plate for incubation
at room temperature
(e.g., for one hour). The solution is then removed and the plate washed eight
times with 0.1%
polysorbate 20 (TWEEN-20 ) in PBS. When the plates have dried, 150 l/well of
scintillant is
added, and the plates are counted on a TOPCOUNTTm gamma counter (Packard) for
ten minutes.
Concentrations of each Fab that give less than or equal to 20% of maximal
binding are chosen for
use in competitive binding assays.
EXAMPLE 24
Determination of Ka Values
[0489] Kd is measured using surface plasmon resonance assays using a BIACOREC)-
2000 or a
BIACOREC)-3000 (BIAcore, Inc., Piscataway, N.J.) at 25 C with immobilized
antigen CM5
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chips at -10 response units (RU). Briefly, carboxymethylated dextran biosensor
chips (CM5,
BIACORE, Inc.) are activated with N-ethyl-N'-(3-dimethylaminopropy1)-
carbodiimide
hydrochloride (EDC) and N-hydroxysuccinimide (NHS) according to the supplier's
instructions.
Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 pg/mL (0.2 M)
before injection at
a flow rate of 5 L/minute to achieve approximately 10 response units (RU) of
coupled protein.
Following the injection of antigen, 1 M ethanolamine is injected to block
unreacted groups. For
kinetics measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM)
are injected in PBS
with 0.05% polysorbate 20 (TWEEN-20Tm) surfactant (PBST) at 25 C at a flow
rate of
approximately 25 L/min. Association rates (lc.) and dissociation rates (kat-)
are calculated using
a simple one-to-one Langmuir binding model (BIACORE Evaluation Software
version 3.2) by
simultaneously fitting the association and dissociation sensorgrams. The
equilibrium dissociation
constant (Kd) is calculated as the ratio koftlkon. See, e.g., Chen et al., J.
Mol. Biol. 293:865-881
(1999). If the on-rate exceeds 106 M-1 s-1 by the surface plasmon resonance
assay above, then
the on-rate can be determined by using a fluorescent quenching technique that
measures the
increase or decrease in fluorescence emission intensity (excitation=295 nm;
emission=340 nm,
16 nm band-pass) at 25 C of a 20 nM anti-antigen antibody (Fab form) in PBS,
pH 7.2, in the
presence of increasing concentrations of antigen as measured in a
spectrometer, such as a stop-
flow equipped spectrophometer (Aviv Instruments) or a 8000-series SLM-AMINCOTm
spectrophotometer (ThermoSpectronic) with a stirred cuvette.
EXAMPLE 25
Lysine-based Bioconjugation
[0296] The antibody construct is exchanged into an appropriate buffer, for
example, phosphate,
borate, PBS, Tris-Acetate at a concentration of about 2 mg/mL to about 10
mg/mL. An
appropriate number of equivalents of the immune stimulatory compound-linker
construct (ATAC)
were added as a solution with stirring. Dependent on the physical properties
of the immune
stimulatory compound-linker construct, a co-solvent was introduced prior to
the addition of the
immune stimulatory compound-linker construct to facilitate solubility. The
reaction was stirred at
room temperature for 2 hours to about 12 hours depending on the observed
reactivity. The
progression of the reaction was monitored by LC-MS. Once the reaction was
deemed complete,
the remaining immune stimulatory compound-linker constructs were removed by
applicable
methods and the antibody construct-immune stimulatory compound conjugate was
exchanged
into the desired formulation buffer. Lysine-linked conjugates were synthesized
starting with 10
mg of antibody (mAb) and 10 equivalents of ATAC1, ATAC2, ATAC3, ATAC4, ATAC5,
ATAC6, ATAC7, ATAC8, ATAC9, or ATAC10 using the conditions described in Scheme
34
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below (ADC = antibody immune-stimulatory compound conjugate). Monomer content
and
immune-stimulatory compound-antibody construct ratios (molar ratios) were
determined by
methods described in EXAMPLES 27 ¨ 31.
Scheme 34:
equivs of ATAC
sodium phoshate
mAb ADC
pH = 8
20% v/v DMSO
rkOH
0 0
/ =
¨N
H2N
0 0
mAbN
110
I N
NH2
TABLE 7
Scaffold Name Isolated % Monomer DAR
ADC
SBT-040-WT-ATAC4 9.7 mg > 95% 4.6
SBT-040-G2-ATAC4 12.3 mg > 95% 2.9
ii SBT-040-WT-ATAC3 7.8 mg > 95% 5.4
ii SBT-040-G2-ATAC3 8.9 mg > 95% 3.3
ii SBT-040-WT-ATAC1 7.2 mg > 99% 1.9
ii SBT-040-G2-ATAC1 7.1 mg 99% 2.1
EXAMPLE 26
Cysteine-based Bioconjugation
[0297] The antibody construct was exchanged into an appropriate buffer, for
example, phosphate,
borate, PBS, Tris-Acetate at a concentration of about 2 mg/mL to about 10
mg/mL with an
appropriate number of equivalents of a reducing agent, for example,
dithiothreitol or tris(2-
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carboxyethyl)phosphine. The resultant solution was stirred for an appropriate
amount of time and
temperature to effect the desired reduction. The immune stimulatory compound-
linker construct
was added as a solution with stirring. Dependent on the physical properties of
the immune
stimulatory compound-linker construct, a co-solvent was introduced prior to
the addition of the
immune stimulatory compound-linker construct to facilitate solubility. The
reaction was stirred at
room temperature for about 1 hour to about 12 hours depending on the observed
reactivity. The
progression of the reaction was monitored by liquid chromatography-mass
spectrometry (LC-
MS). Once the reaction was deemed complete, the remaining free immune
stimulatory
compound-linker construct was removed by applicable methods and the antibody
construct-
immune stimulatory compound conjugate was exchanged into the desired
formulation buffer.
