Note: Descriptions are shown in the official language in which they were submitted.
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IMMUNO ONCOLOGY COMBINATION THERAPY WITH IL-2
CONJUGATES AND ANTI-EGFR ANTIBODIES
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. Provisional Application No.
63/044,199, filed on
June 25, 2020, and to U.S. Provisional Application No. 63/196,448, filed on
June 3, 2021, the
disclosure of each of which is hereby incorporated by reference in its
entirety.
BACKGROUND OF THE DISCLOSURE
[2] Distinct populations of T cells modulate the immune system to
maintain immune
homeostasis and tolerance. For example, regulatory T (Treg) cells prevent
inappropriate responses
by the immune system by preventing pathological self-reactivity, while
cytotoxic T cells target and
destroy infected cells and/or cancerous cells. In some instances, modulation
of the different
populations of T cells provides an option for treatment of a disease or
indication. Modulation of the
different populations of T cells may be enhanced by the presence of additional
agents or methods in
combination therapy.
P1 Cytokines comprise a family of cell signaling proteins such as
chemokines, interferons,
interleukins, lymphokines, tumor necrosis factors, and other growth factors
playing roles in innate
and adaptive immune cell homeostasis. Cytokines are produced by immune cells
such as
macrophages, B lymphocytes, T lymphocytes and mast cells, endothelial cells,
fibroblasts, and
different stromal cells. In some instances, cytokines modulate the balance
between humoral and
cell-based immune responses.
[4] Interleukins are signaling proteins that modulate the
development and differentiation of T
and B lymphocytes, cells of the monocytic lineage, neutrophils, basophils,
eosinophils,
megakaryocytes, and hematopoietic cells. Interleukins are produced by helper
CD4+ T and B
lymphocytes, monocytes, macrophages, endothelial cells, and other tissue
residents.
[51 In some instances, interleukin 2 (IL-2) signaling is used to
modulate T cell responses and
subsequently for treatment of a cancer. Accordingly, in one aspect, provided
herein are methods of
treating cancer in a subject comprising administering an IL-2 conjugate in
combination with an anti-
EGFR antibody.
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SUMMARY OF THE DISCLOSURE
[6] Described herein are methods of treating a cancer in a subject
in need thereof, comprising
administering to the subject a therapeutically effective amount of (a) an 1L-2
conjugate, and (b) an
anti-EGFR antibody
171 Exemplary embodiments include the following.
[8] Embodiment 1. A method of treating a cancer in a subject in
need thereof, comprising
administering to the subject a therapeutically effective amount of (a) an 1L-2
conjugate, and (b) an
anti-EGFR antibody, wherein the IL-2 conjugate comprises the amino acid
sequence of SEQ ID
NO: 3 in which at least one amino acid residue in the IL-2 conjugate is
replaced by the structure of
Formula (I):
x N
0 N'õ
411
Formula (I);
wherein:
.^1`"
Z is CH2 and Y is 0 0 =
N
Y is CH2 and Z is 0 O=
Z is CH2 and Y is 0 ;or
JL
Y is CH2 and Z is 0
W is a PEG group having an average molecular weight of about 5 kDa, 10 kDa, 15
kDa, 20 kDa, 25
kDa, 30 kDa, 35 kDa, 40 kDa, 45 kDa, 50 kDa, or 60 kDa; and
X is an L-amino acid having the structure:
2
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X-1
cs5sN H
Osss X+ 1
X-1 indicates the point of attachment to the preceding amino acid residue; and
X+1 indicates the point of attachment to the following amino acid residue;
wherein the position of the structure of Formula (I) in the amino acid
sequence of the IL-2
conjugate is selected from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44,
V68, and L71.
1911 Embodiment 2. The method of embodiment 1, wherein in the IL-2
conjugate Z is CH2 and
Y is 0 0
[10] Embodiment 3. The method of embodiment 1, wherein in the IL-2
conjugate Y is CH2 and
kN N
Z i s 0 0
[11] Embodiment 4. The method of embodiment 1, wherein in the IL-2
conjugate Z is CH and
0
Y is 0
1121 Embodiment 5. The method of embodiment 1, wherein in the IL-2
conjugate Y is CH2 and
1-,Now
Z is 0
[13] Embodiment 6. The method of any one of embodiments 1-5, wherein in the
IL-2 conjugate
the PEG group has an average molecular weight of about 25 kDa, 30 kDa, or 35
kDa.
[14] Embodiment 7. The method of embodiment 6, wherein in the IL-2
conjugate the PEG
group has an average molecular weight of about 30 kDa.
[15] Embodiment 8. The method of any one of embodiments 1-7, wherein the
position of the
structure of Formula (I) in the amino acid sequence of the IL-2 conjugate is
P64.
[16] Embodiment 9. The method of embodiment 1, wherein the structure of
Formula (I) has the
structure of Formula (IV) or Formula (V), or is a mixture of the structures of
Formula (IV) and
Formula (V):
3
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X N
0
0 4õ
N
41 0
Formula (IV);
= 0
y 0
0 Nõ
0
Formula (V);
wherein:
W is a PEG group having an average molecular weight of about 25 kDa, 30 kDa,
or 30 kDa;
q is 1, 2, or 3;
X is an L-amino acid having the structure:
ssss NH
=
X-1 indicates the point of attachment to the preceding amino acid residue; and
X+1 indicates the point of attachment to the following amino acid residue.
[17] Embodiment 10. The method of embodiment 9, wherein the
position of the structure of
Formula (IV) or Formula (V) in the amino acid sequence of the IL-2 conjugate
is P64.
1181 Embodiment 11. The method according to any one of embodiments
1-10, wherein the anti-
EGFR antibody is cetuximab.
1191 Embodiment 12. A method of treating a cancer in a subject in
need thereof, comprising
administering to the subject a therapeutically effective amount of (a) an 1L-2
conjugate, and (b)
cetuximab, wherein the IL-2 conjugate comprises the amino acid sequence of SEQ
ID NO: 50,
wherein [AzK _ Ll PEG301(D] has the structure of Formula (XII) or Formula
(XIII), or is a mixture
_
of the structures of Formula (XII) and Formula (XIII):
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NH 0
N es) 11
-7/
NI, I 0
,CH3
n 0
k 0
Formula (XII);
" 0
NTO
N
0
0
eV'
wherein:
n is an integer n is an integer such that -(OCH2CH2)n-OCH.3 has a molecular
weight of about 30
kDa;
q is 1,2, or 3, and
the wavy lines indicate covalent bonds to amino acid residues within SEQ ID
NO: 50 that are not
replaced.
[20] Embodiment 13. The method of any one of embodiments 1-12, wherein q is
1.
[21] Embodiment 14. The method of any one of embodiments 1-12, wherein q is
2.
[22] Embodiment 15. The method of any one of embodiments 1-12, wherein q is
3.
[23] Embodiment 16. The method of any one of embodiments 1-15, wherein the
average
molecular weight is a number average molecular weight.
[24] Embodiment 17. The method of any one of embodiments 1-15, wherein the
average
molecular weight is a weight average molecular weight.
[25] Embodiment 18. The method of any one of embodiments 1-17, wherein the
IL-2 conjugate
is a pharmaceutically acceptable salt, solvate, or hydrate.
[26] Embodiment 19. The method according to any one of embodiments 1-18,
wherein the 1L-2
conjugate is administered to the subject about once every two weeks, about
once every three weeks,
or about once every 4 weeks.
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[27] Embodiment 20. The method according to any one of embodiments 1-19,
wherein the anti-
EGFR antibody is administered to the subject about once every week, about once
every two weeks,
about once every three weeks, or about once every 4 weeks.
[28] Embodiment 21. The method according to any one of embodiments 1-20,
wherein the IL-2
conjugate is administered to a subject by intravenous administration.
[29] Embodiment 22. The method according to any one of embodiments 1-21,
wherein the IL-2
conjugate and the anti-EGFR antibody are administered separately.
1301 Embodiment 23. The method of embodiment 23, wherein the 1L-2
conjugate and the anti-
EGFR antibody are administered sequentially.
[31] Embodiment 24. The method according to any one of embodiments 1-23,
wherein the
cancer is selected from renal cell carcinoma (RCC), non-small cell lung cancer
(NSCLC), head and
neck squamous cell cancer (HNSCC), classical Hodgkin lymphoma (cHL), primary
mediastinal
large B-cell lymphoma (PMBCL), urothelial carcinoma, microsatellite unstable
cancer,
microsatellite stable cancer, gastric cancer, colon cancer, colorectal cancer
(CRC), cervical cancer,
hepatocellular carcinoma (HCC), Merkel cell carcinoma (MCC), melanoma, small
cell lung cancer
(SCLC), esophageal, esophageal squamous cell carcinoma (ESCC), glioblastoma,
mesothelioma,
breast cancer, triple-negative breast cancer, prostate cancer, castrate-
resistant prostate cancer,
metastatic castrate-resistant prostate cancer, or metastatic castrate-
resistant prostate cancer having
DNA damage response (DDR) defects, bladder cancer, ovarian cancer, tumors of
moderate to low
mutational burden, cutaneous squamous cell carcinoma (CSCC), squamous cell
skin cancer
(SCSC), tumors of low- to non-expressing PD-L1, tumors disseminated
systemically to the liver and
CNS beyond their primary anatomic originating site, and diffuse large B-cell
lymphoma.
[32] Embodiment 25. The method of any one of embodiments 1-24, comprising
administering
to the subject about 16 [tg/kg of the IL-2 conjugate.
[33] Embodiment 26. The method of any one of embodiments 1-24, comprising
administering
to the subject about 24 lag/kg of the IL-2 conjugate.
1341 Embodiment 27. The method of any one of embodiments 1-10 or 12-
26, wherein the anti-
EGFR antibody is selected from panitumumab (Vectibix), necitumumab
(Portrazza), JNJ-61186372
(Amivantamab), IMC-C225, ABX-EGF, ICR62, and EMD 55900
151 Embodiment 28. An IL-2 conjugate for use in the method of any
one of embodiments 1-
27.
[36] Embodiment 29. Use of an IL-2 conjugate for the manufacture of
a medicament for the
method of any one of embodiments 1-27.
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BRIEF DESCRIPTION OF THE DRAWINGS
1371 The novel features of the invention are set forth with
particularity in the appended claims.
A better understanding of the features and advantages of the present invention
will be obtained by
reference to the following detailed description that sets forth illustrative
embodiments, in which the
principles of the invention are utilized, and the accompanying drawings of
which.
[38] FIGS. 1A-C show the % cytotoxicity in CAL27 cells co-cultured with 3
separate donor
human PBMCs and varying amounts of an IL-2 conjugate and cetuximab
[39] FIG. 2A shows the % cytotoxicity in CAL27 cells co-cultured with human
PBMCs and
varying amounts of an IL-2 conjugate and cetuximab.
[40] FIG. 2B shows the % cytotoxicity in A431 cells co-cultured with human
PBMCs and
varying amounts of an IL-2 conjugate and cetuximab.
[41] FIG. 3A shows the cytotoxic effect on A431 cells co-cultured with NK92
cells and treated
varying amounts of an IL-2 conjugate and cetuximab.
[42] FIG. 3B shows the % cytotoxicity on DLD-1 cells co-cultured with NK92
cells and
treated varying amounts of an IL-2 conjugate and cetuximab
[43] FIG. 3C shows the % cytotoxicity on FaDu cells co-cultured with NK92
cells and treated
varying amounts of an IL-2 conjugate and cetuximab.
[44] FIG. 3D shows the % cytotoxicity on CAL27 cells co-cultured with NK92
cells and
treated varying amounts of an IL-2 conjugate and cetuximab.
[45] FIG. 4A shows the peripheral CD8+ Teff cell counts in the indicated
subjects treated with
16 ng/kg of the IL-2 conjugate in combination with cetuximab at specified
times following
administration of IL-2 conjugate.
[46] FIG. 4B shows the change in peripheral CD8+ Teff cell counts in the
indicated subjects
treated with 16 ng/kg of the IL-2 conjugate in combination with cetuximab at
specified times
following administration of IL-2 conjugate. Data is normalized to pre-
treatment (C1D1) CD8+ Tar
cell count.
[47] FIG. 5A shows the peripheral NK cell counts in the indicated subjects
treated with 16
jig/kg of the IL-2 conjugate in combination with cetuximab at specified times
following
administration of IL-2 conjugate.
[48] FIG. 5B shows the change in peripheral NK cell counts in the indicated
subjects treated
with 16 jig/kg of the IL-2 conjugate in combination with cetuximab at
specified times following
administration of IL-2 conjugate. Data is normalized to pre-treatment (C1D1)
NK cell count
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[49] FIG. 6A shows the peripheral CD4+ Treg cell counts in the indicated
subjects treated with
16 ug/kg of the IL-2 conjugate in combination with cetuximab at specified
times following
administration of IL-2 conjugate.
[50] FIG. 6B shows the change in peripheral CD4+ Treg cell counts in the
indicated subjects
treated with 16 iig/kg of the IL-2 conjugate in combination with cetuximab at
specified times
following administration of IL-2 conjugate. Data is normalized to pre-
treatment (C1D1) CD4+ Treg
cell count.
1511 FIG. 7A shows the eosinophil cell counts in the indicated
subjects treated with 16 ug/kg
of the IL-2 conjugate in combination with cetuximab at specified times
following administration of
IL-2 conjugate.
1521 FIG. 7B shows the change in eosinophil cell counts in the
indicated subjects treated with
16 mg/kg of the IL-2 conjugate in combination with cetuximab at specified
times following
administration of IL-2 conjugate. Data is normalized to pre-treatment (C1D1)
eosinophil cell count.
[53] FIG. 8A shows the lymphocyte cell counts in the indicated subjects
treated with 16 its/kg
of the IL-2 conjugate in combination with cetuximab at specified times
following administration of
IL-2 conjugate.
[54] FIG. 8B shows the change in lymphocyte cell counts in the indicated
subjects treated with
16 mg/kg of the IL-2 conjugate in combination with cetuximab at specified
times following
administration of IL-2 conjugate. Data is normalized to pre-treatment (C1D1)
lymphocyte cell
count.
DETAILED DESCRIPTION OF THE DISCLOSURE
Definitions
[55] Unless defined otherwise, all technical and scientific terms used
herein have the same
meaning as is commonly understood by one of skill in the art to which the
claimed subject matter
belongs. It is to be understood that the foregoing general description and the
following detailed
description are exemplary and explanatory only and are not restrictive of any
subject matter
claimed. To the extent any material incorporated herein by reference is
inconsistent with the express
content of this disclosure, the express content controls. In this application,
the use of the singular
includes the plural unless specifically stated otherwise. It must be noted
that, as used in the
specification and the appended claims, the singular forms "a," "an" and "the"
include plural
referents unless the context clearly dictates otherwise. In this application,
the use of "or" means
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"and/or" unless stated otherwise. Furthermore, use of the term "including" as
well as other forms,
such as "include", "includes," and "included," is not limiting.
[56] Reference in the specification to "some embodiments", "an
embodiment", "one
embodiment" or "other embodiments" means that a particular feature, structure,
or characteristic
described in connection with the embodiments is included in at least some
embodiments, but not
necessarily all embodiments, of the inventions.
1571 As used herein, ranges and amounts can be expressed as "about"
a particular value or
range. About also includes the exact amount. Hence "about 5 L" means "about 5
tit" and also "5
L." Generally, the term "about" includes an amount that would be expected to
be within
experimental error, such as for example, within 15%, 10%, or 5%.
1581 The section headings used herein are for organizational
purposes only and are not to be
construed as limiting the subject matter described.
[59] As used herein, the terms "individual(s)", "subject(s)" and
"patient(s)" mean any mammal.
In some embodiments, the mammal is a human. In some embodiments, the mammal is
a non-
human. None of the terms require or are limited to situations characterized by
the supervision (e.g.
constant or intermittent) of a health care worker (e.g. a doctor, a registered
nurse, a nurse
practitioner, a physician's assistant, an orderly or a hospice worker).
[60] As used herein, the term "significant" or "significantly" in reference
to binding affinity
means a change in the binding affinity of the cytokine (e.g., IL-2
polypeptide) sufficient to impact
binding of the cytokine (e.g., IL-2 polypeptide) to a target receptor. In some
instances, the term
refers to a change of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%,
95%, or more. In
some instances, the term means a change of at least 2-fold, 3-fold, 4-fold, 5-
fold, 6-fold, 7-fold, 8-
fold, 9-fold, 10-fold, 50-fold, 100-fold, 500-fold, 1000-fold, or more.
[61] In some instances, the term "significant" or "significantly" in
reference to activation of
one or more cell populations via a cytokine signaling complex means a change
sufficient to activate
the cell population. In some cases, the change to activate the cell population
is measured as a
receptor signaling potency. In such cases, an EC50 value may be provided. In
other cases, an ED50
value may be provided. In additional cases, a concentration or dosage of the
cytokine may be
provided
[62] As used herein, the term "potency" refers to the amount of a cytokine
(e.g., IL-2
polypeptide) required to produce a target effect. In some instances, the term
"potency" refers to the
amount of cytokine (e.g., IL-2 polypeptide) required to activate a target
cytokine receptor (e.g., IL-2
receptor). In other instances, the term "potency" refers to the amount of
cytokine (e.g., IL-2
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polypeptide) required to activate a target cell population. In some cases,
potency is measured as
ED50 (Effective Dose 50), or the dose required to produce 50% of a maximal
effect. In other cases,
potency is measured as EC50 (Effective Concentration 50), or the dose required
to produce the
target effect in 50% of the population.
[63] As used herein, the term "unnatural amino acid" refers to an amino
acid other than one of
the 20 naturally occurring amino acids. Exemplary unnatural amino acids are
described in Young et
al., "Beyond the canonical 20 amino acids: expanding the genetic lexicon," I.
of Biological
Chemistry 285(15): 11039-11044 (2010), the disclosure of which is herein
incorporated by
reference.
[64] The term "antibody" herein is used in the broadest sense and
encompasses various
antibody structures, including but not limited to monoclonal antibodies,
polyclonal antibodies,
multispecific antibodies (e.g., bispecific antibodies), and antibody fragments
so long as they exhibit
the desired antigen-binding activity. An "antibody fragment" refers to a
molecule other than an
intact antibody that comprises a portion of an intact antibody that binds the
antigen to which the
intact antibody binds. Examples of antibody fragments include but are not
limited to Fv, Fab, Fab',
Fab'-SH, F(abl)2; diabodies; linear antibodies; single-chain antibody
molecules (e.g. scFv), and
multispecific antibodies formed from antibody fragments. In some embodiments,
the antigen is
EGFR.
[65] The term "monoclonal antibody(ies)" as used herein refers to an
antibody obtained from a
population of substantially homogeneous antibodies, i.e., the individual
antibodies comprising the
population are identical and/or bind the same epitope, except for possible
variant antibodies, e.g.,
containing naturally occurring mutations or arising during production of a
monoclonal antibody
preparation, such variants generally being present in minor amounts. In
contrast to polyclonal
antibody preparations, which typically include different antibodies directed
against different
determinants (epitopes), each monoclonal antibody of a monoclonal antibody
preparation is directed
against a single determinant on an antigen. Thus, the modifier "monoclonal"
indicates the character
of the antibody as being obtained from a substantially homogeneous population
of antibodies and is
not to be construed as requiring production of the antibody by any particular
method. For example,
the monoclonal antibodies to be used in accordance with the present invention
may be made by a
variety of techniques, including but not limited to the hybridoma method,
recombinant DNA
methods, phage-display methods, and methods utilizing transgenic animals
containing all or part of
the human immunoglobulin loci, such methods and other exemplary methods for
making
monoclonal antibodies being described herein.