Such cysteine-based conjugates were synthesized starting with 10 mg of
antibody (mAb) and 7
equivalents of ATAC11¨ATAC45 using the conditions described in Scheme 35 below
(ADC =
antibody immune-stimulatory compound conjugate). Monomer content and drug-
antibody ratios
can be determined by methods described in EXAMPLES 27 ¨ 31.
Scheme 35:
1. reducing agent
mAb ____________________________________________ ,..- ADC
2. 7 equivs of ATAC
sodium phoshate
pH = 8
20% v/v DMSO
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0 0 0 rkOH
NI.,.,KN,.,000AN,frN
0 H Ni / =
mAb¨S ¨N
I H2N
0 rkOH
mAb-S
____IfkA0
0 0 0 OANIN
*
C N /
LN - N
0 H i H
H2N
Fi
0 0 0
0 H H
0
mAb-S ill I
A\I
NH2
mAb-S 0
v\IA0 .rFA
0 0 0 OAN
0
H
N , N
I
0 H = H
iv NH2
TABLE 8
Scaffold Name Isolated ADC % Monomer DAR
i SBT-040-WT-ATAC11 9.0 mg >95% 4.5
i SBT-040-G2-ATAC11 8.9 mg 94% 4.0
ii SBT-040-WT-ATAC22 9.0 mg 93% 4.6
ii SBT-040-G2-ATAC22 9.0 mg > 95% 4.1
ii SBT-040-AAA-ATAC22 8.5 mg 98% 4.1
ii SBT-040-VLPLL-ATAC22 8.5 mg 94%
3.9
iii SBT-040-WT-ATAC12 11.7 mg >95% 4.0
iii SBT-040-G2-ATAC12 11.4 mg 96% 4.1
iv SBT-040-WT-ATAC23 11.5 mg 93% 4.7
i SBT-040-AAA-ATAC11 10.2 mg >95% 2.9
i SBT-040-VLPLL-ATAC11 9.4 mg 96%
2.9
iv SBT-040-AAA-ATAC23 7.1 mg > 95% 1.9
iv SBT-040-G1(VLPLL)-ATAC23 7.7 mg > 95% 2.6
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EXAMPLE 27
Determination of Molar Ratio
[0298] This example illustrates one method by which the molar ratio is
determined. One
microgram of antibody construct immune-stimulatory compound conjugate is
injected into an
LC/MS such as an Agilent 6550 iFunnel Q-TOF equipped with an Agilent Dual Jet
Stream ESI
source coupled with Agilent 1290 Infinity UHPLC system. Raw data is obtained
and is
deconvoluted with software such as Agilent MassHunter Qualitative Analysis
Software with
BioConfirm using the Maximum Entropy deconvolution algorithm. The average mass
of intact
antibody construct immune-stimulatory compound conjugates is calculated by the
software,
which can use top peak height at 25% for the calculation. This data is then
imported into another
program to calculate the molar ratio of the antibody construct immune-
stimulatory compound
conjugate such as Agilent molar ratio calculator.
EXAMPLE 28
Determination of molar ratio for SBT-040-G1WT conjugated to a Cys-targeted
compound
[0490] FIGURE 28 shows HPLC analysis of SBT-040-G1WT conjugated to a Cys-
targeted drug
linker tool compound. First, 10 L of a 5 mg/mL solution of the antibody-drug
conjugate was
injected into an HPLC system set-up with a TOSOH TSKgel Butyl-NPR TM
hydrophobic
interaction chromatography (HIC) column (2.5 [tM particle size, 4.6 mm x 35
mm) attached.
Then, over the course of 18 minutes, a method was run in which the mobile
phase gradient ran
from 100% mobile phase A to 100% mobile phase B over the course of 12 minutes,
followed by
a six minute re-equilibration at 100% mobile phase A. The flow rate was 0.8
mL/min and the
detector was set at 280 nM. Mobile phase A was 1.5 M ammonium sulfate, 25 mM
sodium
phosphate (pH 7). Mobile phase B was 25% isopropanol in 25 mM sodium phosphate
(pH 7).
Post-run, the chromatogram was integrated and the molar ratio was determined
by summing the
weighted peak area. The molar ratio was calculated to be about 4.56 with 7%
unconjugated
antibody.
EXAMPLE 29
Determination of Molar Ratio for SBT-040-G1WT Conjugated to ATAC11
[0491] FIGURE 29 shows HPLC analysis of SBT-040-G1WT conjugated to ATAC11,
which is
a cleavable Maleimide-Val-Ala-PABA-Gardiquimod linker. First, 10 L of a 5
mg/mL solution
of the antibody immune-stimulatory compound conjugate was injected into an
HPLC system set-
up with a TOSOH TSKgel Butyl-NPR TM hydrophobic interaction chromatography
(HIC)
column (2.5 [tM particle size, 4.6 mm x 35 mm) attached. Then, over the course
of 18 minutes, a
method was run in which the mobile phase gradient ran from 100% mobile phase A
to 100%
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mobile phase B over the course of 12 minutes, followed by a six minute re-
equilibration at 100%
mobile phase A. The flow rate was 0.8 mL/min and the detector was set at 280
nM. Mobile phase
A was 1.5 M ammonium sulfate, 25 mM sodium phosphate (pH 7). Mobile phase B
was 25%
isopropanol in 25 mM sodium phosphate (pH 7). Post-run, the chromatogram was
integrated and
the molar ratio was determined by summing the weighted peak area. The molar
ratio was
calculated to be about 4.5.
EXAMPLE 30
Determination of Molar Ratio for SBT-040-G2WT Conjugated to ATAC11
[0492] FIGURE 30 shows HPLC analysis of SBT-040-G2WT conjugated to ATAC11,
which is
a cleavable Maleimide-Val-Ala-PABA-Gardiquimod linker. First, 10 L of a 5
mg/mL solution
of the antibody-immune stimulatory compound conjugate was injected into an
HPLC system set-
up with a TOSOH TSKgel Butyl-NPR TM hydrophobic interaction chromatography
(HIC)
column (2.5 [tM particle size, 4.6 mm x 35 mm) attached. Then, over the course
of 18 minutes, a
method was run in which the mobile phase gradient ran from 100% mobile phase A
to 100%
mobile phase B over the course of 12 minutes, followed by a six minute re-
equilibration at 100%
mobile phase A. The flow rate was 0.8 mL/min and the detector was set at 280
nM. Mobile phase
A was 1.5 M ammonium sulfate, 25 mM sodium phosphate (pH 7). Mobile phase B
was 25%
isopropanol in 25 mM sodium phosphate (pH 7). Post-run, the chromatogram was
integrated and
the molar ratio was determined by summing the weighted peak area. The molar
ratio was
calculated to be about 4Ø
EXAMPLE 31
Additional Method for Determination of Molar Ratio
[0493] Another method for determination of molar ratio is as follows. First,
10 L of a 5 mg/mL
solution of an antibody construct immune-stimulatory compound conjugate is
injected into an
HPLC system set-up with a TOSOH TSKgel Butyl-NPR TM hydrophobic interaction
chromatography (HIC) column (2.5 [tM particle size, 4.6 mm x 35 mm) attached.
Then, over the
course of 18 minutes, a method is run in which the mobile phase gradient is
run from 100%
mobile phase A to 100% mobile phase B over the course of 12 minutes, followed
by a six minute
re-equilibration at 100% mobile phase A. The flow rate is 0.8 mL/min and the
detector is set at
280 nM. Mobile phase A is 1.5 M ammonium sulfate, 25 mM sodium phosphate (pH
7). Mobile
phase B is 25% isopropanol in 25 mM sodium phosphate (pH 7). Post-run, the
chromatogram is
integrated and the molar ratio is determined by summing the weighted peak
area.
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EXAMPLE 32
Affinity Measurement of Unconjugated Anti-CD40 Antibody or Anti-C40 Antibody
Immune-Stimulatory Conjugate to Recombinant CD40 Ectodomain Fry Receptors
Using
BioLayer Interferometry
[0494] This examples shows affinity measurements of unconjugated anti-C40
antibody or anti-
CD40 antibody immune-stimulatory conjugate to Fc receptors (FcRs). Antibody
affinity to its
antigen (such as CD40) and the Fcy Receptors (such as FcyRI, FcyRII, FcyRIII)
was quantitated
using BioLayer Interferometry (BLI). The anti-CD40 antibodies with different
Fc (SBT-040-WT,
SBT-040-VLPLL, SBT-040-AAA and SBT-040-G2) were first produced as described in
EXAMPLE 1 and then conjugated with an immune-stimulatory compound (any one of
ATAC1 ¨
ATAC34 and ATAC 43) as described in EXAMPLE 2. Following successful
conjugation, their
molecular interactions with CD40 Extracellular Domain (ECD) and various human
FcyRs were
quantitated using BLI.
[0495] Analysis of CD40 ECD interaction was performed using Octet Red 96
instrument
(ForteBio). The Octet systems use propriety BLI to analyze biomolecular
interaction.
Unconjugated anti-CD40 antibodies (SBT-040-WT, SBT-040-VLPLL, SBT-040-AAA, SBT-
040-G2) and anti-CD40 antibody immune-stimulatory compound conjugates were
immobilized
on anti-human Fc bio sensors and incubated with varying concentration of
monomeric human or
rhesus CD40 ranging from 1.2 nM to 300 nM in PBS. The experiments were
comprised of 5
steps: (1) baseline acquisition (60 s); (2) antibodies and antibody immune-
stimulatory compound
conjugates loading onto anti-human Fc biosensor (120 s); (3) second baseline
acquisition (60 s);
(4) association of interacting monomeric CD40 ECD protein for lc. measurement
(120 s); (5)
Dissociation of interacting monomeric CD40 ECD for kat- measurement (360 s).
The interacting
monomeric CD40 ECD were used at 5-6 concentrations of 3-fold concentration
series. Data were
analyzed using Octet Data Analysis Sofware 9.0 (ForteBio) and fitted to the
1:1 binding model.
Equilibrium dissociation constants (KD) were calculated by the ratio of lc0 to
kat-. Selected data
are shown in TABLE 9. All the anti-CD40 antibody immune-stimulatory compound
conjugates
had similar binding as unconjugated anti-CD40 antibody to monomeric human or
rhesus CD40
ECD.