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[66] As used herein, "nucleotide" refers to a compound comprising a
nucleoside moiety and a
phosphate moiety. Exemplary natural nucleotides include, without limitation,
adenosine
triphosphate (ATP), uridine triphosphate (UTP), cytidine triphosphate (CTP),
guanosine
triphosphate (GTP), adenosine diphosphate (ADP), uridine diphosphate (UDP),
cytidine
diphosphate (CDP), guanosine diphosphate (GDP), adenosine monophosphate (AMP),
uridine
monophosphate (UMP), cytidine monophosphate (CMP), and guanosine monophosphate
(GMP),
deoxyadenosine triphosphate (dATP), deoxythymi dine triphosphate (dTTP),
deoxycytidine
triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), deoxyadenosine
diphosphate (dADP),
thymidine diphosphate (dTDP), deoxycytidine diphosphate (dCDP), deoxyguanosine
diphosphate
(dGDP), deoxyadenosine monophosphate (dAMP), deoxythymidine monophosphate
(dTMP),
deoxycytidine monophosphate (dCMP), and deoxyguanosine monophosphate (dGMP).
Exemplary
natural deoxyribonucleotides, which comprise a deoxyribose as the sugar
moiety, include dATP,
dTTP, dCTP, dGTP, dADP, dTDP, dCDP, dGDP, dAMP, dTMP, dCMP, and dGMP.
Exemplary
natural ribonucleotides, which comprise a ribose as the sugar moiety, include
ATP, UTP, CTP,
GTP, ADP, UDP, CDP, GDP, AMP, UMP, CMP, and GMP.
[67] As used herein, "base" or "nucleobase" refers to at least the
nucleobase portion of a
nucleoside or nucleotide (nucleoside and nucleotide encompass the ribo or
deoxyribo variants),
which may in some cases contain further modifications to the sugar portion of
the nucleoside or
nucleotide. In some cases, "base" is also used to represent the entire
nucleoside or nucleotide (for
example, a "base" may be incorporated by a DNA polymerase into DNA, or by an
RNA polymerase
into RNA). However, the term "base" should not be interpreted as necessarily
representing the
entire nucleoside or nucleotide unless required by the context. In the
chemical structures provided
herein of a base or nucleobase, only the base of the nucleoside or nucleotide
is shown, with the
sugar moiety and, optionally, any phosphate residues omitted for clarity. As
used in the chemical
structures provided herein of a base or nucleobase, the wavy line represents
connection to a
nucleoside or nucleotide, in which the sugar portion of the nucleoside or
nucleotide may be further
modified. In some embodiments, the wavy line represents attachment of the base
or nucleobase to
the sugar portion, such as a pentose, of the nucleoside or nucleotide. In some
embodiments, the
pentose is a ribose or a deoxyribose
[68] In some embodiments, a nucleobase is generally the heterocyclic base
portion of a
nucleoside. Nucleobases may be naturally occurring, may be modified, may bear
no similarity to
natural bases, and/or may be synthesized, e.g., by organic synthesis. In
certain embodiments, a
nucleobase comprises any atom or group of atoms in a nucleoside or nucleotide,
where the atom or
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group of atoms is capable of interacting with a base of another nucleic acid
with or without the use
of hydrogen bonds. In certain embodiments, an unnatural nucleobase is not
derived from a natural
nucleobase. It should be noted that unnatural nucleobases do not necessarily
possess basic
properties, however, they are referred to as nucleobases for simplicity. In
some embodiments, when
referring to a nucleobase, a "(d)" indicates that the nucleobase can be
attached to a deoxyribose or a
ribose, while "d" without parentheses indicates that the nucleobase is
attached to deoxyribose.
[69] As used herein, a "nucleoside" is a compound comprising a nucleobase
moiety and a sugar
moiety. Nucleosides include, but are not limited to, naturally occurring
nucleosides (as found in
DNA and RNA), abasic nucleosides, modified nucleosides, and nucleosides having
mimetic bases
and/or sugar groups. Nucleosides include nucleosides comprising any variety of
substituents. A
nucleoside can be a glycoside compound formed through glycosidic linking
between a nucleic acid
base and a reducing group of a sugar.
[70] An "analog" of a chemical structure, as the term is used herein,
refers to a chemical
structure that preserves substantial similarity with the parent structure,
although it may not be
readily derived synthetically from the parent structure. In some embodiments,
a nucleotide analog
is an unnatural nucleotide. In some embodiments, a nucleoside analog is an
unnatural nucleoside.
A related chemical structure that is readily derived synthetically from a
parent chemical structure is
referred to as a "derivative."
[71] As used herein, "dose-limiting toxicity" (DLT) is defined as an
adverse event occurring
within Day 1 through Day 29 (inclusive) +1 day of a treatment cycle that was
not clearly or
incontrovertibly solely related to an extraneous cause and that meets the
criteria set forth in
Example 5 for DLT.
[72] As used herein, "cetuximab" refers to the chimeric (mouse/human) anti-
EGFR antibody
marketed under the brand name "Erbitux" by Eli Lilly and Co.
[73] Although various features of the invention may be described in the
context of a single
embodiment, the features may also be provided separately or in any suitable
combination.
Conversely, although the invention may be described herein in the context of
separate embodiments
for clarity, the invention may also be implemented in a single embodiment.
IL-2 Conjugates
[74] Interleukin 2 (IL-2) is a pleiotropic type-1 cytokine whose structure
comprises a 15.5 kDa
four a-helix bundle. The precursor form of IL-2 is 153 amino acid residues in
length, with the first
20 amino acids forming a signal peptide and residues 21-153 forming the mature
form. IL-2 is
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produced primarily by CD4+ T cells post antigen stimulation and to a lesser
extent, by CD8+ cells,
Natural Killer (NK) cells, and Natural killer T (NKT) cells, activated
dendritic cells (DCs), and
mast cells. IL-2 signaling occurs through interaction with specific
combinations of IL-2 receptor
(IL-2R) subunits, IL-2Ra (also known as CD25), IL-2R13 (also known as CD122),
and IL-2Ry (also
known as CD132). Interaction of IL-2 with the IL-2Ra forms the "low-affinity"
IL-2 receptor
complex with a Ka of about 10-8M. Interaction of IL-2 with IL-21113 and IL-2Ry
forms the
"intermediate-affinity" IL-2 receptor complex with a Ka of about 10' M.
Interaction of IL-2 with all
three subunits, IL-2Ra, IL-2R13, and IL-2R1, forms the "high-affinity" IL-2
receptor complex with a
Kd of about >1011M.
[75] In some instances, IL-2 signaling via the "high-affinity" IL-2Rapy
complex modulates the
activation and proliferation of regulatory T cells. Regulatory T cells, or
CD4'CD25+Foxp3-
regulatory T (Treg) cells, mediate maintenance of immune homeostasis by
suppression of effector
cells such as CD4+ T cells, CDS+ T cells, B cells, NK cells, and NKT cells. In
some instances, Treg
cells are generated from the thymus (tTreg cells) or are induced from naïve T
cells in the periphery
(pTreg cells). In some cases, Treg cells are considered as the mediator of
peripheral tolerance.
Indeed, in one study, transfer of CD25-depleted peripheral CD4+ T cells
produced a variety of
autoimmune diseases in nude mice, whereas cotransfer of CD4+CD25+ T cells
suppressed the
development of autoimmunity (Sakaguchi, et al., "Immunologic self-tolerance
maintained by
activated T cells expressing IL-2 receptor alpha-chains (CD25)," 1 Immunol.
155(3): 1151-1164
(1995), the disclosure of each of which is herein incorporated by reference).
Augmentation of the
Treg cell population down-regulates effector T cell proliferation and
suppresses autoimmunity and
T cell anti-tumor responses.
[76] IL-2 signaling via the "intermediate-affinity" IL-2N3y complex
modulates the activation
and proliferation of CD8+ effector T (Teff) cells, NK cells, and NKT cells.
CD8+ Teff cells (also
known as cytotoxic T cells, Tc cells, cytotoxic T lymphocytes, CTLs, T-killer
cells, cytolytic T
cells, Tcon, or killer T cells) are T lymphocytes that recognize and kill
damaged cells, cancerous
cells, and pathogen-infected cells. NK and NKT cells are types of lymphocytes
that, similar to CD8
Teff cells, target cancerous cells and pathogen-infected cells.
[77] In some instances, IL-2 signaling is utilized to modulate T cell
responses and subsequently
for treatment of a cancer. For example, IL-2 is administered in a high-dose
form to induce
expansion of Teff cell populations for treatment of a cancer. However, high-
dose IL-2 further leads
to concomitant stimulation of Treg cells that dampen anti-tumor immune
responses. High-dose IL-2
also induces toxic adverse events mediated by the engagement of IL-2R alpha
chain-expressing
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cells in the vasculature, including type 2 innate immune cells (ILC-2),
eosinophils and endothelial
cells. This leads to eosinophilia, capillary leak and vascular leak syndrome
VLS).
[78] Adoptive cell therapy enables physicians to effectively harness a
patient's own immune
cells to fight diseases such as proliferative disease (e.g., cancer) as well
as infectious disease. The
effect of IL-2 signaling may be further enhanced by the presence of additional
agents or methods in
combination therapy. For example, epidermal growth factor receptor (EGFR) is a
cell surface
receptor overexpressed in many types of cancer. Activation of EGFR promotes
cell proliferation
and survival, as well as angiogenesis, leading to tumor growth and metastasis.
Cell growth and
angiogenesis may be regulated by blocking the binding of EGFR to epidermal
growth factor (EGF).
Anti-EGFR antibodies bind to the extracellular domain of EGFR and prevent EGF
from binding to
EGFR, thereby inhibiting downstream signal transduction cascade and leading to
decreased cell
growth. Anti-EGFR antibodies can cause the same effect by also competitively
inhibiting
transforming growth factor alpha (TGF-a) from binding to EGFR.
[79] Provided herein are methods of treating a cancer in a subject in need
thereof, comprising
administering to the subject a therapeutically effective amount of (a) an IL-2
conjugate, and (b) an
anti-EGFR antibody. Certain exemplary IL-2 polypeptides and IL-2 conjugates
are provided in
Table 1.
Table 1.
SEQ
Name Sequence
ID
NO:
APTSSSTKKTQLQLEEILLLDLQMILNGINNYKNPKLTRM
IL-2
LTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKN
(homo sapiens) 1
FHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEF
(mature form)
LNRWITFCQSIISTLT
IL-2 MYRMQLLSCIALSLALVTNSAPTSSSTKKTQLQLEHLLL
(homo sapiens) DLQMILNGINNYKNPKLTRMLTFKFYMPKKATELKHLQ
(precursor) CLEEELKPLEEVLNLAQSKNFHLRPRDLISNINVIVLELK 2
NCBI Accession No.: GSETTFMCEYADETATIVEFLNRWITFCQSIISTLT
AAB46883.1
PTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRML
TFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNF
aldesleuldn
HLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFL
NRWITFSQSIISTLT
APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRM
IL-2 C125S LTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKN
4
FHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEF
LNRWITFSQSIISTLT
IL-2 P65X APTSSSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRM 5
14
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LTFKFYMPKKATELKHLQCLEEELKXLEEVLNLAQSKN
FHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEF
LNRWITF SQ SITS TLT
APT S SSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRM
LTFKFYMPKKATELKHLQCLEEXLKPLEEVLNLAQ SKN
IL-2 E62X 6
FHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEF
LNRWITF SQ SITS TLT
APT S SSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRM
LTXKFYMPKKATELKEILQCLEEELKPLEEVLNLAQ SKN
IL-2 F42X 7
FHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEF
LNRWITF SQ SITS TLT
APT S SSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRM
LTFXFYMPKKATELKHLQCLEEELKPLEEVLNLAQ SKN
IL-2 K43 X 8
FHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEF
LNRWITF SQSIISTLT
APT S S STKKTQLQLEHLLLDLQMILNGINNYKNPXLTRM
LTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQ SKN
IL-2 K35X 9
FHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEF
LNRWITF SQSIISTLT
APT S SSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRM
LTFKFYMAKKATELMILQCLEEELK [AzK1LEEVLNLAQ
IL-2P65 [AzK] 10
SKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATI
VEFLNRWITF SQ SIISTLT
APT S SSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRM
LTFKF YMPKKATELKHLQCLEE [AzK I LKPLEEVLNLAQ
IL-2 E62[AzK] 11
SKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATI
VEFLNRWITF SQ SIISTLT
APT S SSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRM
LT [AzK1KF YMPKKATELKHL Q CLEEELKPLEEVLNL AQ
IL-2F42 [AzK] 12
SKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATI
VEFLNRWITF SQ SIISTLT
APT S SSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRM
LTF [AzK]FYMPKKATELKHLQCLEEELKPLEEVLNLAQ S
IL-2_1(43 [AzK] 13
KNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIV
EFLNRWITF SQ SIISTLT
APT S S STKKTQLQLEHLLLDLQMILNGINNYKNP [AzK1L
TRMLIFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQ
IL-2 K35 [AzK] 14
SKNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATI
VEFLNRWITF SQ SIISTLT
APT S S STKKTQLQLEHLLLDL QMILNGINNYKNPKLTRM
LTFKFYMPKKATELKHLQCLEEELK [AzK PE G1LEEVL
IL-2P65 [AzK PEG]
NLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYAD
15
ETATIVEFLNRWITF SQ SIISTLT
APT S SSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRM
LTFKFYMPKK A TELKHLQCLEE IA zK PE G1LKPLEEVL
IL-2E62 [AzK PEG] 16
NLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYAD
ETATIVEFLNRWITF SQ SIISTLT
IL-2F42 [AzK PEG] APT S S STKKTQLQLEHLLLDLQMILNGINNYKNPKLTRM 17
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LT[AzK PEG1KFYMPKKATELKHLQCLEEELKPLEEVL
NLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYAD
ETATIVEFLNRWITF SQ SIISTLT
APT S SSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRM
IL 2 K43 [A K PEG] LTF [AzK PE G[FYMPKKATELKHLQ CLEEELKPLEEVLN 18
- z
LAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADET
ATIVEFLNRWITF S Q S II S TL T
APT S S STKKTQLQLEHLLLDLQMILNGINNYKNP [AzK P
IL-2 K35 AzK PEG]
EG1LTRMLTFKFYMPKKATELKEILQCLEEELKPLEEVL
[
NLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYAD
19
ETATIVEFLNRWITF SQ SIISTLT
APT S SSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRM
IL- LTFKFYMPKKATELKHLQCLEEELK [AzK PE G51(1)1LEE
2 P 65 [AzK PEGS kD] VLNLAQ SKNFHLRPRDLISNINVIVLELKGSETTFMCEY
ADETATIVEFLNRWITF SQ S II S TL T
APT S SSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRM
IL- LTFKF YMPKKATELKHLQCLEE [AzK PEG5kaILKPLEE
2 E62[AzK PEG51(13] VLNLAQ SKNFHLRPRDLISNINVIVLELKGSETTFMCEY
21
ADETATIVEFLNRWITF SQ S II S TL T
APT S SSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRM
LT[AzK PE G5liD1KF YMAKKATELKIIL Q CLEEELKPLEE
2 F 42 [AzK PEG5kD] VLNLAQ SKNFHLRPRDLISNINVIVLELKGSETTFMCEY
22
ADETATIVEFLNRWITF SQ S II S TL T
APT S SSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRM
IL- LTF [AzK PE G5kD I F Y MPKKATELKHLQ CLEEELKPLEE 23
2_K43 [AzK PEGS kD] VLNLAQ SKNFHLRPRDLISNINVIVLELKGSETTFMCEY
ADETATIVEFLNRWITF SQ S II S TL T
APT SS STKKTQLQLEHLLLDLQMILNGINNYKNPIAzK P
IL- E G5kD1 L TRMLTFKF YMPKKATELKEIL Q CLEEELKFLEE
24
2 K35 [AzK PEGS kD] VLNLAQ SKNFHLRPRDLISNINVIVLELKGSETTFMCEY
ADETATIVEFLNRWITF SQ S II S TL T
APT S SSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRM
IL- LTFKFYMPKKATELKHLQCLEEELK [AzK PE G3Okal LE 25
2 P 65 [AzK PEG3 OkD] EVLNLAQ SKNFHLRPRDLISNINVIVLELKGSETTFMCEY
ADETATIVEFLNRWITF SQ S II S TL T
APT S SSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRM
IL- LTFKFYMPKKATELKHLQCLEE[AzK PEG30kD1LKPLE
26
2E62 [AzK PEG3 Old)] EVLNLAQ SKNFHLRPRDLISNINVIVLELKGSETTFMCEY
ADETATIVEFLNRWITF SQ S II S TL T
APT S S STKKTQL QLEHLLLDL QMILNGINNYKNPKLTRM
IL- LTIAzK PEG30kDiKFYIVIPKKATELKEILQCLEEELKPLE 27
2_F42 [AzK_PEG3 Old)] EVLNLAQ SKNFHLRPRDLISNINVIVLELKGSETTFMCEY
ADETATIVEFLNRWITF SQ S II S TL T
APT S SSTKKTQLQLHILLLDLQMILNGINNYKNPKLTRM
IL- LTF IA zK PE G30k1/1 F YMPKK A TELKHL Q
CLEEELKPLE
28
2 K43 [AzK PEG3 OW] EVLNLAQ SKNFHLRPRDLISNINVIVLELKGSETTFMCEY
ADETATIVEFLNRWITF SQ S II S TL T
IL- APT S SSTKKTQLQLEHLLLDLQMILNGINNYKNP [AzK P 29
16
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2 K35[AzK PEG30kD] EG30kDILTRMLTFKFYMPKKATELKHLQCLEEELKPLE
EVLNLAQ SKNFHLRPRDLISNINVIVLELKGSETTFMCEY
ADETATIVEFLNRWITF SQ SIISTLT
PT S SSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRML
TFKFYMPKKATELKHLQCLEEELKXLEEVLNLAQ SKNF
IL-2 P65X-1 30
HLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFL
NRWITF SQ SIISTLT
PT S SSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRML
TFKFYMPKKATELKHLQCLEEXLKPLEEVLNLAQSKNF
IL-2 E62X-1 31
HLRPRDLISNINVIVLELKGS ET TFMCEYADETATIVEFL
NRWITF SQ SITS TL T
PT S SSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRML
TXKFYMPKKATELKHLQCLEEELKPLEEVLNLAQ SKNF
IL-2 F42X-1 32
HLRPRDLISNINVIVLELKGS ET TFMCEYADETATIVEFL
NRWITF SQ SIISTLT
PT S SSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRML
TFXFYMPKKATELKHLQCLEEELKPLEEVLNLAQ SKNF
IL-2 K43X-1 33
EILRPRDLISNINVIVLELKGS ET TFMCEYADETATIVEFL
NRWITF SQ SIISTLT
PT S SSTKKTQLQLEHLLLDLQMILNGINNYKNPXLTRML
TFKFYMPKKATELKIILQCLEEELKPLEEVLNLAQ SKNF
IL-2 K35X-1 34
HLRPRDLISNINVIVLELKGS ET TFMCEYADETATIVEFL
NRWITF SQ SIISTLT
PT S SSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRML
TFKF YMPKKATELKHL Q CLEEELK I AzK I LEE VLNLAQ S
IL-2 P 65 [AzKl -1
35
KNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIV
EFLNRWITF SQ SIISTLT
PT S SSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRML
TFKFYMPKKATELKHLQCLEE[AzKILKPLEEVLNLAQ S
1L-2E62 [AzK] -1
36
KNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIV
EFLNRWITF SQ SIISTLT
PT S SSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRML
T[AzKIKFYMPKKATELKHLQCLEEELKPLEEVLNLAQ S
IL-2F42 [AzK]-1 37
KNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIV
EFLNRWITF SQ SIISTLT
PT S SSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRML
IF [AzKlFYMPKKATELKHLQCLEEELKPLEEVLNLAQS
IL-2 K43 [AzK] -1
38
KNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIV
EFLNRWITF SQ SIISTLT
PT S S STKKTQLQLEHLLLDL QMILNGINNYKNP [AzK1LT
RMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQ S
IL-2 K35 [AzK] -1
39
KNFHLRPRDLISNINVIVLELKGSETTFMCEYADETATIV
EFLNRWITF SQ SIISTLT
PT S SSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRML
IL- TFKFYMPKK A TELKHLQ CLEEELK 1-AzK Ll PEG] LEEV
2 P65 [AzK Ll PEG]-1 LNLAQ SKNFHLRPRDL1SNINVIVLELKGSETTFMCEYA
DETATIVEFLNRWITF SQ SIISTLT
IL-2_E62 [AzK PT S
SSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRML 41
17
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Ll PEGH TFKFYMPKKATELKHLQCLEE1AzK Li PEGILKPLEEV
LNLAQ SKNFHLRPRDLISNINVIVLELKGSETTFMCEYA
DETATIVEFLNRWITFSQSIISTLT
PT S SSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRML
IL-2 F42 [AzK TI-AzK Li PEG1KFYMPKKATELKHLQCLEEELKPLEEV
42
Li PEGH LNLAQSKNFHLRPRDL1SNINVIVLELKGSETTFMCEYA
DETATIVEFLNRWITFSQSIISTLT
PT S SSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRML
IL-2 K43 [AzK TF [AzK Li PEGIF YMPKKATELKHLQCLEEELKPLEEV
43
Ll PEG] -1 LNLAQ SKNFHLRPRDLISNINVIVLELKGSETTFMCEYA
DETATIVEFLNRWITFSQSIISTLT
PT S SSTKKTQLQLEHLLLDLQMILNGINNYKNP [AzK Li
IL-2 K35 [AzK PE GI LTRMLTFKFYMPKKATELKHLQ CLEEELKPLEEV
44
L 1 PEG] -1 LNLAQ SKNFHLRPRDLISNINVIVLELKGSETTFMCEYA
DETATIVEFLNRWITFSQSIISTLT
PT S SSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRML
IL-2P65 [AzK TFKFYMPKKATELKHLQCLEEELKIAzK Li PE G5kal L
Ll PEG51cD] -1 EEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCE
YADETATIVEFLNRWITF SQSIISTLT
PT S SSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRML
IL-2 E62[AzK TFKF YMPKKATELKIILQ CLEF [AzK Li PE G5kal LKPL
46
Ll PEG5kID] -1 EEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCE
YADETATIVEFLNRWITF SQSIISTLT
PT S SSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRML
IL-2F42 [AzK T [AzK Li PE G5 kD I KF YMPKKATELKHLQCLEEELKPL
47
Ll PEG5k1D] -1 EEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCE
YADETATIVEFLNRWITF SQSIISTLT
PT S SSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRML
IL-2 K43 [AzK TF [AzK Li PEG5kUIFYMPKKATELKFILQCLEEELKPL
48
Ll PEG5k1D] -1 EEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCE
YADETATIVEFLNRWITF SQSIISTLT
PT S SSTKKTQLQLEHLLLDLQMILNGINNYKNP [AzK Li
IL-2 K35 [AzK PE G5kD1LTRML TFKF YMPKKATELKHLQ C LEEELKPL
49
Ll PEG5kID] -1 EEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCE
YADETATIVEFLNRWITF SQ SIISTLT
PT S SSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRML
IL-2_P65 [AzK TFKEYMPKKATELKHL Q CLEEELK [AzK Ll PE G30kD1
50
Ll PEG301(13]-1 LEEVLNLAQ SKNFHLRPRDLISNINVIVLELKGSETTFMC
EYADETATIVEFLNRWITF SQ SIISTLT
PT S S STKKTQL QLEHLLLDL QMILNGINNYKNPKLTRML
IL-2 E62[AzK TFKF YMPKKATELKHLQ CLEE [AzK L1 PEG3OlinlLKP
51
Ll_PEG301(D] -1 LEEVLNLAQ SKNFHLRPRDLISNINVIVLELKGSETTFMC
EYADETATIVEFLNRWITF SQ SIISTLT
PT S SSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRML
IL-2 F42 [ AzK T [AzK L 1 PEG30kD1KFYMPKK A TELKHLQCLEEELKP
52
Ll PEG30k13]-1 LEEVLNLAQ SKNFHLRPRDLISNINVIVLELKGSETTFMC
EYADETATIVEFLNRWITF SQ SIISTLT
IL-2_K43 [AzK PT S SSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRML 53
18
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Li PEG30kD]-1 TFIAzKLJPEG3OkDIFYMPKKATELKHLQCLEEELKPL
EEVLNLAQ SKNFHLRPRDLISNINVIVLELKGSETTFMCE
YADETATIVEFLNRWITF SQ SIISTLT
PT S SSTKKTQLQLEHLLLDLQMILNGINNYKNP [AzK Li
IL-2 K35 [AzK PEG30kUILTRMLTFKFYMPKKATELKHLQCLEEELKP
54
Li PEG30kD]-1 LEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMC
EYADETATIVEFLNRWITF SQ SIISTLT
APT S SSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRM
IL- LTFKEYMPKKATELKEILQCLEEELK [AzK Li PEG-I LEE
2P65 [AzK Ll PEG]-2 VLNLAQ SKNFHLRPRDLISNINVIVLELKGSETTFMCEY
ADETATIVEFLNRWITF SQ SIISTLT
APT S SSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRM
IL-2 E62[AzK L TFKF YMPKKA TELKHLQCLEE [AzK Li PEG] LKPLEE
Ll PEG] -2 VLNLAQ
SKNFHLRPRDLISNINVIVLELKGSETTFMCEY 56
ADETATIVEFLNRWITF SQ SIISTLT
APT S SSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRM
IL-2 F42 [AzK LT[AzK Li PEGIKFYMPKKATELKHLQCLEEELKPLEE
57
Li _PEG] -2 VLNLAQ SKNFHLRPRDLISNINVIVLELKGSETTFMCEY
ADETATIVEFLNRWITF SQ SIISTLT
APT S SSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRM
IL-2 K43 [AzK LTF [AzK Li PEGWYMPKKATELKITLQCLEEELKPLEE
58
Li _PEG] -2 VLNLAQ SKNFHLRPRDLISNINVIVLELKGSETTFMCEY
ADETATIVEFLNRWITF SQ SIISTLT
APT S S STKKTQLQLEHLLLDLQMILNGINNYKNP [AzK L
IL -2 K35 [AzK 1 PEG I LTRMLTFKF Y MPKKATELKHLQCLEEELKPLEE
59
Li _PEG] -2 VLNLAQ SKNFHLRPRDLISNINVIVLELKGSETTFMCEY
ADETATIVEFLNRWITF SQ SIISTLT
APT S SSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRM
IL-2 P65 [AzK LTFKFYMPKKATELKHLQCLEEELK[AzK Li PEG5kD1
Li PEG5kD] -2 LEE VLNLAQ SKNFHLRPRDLISNINVIVLELKGSETTFMC
EYADETATIVEFLNRWITF SQ SIISTLT
APT S SSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRM
IL-2 E62[AzK LTFKF YMPKKATELKHL Q CLEE [AzK Li PEG5kIVILKP
61
Li PEG5kD] -2 LEE VLNLAQ SKNFHLRPRDLISNINVIVLELKGSETTFMC
EYADETATIVEFLNRWITF SQ SIISTLT
APT S SSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRM
IL-2 F42 [AzK LT[AzK L 1 PEG 5kD1KF YMPKKATELKHL QCLEEELKP
62
Li PEG5kD] -2 LEE VLNLAQ SKNFHLRPRDLISNINVIVLELKGSETTFMC
EYADETATIVEFLNRWITF SQ SIISTLT
APT S S STKKTQLQLEHLLLDL QMILNGINNYKNPKLTRM
IL-2K43 [AzK LTF [AzK L1 PEGS kD1FYMPKKATELKHLQ CLEEELKP
63
Li _PEG5kD] -2 LEE VLNLAQ SKNFHLRPRDLISNINVIVLELKGSETTFMC
EYADETATIVEFLNRWITF SQ SIISTLT
APT S S STKKTQLQLEULLLDLQMILNGINNYKNP [AzK L
IL-2 K35 [AzK 1 PEG5ka1LTRMLTFKEYMPKK A TELKHLQ CLEEELKP
64
Li PEG5kD] -2 LEE VLNLAQ SKNFHLRPRDLISNINVIVLELKGSETTFMC
EYADETATIVEFLNRWITF SQ SIISTLT
IL-2 P65 [AzK APT S SSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRM 65
19
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Li PEG30kD]-2 LTFKFYMPKKATELKHLQCLEEELKIAzK Li PEG30kD
1LEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFM
CEYADETATIVEFLNRWITF SQSIISTLT
APT S SSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRM
IL-2 E62 [AzK LTFKFYMPKKATELKHLQCLEE [AzK Li PEG30k1/1 LK
66
Li PEG30kD]-2 PLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFM
CEYADETATIVEFLNRWITF SQSIISTLT
APT S SSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRM
IL-2 F42 [AzK LT [AzK Li PEG30kD1KFYMPKKATELKHLQCLEEELK
67
L 1 PEG301(13]-2 PLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFM
CEYADETATIVEFLNRWITF SQSIISTLT
APT S SSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRM
IL-2 K43 [AzK LTF [AzK Li PE G30 kDiFYMPKKATELKEILQ C LEEELKP
68
L 1 PEG30kD]-2 LEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMC
EYADETATIVEFLNRWITF SQSIISTLT
APT S S STKKTQLQLEHLLLDLQMILNGINNYKNP [AzK L
IL-2 K35 [AzK 1 PEG301iD1LTRMLTFKFYMPKKATELKHLQCLEEELK
69
L 1 PEG301cD]-2 PLEEVLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFM
CEYADETATIVEFLNRWITF SQSIISTLT
PT S SSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRML
TFKFYMPKKATELKITL Q C LEEFLK [AzK PE Gl LEEVLN
IL-2P65 [AzK PEG] -1
LAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADET 70
ATIVEFLNRWITF SQSIISTLT
PT S SSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRML
IL -2 E62 [AzK PEG TFKF YMPKKATELKHL Q CLEE [Az K PE GILKPLEEVLN
LAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADET 71
]-1
ATIVEFLNRWITF SQSIISTLT
PT S SSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRML
IL- F42 T[AzK PEG1KFYMPKKATELKELLQCLEEELKPLEEVLN
2 [AzK PEG]-1
LAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYADET 72
ATIVEFLNRWITF SQSIISTLT
PT S SSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRML
IL -2 K43 [A K PEG 1 TF [AzK PE G1F YMPKKATELKEILQ CLEEELKPLEEVLNL
_ z _ ]-
AQ SKNFHLRPRDLISNINVIVLELKGSETTFMCEYADET
ATIVEFLNRWITF SQSIISTLT
PT S SSTKKTQLQLEHLLLDLQMILNGINNYKNP [Az K PE
WLTRMLTFKFYMPKKATELKHLQCLEEELKPLEEVLNL
IL-2 K35 [AzK PEG] -1 74
AQ SKNFHLRPRDLISNINVIVLELKGSETTFMCEYADET
ATIVEFLNRWITF SQSIISTLT
PT S S STKKTQL QLEHLLLDL QMILNGINNYKNPKLTRML
IL-2P65 [AzK TFKFYMPKKATELKHLQCLEEELK[AzK PE G 5kDl LEEV 75
PEG51(1)]-1 LNLAQ SKNFHLRPRDLISNINVIVLELKGSETTFMCEYA
DETATIVEFLNRWITF SQSIISTLT
PT S SSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRML
IL- TFKFYMPKK A TELKHL Q CLEE [A z K PE G5k1/ILKPLEEV 76
2 E62[AzK PEGS kD] -1 LNLAQ SKNFHLRPRDLISNINVIVLELKGSETTFMCEYA
DETATIVEFLNRWITF SQSIISTLT
IL- PT SS STKKTQLQLEHLLLDLQMILNGINNYKNPKLTRML 77
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2 F42[AzK PEG5kID]-1 TIAz K PEG5kDIKFYMPKKATELKHLQCLEEELKPLEEV
LNLAQ SKNFHLRPRDLISNINVIVLELKGSETTFMCEYA
DETATIVEFLNRWITF SQSIISTLT
PT S SSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRML
IL- TF [AzK PE G5k1)1F YMPKKATELKHL Q CLEEELKPLEEV 78
2 K43[AzK PEG5kM-1 LNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEYA
DETATIVEFLNRWITF SQSIISTLT
PT S SSTKKTQLQLEHLLLDLQMILNGINNYKNP [Az K PE
IL-
G5ka1LTRMLTFKFYMPKKATELKHLQCLEEELKPLEEV 79
2 K35 [AzK PEG51(D1-1 LNLAQ SKNFHLRPRDLISNINVIVLELKGSETTFMCEYA
DETATIVEFLNRWITF SQSIISTLT
PT S SSTKKTQLQLETILLLDLQMILNGINNYKNPKLTRML
IL-
TFKFYMPKKATELKHLQCLEEELK[AzK PE G30 kill LEE
2 1365[AzK PEG30kD]-
80
1 VLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEY
ADETATIVEFLNRWITF SQ S II S TL T
PT S S STKKTQL QLEIILLLDL QMILNGINNYKNPKLTRML
IL-
TFKFYMPKKATELKHLQCLEE [Az K PE G30kEll LKPLEE
2 E62[AzK PEG30k13]-
VLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEY
81
1
ADETATIVEFLNRWITF SQSIISTLT
PT S SSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTRML
IL-
T [AzK PEG30kD1KFYMPKKATELKIILQCLEEELKPLEE
2F 42 [AzK PEG3 Oki]-
1
82
VLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEY
ADETATIVEFLNRWITF SQSIISTLT
PT S S STKKTQL QLEIILLLDL QMILNGINNYKNPKLTRML
IL-
TF [AzK PE G30 kD IF YMPKKATELKHLQCLEEELKPLEE
2K43 [AzK PEG301c13]-83
1 VLNLAQSKNFHLRPRDLISNINVIVLELKGSETTFMCEY
ADETATIVEFLNRWITF SQSIISTLT
PT S SSTKKTQLQLEHLLLDLQMILNGINNYKNP [Az K PE
IL-
G30 kal LTRMLTFKFYMPKKATELKELLQ CLEEELKPLEE
2 K35 [AzK PEG301(13]-
84
VLNLAQ SKNFHLRPRDLISNINVIVLELKGSETTFMCEY
1
ADETATIVEFLNRWITF SQ S II S TLT
APT S S STKKTQLQLEHLLLDLQMILNGINNYKNPKLTRM
LTFKXYMPKKATELKHLQCLEEELKPLEEVLNLAQSKN
IL-2 _F44X 85
FHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEF
LNRWITFCQ SIISTLT
PT S S STKKTQLQLEHLLLDLQMILNGINNYKNPKLTRML
TFKXYMPKKATELKHLQCLEEELKPLEEVLNLAQ SKNF
IL-2 F44X-1 86
EILRPRDLI SNINVIVLELKGS ET TFMCEYADETATIVEFL
NRWITFCQ SIISTLT
APT S S STKKTQLQLEHLLLDLQMILNGINNYKNPKLTXM
LTFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQ SKN
IL-2 R38X 87
FHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEF
LNRWITFCQSIISTLT
PT S SSTKKTQLQLEHLLLDLQMILNGINNYKNPKLTXML
TFKFYMPKK A TELKHL Q CLEEELKPLEEVLNL A Q SKNF
IL-2 R38X-1 88
HLRPRDLI SNINVIVLELKGS ET TFMCEYADETATIVEFL
NRWITFCQ SIISTLT
IL-2 T41X APT S S
STKKTQLQLEHLLLDLQMILNGINNYKNPKLTRM 89
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LXFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKN
FHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEF
LNRWITFCQ SIISTLT
PT S S STKKTQLQLEHLLLDLQMILNGINNYKNPKLTRML
IL 2 T41X 1
XFKFYMPKKATELKHLQCLEEELKPLEEVLNLAQSKNF 90
-
- HLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEFL
NRWITFCQ SIISTLT
APT S S STKKTQLQLEHLLLDLQMILNGINNYKNPKLTRM
LTFKFYMPKKATELKHLQCLEEELKPLEXVLNLAQ SKN
IL-2 E68X 91
FHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEF
LNRWITFCQ SIISTLT
PT S S STKKTQLQLEHLLLDLQMILNGINNYKNPKLTRML
IL 2 TFKFYMPKKATELKHLQCLEEELKPLEXVLNLAQSKNF 92
E68X- 1 -
HLRPRDLI SNINVIVLELKGS ET TFMCEYADETATIVEFL
NRWITFCQ SIISTLT
APT S S STKKTQLQLEFILLLDLQMILNGINNYKNPKLTRM
LTFKFXMPKKATELKHLQCLEEELKPLEEVLNLAQ SKN
IL-2 Y45X 93
FHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEF
LNRWITFCQ SIISTLT
PT S S STKKTQLQLEHLLLDLQMILNGINNYKNPKLTRIVIL
IL -2 Y45X 1 TFKFXMAKKATELKULQCLEEELKPLEEVLNLAQ SKNF 94
- HLRPRDLI SNINVIVLELKGS ET TFMCEYADETATIVEFL
NRWITFCQ SIISTLT
APT S S STKKTQLQLEHLLLDLQMILNGINNYKNPKLTRIVI
IL -2 69X LTFKF YMPKKATELKHLQCLEEELKPLEEXLNLAQ SKN 95
V
FHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEF
LNRWITFCQ SIISTLT
PT SS STKKTQLQLEHLLLDLQMILNGINNYKNPKLTRML
IL -2 V69X 1 TFKFYMPKKATELKHLQCLEEELKPLEEXLNLAQ SKNF 96
- HLRPRDLI SNINVIVLELKGS ET TFMCEYADETATIVEFL
NRWITFCQ SIISTLT
APT S S STKKTQLQLEHLLLDLQMILNGINNYKNPKLTRM
IL -2 L72X
LTFKFYMPKKATELKHLQCLEEELKPLEEVLNXAQ SKN
_ 97
FHLRPRDLISNINVIVLELKGSETTFMCEYADETATIVEF
LNRWITFCQ SIISTLT
PT S S STKKTQLQLEHLLLDLQMILNGINNYKNPKLTRML
1L L72X 1 TFKFYMPKKATELKHLQCLEEELKPLEEVLNXAQSKNF 98
-2
- HLRPRDLI SNINVIVLELKGS ET TFMCEYADETATIVEFL
NRWITFCQ SIISTLT
X = site comprising an unnatural amino acid.