TABLE 9
Human CD40 ECD monomer Rhesus CD40 ECD monomer
Antibody/Conjugate KD (nM)
Ka (1/Ms) Kd (1/s) KD (nM) Ka (1/Ms) Kd (1/s)
SBT-040-WT 8.4 1.35E+5 1.13E-3 14.0
7.03E+4 9.87E-4
SBT-040-WT-ATAC11 8.3 1.24E+5 1.03E-3 13.5
7.56E+4 1.02E-3
SBT-040-WT-ATAC22 8.0 1.37E+5 1.09E-3 13.6
7.61E+4 1.04E-3
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SBT-040-WT-ATAC4 9.3 1.04E+5 9.68E-4 12.9 6.55E+4
8.46E-4
SBT-040-WT-ATAC3 9.0 9.97E+5 8.97E-4 13.1 6.31E+4
8.23E-4
SBT-040-G2 7.7 1.34E+5 1.04E-3 12.4 7.61E+4
9.41E-4
SBT-040-G2-ATAC11 7.4 1.47E+5 1.09E-3 14.0 8.16E+4
1.14E-3
SBT-040-G2-ATAC22 7.2 1.42E+5 1.02E-3 15.2 7.20E+4
1.09E-3
SBT-040-G2-ATAC4 8.7 9.97E+5 8.67E-4 13.6 5.80E+4
7.88E-4
SBT-040-G2-ATAC3 7.3 1.15E+5 8.97E-4 11.5 7.21E+4
8.33E-4
SBT040-VLPLL 5.6 9.37E+4 5.26E-4 Not done
SBT040-VLPLL ATAC11 7.6 9.75E+4 7.37E-4 Not done
SBT040-VLPLL ATAC22 7.5 1.02E+5 7.71E-4 Not done
SBT040-AAA 6.0 9.89E+4 5.97E-4 Not done
SBT040-AAA 4.7 1.61E+5 7.53E-4 Not done
ATAC11
SBT040-AAA 5.0 1.36E+5 6.81E-4 Not done
ATAC22
[0496] Human Fcy R interaction analysis was also performed using Octet Red 96
instrument. For
human FcyRI and FcyRIIA interactions, unconjugated anti-CD40 antibodies or
anti-CD40
antibody immune-stimulatory compound conjugates were immobilized on anti-human
Fc
biosensors and incubated with varying concentration of monomeric FcyR ranging
from 1.2 nM to
1 M in PBS. The experiments were comprised of 5 steps: (1) baseline
acquisition (60 s); (2)
anti-CD40 antibodies or anti-CD40 antibody immune-stimulatory compound
conjugates loading
onto anti-human Fc biosensor (120 s); (3) second baseline acquisition (60 s);
(4) association of
interacting protein for lc. measurement (120 s); (5) dissociation of
interacting FcyR for kat-
measurement (300 s). The interacting monomeric FcyR were used at 5-6
concentrations of 3-fold
concentration series. Data were analyzed using Octet Data Analysis Software
9.0 (ForteBio) and
fitted to the 1:1 binding model. Equilibrium dissociation constants (KD) were
calculated by the
ratio of lc0 to kat-. Selected data are shown in TABLE 10. There is very
little to no changes in
anti-CD40 antibody immune-stimulatory compound conjugate interaction with
human FcyRI and
FcyRIIA as compared to unconjugated anti-CD40 antibody interactions with the
respective FcyR
monomeric protein.
[0497] For human FcyRIIB/C, FcyRIIIA F158, FcyRIIIA V158 and FcyRIIIB
interaction studies,
the proteins were immobilized on anti-his tag bio sensors and incubated with
varying
concentration of unconjugated anti-CD40 antibodies or anti-CD40 antibody
immune-stimulatory
compound conjugates ranging from 0.04 M to 8 M. This format was chosen
because of weak
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interactions if antibodies were captured first and FcyR added afterwards. The
experiment
comprised of 5 steps: (1) baseline acquisition (60 s); (2) anti-CD40
antibodies or anti-CD40
antibody immune-stimulatory compound conjugates loading onto anti-human Fc
biosensor (120
s); (3) second baseline acquisition (60 s); (4) association of interacting
protein for lc.
measurement (120 s); (5) dissociation of interacting FcyR for kat- measurement
(300 s). The
interacting anti-CD40 antibodies or anti-CD40 antibody immune-stimulatory
compound
conjugates were used at 4 concentrations of 2 fold concentration series. Data
were analyzed using
Octet Data Analysis Software 9.0 (ForteBio) and \ fitted to the avidity
binding model.
Equilibrium dissociation constants (KD) were calculated by the ratio of lc0 to
kat-. Selected data
are shown in TABLE 10. In most cases, there were no changes with the antibody
immune-
stimulatory compound conjugates with human FcyRIIB/C, FcyRIIIA F158, FcyRIIIA
V158 and
FcyRIIIB when compared to the parental unconjugated antibody. In some cases,
there were small
changes usually within 2 fold such as SBT040-G2 ATAC11 interaction with
FcyRIIIA F158
when compared to the unconjugated SBT040-G2.