[AA(' = N6-((2-azidoethoxy)-carbonyl)-L-lysine, haying Chemical Abstracts
Registry No.
1167421-25-1.
[AzK_PEGI = N6-((2-azidoethoxy)-carbonyl)-L-lysine stably-conjugated to PEG
via DBCO-
mediated click chemistry, to form a compound comprising a structure of Formula
(II) or Formula
(III). For example, if specified, PEG5kD indicates a linear polyethylene
glycol chain with an
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average molecular weight of 5 kiloDaltons, capped with a methoxy group. The
ratio of regioisomers
generated from the click reaction is about 1:1 or greater than 1:1. The term
"DBCO" means a
chemical moiety comprising a dibenzocyclooctyne group.
[AzK_ Ll _ PEG] = N6-((2-azidoethoxy)-carbonyl)-L-lysine stably-conjugated to
PEG via DBCO-
mediated click chemistry to form a compound comprising a structure of Formula
(IV) or Formula
(V). For example, if specified, PEG5kD indicates a linear polyethylene glycol
chain with an average
molecular weight of 5 kiloDaltons, capped with a methoxy group. The ratio of
regioisomers
generated from the click reaction is about 1:1 or greater than 1:1. The term
"DBCO" means a
chemical moiety comprising a dibenzocyclooctyne group.
[80] In some embodiments, the IL-2 conjugate comprises the amino
acid sequence of SEQ ID
NO: 3 in which at least one amino acid residue in the IL-2 conjugate is
replaced by the structure of
Formula (I):
x N N
0 1\1õ
(I)
wherein:
N N
Z is CH2 and Y is 0 0
N N
Y is CH2 and Z is 0 0ow
=
0
Z i s CH2 and Y is 0 ;or
ow
Y is CH2 and Z is 0 =
W is a PEG group having an average molecular weight selected from about 5kDa,
10kDa, 15kDa,
20kDa, 25kDa, 30kDa, 351(Da, 40kDa, 45kDa, 50kDa, and 60Wa;
q is 1, 2, or 3;
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X has the structure:
N H
"µo.ss
=
X-1 indicates the point of attachment to the preceding amino acid residue; and
X+1 indicates the point of attachment to the following amino acid residue;
wherein the position of the structure of Formula (I) in the amino acid
sequence of the IL-2
conjugate is selected from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44,
V68, and L71.
[81] In any of the embodiments or variations of Formula (I) described
herein, the IL-2
conjugate is a pharmaceutically acceptable salt, solvate, or hydrate. In some
embodiments, the IL-2
conjugate is a pharmaceutically acceptable salt. In some embodiments, the IL-2
conjugate is a
solvate. In some embodiments, the IL-2 conjugate is a hydrate.
[82] In some embodiments or variations of Formula (I) described herein, X
is an L-amino acid.
N' N
[83] In
some embodiments of Formula (I), Z is CH2 and Y is 0 0
In some embodiments of Formula (I), Y is CH2 and Z is 0 0
. In some
ow
embodiments of Formula (I), Z is CH2 and Y is 0 . In
some
0
11=))1
embodiments of Formula (I), Y is CH2 and Z is 0
[84] In some embodiments of Formula (I), q is 1. In some embodiments of
Formula (I), q is 2.
In some embodiments of Formula (I), q is 3.
[85] In some embodiments of Formula (I), q is 1 and the structure of
Formula (I) is the
structure of Formula (Ia):
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0 Nõ
`I(
Formula (Ia);
wherein.
4 "
Z is CH2 and Y is 0 0 =
Y is CH2 and Z is 0 0 =
0
Z is CH2 and Y is 0 ;or
'sr 0
Y is CH2 and Z is 0 =
W is a PEG group having an average molecular weight selected from about 5kDa,
10kDa, 15kDa,
20kDa, 251(Da, 30kDa, 35kDa, 40kDa, 45kDa, 50kDa, and 60kDa; and
X has the structure:
1E1
X-1 indicates the point of attachment to the preceding amino acid residue; and
X+1 indicates the point of attachment to the following amino acid residue;
wherein the position of the structure of Formula (Ia) in the amino acid
sequence of the IL-2
conjugate is selected from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44,
V68, and L71.
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414s1
N
[86] In some embodiments of Formula (Ia), Z is CH2 and Y is 0
7-0 N
. In some embodiments of Formula (Ia), Y is CH2 and Z is 0 0
. In
.rt'd 0
17x N 1r-A N ()W
some embodiments of Formula (Ia), Z is CH2 and Y is 0 .
In some
Jt" 0
N
embodiments of Formula (Ia), Z is CH2 and Y is 0 . In
some
0
1,.xN
embodiments of Formula (Ia), Y is CH2 and Z is 0
[87] In some embodiments, the PEG group has an average molecular weight
selected from
about 5kDa, 10kDa, 20 kDa and 30kDa. In some embodiments, the PEG group has an
average
molecular weight of about 5kDa. In some embodiments, the PEG group has an
average molecular
weight of about 10kDa. In some embodiments, in the PEG group has an average
molecular weight
of about 15kDa. In some embodiments, the PEG group has an average molecular
weight of about
20kDa. In some embodiments, the PEG group has an average molecular weight of
about 25kDa. In
some embodiments, the PEG group has an average molecular weight of about
30kDa. In some
embodiments, the PEG group has an average molecular weight of about 35kDa. In
some
embodiments, the PEG group has an average molecular weight of about 40kDa. In
some
embodiments, the PEG group has an average molecular weight of about 45kDa. In
some
embodiments, the PEG group has an average molecular weight of about 50kDa. In
some
embodiments, the PEG group has an average molecular weight of about 60kDa.
[88] In some embodiments, the structure of Formula (I) in the amino acid
sequence of the IL-2
conjugate is selected from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44,
V68, and L71,
wherein the position of the structure of Formula (I) in the amino acid
sequence of the IL-2
conjugate is in reference to the positions in SEQ ID NO: 3. In some
embodiments, the position of
the structure of Formula (I) in the amino acid sequence of the IL-2 conjugate
is selected from F41,
E61, and P64, wherein the position of the structure of Formula (I) in the
amino acid sequence of the
IL-2 conjugate is in reference to the positions in SEQ ID NO: 3. In some
embodiments, the structure
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of Formula (I) in the amino acid sequence of the IL-2 conjugate is K34,
wherein the position of the
structure of Formula (I) in the amino acid sequence of the IL-2 conjugate is
in reference to the
positions in SEQ ID NO: 3. In some embodiments, the structure of Formula (I)
in the amino acid
sequence of the IL-2 conjugate is F41, wherein the position of the structure
of Formula (I) in the
amino acid sequence of the IL-2 conjugate is in reference to the positions in
SEQ ID NO: 3. In
some embodiments, the structure of Formula (I) in the amino acid sequence of
the IL-2 conjugate is
selected from F43, wherein the position of the structure of Formula (I) in the
amino acid sequence
of the IL-2 conjugate is in reference to the positions in SEQ ID NO: 3. In
some embodiments, the
structure of Formula (I) in the amino acid sequence of the IL-2 conjugate is
K42, wherein the
position of the structure of Formula (I) in the amino acid sequence of the IL-
2 conjugate is in
reference to the positions in SEQ ID NO: 3. In some embodiments, the structure
of Formula (I) in
the amino acid sequence of the IL-2 conjugate is E61, wherein the position of
the structure of
Formula (I) in the amino acid sequence of the IL-2 conjugate is in reference
to the positions in SEQ
ID NO: 3. In some embodiments, the structure of Formula (I) in the amino acid
sequence of the IL-
2 conjugate is P64, wherein the position of the structure of Formula (I) in
the amino acid sequence
of the IL-2 conjugate is in reference to the positions in SEQ ID NO: 3. In
some embodiments, the
structure of Formula (I) in the amino acid sequence of the IL-2 conjugate is
R37, wherein the
position of the structure of Formula (I) in the amino acid sequence of the IL-
2 conjugate is in
reference to the positions in SEQ ID NO: 3. In some embodiments, the structure
of Formula (I) in
the amino acid sequence of the IL-2 conjugate is T40, wherein the position of
the structure of
Formula (I) in the amino acid sequence of the IL-2 conjugate is in reference
to the positions in SEQ
ID NO: 3. In some embodiments, the structure of Formula (I) in the amino acid
sequence of the IL-
2 conjugate is selected from E67, wherein the position of the structure of
Formula (I) in the amino
acid sequence of the IL-2 conjugate is in reference to the positions in SEQ ID
NO: 3. In some
embodiments, the structure of Formula (I) in the amino acid sequence of the IL-
2 conjugate is Y44,
wherein the position of the structure of Formula (I) in the amino acid
sequence of the IL-2
conjugate is in reference to the positions in SEQ ID NO: 3. In some
embodiments, the structure of
Formula (I) in the amino acid sequence of the IL-2 conjugate is V68, wherein
the position of the
structure of Formula (I) in the amino acid sequence of the IL-2 conjugate is
in reference to the
positions in SEQ ID NO: 3. In some embodiments, the structure of Formula (I)
in the amino acid
sequence of the IL-2 conjugate is L71, wherein the position of the structure
of Formula (I) in the
amino acid sequence of the IL-2 conjugate is in reference to the positions in
SEQ ID NO: 3.
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1891 In some embodiments, the IL-2 conjugate comprises the amino
acid sequence of any one
of SEQ ID NOS: 15-19, wherein [AzK PEG] has the structure of Formula (II) or
Formula (III), or a
mixture of Formula (II) and Formula
X N
0 NI H
N N
_ w
0 0
Formula (II);
0 0
y0 N
Formula (III);
W is a PEG group having an average molecular weight selected from about 5kDa,
10kDa, 15kDa,
20kDa, 25kDa, 30kDa, 35kDa, 40kDa, 45kDa, 50kDa, and 60kDa;
q is 1,2, or 3; and
X has the structure:
ell
,s
0 is's-
=
X-1 indicates the point of attachment to the preceding amino acid residue; and
X+1 indicates the point of attachment to the following amino acid residue.
1901 Here and throughout, the structure of Formula (H) encompasses
pharmaceutically
acceptable salts, solvates, or hydrates thereof. Here and throughout, the
structure of Formula (III)
encompasses pharmaceutically acceptable salts, solvates, or hydrates thereof
In some
embodiments, the IL-2 conjugate is a pharmaceutically acceptable salt,
solvate, or hydrate.
1911 In some embodiments, q is 1 and the structures of Formula (II)
and Formula (HI) are the
structures of Formula (Ha) and Formula (Ma):
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0 Nõ
N N
0 0
Formula (Ha);
0 0
x N
I I N N
0 NI H
Formula (IIIa);
wherein:
W is a PEG group having an average molecular weight selected from about 5kDa,
10kDa, 15kDa,
20kDa, 25kDa, 30kDa, 35kDa, 40kDa, 45kDa, 50kDa, and 60kDa; and
X has the structure:
ell
cs 47"
N H
04 X + 1
=
X-1 indicates the point of attachment to the preceding amino acid residue; and
X+1 indicates the point of attachment to the following amino acid residue.
[92] In some embodiments, the [A7K_PEG] is a mixture of Formula (II) and
Formula (III). In
some embodiments, the [AzK PEG] is a mixture of Formula (Ha) and Formula (Ma).
[93] In some embodiments, the [AzK PEG] has the structure of Formula (II).
In some
embodiments, the [AzK PEG] has the structure of Formula (Ha).
[94] In some embodiments, the [AzK PEG] has the structure of Formula (III).
In some
embodiments, the [AzK_PEG] has the structure of Formula (Ma).
[95] In some embodiments, the IL-2 conjugate has the amino acid sequence of
SEQ ID NO: 15.
In some embodiments, W in the structure of Formula (II) is a PEG group having
an average
molecular weight selected from about 5kDa, 10kDa, 15kDa, 20kDa, 25kDa, and
30kDa. In some
embodiments, W in the structure of Formula (II) is a PEG group having an
average molecular
weight selected from about 5kDa and 30kDa. In some embodiments, W in the
structure of Formula
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(II) is a PEG group having an average molecular weight of about 5kDa. In some
embodiments, W in
the structure of Formula (II) is a PEG group having an average molecular
weight of about 30kDa.
[96] In some embodiments, the IL-2 conjugate has the amino acid sequence of
SEQ ID NO: 16.
In some embodiments, W in the structure of Formula (II) is a PEG group having
an average
molecular weight selected from about 5kDa, 10kDa, 15kDa, 20kDa, 25kDa, and
30kDa. In some
embodiments, W in the structure of Formula (II) is a PEG group having an
average molecular
weight selected from 5 about kDa and 30kDa. In some embodiments, W in the
structure of Formula
(II) is a PEG group having an average molecular weight of about 5kDa. In some
embodiments, W
in the structure of Formula (II) is a PEG group having an average molecular
weight of about 30kDa.
[97] In some embodiments, the IL-2 conjugate has the amino acid sequence of
SEQ ID NO: 17.
In some embodiments, W in the structure of Formula (II) is a PEG group having
an average
molecular weight selected from about 5kDa, 10kDa, 15kDa, 20kDa, 25kDa, and
30kDa. In some
embodiments, W in the structure of Formula (II) is a PEG group having an
average molecular
weight selected from about 5kDa and 30kDa. In some embodiments, W in the
structure of Formula
(II) is a PEG group having an average molecular weight of about 5kDa. In some
embodiments, W in
the structure of Formula (II) is a PEG group having an average molecular
weight of about 30kDa.
[98] In some embodiments, the IL-2 conjugate has the amino acid sequence of
SEQ ID NO: 18.
In some embodiments, W in the structure of Formula (II) is a PEG group having
an average
molecular weight selected from about 5kDa, 10kDa, 15kDa, 20kDa, 25kDa, and
30kDa. In some
embodiments, W in the structure of Formula (II) is a PEG group having an
average molecular
weight selected from about 5kDa and 30kDa. In some embodiments, W in the
structure of Formula
(II) is a PEG group having an average molecular weight of about 5kDa. In some
embodiments, W in
the structure of Formula (II) is a PEG group having an average molecular
weight of about 30Wa.
[99] In some embodiments, the IL-2 conjugate has the amino acid sequence of
SEQ ID NO: 19.
In some embodiments, W in the structure of Formula (II) is a PEG group having
an average
molecular weight selected from about 5kDa, 10kDa, 15kDa, 20kDa, 25kDa, and
30kDa. In some
embodiments, W in the structure of Formula (II) is a PEG group having an
average molecular
weight selected from about 5kDa and 30kDa. In some embodiments, W in the
structure of Formula
(II) is a PEG group having an average molecular weight of about 5kDa. In some
embodiments, Win
the structure of Formula (II) is a PEG group having an average molecular
weight of about 30kDa.
11001 In some embodiments, the [AzK_PEG] has the structure of
Formula (Ill). In some
embodiments, the [AzK PEG] has the structure of Formula (IIIa).
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1101] In some embodiments, the IL-2 conjugate has the amino acid
sequence of SEQ ID NO: 15.
In some embodiments, W in the structure of Formula (III) is a PEG group having
an average
molecular weight selected from about 5kDa, 10kDa, 15kDa, 20kDa, 25kDa, and
30kDa. In some
embodiments, W in the structure of Formula (III) is a PEG group having an
average molecular
weight selected from about 5kDa and 30kDa. In some embodiments, W in the
structure of Formula
(III) is a PEG group having an average molecular weight of about 5kDa. In some
embodiments, W
in the structure of Formula (III) is a PEG group having an average molecular
weight of about
30kDa.
[102] In some embodiments, the IL-2 conjugate has the amino acid sequence
of SEQ ID NO: 16.
In some embodiments, W in the structure of Formula (III) is a PEG group having
an average
molecular weight selected from about 5kDa, 10kDa, 15kDa, 20kDa, 25kDa, and
30kDa. In some
embodiments, W in the structure of Formula (III) is a PEG group having an
average molecular
weight selected from about 5kDa and 30kDa. In some embodiments, W in the
structure of Formula
(III) is a PEG group having an average molecular weight of about 5kDa. In some
embodiments, W
in the structure of Formula (III) is a PEG group having an average molecular
weight of about
30kDa.
[103] In some embodiments, the IL-2 conjugate has the amino acid sequence
of SEQ ID NO: 17.
In some embodiments, W in the structure of Formula (III) is a PEG group having
an average
molecular weight selected from about 5kDa, 10kDa, 15kDa, 20kDa, 25kDa, and
30kDa. In some
embodiments, W in the structure of Formula (III) is a PEG group having an
average molecular
weight selected from about 5kDa and 30kDa. In some embodiments, W in the
structure of Formula
(III) is a PEG group having an average molecular weight of about 5kDa. In some
embodiments, W
in the structure of Formula (III) is a PEG group having an average molecular
weight of about
30kDa.
[104] In some embodiments, the IL-2 conjugate has the amino acid sequence
of SEQ ID NO: 18.
In some embodiments, W in the structure of Formula (III) is a PEG group having
an average
molecular weight selected from about 5kDa, 10kDa, 15kDa, 20kDa, 25kDa, and
30kDa. In some
embodiments, W in the structure of Formula (III) is a PEG group having an
average molecular
weight selected from about 5kDa and 30kDa In some embodiments, W in the
structure of Formula
(III) is a PEG group having an average molecular weight of about 5kDa. In some
embodiments, W
in the structure of Formula (III) is a PEG group having an average molecular
weight of about
30kDa.
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[105] In some embodiments, the IL-2 conjugate has the amino acid sequence
of SEQ ID NO: 19.
In some embodiments, W in the structure of Formula (III) is a PEG group having
an average
molecular weight selected from about 5kDa, 10kDa, 15kDa, 20kDa, 25kDa, and
30kDa. In some
embodiments, W in the structure of Formula (III) is a PEG group having an
average molecular
weight selected from about 5kDa and 30kDa. In some embodiments, W in the
structure of Formula
(III) is a PEG group having an average molecular weight of about 5kDa. In some
embodiments, W
in the structure of Formula (Ill) is a PEG group having an average molecular
weight of about
30kDa.