TABLE 10
Antibody/Conjugate Fcl/RI Fc7RITA Fcl/RITB/C FcyRIIIA FcyRIIIA Fc7RITIB
KD ( 1 - l ) KD ( 1 - l ) KD (avidity) F158
V158 KD
KD KD (avidity)
(avidity) (avidity)
SBT-040-WT 0.68 nM 27 nM 1.60 uM 0.86 uM 0.51 uM 3.60 uM
SBT-040-WT- 0.79 nM 35 nM 2.20 uM 1.21 uM 0.62 uM 5.00 uM
ATAC11
SBT-040-WT- 0.80 nM 30 nM 1.68 uM 0.82 uM 0.41 uM 2.71 uM
ATAC22
SBT-040-WT- 0.63 nM 22 nM 1.83 uM 0.83 uM 0.46 uM 3.24 uM
ATAC4
SBT-040-WT- 0.77 nM 22 nM 0.97 uM 0.57 uM 0.30 uM 1.50 uM
ATAC3
SBT-040-G2 No binding 22 nM 2.62 uM 6.50 uM 3.65 uM No
binding
SBT-040-G2- No binding 23 nM 3.78 uM 10.6 uM 4.11 uM No
binding
ATAC11
SBT-040-G2- No binding 22 nM 3.16 uM 7.06 uM 3.57 uM No
binding
ATAC22
SBT-040-G2- No binding 22 nM 3.49 uM 7.00 uM 3.84 uM No
binding
ATAC4
SBT-040-G2- No binding 17 nM 1.85 uM 8.09 uM 4.86 uM 3.93 uM
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ATAC3
SBT040-VLPLL 1.5 nM 16 nM 2.30 uM 0.16 uM 0.08 uM 1.73 uM
SBT040-VLPLL 1.3 nM 24 nM 3.36 uM 0.16 uM 0.08 uM 2.34 uM
ATAC11
SBT040-VLPLL 1.2 nM 22 nM 2.14 uM 0.14 uM 0.08 uM 1.65 uM
ATAC22
SBT040-AAA 0.7 nM 64 nM 4.84 uM 0.47 uM 0.27 uM 2.44 uM
SBT040-AAA 0.5 nM 97 nM 3.77 uM 0.59 uM 0.29 uM 3.83 uM
ATAC11
SBT040-AAA 0.5 nM 116 nM 2.93 uM 0.45 uM 0.25 uM 2.56 uM
ATAC22
EXAMPLE 33
Stability of Anti-C40 Antibody Immune-Stimulatory Conjugates in IgG Depleted
Human
Serum
[0498] Stability of the anti-CD40 antibody immune-stimulatory conjugates in
human serum (IgG
depleted) were measured over 96 hours at 37 C using either a direct HIC-UV
analysis approach
(Method A) or an affinity capture approach (Method B). SBT-040-G1-ATAC12, SBT-
040-G2-
ATAC12, or SBT-040-G1-ATAC30 were spiked in IgG-depleted human serum (BBI
solutions #
5F142-2) in sterile tubes (75% final serum concentration) and samples were
split into 4 aliquots
of equal size then transferred to a 37 C incubator. One of the aliquots of
each sample was taken
from the incubator at each time-point (T = Oh, 24h, 48h, 96h) and average drug-
antibody ratios
(DAR) were recorded.
Method A: Direct HIC-UV analysis
[0499] At 0, 24, 48 and 96 hours after the beginning of incubation, the anti-
CD40 antibody
immune-stimulatory conjugates spiked in IgG depleted human serum were analyzed
by analytical
hydrophobic interaction chromatography (HIC) using a TOSOH TSKgel Butyl-NPR
4.6 mm x
35 mm HIC column (TOSOH Bioscience, # 14947) connected to a Dionex Ultimate
3000R5
HPLC system (ThermoFisher Scientific, Hemel Hemstead, UK). Results are
reported below in
TABLE 11.
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TABLE 11
Average DAR
Time (h) SBT-040-G1- SBT-040-G2- SBT-040-G1-
ATAC12 ATAC12 ATAC30
0 4.3 3.5 3.7
24 4.1 3.5 3.6
48 3.9 3.5 3.4
96 3.8 3.4 3.0
Method B: Affinity capture, de-glycosylation and RP-ESI-MS analysis
[0500] ADCs were immunocaptured from the IgG depleted human serum using an
anti-Human
IgG (Fc specific) biotin antibody immobilized on streptavidin beads at 0, 24,
48 and 96 hours
after the beginning of incubation. After elution from the beads, the samples
were de-glycosylated
using agarose-immobilized EndoS (Genovis Inc, USA). The de-glycosylated ADCs
were
analyzed by reverse phase chromatography hyphenated to electrospray ionization
mass
spectrometry (RP-ESI-MS) using an Acquity nano UPLC in line with a Xevo G25 Q-
TOF
(Waters, Elstree, UK). The separation was performed using an Acquity UPLC
online coupled to
an ESI-MS mass spectrometer. Mass spectrometric analysis was performed in
positive ion mode,
scanning from 1000 to 4000 m/z in high mass operating mode. The ion envelope
produced by
each sample was deconvoluted using the MaxEntl algorithm provided within the
MassLynx
software (Waters, Elstree, UK). Results are reported in the table below.
Average DAR
Time (h)
SBT-040-G1-ATAC4 SBT-
040-G1-ATAC3
0 4.4 3.8
24 4.7 3.8
48 4.6 3.7
96 4.5 3.7
EXAMPLE 34
Synthesis of ATAC 18
[0501] This example shows the synthesis of (1R,6R,8R,95,10S,15R,17R,185)-8,17-
Bis(2-amino-
6-oxo-1,9-dihydropurin-9-y1)-18-(3-1242-(2-1243-(2,5-dioxo-1H-pyrrol-1-
yl)propionylaminolethoxy}ethoxy)-ethoxylethoxy }propionylamino)-9-hydroxy-3,12-
dioxy-
2.4.7.11.13.16-hexaoxa-3k5.1215-diphosphatricyclo [13.3 Ø06,10]octadecane-
3,12-dione,
triethylammonium salt (ATAC 18).
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0
N
0 DeNe
- II
0-p-0 0 N N NH2
OH 6 0
0 0
I
H2N (-) ¨ ¨
0
HN N
0 NI-1-!) \) NI-1-:
ATAC 1 8
Step A. Preparation of Int ATAC 18-1
0
0
0
-0-P-0 N N N 1 HOBT / DIC
0
H H
TKO7 0 HOIH01,11HBoc
0
N.... ...
\
.., ii ,-, 2 TFA
y
0 - 1 0
0 HN y-- //
N I-1)
N H I N"---) NH 0
0 0I *L
) N HO-P-0 N---N N
Compound 21 ) TBSO c; ----y) "
0 HN 0 0
NH
H 0
NyNT....N \
(:).-Ti -OH 0
0
0 HN .(----N//
0 Int ATAC18-1
[0502] To a solution containing 100 mg (0.106 mmol) of Compound 21 in 5 mL of
DMSO and
39 mg (0.106 mmol) of 3-[2-(2-12-[2-(tert-
butoxycarbonylamino)ethoxy]ethoxy}ethoxy)ethoxyl
propionic acid was added 22 mg (0.16 mmol) of HOBT and 27 mg (0.212 mmol) of
DIC. The
reaction mixture was stirred at room temperature for 16h then purified by
reverse phase
chromatography without work-up. The resulting product fractions were
lyophilized to afford 70
mg of product which was covered with 7 mL of a 1:2 mixture of TFA and CH2C12.
The mixture
was stirred for 2h at ambient temperature before the solvent was removed. The
resulting residue
Int ATAC18-1 was used directly in the next step without purification.
Step B: Preparation of Int ATAC 18-2
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0
1 :1L-1 L
0
HO-T,-0.....10)
TBSO 0 H
CH3NH2
0 HN 0 0 NH
H 0
N NY EtON
T...
.........ii, N ...T.:, \
(:)---- Yi -OH 0
0
OHNy-----N/ 0
0 N----A NH
9
-0-1,--0 N N NH2
Int ATAC18-1 0
TBSO 0
NH2
H2N N N
I 1 0 -
NNy.---N
0 2 CH3NH3+
Int ATAC18-2
[0503] A solution containing Int ATAC18-1 (50 mg, 0.04 mmol) and 3.0 mL of
methylamine
(33% in anhydrous ethanol) was stirred for 16h at room temperature. The
reaction mixture was
concentrated to provide Int ATAC18-2 as a white foam which used directly in
the next step.
LCMS (ESI, m/z): 1051 [M+H].
Step C: Preparation of Int ATAC 18-3
0
N w
-.....--1-m
0 1 7.
---N.4.1...-NH2
-0-,g-0 N
----y_0_
TBSO (3
NH2
Et3N-HF
H2N ...,Nk....-N (D---Ti '0 0
'T/ 1 0 - 0
HNI(---N
N')LNH
0
2 CH3NH3+0
-04-0 N N NH2
0
Int ATAC18-2 HO 0
0
0 I
H2NyN,...,-N (:)---Ti -0 0
I 0
HN ----
,(N -
NI-1>
0 ,..,.--
ATAC18-3
[0504] The above crude Int ATAC18-2 was azeotroped with 3:1
pyridine/triethylamine three
times then dissolved in 0.8 mL pyridine. To this solution at 55 C was added 2
mL triethylamine
and lmL triethylamine trihydrofluride simultaneously. After stirring lh, the
bath was removed
and anhydrous acetone was added immediately. The mixture was stirred for 20
min and the white
solid was collected by filtration. The precipitate was washed with anhydrous
acetone. The
product was purified by preparative flash chromatography. The resulting
solution was lyophilized
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to provide 3 mg of Int ATAC18-3 as a white solid. LCMS: (ESI, m/z): 937.6
[M+H] . 1H NMR
(DMSO-d6): 6 7.99 (s, 1H), 7.89 (s, 1H), 5.73 (m, 2H), 5.06 (m, 1H); 4.84-4.74
(m, 2H); 4.53 (t,
J=5.1 Hz, 1H), 4.21 (m, 1H), 2.96 (m, 2H), 3.73-3.29 (m, 16H), 2.87 (q, J=7.2
Hz, 8H), 2.35 (m,
2H), 1.10 (t, J=7.2 Hz, 12H). 31P-NMR (DMSO-d6) 6 0.25, -1.32.
Step D: Preparation of ATAC18
0
0 0
04-0 N N NH2 ,0
0
OH 0
0
? H N N H2
DIPEA / DCM
N N
0 NH-2 ====,õõ NH-2 0
XI
Int ATAC18-3 0 0 NH2
OH 0 --10 0
?