[106] In some embodiments, the IL-2 conjugate has the amino acid sequence
selected from any
one of SEQ ID NO: 15, 16, 17, 18, and 19, wherein [AzK PEG] contains a PEG
group having an
average molecular weight selected from about 5kDa, 10kDa, 15kDa, 20kDa, 25kDa,
30kDa, 35kDa,
40kDa, 45kDa, 50kDa, and 60kDa. In some embodiments, the IL-2 conjugate has
the amino acid
sequence selected from any one of SEQ ID NO: 15, 16, 17, 18, and 19, wherein
[AzK PEG]
contains a PEG group having an average molecular weight of about 5kDa. In some
embodiments,
the IL-2 conjugate has the amino acid sequence selected from any one of SEQ ID
NO: 15, 16, 17,
18, and 19, wherein [AzK PEG] contains a PEG group having an average molecular
weight of
about 10kDa. In some embodiments, the IL-2 conjugate has the amino acid
sequence selected from
any one of SEQ ID NO: 15, 16, 17, 18, and 19, wherein [AzK PEG] contains a PEG
group having
an average molecular weight of about 15kDa. In some embodiments, the IL-2
conjugate has the
amino acid sequence selected from any one of SEQ ID NO: 15, 16, 17, 18, and
19, wherein
[AzK PEG] contains a PEG group having an average molecular weight of about
20kDa. In some
embodiments, the IL-2 conjugate has the amino acid sequence selected from any
one of SEQ ID
NO: 15, 16, 17, 18, and 19, wherein [AzK PEG] contains a PEG group having an
average
molecular weight of about 25kDa. In some embodiments, the IL-2 conjugate has
the amino acid
sequence selected from any one of SEQ ID NO: 15, 16, 17, 18, and 19, wherein
[AzK_PEG]
contains a PEG group having an average molecular weight of about 30kDa. In
some embodiments,
the IL-2 conjugate has the amino acid sequence selected from any one of SEQ ID
NO: 15, 16, 17,
18, and 19, wherein [AzK PEG] contains a PEG group having an average molecular
weight of
about 35kDa. In some embodiments, the IL-2 conjugate has the amino acid
sequence selected from
any one of SEQ ID NO: 15, 16, 17, 18, and 19, wherein [AzK PEG] contains a PEG
group having
an average molecular weight of about 40kDa. In some embodiments, the IL-2
conjugate has the
amino acid sequence selected from any one of SEQ ID NO: 15, 16, 17, 18, and
19, wherein
[AzK PEG] contains a PEG group having an average molecular weight of about
45kDa. In some
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embodiments, the IL-2 conjugate has the amino acid sequence selected from any
one of SEQ ID
NO: 15, 16, 17, 18, and 19, [AzK PEG] contains a PEG group having an average
molecular weight
of about 50kDa. In some embodiments, the IL-2 conjugate has the amino acid
sequence selected
from any one of SEQ ID NO: 15, 16, 17, 18, and 19, wherein [AzK PEG] contains
a PEG group
having an average molecular weight of about 60kDa. In some embodiments, the IL-
2 conjugate has
the amino acid sequence selected from any one of SEQ ID NO: 15, 16, 17, 18,
and 19, wherein
[AzK PEG] contains a PEG group having an average molecular weight selected
from about 5kDa,
10kDa, 15kDa, 20kDa, 25kDa, 30kDa, 35kDa, 40kDa, 451(Da, 50kDa, and 60kDa, and
the PEG
group is a methoxy PEG group, a linear methoxy PEG group, or a branched
methoxy PEG group.
[107] In some embodiments, the IL-2 conjugate comprises the amino
acid sequence of any one
of SEQ ID NOS: 20-24, wherein rAzK PEG5kD] has the structure of Formula (II)
or Formula (III),
or a mixture of Formula (II) and Formula
X N N
0 N.
N N
0 0
Formula (II);
0 0
X N
N N
0
Formula (III);
wherein:
W is a PEG group having an average molecular weight of about 5kDa;
q is 1,2, or 3; and
X has the structure:
ssc,lV H
0 F-,Em
X- 1 indicates the point of attachment to the preceding amino acid residue;
and
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X+1 indicates the point of attachment to the following amino acid residue.
In some embodiments, q is 1 and the [AzK_PEG5kID] has the structure of Formula
(Ha) or Formula
(Ma), or a mixture of Formula (Ha) and Formula (Ina).
[108] In some embodiments, the IL-2 conjugate has the amino acid sequence
of SEQ ID NO: 20.
In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID
NO: 21. In some
embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 22.
In some
embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 23.
In some
embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 24.
[109] In some embodiments, the [AzK PEG51(D] has the structure of Formula
(II)
N
O NI H
N N
0
Formula (II).
In some embodiments, q is 1 and the [AzK PEG5kD1 has the structure of Formula
(Ha).
[110] In some embodiments, the [AzK PEG5k13] has the structure of
Formula (III):
0 0
N
X N
O NI q H
Formula (HI).
In some embodiments, q is 1 and the [AzK PEG5k13] has the structure of Formula
(Ma).
[111] In some embodiments, the IL-2 conjugate comprises the amino
acid sequence of any one
of SEQ ID NOS: 25-29, wherein [AzK PEG30k1)] has the structure of Formula (II)
or Formula
(III), or is a mixture of the structures of Formula (II) and Formula (III).
411
O NI H
N N
44/ 0 0
Formula (II);
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1, 0 0
N
N
0
NI qH
Formula (III);
wherein:
W is a PEG group having an average molecular weight of about 30kDa;
q is 1,2, or 3; and
X has the structure:
ss
0.1"µ ____________
x+1
=
X-1 indicates the point of attachment to the preceding amino acid residue; and
X+1 indicates the point of attachment to the following amino acid residue.
In some embodiments, q is 1 and the [AzK PEG30kD] has the structure of Formula
(Ha) or
Formula (Ma), or is a mixture of the structures of Formula (Ha) and Formula
(Ma).
[112] In some embodiments, the IL-2 conjugate has the amino acid sequence
of SEQ ID NO: 25.
In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID
NO: 26 In some
embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 27.
In some
embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 28.
In some
embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 29.
[113] In some embodiments, the [AzK PEG30kD] has the structure of Formula
(II):
=N
0 NI H
N
40 0 0
Formula (II).
In some embodiments, q is 1 and the [AzK PEG30kD] has the structure of Formula
(Ha).
[114] In some embodiments, the methods use an IL-2 conjugate in
which the [AzK PEG30kD]
has the structure of Formula (III):
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1, 0 0
X N
N
N
O NI q H
Formula
In some embodiments, q is 1 and the [AzK PEG30kD] has the structure of Formula
(Ma).
[115] In some embodiments, the [AzK PEG] is a mixture of the
structures of Formula (II) and
Formula (III):
x N N
O N.
Formula N N
0 0
Formula (II);
I* 0 0
X N
N
O NI q H
Formula (III);
wherein:
W is a PEG group having an average molecular weight selected from 5kDa, 10kDa,
15kDa, 20kDa,
25kDa, 30kDa, 35kDa, 40kDa, 45kDa, 50kDa, and 60kDa;
q is 1,2, or 3; and
X has the structure:
H
0 e
X-1 indicates the point of attachment to the preceding amino acid residue; and
X+1 indicates the point of attachment to the following amino acid residue.
In some embodiments, q is 1 and the [AzK PEG] is a mixture of the structures
of Formula (IIa) and
Formula (Ma).
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[116] In some embodiments, the ratio of the amount of the structure of
Formula (II) to the
amount of the structure of Formula (III) comprising the total amount of
[AzK_PEG] in the IL-2
conjugate is about 1:1. In some embodiments, the ratio of the amount of the
structure of Formula
(II) to the amount of the structure of Formula (III) comprising the total
amount of [AzK_PEG] in
the IL-2 conjugate is greater than 1:1. In some embodiments, the ratio of the
amount of the structure
of Formula (II) to the amount of the structure of Formula (III) comprising the
total amount of
[AzK PEG] in the IL-2 conjugate is less than 1:1.
[117] In some embodiments, W is a linear or branched PEG group. In some
embodiments, W is
a linear PEG group. In some embodiments W is a branched PEG group. In some
embodiments, W is
a methoxy PEG group. In some embodiments, the methoxy PEG group is linear or
branched. In
some embodiments, the methoxy PEG group is linear. In some embodiments, the
methoxy PEG
group is branched.
[118] In some embodiments, the IL-2 conjugate comprises the amino acid
sequence of any one
of SEQ ID NOS: 20 to 24, wherein [AzK_PEG5kD] is a mixture of the structures
of Formula (II)
and Formula (III):
X N
I I
0 qõ
N N
0 0
Formula (II);
0 0
N
N N
0 Nis, I q H
Formula (III);
wherein:
W is a PEG group having an average molecular weight of 5kDa;
q is 1, 2, or 3; and
X has the structure:
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cs5sN H
0 e,.110
X-1 indicates the point of attachment to the preceding amino acid residue; and
X+1 indicates the point of attachment to the following amino acid residue.
In some embodiments, q is 1 and the [AzK PEG5kD] is a mixture of the
structures of Formula (Ha)
and Formula (IIIa).
[119] In some embodiments, the ratio of the amount of the structure of
Formula (II) to the
amount of the structure of Formula (III) comprising the total amount of [AzK
PEG5kID] in the IL-2
conjugate is about 1:1. In some embodiments, the ratio of the amount of the
structure of Formula
(II) to the amount of the structure of Formula (III) comprising the total
amount of [AzK PEG5kD]
in the IL-2 conjugate is greater than 1:1. In some embodiments, the ratio of
the amount of the
structure of Formula (II) to the amount of the structure of Formula (III)
comprising the total amount
of [AzK PEG5kD] in the IL-2 conjugate is less than 1:1.
[120] In some embodiments, the IL-2 conjugate comprises the amino acid
sequence of any one
of SEQ ID NOS: 25-29, wherein [AzK PEG30kD] is a mixture of the structures of
Formula (II) and
Formula (III):
x N N
0 NI H
N , N
w
41 0 0
Formula (II);
= 0 0
N
N N
0
NI q H
Formula (III);
wherein:
W is a PEG group having an average molecular weight of 30kDa;
q is 1, 2, or 3; and
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X has the structure:
x-1
Ocs( X+1
=
X-1 indicates the point of attachment to the preceding amino acid residue; and
X+1 indicates the point of attachment to the following amino acid residue.
In some embodiments, q is 1 and the [AzK PEG30k1D] is a mixture of the
structures of Formula
(Ha) and Formula (Ma).
[121] In some embodiments, the ratio of the amount of the structure of
Formula (II) to the
amount of the structure of Formula (III) comprising the total amount of [AzK
PEG30kID] in the IL-
2 conjugate is about 1:1. In some embodiments, the ratio of the amount of the
structure of Formula
(II) to the amount of the structure of Formula (III) comprising the total
amount of [AzK PEG30k1D]
in the IL-2 conjugate is greater than 1:1. In some embodiments, the ratio of
the amount of the
structure of Formula (II) to the amount of the stnicture of Formula (III)
comprising the total amount
of [AzK PEG30k1D] in the IL-2 conjugate is less than 1:1.
[122] In some embodiments, the IL-2 conjugate comprises the structure of
Formula (II) or
Formula (III), or a mixture of Formula (II) and Formula (III), wherein W is a
linear or branched
PEG group, or a pharmaceutically acceptable salt, solvate, or hydrate thereof.
In some
embodiments, W in the structure of Formula (II) or Formula (III) is a linear
PEG group. In some
embodiments, W in the structure of Formula (II) or Formula (III) is a branched
PEG group. In some
embodiments, W in the structure of Formula (II) or Formula (III) is a methoxy
PEG group. In some
embodiments, W in the structure of Formula (II) or Formula (III) is a methoxy
PEG group that is
linear or branched. In some embodiments, the methoxy PEG group in the
structure of Formula (II)
or Formula (III) is linear. In some embodiments, the methoxy PEG group in the
structure of
Formula (II) or Formula (III) is branched.
[123] In some embodiments, the IL-2 conjugate comprises the amino acid
sequence of any one
of SEQ ID NOS: 40-44, wherein [AA( Ll _PEG] has the structure of Formula (IV)
or Formula (V),
_
or a mixture of Formula (IV) and Formula (V):
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x N 0
I I
0
0 14õ
N N
0
Formula (IV);
0
X
jt,Hryi H N 0
I I N
0
0 Niõ I
0
Formula (V);
wherein:
W is a PEG group having an average molecular weight selected from 5kDa, 10kDa,
15kDa, 20kDa,
25kDa, 30kDa, 35kDa, 40kDa, 45kDa, 50kDa, and 60kDa;
q is 1,2, or 3; and
X has the structure:
X-1
N H
Oqs( __________ x+1
=
X-1 indicates the point of attachment to the preceding amino acid residue; and
X+1 indicates the point of attachment to the following amino acid residue.
[124] In some embodiments of Formula (IV) or Formula (V), or a mixture of
Formula (IV) or
Formula (V), q is 1. In some embodiments of Formula (IV) or Formula (V), or a
mixture of Formula
(IV) or Formula (V), q is 2. In some embodiments of Formula (IV) or Formula
(V), or a mixture of
Formula (IV) or Formula (V), q is 3.
[125] Here and throughout, the structure of Formula (IV) encompasses
pharmaceutically
acceptable salts, solvates, or hydrates thereof. Here and throughout, the
structure of Formula (V)
encompasses pharmaceutically acceptable salts, solvates, or hydrates thereof
In some
embodiments, the IL-2 conjugate is a pharmaceutically acceptable salt,
solvate, or hydrate.
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[126] In some embodiments, q is 1 and the structures of Formula (IV)
and Formula (V) are
Formula (IVa) and Formula (Va):
x
0
0 Nõ
N
0
Formula (IVa);
411 0
x TO
N IR11
0 Nõ 0
Formula (Va);
wherein:
W is a PEG group having an average molecular weight selected from about 5kDa,
10kDa, 15kDa,
20kDa, 251(Da, 30Wa, 351(Da, 40kDa, 45kDa, 50kDa, and 60Wa; and
X has the structure:
x-1
X-1 indicates the point of attachment to the preceding amino acid residue; and
X+1 indicates the point of attachment to the following amino acid residue.
[127] In some embodiments, the [AzK Ll PEG] is a mixture of Formula
(IV) and Formula (V).
In some embodiments, the [AzK LI PEG] is a mixture of Formula (IVa) and
Formula (Va).
[128] In some embodiments, the [AzK Li PEG] has the structure of
Formula (IV):
II x
0
0 N
N
N
Formula (IV).
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In some embodiments, q is 1 and the [AzK Li PEG] has the structure of Formula
(IVa).
[129] In some embodiments, the IL-2 conjugate has the amino acid sequence
of SEQ ID NO: 40.
In some embodiments, W in the structure of Formula (IV) is a PEG group having
an average
molecular weight selected from about 5kDa, 10kDa, 15kDa, 20kDa, 25kDa, and
30kDa. In some
embodiments, W in the structure of Formula (IV) is a PEG group having an
average molecular
weight selected from about 5kDa and 30kDa. In some embodiments, W in the
structure of Formula
(IV) is a PEG group having an average molecular weight of about 5kDa. In some
embodiments, W
in the structure of Formula (IV) is a PEG group having an average molecular
weight of about
30kDa.
[130] In some embodiments, the IL-2 conjugate has the amino acid sequence
of SEQ ID NO: 41.
In some embodiments, W in the structure of Formula (IV) is a PEG group having
an average
molecular weight selected from about 5kDa, 10kDa, 15kDa, 20kDa, 25kDa, and
30kDa. In some
embodiments, W in the structure of Formula (IV) is a PEG group having an
average molecular
weight selected from about 5kDa and 30kDa. In some embodiments, W in the
structure of Formula
(IV) is a PEG group having an average molecular weight of about 5kDa. In some
embodiments, W
in the structure of Formula (IV) is a PEG group having an average molecular
weight of about
30kDa.
[131] In some embodiments, the IL-2 conjugate has the amino acid sequence
of SEQ ID NO: 42.
In some embodiments, W in the structure of Formula (IV) is a PEG group having
an average
molecular weight selected from about 5kDa, 10kDa, 15kDa, 20kDa, 25kDa, and
30kDa. In some
embodiments, W in the structure of Formula (IV) is a PEG group having an
average molecular
weight selected from about 5kDa and 30kDa. In some embodiments, W is a PEG
group having an
average molecular weight of about 5kDa. In some embodiments, W in the
structure of Formula (IV)
is a PEG group having an average molecular weight of about 30kDa.
[132] In some embodiments, the IL-2 conjugate has the amino acid sequence
of SEQ ID NO: 43.
In some embodiments, W in the structure of Formula (IV) is a PEG group having
an average
molecular weight selected from about 5kDa, 10kDa, 15kDa, 20kDa, 25kDa, and
30kDa. In some
embodiments, W in the structure of Formula (IV) is a PEG group having an
average molecular
weight selected from about 5kDa and 30kDa In some embodiments, W in the
structure of Formula
(IV) is a PEG group having an average molecular weight of about 5kDa. In some
embodiments, W
in the structure of Formula (IV) is a PEG group having an average molecular
weight of about
30kDa.
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[133] In some embodiments, the IL-2 conjugate has the amino acid
sequence of SEQ ID NO: 44.
In some embodiments, W in the structure of Formula (IV) is a PEG group having
an average
molecular weight selected from about 5kDa, 10kDa, 15kDa, 20kDa, 25kDa, and
30kDa. In some
embodiments, W in the structure of Formula (IV) is a PEG group having an
average molecular
weight selected from about 5kDa and 30kDa. In some embodiments, W in the
structure of Formula
(IV) is a PEG group having an average molecular weight of about 5kDa. In some
embodiments, W
in the structure of Formula (IV) is a PEG group having an average molecular
weight of about
30kDa.
[134] In some embodiments, the [AzK LI PEG] has the structure of
Formula (V):
0
H
0
0
Formula (V).
In some embodiments, q is 1 and the [AzK Li PEG] has the structure of Formula
(V).
[135] In some embodiments, the IL-2 conjugate has the amino acid
sequence of SEQ ID NO: 40.
In some embodiments, W in the structure of Formula (V) is a PEG group having
an average
molecular weight selected from about 5kDa, 10kDa, 15kDa, 20kDa, 25kDa, and
30kDa. In some
embodiments, W in the structure of Formula (V) is a PEG group having an
average molecular
weight selected from about 5kDa and 30kDa. In some embodiments, W in the
structure of Formula
(V) is a PEG group having an average molecular weight of about 5kDa. In some
embodiments, W
in the structure of Formula (V) is a PEG group having an average molecular
weight of about 30kDa.
[136] In some embodiments, the IL-2 conjugate has the amino acid
sequence of SEQ ID NO: 41.
In some embodiments, W in the structure of Formula (V) is a PEG group having
an average
molecular weight selected from about 5kDa, 10kDa, 15kDa, 20kDa, 25kDa, and
30kDa. In some
embodiments, W in the structure of Formula (V) is a PEG group having an
average molecular
weight selected from about 5kDa and 30kDa. In some embodiments, W in the
structure of Formula
(V) is a PEG group having an average molecular weight of about 5kDa. In some
embodiments, W
in the structure of Formula (V) is a PEG group having an average molecular
weight of about 30kDa.
[137] In some embodiments, the IL-2 conjugate has the amino acid
sequence of SEQ ID NO: 42.
In some embodiments, W in the structure of Formula (V) is a PEG group having
an average
molecular weight selected from about 5kDa, 10kDa, 15kDa, 20kDa, 25kDa, and
30kDa. In some
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embodiments, W in the structure of Formula (V) is a PEG group having an
average molecular
weight selected from about 5kDa and 30kDa. In some embodiments, W in the
structure of Formula
(V) is a PEG group having an average molecular weight of about 5kDa. In some
embodiments W in
the structure of Formula (V) is a PEG group having an average molecular weight
of about 30kDa.
[138] In some embodiments, the IL-2 conjugate has the amino acid sequence
of SEQ ID NO: 43.
In some embodiments, W in the structure of Formula (V) is a PEG group having
an average
molecular weight selected from about 5kDa, 10kDa, 15kDa, 20kDa, 25kDa, and
30kDa. In some
embodiments, W in the structure of Formula (V) is a PEG group having an
average molecular
weight selected from about 5kDa and 30kDa. In some embodiments, W in the
structure of Formula
(V) is a PEG group having an average molecular weight of about 5kDa. In some
embodiments W in
the structure of Formula (V) is a PEG group having an average molecular weight
of about 30kDa.
[139] In some embodiments, the IL-2 conjugate has the amino acid sequence
selected from any
one of SEQ ID NO: 40, 41, 42, 43, and 44, wherein [AzK Li PEG] contains a PEG
group having
an average molecular weight selected from about 5kDa, 10kDa, 15kDa, 20kDa,
25kDa, 30kDa,
35kDa, 40kDa, 45kDa, 50kDa, and 60kDa. In some embodiments, the IL-2 conjugate
has the
amino acid sequence selected from any one of SEQ ID NO: 40, 41, 42, 43, and
44, wherein
[AzK Ll PEG] contains a PEG group having an average molecular weight of about
5kDa. In
some embodiments, the IL-2 conjugate has the amino acid sequence selected from
any one of SEQ
ID NO: 40, 41, 42, 43, and 44, wherein [AzK Ll PEG] contains a PEG group
having an average
molecular weight of about 10kDa. In some embodiments, the IL-2 conjugate has
the amino acid
sequence selected from any one of SEQ ID NO: 40, 41, 42, 43, and 44, wherein
[AzK Li PEG]
contains a PEG group having an average molecular weight of about 15kDa. In
some embodiments,
the IL-2 conjugate has the amino acid sequence selected from any one of SEQ ID
NO: 40, 41, 42,
43, and 44, wherein [AzK Ll PEG] contains a PEG group having an average
molecular weight of
about 20kDa. In some embodiments, the IL-2 conjugate has the amino acid
sequence selected from
any one of SEQ ID NO: 40, 41, 42, 43, and 44, wherein [AzK LI PEG] contains a
PEG group
having an average molecular weight of about 25kDa. In some embodiments, the IL-
2 conjugate has
the amino acid sequence selected from any one of SEQ ID NO: 40, 41, 42, 43,
and 44, wherein
[AzK Li PEG] contains a PEG group having an average molecular weight of about
30kDa. In
some embodiments, the IL-2 conjugate has the amino acid sequence selected from
any one of SEQ
ID NO: 40, 41, 42, 43, and 44, wherein [AzK_Ll_PEG] contains a PEG group
having an average
molecular weight of about 35kDa. In some embodiments, the IL-2 conjugate has
the amino acid
sequence selected from any one of SEQ ID NO: 40, 41, 42, 43, and 44, wherein
[AzK Ll PEG]
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contains a PEG group having an average molecular weight of about 40kDa. In
some embodiments,
the IL-2 conjugate has the amino acid sequence selected from any one of SEQ ID
NO: 40, 41, 42,
43, and 44, wherein [AzK Ll PEG] contains a PEG group having an average
molecular weight of
about 45kDa In some embodiments, the IL-2 conjugate has the amino acid
sequence selected from
any one of SEQ ID NO: 40, 41, 42, 43, and 44, wherein [AzK Li PEG] contains a
PEG group
having an average molecular weight of about 50kDa. In some embodiments, the IL-
2 conjugate has
the amino acid sequence selected from any one of SEQ ID NO: 40, 41, 42, 43,
and 44, wherein
[AzK Ll PEG] contains a PEG group having an average molecular weight of about
60kDa. In
some embodiments, the IL-2 conjugate has the amino acid sequence selected from
any one of SEQ
ID NO: 40, 41, 42, 43, and 44, wherein [AzK Li PEG] contains a PEG group
having an average
molecular weight selected from about 5kDa, 10kDa, 15kDa, 20kDa, 25kDa, 30kDa,
35kDa, 40kDa,
45kDa, 50kDa, and 60kDa, and wherein the PEG group is a methoxy PEG group, a
linear methoxy
PEG group, or a branched methoxy PEG group.
[140] In some embodiments, the IL-2 conjugate comprises the amino
acid sequence of any one
of SEQ ID NOS: 45-49, wherein [AzK Li PEG51cD] has the structure of Formula
(IV) or Formula
(V), or a mixture of Formula (IV) and Formula (V):
X Ny N
0 Nis, I 0
N
41 0
Formula (IV);
= 0
H
N
0
0
Formula (V);
wherein:
W is a PEG group having an average molecular weight of about 5kDa; and
X has the structure:
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cs5SNH
0 e,.cal
X-1 indicates the point of attachment to the preceding amino acid residue; and
X+1 indicates the point of attachment to the following amino acid residue.
In some embodiments, q is 1 and the [AzK Li PEG5kD] has the structure of
Formula (IVa) or
Formula (Va), or a mixture of Formula (IVa) and Formula (Va).
[141] In some embodiments, the IL-2 conjugate has the amino acid sequence
of SEQ ID NO:
45. In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ
ID NO: 46. In
some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO:
47. In some
embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 48.
In some
embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 49.
[142] In some embodiments, the [AzK Li PEG5kD] has the structure of Formula
(IV)
II
0 N I
0
41 0
NLLo
Formula (IV).
In some embodiments, q is 1 and the [AzK Li PEG5kD] has the structure of
Formula (IVa).
[143] In some embodiments, the [AzK Li PEG51cD] has the structure of
Formula (V)
0
H
0 Nõ
0
411
Formula (V).
In some embodiments, q is 1 and the [AzK Li PEG5kD] has the structure of
Formula (Va).
[144] In some embodiments, the IL-2 conjugate comprises the amino acid
sequence of any one
of SEQ ID NOS: 50-54, wherein [AzK Li PEG30kD] has the structure of Formula
(IV) or
Formula (V), or is a mixture of the structures of Formula (IV) and Formula
(V):
46
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0
0 N'õ
N
0
Formula (IV);
H
N
0
0 Nõ
0
Formula (V);
wherein:
W is a PEG group having an average molecular weight of about 301(Da;
q is 1,2, or 3; and
X has the structure:
x-1
ck,NH
Ocs( X + 1
=
X-1 indicates the point of attachment to the preceding amino acid residue; and
X+1 indicates the point of attachment to the following amino acid residue.
In some embodiments, q is 1 and the [AzK Li PEG3OLD] has the structure of
Formula (IVa) or
Formula (Va), or is a mixture of the structures of Formula (IVa) and Formula
(Va).
[145] In some embodiments, the 1L-2 conjugate has the amino acid sequence
of SEQ ID NO:
50. In some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ
ID NO: 51. In
some embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO:
52. In some
embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 53.
In some
embodiments, the IL-2 conjugate has the amino acid sequence of SEQ ID NO: 54.
[146] In some embodiments, the [AzK_Ll_PEG30kID] has the structure of
Formula (IV):
47
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0 N
0
0
Formula (IV).
In some embodiments, q is 1 and the [AzK Li PEG301cD] has the structure of
Formula (IVa).
[147] In some embodiments, the [AzK Li PEG301cD] has the structure of
Formula (V):
II 0
H
x N
N
OW
0 Nõ
0
Formula (V).
In some embodiments, q is 1 and the [AzK Li PEG301cD] has the structure of
Formula (Va).
[148] In some embodiments, the IL-2 conjugate comprises the amino acid
sequence of any one of
SEQ ID NOS: 40-44, wherein [Azk LI PEG] is a mixture of the structures of
Formula (IV) and
Formula (V):
NyO
0 N
0
= 0 H
Formula (IV);
= 0
r H
N
0 N
0
411
Formula (V);
wherein:
W is a PEG group having an average molecular weight selected from about 5kDa,
10kDa, 15kDa,
20kDa, 25kDa, 30kDa, 35kDa, 40kDa, 45kDa, 50kDa, and 60kDa;
q is 1, 2, or 3; and
X has the structure:
48
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cs'ss NH
0 c3 .131
X-1 indicates the point of attachment to the preceding amino acid residue; and
X+1 indicates the point of attachment to the following amino acid residue.
[149] In some embodiments, the ratio of the amount of the structure of Formula
(IV) to the amount
of the structure of Formula (V) comprising the total amount of [AzK Ll PEG] in
the IL-2
conjugate is about 1:1. In some embodiments, the ratio of the amount of the
structure of Formula
(IV) to the amount of the structure of Formula (V) comprising the total amount
of [AzK Ll PEG]
in the IL-2 conjugate is greater than 1:1. In some embodiments, the ratio of
the amount of the
structure of Formula (IV) to the amount of the structure of Formula (V)
comprising the total amount
of [AzK Li PEG] in the IL-2 conjugate is less than 1:1.
[150] In some embodiments, the IL-2 conjugate comprises the amino acid
sequence of any one of
SEQ ID NOS. 45 to 49, wherein [AzK Li PEG5kD] is a mixture of the stnictures
of Formula (IV)
and Formula (V):
X
0
0 qõ
N
410' 0
Formula (IV);
=0
H
N
0 Nõ
0
41/
Formula (V);
wherein:
W is a PEG group having an average molecular weight of about 5kDa;
q is 1, 2, or 3; and
X has the structure:
49
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X-1
cs5sNH
Osss X+1
X-1 indicates the point of attachment to the preceding amino acid residue; and
X+1 indicates the point of attachment to the following amino acid residue.
In ome embodiments, q is 1 and the [AzK Li PEG.51(D] is a mixture of the
structures of Formula
(IVa) and Formula (Va).
[151] In some embodiments, the ratio of the amount of the structure of
Formula (IV) to the
amount of the structure of Formula (V) comprising the total amount of [AzK L1
PEG5kD] in the
IL-2 conjugate is about 1:1. In some embodiments, the ratio of the amount of
the structure of
Formula (IV) to the amount of the structure of Formula (V) comprising the
total amount of
[AzK Ll PEG5kD] in the IL-2 conjugate is greater than 1:1. In some
embodiments, the ratio of the
amount of the structure of Formula (IV) to the amount of the structure of
Formula (V) comprising
the total amount of [AzK Li PEG5k1)] in the IL-2 conjugate is less than 1:1.
[152] In some embodiments, the IL-2 conjugate comprises the amino acid
sequence of any one
of SEQ ID NOS: 50-54, wherein [AzK Li PEG301cD] is a mixture of the structures
of Formula
(IV) and Formula (V):
NO-
0 N.
4111 0
Formula (IV);
=0
FINoW
c11-4"nii
0
0
Formula (V);
wherein:
W is a PEG group having an average molecular weight of about 30kDa;
q is 1, 2, or 3; and
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X has the structure:
N H
Ian =
X-1 indicates the point of attachment to the preceding amino acid residue; and
X+1 indicates the point of attachment to the following amino acid residue.
In some embodiments, q is 1 and the [AzK Li PEG30kD] is a mixture of the
structures of Formula
(IVa) and Formula (Va).
[153] In some embodiments, the ratio of the amount of the structure of
Formula (IV) to the
amount of the structure of Formula (V) comprising the total amount of [AzK Ll
PEG30k1D] in the
IL-2 conjugate is about 1:1. In some embodiments, the ratio of the amount of
the structure of
Formula (IV) to the amount of the structure of Formula (V) comprising the
total amount of
[AzK Ll PEG30k1D] in the IL-2 conjugate is greater than 1:1. In some
embodiments, the ratio of
the amount of the structure of Formula (IV) to the amount of the structure of
Formula (V)
comprising the total amount of [AzK Li PEG30kID] in the IL-2 conjugate is less
than 1:1.
[154] In some embodiments, W in the structure of Formula (IV) or (V) is a
PEG group having an
average molecular weight selected from about 5kDa, 10kDa, 15kDa, 20kDa, 25kDa,
and 30kDa. In
some embodiments, W in the structure of Formula (IV) or (V) is a PEG group
having an average
molecular weight of about 51(Da. In some embodiments, W in the structure of
Formula (IV) or (V)
is a PEG group having an average molecular weight of about 30kDa. In some
embodiments, W in
the structure of Formula (IV) or (V) is a PEG group having an average
molecular weight of about
10kDa. In some embodiments, W in the structure of Formula (IV) or (V) is a PEG
group having an
average molecular weight of about 15kDa. In some embodiments, W in the
structure of Formula
(IV) or (V) is a PEG group having an average molecular weight of about 20kDa.
In some
embodiments, W in the structure of Formula (IV) or (V) is a PEG group having
an average
molecular weight of about 25kDa. In some embodiments, W in the structure of
Formula (IV) or (V)
is a PEG group having an average molecular weight of about 30kDa. In some
embodiments, W in
the structure of Formula (IV) or (V) is a PEG group having an average
molecular weight of about
35kDa. In some embodiments, W in the structure of Formula (IV) or (V) is a PEG
group having an
average molecular weight of about 40kDa. In some embodiments, W in the
structure of Formula
(IV) or (V) is a PEG group having an average molecular weight of about 45kDa.
In some
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embodiments, W in the structure of Formula (IV) or (V) is a PEG group having
an average
molecular weight of about 50kDa. In some embodiments, W in the structure of
Formula (IV) or (V)
is a PEG group having an average molecular weight of about 55kDa. In some
embodiments, W in
the structure of Formula (IV) or (V) is a PEG group having an average
molecular weight of about
60kDa.
[155] In some embodiments, the IL-2 conjugate described herein comprises
the structure of
Formula (IV) or Formula (V), or a mixture of Formula (II) and Formula (III),
wherein W is a linear
or branched PEG group. In some embodiments, W in the structure of Formula (IV)
or Formula (V)
is a linear PEG group. In some embodiments, W in the structure of Formula (IV)
or Formula (V) is
a branched PEG group. In some embodiments, W in the structure of Formula (IV)
or Formula (V) is
a methoxy PEG group. In some embodiments, W in the structure of Formula (IV)
or Formula (V) is
a methoxy PEG group that is linear or branched. In some embodiments, the
methoxy PEG group in
the structure of Formula (IV) or Formula (V) is linear. In some embodiments,
the methoxy PEG
group in the structure of Formula (IV) or Formula (V) is branched.
[156] With respect to the IL-2 conjugates used in the methods described
herein, an exemplary
structure of a methoxy PEG group is illustrated in the mPEG-DBCO structures
below.
0 0
N
- n
N N
N H
0
Cfr,
N N N
H
n 0 0
0
0
oss
[157] In some embodiments, the IL-2 conjugate comprises the amino acid
sequence of SEQ ID
NO: 3 in which at least one amino acid residue in the IL-2 conjugate is
replaced by the structure of
Formula (VI) or (VII), or a mixture of (VI) and (VII):
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XoTh0
N 411
NI's, I
N N
II 0 0
Formula (VI);
NI: I
N N 0CH 3
x N 0 0
0
Formula (VII);
wherein:
n is an integer in the range from about 2 to about 5000;
q is 1,2, or 3; and
X has the structure:
X-1
0 c-VN
=
X-1 indicates the point of attachment to the preceding amino acid residue; and
X+1 indicates the point of attachment to the following amino acid residue.
11581 In some embodiments of Formula (VI) or Formula (VII), or a
mixture of Formula (VI) or
Formula (VII), q is 1. In some embodiments of Formula (VI) or Formula (VII),
or a mixture of
Formula (VI) or Formula (VII), q is 2. In some embodiments of Formula (VI) or
Formula (VII), or a
mixture of Formula (VT) or Formula (VII), q is 3
11591 Here and throughout, the structure of Formula (VI) encompasses
pharmaceutically
acceptable salts, solvates, or hydrates thereof. Here and throughout, the
structure of Formula (VII)
encompasses pharmaceutically acceptable salts, solvates, or hydrates thereof.
In some
embodiments, the IL-2 conjugate is a pharmaceutically acceptable salt,
solvate, or hydrate.
[160] In some embodiments, q is 1 and the structures of Formula (VI)
and Formula (VII) are the
structures of Formula (VIa) and Formula (VIIa):
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0
N'õ
3
/n
0 0
Formula (Via);
=
NI I
N 3
0 0ii0
Formula (Vila);
wherein:
n is an integer in the range from about 2 to about 5000; and
X has the structure:
"s=.,NH
0 cs--, cal
X-1 indicates the point of attachment to the preceding amino acid residue; and
X+1 indicates the point of attachment to the following amino acid residue.
[161] In some embodiments, n in the compounds of Formula (VI) and
(VII) is in the range from
about 5 to about 4600, or from about 10 to about 4000, or from about 20 to
about 3000, or from
about 100 to about 3000, or from about 100 to about 2900, or from about 150 to
about 2900, or
from about 125 to about 2900, or from about 100 to about 2500, or from about
100 to about 2000,
or from about 100 to about 1900, or from about 100 to about 1850, or from
about 100 to about
1750, or from about 100 to about 1650, or from about 100 to about 1500, or
from about 100 to
about 1400, or from about 100 to about 1300, or from about 100 to about 1250,
or from about 100
to about 1150, or from about 100 to about 1100, or from about 100 to about
1000, or from about
100 to about 900, or from about 100 to about 750, or from about 100 to about
700, or from about
100 to about 600, or from about 100 to about 575, or from about 100 to about
500, or from about
100 to about 450, or from about 100 to about to about 350, or from about 100
to about 275, or from
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about 100 to about 230, or from about 150 to about 475, or from about 150 to
about 340, or from
about 113 to about 340, or from about 450 to about 800, or from about 454 to
about 796, or from
about 454 to about 682, or from about 340 to about 795, or from about 341 to
about 682, or from
about 568 to about 909, or from about 227 to about 1500, or from about 225 to
about 2280, or from
about 460 to about 2160, or from about 460 to about 2050, or from about 341 to
about 1820, or
from about 341 to about 1710, or from about 341 to about 1250, or from about
225 to about 1250,
or from about 341 to about 1250, or from about 341 to about 1136, or from
about 341 to about
1023, or from about 341 to about 910, or from about 341 to about 796, or from
about 341 to about
682, or from about 341 to about 568, or from about 114 to about 1000, or from
about 114 to about
950, or from about 114 to about 910, or from about 114 to about 800, or from
about 114 to about
690, or from about 114 to about 575. In some embodiments, n in the compounds
of Formula (VI)
and (VII) is an integer selected from 2,5, 10, 11, 22, 23, 113, 114, 227, 228,
340, 341, 454, 455,
568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135,
1136, 1137, 1249,
1250, 1251, 1362, 1363, 1364, 1476, 1477, 1478, 1589, 1590, 1591, 1703, 1704,
1705, 1817, 1818,
1819, 1930, 1931, 1932, 2044, 2045, 2046, 2158, 2159, 2160, 2271, 2272, 2273,
2839, 2840, 2841,
2953, 2954, 2955, 3408, 3409, 3410, 3976, 3977, 3978, 4544, 4545, and 4546.