0-1-H2N0_ 0 0
0
N N
0 NH>, ====.õ,õ. NH2
ATAC18
[0505] To a solution containing 2.0 mg (0.002 mmol) of Int ATAC18-3 in 0.2 mL
of DCM was
added 0.5 mg (0.004 mmol) of DIPEA then 1.06 mg (0.004 mmol) of 2,5-dioxo-1-
pyrrolidinyl 3-
(2,5-dioxo-1H-pyrrol-1-yl)propionate. The resulting solution was stirred
overnight then the
solvent was evaporated. Reverse phase column chromatography afforded the
desired compound
ATAC18 as a white solid. LCMS: (ESI, m/z): 1088 [M+H] . 1H-NMR (D20) 6 8.15
(s, 1H),
8.05 (s, 1H), 7.07 (s, 2H), 5.88-5.84 (m, 2H), 5.26 (m, 1H), 5.05-4.91 (m,
2H), 4.76 (m, 1H),
4.35 (m, 1H), 4.29-4.20 (m, 5H), 3.77 (t, 2H), 3.7-3.5 (m, 16H), 3.33 (m, 2H),
3.15 (q, J=7.2 Hz,
15H), 2.52 (m, 1H), 1.25 (t, J=7.2 Hz, 21H). 31P-NMR (DMSO-d6) 6 1.51, 0.62.
EXAMPLE 35
Synthesis of ATAC 43
[0506] This example shows the synthesis of 2,3,4,5,6-Pentafluorophenyl 5-
{(1S,6R,8R,9S,10R,15R,17R,18S)-8,17-bis(2-amino-6-oxo-1,9-dihydropurin-9-y1)-
18-hydroxy-
3,12-dioxo-3,12-dioxy-2.4.7.11.13.16-hexaoxa-3k5.1215-
diphosphatricyclo[13.3Ø06,10]octadec-9-ylamino }-5-oxovalerate
triethylammonium salt
(ATAC 43)
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0
N
0 De(1H
O-A-0 N Nr NH2
LC)4
OH O
F
HN 0
HN NN ====.
2
0 11....._p ir0 F
-0 0 0
HT1N 0 - F F
F
0 ATAC43
Step A: Preparation of Int ATAC 43-1
o
0
NNH
N"'"-)NH o
-04-o-...); N NH2
1
04-.0 =,,.]:) 1\i----N NH2 i
OH 0
0
0 NH2 _________________________________ ).
0 ? HNOH
0 I H2N N
H2N N N ====.,__p..., C)---t¨C ) 0 0
r N I 0
r
0 HN .r--N
HN,r-N
I
0
0 .,NFI-,. .,N1-1-, .,N1-1-.
.,NH->
Compound 2 Int ATAC 43-1
[0507] To a solution containing 50 mg (0.072 mmol) of Compound 2 in 2.5 mL of
DMSO was
added 57 mg (0.72 mmol) of pyridine and 5 mg of DMAP. The resulting solution
was stirred for
minutes then treated with 82 mg (0.72 mmol) of glutaric anhydride. The
reaction mixture was
stirred at room temperature for 16h then purified by reverse phase
chromatography without
work-up. Fractions containing product were lyophilized over 48h to provide 10
mg of Int ATAC
43-1 as a white solid. LCMS (ESI, m/z): 804 [M+H]. 1H NMR (DMSO-d6) 8 10.6
(bs, 2H), 9.20
(bs, 1H), 7.99 (s, 1H), 7.92 (s, 1H), 7.56 (bs, 1H), 6.56 (m, 4H), 5.76 (dd,
J=8.1, 17.7 Hz, 2H),
4.94 (m, 1H), 4.76-4.68 (m, 2H), 4.58 (m, 1H), 4.22 (m, 1H), 4.03-3.91 (m,
6H), 3.97 (q,
J=7.0Hz, 12H), 2.21 (t, J=7.2Hz, 2H), 2.09-1.99 m, 2H), 1.67-1.60 (m, 2H),
1.20 (t, J=7.2Hz,
18H). 31P NMR (DMSO-d6) d 0.44, -1.3.
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Step B. Preparation of ATAC43
0
0
0- 0 N NH2 ;.-CD -
N NH2 HO 0 N F
OH 0 F 4411'P F OH 0
?
EDC / DMSO ?
4/0 F
--"Yi."0 0 0 0
I I (:) 0 - I 0 0 0
-
HNy----NHN 0
F
0 0
Int ATAC 43-1 ATAC 43
[0508] To a solution containing 7.0 mg (0.009 mmol) of Int ATAC 43-1 and 2.6
mg (0.013
mmol, 1.5 eq) of pentafluorophenol in 300 uL of DMSO was added 1.8 mg (0.012
mmol, 1.3 eq)
of EDC and the reaction mixture was stirred for 16h at room temperature then
purified by reverse
phase chromatography without work-up. Fractions containing product were
lyophilized to
provide ATAC 43 as a white solid. LCMS (ESI, m/z): 970 [M+H].