[162] In some embodiments, the position of the structure of Formula
(VI), Formula (VII), or a
mixture of Formula (VI) and (VII) in the amino acid sequence of the IL-2
conjugate is selected from
K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68, and L71, wherein the
position of the
structure of Formula (I) in the amino acid sequence of the IL-2 conjugate is
in reference to the
positions in SEQ ID NO: 3. In some embodiments, the position of the structure
of Formula (VI),
Formula (VII), or a mixture of Formula (VI) and (VII) in the amino acid
sequence of the IL-2
conjugate of SEQ ID NO: 3 is selected from K34, F41, F43, K42, E61, P64, R37,
T40, E67, Y44,
V68, and L71. In some embodiments, the position of the structure of Formula
(VI), Formula (VII),
or a mixture of Formula (VI) and (VII) in the amino acid sequence of the IL-2
conjugate of SEQ ID
NO: 3 is at position K34. In some embodiments, the position of the structure
of Formula (VI),
Formula (VII), or a mixture of Formula (VI) and (VII) in the amino acid
sequence of the IL-2
conjugate of SEQ ID NO: 3 is at position F41. In some embodiments, the
position of the structure
of Formula (VI), Formula (VII), or a mixture of Formula (VI) and (VII) in the
amino acid sequence
of the IL-2 conjugate of SEQ ID NO: 3 is at position F43. In some embodiments,
the position of
the structure of Formula (VI), Formula (VII), or a mixture of Formula (VI) and
(VII) in the amino
acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is at position 1(42. In
some embodiments, the
position of the structure of Formula (VI), Formula (VII), or a mixture of
Formula (VI) and (VII) in
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the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is at position
E61. In some
embodiments, the position of the structure of Formula (VI), Formula (VII), or
a mixture of Formula
(VI) and (VII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO:
3 is at position P64.
In some embodiments, the position of the structure of Formula (VI), Formula
(VII), or a mixture of
Formula (VI) and (VII) in the amino acid sequence of the IL-2 conjugate of SEQ
ID NO 3 is at
position R37. In some embodiments, the position of the structure of Formula
(VI), Formula (VII),
or a mixture of Formula (VI) and (VII) in the amino acid sequence of the IL-2
conjugate of SEQ ID
NO: 3 is at position T40. In some embodiments, the position of the structure
of Formula (VI),
Formula (VII), or a mixture of Formula (VI) and (VII) in the amino acid
sequence of the IL-2
conjugate of SEQ ID NO: 3 is at position E67. In some embodiments, the
position of the structure
of Formula (VI), Formula (VII), or a mixture of Formula (VI) and (VII) in the
amino acid sequence
of the IL-2 conjugate of SEQ ID NO: 3 is at position Y44. In some embodiments,
the position of
the structure of Formula (VI), Formula (VII), or a mixture of Formula (VI) and
(VII) in the amino
acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is at position V68. In
some embodiments, the
position of the structure of Formula (VI), Formula (VII), or a mixture of
Formula (VI) and (VII) in
the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is at position
L71.
[163] In some embodiments, the ratio of the amount of the structure of
Formula (VI) to the
amount of the structure of Formula (VII) comprising the total amount of the IL-
2 conjugate is about
1:1. In some embodiments, the ratio of the amount of the structure of Formula
(VI) to the amount
of the structure of Formula (VII) comprising the total amount of the IL-2
conjugate is greater than
1:1. In some embodiments, the ratio of the amount of the structure of Formula
(VI) to the amount of
the structure of Formula (VII) comprising the total amount of the IL-2
conjugate is less than 1:1.
[164] In some embodiments, the IL-2 conjugate comprises the amino acid
sequence of SEQ ID
NO: 3 in which at least one amino acid residue in the IL-2 conjugate is
replaced by the structure of
Formula (VI) or (VII), or a mixture of (VI) and (VII), wherein the amino acid
residue in SEQ ID
NO: 3 that is replaced is selected from K34, F41, F43, K42, E61, P64, R37,
T40, E67, Y44, V68,
and L71, and wherein n is an integer from 100 to about 1150, or from about 100
to about 1100, or
from about 100 to about 1000, or from about 100 to about 900, or from about
100 to about 750, or
from about 100 to about 700, or from about 100 to about 600, or from about 100
to about 575, or
from about 100 to about 500, or from about 100 to about 450, or from about 100
to about to about
350, or from about 100 to about 275, or from about 100 to about 230, or from
about 150 to about
475, or from about 150 to about 340, or from about 113 to about 340, or from
about 450 to about
800, or from about 454 to about 796, or from about 454 to about 682, or from
about 340 to about
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795, or from about 341 to about 682, or from about 568 to about 909, or from
about 227 to about
1500, or from about 225 to about 2280, or from about 460 to about 2160, or
from about 460 to
about 2050, or from about 341 to about 1820, or from about 341 to about 1710,
or from about 341
to about 1250, or from about 225 to about 1250, or from about 341 to about
1250, or from about
341 to about 1136, or from about 341 to about 1023, or from about 341 to about
910, or from about
341 to about 796, or from about 341 to about 682, or from about 341 to about
568, or from about
114 to about 1000, or from about 114 to about 950, or from about 114 to about
910, or from about
114 to about 800, or from about 114 to about 690, or from about 114 to about
575. In some
embodiments, n in the compounds of Formula (VI) and (VII) is an integer
selected from 2, 5, 10,
11, 22, 23, 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682,
794, 795, 796, 908,
909, 910, 1021, 1022, 1023, 1135, 1136, 1137, 1249, 1250, 1251, 1362, 1363,
1364, 1476, 1477,
1478, 1589, 1590, 1591, 1703, 1704, 1705, 1817, 1818, 1819, 1930, 1931, 1932,
2044, 2045, 2046,
2158, 2159, 2160, 2271, 2272, 2273, 2839, 2840, 2841, 2953, 2954, 2955, 3408,
3409, 3410, 3976,
3977, 3978, 4544, 4545, and 4546.
[165] In some embodiments, the IL-2 conjugate comprises the amino acid
sequence of SEQ ID
NO: 3 in which at least one amino acid residue in the IL-2 conjugate is
replaced by the structure of
Formula (VI) or (VII), or a mixture of (VI) and (VII), wherein the amino acid
residue in SEQ ID
NO: 3 that is replaced is selected from F41, F43, K42, E61, and P64, and
wherein n is an integer
from about 450 to about 800, or from about 454 to about 796, or from about 454
to about 682, or
from about 568 to about 909. In some embodiments, n in the compounds of
Formula (VI) and (VII)
is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796,
908, 909, 910, 1021,
1022, 1023, 1135, 1136, 1137, and 1249.
[166] In some embodiments, the IL-2 conjugate comprises the amino acid
sequence of SEQ ID
NO: 3 in which at least one amino acid residue in the IL-2 conjugate is
replaced by the structure of
Formula (VI) or (VII), or a mixture of (VI) and (VII), wherein the amino acid
residue in SEQ ID
NO: 3 that is replaced is selected from E61 and P64, and wherein n is an
integer from about 450 to
about 800, or from about 454 to about 796, or from about 454 to about 682, or
from about 568 to
about 909. In some embodiments, n in the compounds of Formula (VI) and (VII)
is an integer
selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and
910
[167] In some embodiments, the IL-2 conjugate comprises the amino acid
sequence of SEQ ID
NO: 3 in which at least one amino acid residue in the 1L-2 conjugate is
replaced by the structure of
Formula (VI) or (VII), or a mixture of (VI) and (VII), wherein the amino acid
residue in SEQ ID
NO: 3 that is replaced is E61, and wherein n is an integer from about 450 to
about 800, or from
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about 454 to about 796, or from about 454 to about 682, or from about 568 to
about 909. In some
embodiments, n in the compounds of Formula (VI) and (VII) is an integer
selected from 454, 455,
568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910. In some
embodiments, n is from about
500 to about 1000. In some embodiments, n is from about 550 to about 800. In
some
embodiments, n is about 681.
[168] In some embodiments, the IL-2 conjugate comprises the amino acid
sequence of SEQ ID
NO: 3 in which at least one amino acid residue in the IL-2 conjugate is
replaced by the structure of
Formula (VI) or (VII), or a mixture of (VI) and (VII), wherein the amino acid
residue in in SEQ ID
NO: 3 that is replaced is P64, and wherein n is an integer from about 450 to
about 800, or from
about 454 to about 796, or from about 454 to about 682, or from about 568 to
about 909. In some
embodiments, n in the compounds of Formula (VI) and (VII) is an integer
selected from 454, 455,
568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910. In some
embodiments, n is from about
500 to about 1000. In some embodiments, n is from about 550 to about 800. In
some
embodiments, n is about 681.
[169] In some embodiments n in the structures of Formula (VI) and (VII) is
an integer such that
the molecular weight of the PEG moiety is in the range from about 1,000
Daltons to about 200,000
Daltons, or from about 2,000 Daltons to about 150,000 Daltons, or from about
3,000 Daltons to
about 125,000 Daltons, or from about 4,000 Daltons to about 100,000 Daltons,
or from about 5,000
Daltons to about 100,000 Daltons, or from about 6,000 Daltons to about 90,000
Daltons, or from
about 7,000 Daltons to about 80,000 Daltons, or from about 8,000 Daltons to
about 70,000 Daltons,
or from about 5,000 Daltons to about 70,000 Daltons, or from about 5,000
Daltons to about 65,000
Daltons, or from about 5,000 Daltons to about 60,000 Daltons, or from about
5,000 Daltons to about
50,000 Daltons, or from about 6,000 Daltons to about 50,000 Daltons, or from
about 7,000 Daltons
to about 50,000 Daltons, or from about 7,000 Daltons to about 45,000 Daltons,
or from about 7,000
Daltons to about 40,000 Daltons, or from about 8,000 Daltons to about 40,000
Daltons, or from
about 8,500 Daltons to about 40,000 Daltons, or from about 8,500 Daltons to
about 35,000 Daltons,
or from about 9,000 Daltons to about 50,000 Daltons, or from about 9,000
Daltons to about 45,000
Daltons, or from about 9,000 Daltons to about 40,000 Daltons, or from about
9,000 Daltons to about
35,000 Daltons, or from about 9,000 Daltons to about 30,000 Daltons, or from
about 9,500 Daltons
to about 35,000 Daltons, or from about 9,500 Daltons to about 30,000 Daltons,
or from about
10,000 Daltons to about 50,000 Daltons, or from about 10,000 Daltons to about
45,000 Daltons, or
from about 10,000 Daltons to about 40,000 Daltons, or from about 10,000
Daltons to about 35,000
Daltons, or from about 10,000 Daltons to about 30,000 Daltons, or from about
15,000 Daltons to
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about 50,000 Daltons, or from about 15,000 Daltons to about 45,000 Daltons, or
from about 15,000
Daltons to about 40,000 Daltons, or from about 15,000 Daltons to about 35,000
Daltons, or from
about 15,000 Daltons to about 30,000 Daltons, or from about 20,000 Daltons to
about 50,000
Daltons, or from about 20,000 Daltons to about 45,000 Daltons, or from about
20,000 Daltons to
about 40,000 Daltons, or from about 20,000 Daltons to about 35,000 Daltons, or
from about 20,000
Daltons to about 30,000 Daltons.
11701 In some embodiments, the IL-2 conjugate comprises the amino
acid sequence of SEQ ID
NO: 3 in which at least one amino acid residue in the IL-2 conjugate is
replaced by the structure of
Formula (VI) or (VII), or a mixture of (VI) and (VII), wherein n is an integer
such that the
molecular weight of the PEG moiety is about 5,000 Daltons, about 7,500
Daltons, about 10,000
Daltons, about 15,000 Daltons, about 20,000 Daltons, about 25,000 Daltons,
about 30,000 Daltons,
about 35,000 Daltons, about 40,000 Daltons, about 45,000 Daltons, about 50,000
Daltons, about
60,000 Daltons, about 70,000 Daltons, about 80,000 Daltons, about 90,000
Daltons, about 100,000
Daltons, about 125,000 Daltons, about 150,000 Daltons, about 175,000 Daltons
or about 200,000
Daltons.
[171] In some embodiments, the IL-2 conjugate comprises the amino acid
sequence of SEQ ID
NO: 3 in which at least one amino acid residue in the IL-2 conjugate is
replaced by the structure of
Formula (VI) or (VII), or a mixture of (VI) and (VII), wherein n is an integer
such that the
molecular weight of the PEG moiety is about 5,000 Daltons, about 7,500
Daltons, about 10,000
Daltons, about 15,000 Daltons, about 20,000 Daltons, about 25,000 Daltons,
about 30,000 Daltons,
about 35,000 Daltons, about 40,000 Daltons, about 45,000 Daltons, or about
50,000 Daltons.
[172] In some embodiments, the IL-2 conjugate comprises the amino acid
sequence of SEQ ID
NO: 3 in which at least one amino acid residue in the IL-2 conjugate is
replaced by the structure of
Formula (VIII) or (IX), or a mixture of (VIII) and (IX):
0
Niõ I 0
N
CH3
40 0
Formula (VIII);
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0
N cr,H.T N
H3
X N 0
0
Formula (IX);
wherein:
n is an integer in the range from about 2 to about 5000;
q is 1, 2, or 3; and
X has the structure:
X-1
cs's H
OA X+ 1
X-1 indicates the point of attachment to the preceding amino acid residue; and
X+1 indicates the point of attachment to the following amino acid residue.
[173] In some embodiments of Formula (VIII) or Formula (IX), or a mixture
of Formula (VIII)
or Formula (IX), q is 1. In some embodiments of Formula (VIII) or Formula
(IX), or a mixture of
Formula (VIII) or Formula (IX), q is 2. In some embodiments of Formula (VIII)
or Formula (IX), or
a mixture of Formula (VIII) or Formula (IX), q is 3.
[174] Here and throughout, the structure of Formula (VIII) encompasses
pharmaceutically
acceptable salts, solvates, or hydrates thereof. Here and throughout, the
structure of Formula (IX)
encompasses pharmaceutically acceptable salts, solvates, or hydrates thereof
In some
embodiments, the IL-2 conjugate is a pharmaceutically acceptable salt,
solvate, or hydrate.
[175] In some embodiments, q is 1 and the structures of Formula (VIII) and
Formula (IX) are the
structures of Formula (Villa) or (IXa):
0
0
N'õ
N N C H
3
/ n
0
Formula (Villa);
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0
NI' I
0
0
Formula (IXa);
wherein:
n is an integer in the range from about 2 to about 5000; and
X has the structure:
,5
NH
0 5-5-
=
X-1 indicates the point of attachment to the preceding amino acid residue; and
X+1 indicates the point of attachment to the following amino acid residue.
[176] In some embodiments, n in the compounds of Formula (VIII) and
(IX) is in the range from
about 5 to about 4600, or from about 10 to about 4000, or from about 20 to
about 3000, or from
about 100 to about 3000, or from about 100 to about 2900, or from about 150 to
about 2900, or
from about 125 to about 2900, or from about 100 to about 2500, or from about
100 to about 2000,
or from about 100 to about 1900, or from about 100 to about 1850, or from
about 100 to about
1750, or from about 100 to about 1650, or from about 100 to about 1500, or
from about 100 to
about 1400, or from about 100 to about 1300, or from about 100 to about 1250,
or from about 100
to about 1150, or from about 100 to about 1100, or from about 100 to about
1000, or from about
100 to about 900, or from about 100 to about 750, or from about 100 to about
700, or from about
100 to about 600, or from about 100 to about 575, or from about 100 to about
500, or from about
100 to about 450, or from about 100 to about to about 350, or from about 100
to about 275, or from
about 100 to about 230, or from about 150 to about 475, or from about 150 to
about 340, or from
about 113 to about 340, or from about 450 to about 800, or from about 454 to
about 796, or from
about 454 to about 682, or from about 340 to about 795, or from about 341 to
about 682, or from
about 568 to about 909, or from about 227 to about 1500, or from about 225 to
about 2280, or from
about 460 to about 2160, or from about 460 to about 2050, or from about 341 to
about 1820, or
from about 341 to about 1710, or from about 341 to about 1250, or from about
225 to about 1250,
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or from about 341 to about 1250, or from about 341 to about 1136, or from
about 341 to about
1023, or from about 341 to about 910, or from about 341 to about 796, or from
about 341 to about
682, or from about 341 to about 568, or from about 114 to about 1000, or from
about 114 to about
950, or from about 114 to about 910, or from about 114 to about 800, or from
about 114 to about
690, or from about 114 to about 575. In some embodiments, n in the compounds
of Formula (VIII)
and (IX) is an integer selected from 2, 5, 10, 11, 22, 23, 113, 114, 227, 228,
340, 341, 454, 455,
568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135,
1136, 1137, 1249,
1250, 1251, 1362, 1363, 1364, 1476, 1477, 1478, 1589, 1590, 1591, 1703, 1704,
1705, 1817, 1818,
1819, 1930, 1931, 1932, 2044, 2045, 2046, 2158, 2159, 2160, 2271, 2272, 2273,
2839, 2840, 2841,
2953, 2954, 2955, 3408, 3409, 3410, 3976, 3977, 3978, 4544, 4545, and 4546.
11771 In some embodiments, the position of the structure of Formula
(VIII), Formula (IX), or a
mixture of Formula (VIII) and (IX) in the amino acid sequence of the IL-2
conjugate is selected
from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68, and L71, wherein
the position of
the structures of Formula (VIII), Formula (IX), or mixture thereof in the
amino acid sequence of the
IL-2 conjugate is in reference to the positions in SEQ ID NO: 3. In some
embodiments, the position
of the structure of Formula (VIII), Formula (IX), or a mixture of Formula
(VIII) and Formula (IX)
in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is selected
from K34, F41, F43,
K42, E61, P64, R37, T40, E67, Y44, V68, and L71. In some embodiments, the
position of the
structure of Formula (VIII), Formula (IX), or a mixture of Formula (VIII) and
Formula (IX) in the
amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is at position K34.
In some
embodiments, the position of the structure of Formula (VIII), Formula (IX), or
a mixture of
Formula (VIII) and Formula (IX) in the amino acid sequence of the IL-2
conjugate of SEQ ID NO:
3 is at position F41. In some embodiments, the position of the structure of
Formula (VIII), Formula
(IX), or a mixture of Formula (VIII) and Formula (IX) in the amino acid
sequence of the IL-2
conjugate of SEQ ID NO: 3 is at position F43. In some embodiments, the
position of the structure
of Formula (VIII), Formula (IX), or a mixture of Formula (VIII) and Formula
(IX) in the amino acid
sequence of the IL-2 conjugate of SEQ ID NO: 3 is at position K42. In some
embodiments, the
position of the structure of Formula (VIII), Formula (IX), or a mixture of
Formula (VIII) and
Formula (IX) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3
is at position E61.
In some embodiments, the position of the structure of Formula (VIII), Formula
(IX), or a mixture of
Formula (VIII) and Formula (IX) in the amino acid sequence of the IL-2
conjugate of SEQ ID NO:
3 is at position P64. In some embodiments, the position of the structure of
Formula (VIII), Formula
(IX), or a mixture of Formula (VIII) and Formula (IX) in the amino acid
sequence of the IL-2
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conjugate of SEQ ID NO: 3 is at position R37. In some embodiments, the
position of the structure
of Formula (VIII), Formula (IX), or a mixture of Formula (VIII) and Formula
(IX) in the amino acid
sequence of the IL-2 conjugate of SEQ ID NO: 3 is at position T40. In some
embodiments, the
position of the structure of Formula (VIII), Formula (IX), or a mixture of
Formula (VIII) and
Formula (IX) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3
is at position E67.