EXAMPLE 36
STING Agonist Screening Assay
[0509] Biology materials and general procedures. The following reporter cell
lines, reagents and
ligands were obtained from InvivoGen: THP-1 Dual cells (thpd-nfis); THP-1 Dual
KO-STING
cells (thpd-kostg); Quanti-Blue (rep-qbl); Quanti-Luc (rep-q1c1); Normocin
(ant-nr-1); Zeocin
(ant-zn-1); Blasticidin (ant-bl-1); PMA (tlrl-pma); 2',3'-cGAMP (tlrl-nacga);
and 2'3'-c-di-
AM(PS)2 (Rp,Rp) (tlrl-nacda2r). 3',3'-cGAMP (SML 1232) and 3',5'-cyclic-di-GMP
(SML
1228) were purchased from Sigma. THP-1 Dual cells were cultured in RPMI 1640
(Lonza)
supplemented with 10% fetal bovine serum, 2mM glutamine, 50m/mL penicillin, 50
U/mL
streptomycin (all from Gibco). Cells were passaged at 0.7 x 106 cell/mL every
2-3 days. 100
1.tg/mL Normocin, 100m/mL Zeocin, and 10m/mL Blasticidin were added every
other passage
to maintain reporter expression according to the manufacturer's instructions.
[0510] General procedure for in vitro screening of CDNs for cytokine induction
activity.
THP-1 Dual or THP-1 Dual KO-STING cells were plated at 50,000 cells per well
in 200 [IL of
culture media in 96-well plates and matured with 150 nM PMA for 16-18 hours.
Cells were
washed in culture media the following day and supernatants were removed. Cells
were stimulated
for 30 min at 37 C in a 5% CO2 incubator with 150 [IL of CDNs prepared in
permeabilization
buffer with 2m/mL digitonin (CalBiochem) at four different concentrations (1,
0.1, 0.01 and
0.001 p,M). After the incubation, the permeabilization buffer and CDNs were
removed and
replaced with 150 [IL culture media. Cells were incubated an additional 23.5 h
at 37 C in a 5%
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CO2 incubator. Prior to supernatnant harvest, cells were spun at 300 x g for 5
min to remove cell
debris. ISG54 activity was indirectly quantified from supernatants using
QUANTI-Luc, which
was prepared and used according to the manufacturer's instructions. NF-KB
secreted alkaline
phosphatase pathway activity was indirectly quantified from supernatants using
QUANTI-Blue,
which was prepared and used according to the manufacturer's instructions.
CellTiterGlo
(Promega) was used to lyse and assess viability according to the
manufacturer's instructions.
Plates were analyzed on Envision (Perkin Elmer) or Synergy (BioTek) plate
readers.
EXAMPLE 37
Tumor Size in a Mouse Tumor Model Is Reduced by Anti-C40 Antibody Immune-
Stimulatory Compound Conjugates
[0511] The example shows tumor size is reduced in a mouse tumor model after
administration of
an anti-CD40 antibody immune-stimulatory compound conjugate. The
immunocompromised
mouse strain NSG (NOD.Cg-PrkdcscidIL-2reiwil/SzJ) into which human tumor cells
are co-
injected with human T cells and myeloid dendritic cells (mDC) is used as the
mouse tumor
model. In vivo immune-mediated activity of the anti-CD40 antibody immune-
stimulatory
compound conjugate is assessed by examining human mDCs in this model.
[0512] Anti-CD40 antibody immune-stimulatory compound conjugates or anti-CD40
antibody
(control) are injected intraperitoneally to mice. Immediately after this
injection, Raji cells (a B
cell lymphoma tumor cell line), human T cells, and mDCs from the same human
donor are co-
injected into the mice. The Raji cells are injected subcutaneously at a
concentration of lx10^7
cells/mouse. The human T cells are injected at a concentration of lx10^6
cells/mouse, and mDCs
are injected at a concentration of 5x10^5 cells/mouse. Tumor growth is
measured using calipers
twice per week, beginning seven days post tumor cell transfer and ending at
study termination,
approximately 3 weeks after tumor cell inoculation. Tumor size is reduced in
mice that received
the anti-CD40 antibody immune-stimulatory compound conjugates in comparison to
mice that
received the anti-CD40 antibody.
EXAMPLE 38
Dendritic Cell Priming of T Cells Is Enhanced by Anti-C40 Antibody Immune-
Stimulatory
Compound Conjugates
[0513] This example shows that dendritic cell priming of T cells is enhanced
by anti-CD40
antibody immune-stimulatory compound conjugates. After anti-CD40 antibody
immune-
stimulatory compound conjugates is administered, the upregulation of co-
stimulatory molecules
and cytokine production by dendritic cells is induced. Priming of a T cell
response is therefore
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enhanced by the stimulation of dendritic cells in this manner. An in vitro
human mDC and T cell
co-culture assay is used to demonstrate this.
[0514] Myeloid dendritic cells (mDCs) and allogeneic T cells are isolated from
peripheral blood
mononuclear cells (PBMCs), are labeled with a dye to monitor cell division,
and are co-cultured
for 5 days in the presence of the anti-CD40 antibody immune-stimulatory
compound conjugates
or an isotype control. T cell activation is assessed as percent dividing cells
as measured by flow
cytometry using the indicator dye. The percent of dividing T cells is
increased in T cells co-
cultured with the anti-CD40 antibody immune-stimulatory compound conjugates in
comparison
to the percent of dividing T cells for T cells co-cultured with the isotype
control.
[0515] While aspects of the present disclosure have been shown and described
herein, it will be
apparent to those skilled in the art that such aspects are provided by way of
example only.
Numerous variations, changes, and substitutions will now occur to those
skilled in the art without
departing from the disclosure. It should be understood that various
alternatives to the aspects of
the disclosure described herein may be employed in practicing the disclosure.
It is intended that
the following claims define the scope of the disclosure and that methods and
structures within the
scope of these claims and their equivalents be covered thereby.
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