In some embodiments, the position of the structure of Formula (VIII), Formula
(IX), or a mixture of
Formula (VIII) and Formula (IX) in the amino acid sequence of the IL-2
conjugate of SEQ ID NO:
3 is at position Y44. In some embodiments, the position of the structure of
Formula (VIII), Formula
(IX), or a mixture of Formula (VIII) and Formula (IX) in the amino acid
sequence of the IL-2
conjugate of SEQ ID NO: 3 is at position V68. In some embodiments, the
position of the structure
of Formula (VIII), Formula (IX), or a mixture of Formula (VIII) and Formula
(IX) in the amino acid
sequence of the IL-2 conjugate of SEQ ID NO: 3 is at position L71.
[178] In some embodiments, the ratio of the amount of the structure of
Formula (VIII) to the
amount of the structure of Formula (IX) comprising the total amount of the IL-
2 conjugate is about
1:1. In some embodiments, the ratio of the amount of the structure of Formula
(VIII) to the amount
of the structure of Formula (IX) comprising the total amount of the IL-2
conjugate is greater than
1:1. In some embodiments, the ratio of the amount of the structure of Formula
(VIII) to the amount
of the structure of Formula (IX) comprising the total amount of the IL-2
conjugate is less than 1:1.
[179] In some embodiments, the IL-2 conjugate comprises the amino acid
sequence of SEQ ID
NO: 3 in which at least one amino acid residue in the IL-2 conjugate is
replaced by the structure of
Formula (VIII) or (IX), or a mixture of (VIII) and (IX), wherein the amino
acid residue in in SEQ
ID NO: 3 that is replaced is selected from K34, F41, F43, K42, E61, P64, R37,
T40, E67, Y44,
V68, and L71, and wherein n is an integer from 100 to about 1150, or from
about 100 to about
1100, or from about 100 to about 1000, or from about 100 to about 900, or from
about 100 to about
750, or from about 100 to about 700, or from about 100 to about 600, or from
about 100 to about
575, or from about 100 to about 500, or from about 100 to about 450, or from
about 100 to about to
about 350, or from about 100 to about 275, or from about 100 to about 230, or
from about 150 to
about 475, or from about 150 to about 340, or from about 113 to about 340, or
from about 450 to
about 800, or from about 454 to about 796, or from about 454 to about 682, or
from about 340 to
about 795, or from about 341 to about 682, or from about 568 to about 909, or
from about 227 to
about 1500, or from about 225 to about 2280, or from about 460 to about 2160,
or from about 460
to about 2050, or from about 341 to about 1820, or from about 341 to about
1710, or from about
341 to about 1250, or from about 225 to about 1250, or from about 341 to about
1250, or from
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about 341 to about 1136, or from about 341 to about 1023, or from about 341 to
about 910, or from
about 341 to about 796, or from about 341 to about 682, or from about 341 to
about 568, or from
about 114 to about 1000, or from about 114 to about 950, or from about 114 to
about 910, or from
about 114 to about 800, or from about 114 to about 690, or from about 114 to
about 575. In some
embodiments, n in the compounds of Formula (VIII) and (IX) is an integer
selected from 2, 5, 10,
11, 22, 23, 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682,
794, 795, 796, 908,
909, 910, 1021, 1022, 1023, 1135, 1136, 1137, 1249, 1250, 1251, 1362, 1363,
1364, 1476, 1477,
1478, 1589, 1590, 1591, 1703, 1704, 1705, 1817, 1818, 1819, 1930, 1931, 1932,
2044, 2045, 2046,
2158, 2159, 2160, 2271, 2272, 2273, 2839, 2840, 2841, 2953, 2954, 2955, 3408,
3409, 3410, 3976,
3977, 3978, 4544, 4545, and 4546.
[180] In some embodiments, the IL-2 conjugate comprises the amino acid
sequence of SEQ ID
NO: 3 in which at least one amino acid residue in the IL-2 conjugate is
replaced by the structure of
Formula (VIII) or (IX), or a mixture of Formula (VIII) and Formula (IX),
wherein the amino acid
residue in in SEQ ID NO: 3 that is replaced is selected from F41, F43, K42,
E61, and P64, and
wherein n is an integer from about 450 to about 800, or from about 454 to
about 796, or from about
454 to about 682, or from about 568 to about 909. In some embodiments, n in
the compounds of
Formula (VIII) and Formula (IX) is an integer selected from 454, 455, 568,
569, 680, 681, 682, 794,
795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137, and 1249.
[181] In some embodiments, the IL-2 conjugate comprises the amino acid
sequence of SEQ ID
NO: 3 in which at least one amino acid residue in the IL-2 conjugate is
replaced by the structure of
Formula (VIII) or (IX), or a mixture of Formula (VIII) and Formula (IX),
wherein the amino acid
residue in SEQ ID NO: 3 that is replaced is selected from E61 and P64, and
wherein n is an integer
from about 450 to about 800, or from about 454 to about 796, or from about 454
to about 682, or
from about 568 to about 909. In some embodiments, n in the compounds of
Formula (VIII) and
Formula (IX) is an integer selected from 454, 455, 568, 569, 680, 681, 682,
794, 795, 796, 908, 909,
and 910.
[182] In some embodiments, the IL-2 conjugate comprises the amino acid
sequence of SEQ ID
NO: 3 in which at least one amino acid residue in the IL-2 conjugate is
replaced by the structure of
Formula (VIII) or (IX), or a mixture of Formula (VIII) and Formula (IX),
wherein the amino acid
residue in SEQ ID NO: 3 that is replaced is E61, and wherein n is an integer
from about 450 to
about 800, or from about 454 to about 796, or from about 454 to about 682, or
from about 568 to
about 909. In some embodiments, n in the compounds of Formula (VIII) and
Formula (IX) is an
integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908,
909, and 910. In some
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embodiments, n is from about 500 to about 1000. In some embodiments, n is from
about 550 to
about 800. In some embodiments, n is about 681.
[183] In some embodiments, the IL-2 conjugate comprises the amino acid
sequence of SEQ ID
NO: 3 in which at least one amino acid residue in the IL-2 conjugate is
replaced by the structure of
Formula (VIII) or Formula (IX), or a mixture of Formula (VIII) and Formula
(IX), wherein the
amino acid residue in in SEQ ID NO: 3 that is replaced is P64, and wherein n
is an integer from
about 450 to about 800, or from about 454 to about 796, or from about 454 to
about 682, or from
about 568 to about 909. In some embodiments, n in the compounds of Formula
(VIII) and Formula
(IX) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795,
796, 908, 909, and
910. In some embodiments, n is from about 500 to about 1000. In some
embodiments, n is from
about 550 to about 800. In some embodiments, n is about 681.
[184] In some embodements, the IL-2 conjugate comprises the amino acid
sequence of SEQ ID
NO: 3 in which at least one amino acid residue in the IL-2 conjugate is
replaced by the structure of
Formula (VIII) or Formula (IX), or a mixture of Formula (VIII) and Formula
(IX), wherein n is an
integer such that the molecular weight of the PEG moiety is in the range from
about 1,000 Daltons
about 200,000 Daltons, or from about 2,000 Daltons to about 150,000 Daltons,
or from about 3,000
Daltons to about 125,000 Daltons, or from about 4,000 Daltons to about 100,000
Daltons, or from
about 5,000 Daltons to about 100,000 Daltons, or from about 6,000 Daltons to
about 90,000
Daltons, or from about 7,000 Daltons to about 80,000 Daltons, or from about
8,000 Daltons to about
70,000 Daltons, or from about 5,000 Daltons to about 70,000 Daltons, or from
about 5,000 Daltons
to about 65,000 Daltons, or from about 5,000 Daltons to about 60,000 Daltons,
or from about 5,000
Daltons to about 50,000 Daltons, or from about 6,000 Daltons to about 50,000
Daltons, or from
about 7,000 Daltons to about 50,000 Daltons, or from about 7,000 Daltons to
about 45,000 Daltons,
or from about 7,000 Daltons to about 40,000 Daltons, or from about 8,000
Daltons to about 40,000
Daltons, or from about 8,500 Daltons to about 40,000 Daltons, or from about
8,500 Daltons to about
35,000 Daltons, or from about 9,000 Daltons to about 50,000 Daltons, or from
about 9,000 Daltons
to about 45,000 Daltons, or from about 9,000 Daltons to about 40,000 Daltons,
or from about 9,000
Daltons to about 35,000 Daltons, or from about 9,000 Daltons to about 30,000
Daltons, or from
about 9,500 Daltons to about 35,000 Daltons, or from about 9,500 Daltons to
about 30,000 Daltons,
or from about 10,000 Daltons to about 50,000 Daltons, or from about 10,000
Daltons to about
45,000 Daltons, or from about 10,000 Daltons to about 40,000 Daltons, or from
about 10,000
Daltons to about 35,000 Daltons, or from about 10,000 Daltons to about 30,000
Daltons, or from
about 15,000 Daltons to about 50,000 Daltons, or from about 15,000 Daltons to
about 45,000
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Daltons, or from about 15,000 Daltons to about 40,000 Daltons, or from about
15,000 Daltons to
about 35,000 Daltons, or from about 15,000 Daltons to about 30,000 Daltons, or
from about 20,000
Daltons to about 50,000 Daltons, or from about 20,000 Daltons to about 45,000
Daltons, or from
about 20,000 Daltons to about 40,000 Daltons, or from about 20,000 Daltons to
about 35,000
Daltons, or from about 20,000 Daltons to about 30,000 Daltons. In some
embodiments, the IL-2
conjugate comprises the amino acid sequence of SEQ ID NO: 3 in which at least
one amino acid
residue in the IL-2 conjugate is replaced by the structure of Formula (VIII)
or Formula (IX), or a
mixture of Formula (VIII) and Formula (IX), wherein n is an integer such that
the molecular weight
of the PEG moiety is about 5,000 Daltons, about 7,500 Daltons, about 10,000
Daltons, about 15,000
Daltons, about 20,000 Daltons, about 25,000 Daltons, about 30,000 Daltons,
about 35,000 Daltons,
about 40,000 Daltons, about 45,000 Daltons, about 50,000 Daltons, about 60,000
Daltons, about
70,000 Daltons, about 80,000 Daltons, about 90,000 Daltons, about 100,000
Daltons, about 125,000
Daltons, about 150,000 Daltons, about 175,000 Daltons or about 200,000
Daltons. In some
embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID
NO: 3 in which at
least one amino acid residue in the IL-2 conjugate is replaced by the
structure of Formula (VIII) or
Formula (IX), or a mixture of Formula (VIII) and Formula (IX), wherein n is an
integer such that
the molecular weight of the PEG moiety is about 5,000 Daltons, about 7,500
Daltons, about 10,000
Daltons, about 15,000 Daltons, about 20,000 Daltons, about 25,000 Daltons,
about 30,000 Daltons,
about 35,000 Daltons, about 40,000 Daltons, about 45,000 Daltons, or about
50,000 Daltons.
[185] In some embodiments, the IL-2 conjugate comprises the amino
acid sequence of SEQ ID
NO: 3 in which at least one amino acid residue in the IL-2 conjugate is
replaced by the structure of
Formula (X) or Formula (XI), or a mixture of Formula (X) and Formula (XI):
411
NI: I
y0 1,c; n
0 11- 0
Formula (X);
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'cisNH 0
NI's, I
0 0
Formula (XI);
wherein:
n is an integer in the range from about 2 to about 5000;
q is 1, 2, or 3; and
the wavy lines indicate covalent bonds to amino acid residues within SEQ ID
NO: 3 that are not
replaced.
[186] In some embodiments of Formula (X) or Formula (XI), or a mixture of
Formula (X) or
Formula (XI), q is 1. In some embodiments of Formula (X) or Formula (XI), or a
mixture of
Formula (X) or Formula (XI), q is 2. In some embodiments of Formula (X) or
Formula (XI), or a
mixture of Formula (X) or Formula (XI), q is 3
[187] Here and throughout, the structure of Formula (X) encompasses
pharmaceutically
acceptable salts, solvates, or hydrates thereof. Here and throughout, the
structure of Formula (XI)
encompasses pharmaceutically acceptable salts, solvates, or hydrates thereof.
In some
embodiments, the IL-2 conjugate is a pharmaceutically acceptable salt,
solvate, or hydrate.
[188] In some embodiments, q is 1 and the structures of Formula (X) and
Formula (XI) are the
structures of Formula (Xa) and Formula (XIa):
N's' I
CH _3
0 0
0 / 0
Formula (Xa);
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NH 0
L'-'''N-j'L'eN1 =
,N
Nõ
N
11 0 0 /fl
Formula (X Ia);
wherein:
n is an integer in the range from about 2 to about 5000; and
the wavy lines indicate covalent bonds to amino acid residues within SEQ ID
NO: 3 that are not
replaced.
[189] In some embodiments, the stereochemistry of the chiral center within
Formula (X) and
Formula (XI) is racemic, is enriched in (R), is enriched in (S), is
substantially (R), is substantially
(S), is (R) or is (S). In some embodiments, the stereochemistry of the chiral
center within Formula
(X) and Formula (XI) is racemic. In some embodiments, the stereochemistry of
the chiral center
within Formula (X) and Formula (XI) is enriched in (R). In some embodiments,
the
stereochemistry of the chiral center within Formula (X) and Foimula (XI) is
enriched in (S). In
some embodiments, the stereochemistry of the chiral center within Formula (X)
and Formula (XI) is
substantially (R). In some embodiments, the stereochemistry of the chiral
center within Formula
(X) and Formula (XI) is substantially (S). In some embodiments, the
stereochemistry of the chiral
center within Formula (X) and Formula (XI) is (R). In some embodiments, the
stereochemistry of
the chiral center within Formula (X) and Formula (XI) is (S).
[190] In some embodiments, n in the compounds of Formula (X) and (XI) is in
the range from
about 5 to about 4600, or from about 10 to about 4000, or from about 20 to
about 3000, or from
about 100 to about 3000, or from about 100 to about 2900, or from about 150 to
about 2900, or
from about 125 to about 2900, or from about 100 to about 2500, or from about
100 to about 2000,
or from about 100 to about 1900, or from about 100 to about 1850, or from
about 100 to about
1750, or from about 100 to about 1650, or from about 100 to about 1500, or
from about 100 to
about 1400, or from about 100 to about 1300, or from about 100 to about 1250,
or from about 100
to about 1150, or from about 100 to about 1100, or from about 100 to about
1000, or from about
100 to about 900, or from about 100 to about 750, or from about 100 to about
700, or from about
100 to about 600, or from about 100 to about 575, or from about 100 to about
500, or from about
100 to about 450, or from about 100 to about to about 350, or from about 100
to about 275, or from
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about 100 to about 230, or from about 150 to about 475, or from about 150 to
about 340, or from
about 113 to about 340, or from about 450 to about 800, or from about 454 to
about 796, or from
about 454 to about 682, or from about 340 to about 795, or from about 341 to
about 682, or from
about 568 to about 909, or from about 227 to about 1500, or from about 225 to
about 2280, or from
about 460 to about 2160, or from about 460 to about 2050, or from about 341 to
about 1820, or
from about 341 to about 1710, or from about 341 to about 1250, or from about
225 to about 1250,
or from about 341 to about 1250, or from about 341 to about 1136, or from
about 341 to about
1023, or from about 341 to about 910, or from about 341 to about 796, or from
about 341 to about
682, or from about 341 to about 568, or from about 114 to about 1000, or from
about 114 to about
950, or from about 114 to about 910, or from about 114 to about 800, or from
about 114 to about
690, or from about 114 to about 575. In some embodiments, n in the compounds
of Formula (X)
and (XI) is an integer selected from 2, 5, 10, 11, 22, 23, 113, 114, 227, 228,
340, 341, 454, 455,
568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135,
1136, 1137, 1249,
1250, 1251, 1362, 1363, 1364, 1476, 1477, 1478, 1589, 1590, 1591, 1703, 1704,
1705, 1817, 1818,
1819, 1930, 1931, 1932, 2044, 2045, 2046, 2158, 2159, 2160, 2271, 2272, 2273,
2839, 2840, 2841,
2953, 2954, 2955, 3408, 3409, 3410, 3976, 3977, 3978, 4544, 4545, and 4546.
[191] In some embodiments, the position of the structure of Formula
(X), Formula (XI), or a
mixture of Formula (X) and Formula (XI) in the amino acid sequence of the IL-2
conjugate is
selected from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68, and L71,
wherein the
position of the structure of Formula (X), Formula (XI), or a mixture thereof,
in the amino acid
sequence of the IL-2 conjugate is in reference to the positions in SEQ ID NO:
3. In some
embodiments, the IL-2 conjugate in which the position of the structure of
Formula (X), Formula
(XI), or a mixture of Formula (X) and Formula (XI) in the amino acid sequence
of the IL-2
conjugate of SEQ ID NO: 3 is selected from K34, F41, F43, K42, E61, P64, R37,
T40, E67, Y44,
V68, and L71. In some embodiments, the position of the structure of Formula
(X), Formula (XI), or
a mixture of Formula (X) and Formula (XI) in the amino acid sequence of the IL-
2 conjugate of
SEQ ID NO: 3 is at position K34. In some embodiments, the position of the
structure of Formula
(X), Formula (XI), or a mixture of Formula (X) and Formula (XI) in the amino
acid sequence of the
IL-2 conjugate of SEQ ID NO: 3 is at position F41. In some embodiments, the
position of the
structure of Formula (X), Formula (XI), or a mixture of Formula (X) and
Formula (XI) in the amino
acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is at position F43. In
some embodiments, the
position of the structure of Formula (X), Formula (XI), or a mixture of
Formula (X) and Formula
(XI) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is at
position K42. In some
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embodiments, the position of the structure of Formula (X), Formula (XI), or a
mixture of Formula
(X) and Formula (XI) in the amino acid sequence of the IL-2 conjugate of SEQ
ID NO: 3 is at
position E61. In some embodiments, the position of the structure of Formula
(X), Formula (XI), or
a mixture of Formula (X) and Formula (XI) in the amino acid sequence of the IL-
2 conjugate of
SEQ ID NO: 3 is at position P64. In some embodiments, the position of the
structure of Formula
(X), Formula (XI), or a mixture of Formula (X) and Formula (XI) in the amino
acid sequence of the
IL-2 conjugate of SEQ ID NO: 3 is at position R37. In some embodiments, the
position of the
structure of Formula (X), Formula (XI), or a mixture of Formula (X) and
Formula (XI) in the amino
acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is at position 140. In
some embodiments, the
position of the structure of Formula (X), Formula (XI), or a mixture of
Formula (X) and Formula
(XI) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is at
position E67. In some
embodiments, the position of the structure of Formula (X), Formula (XI), or a
mixture of Formula
(X) and Formula (XI) in the amino acid sequence of the IL-2 conjugate of SEQ
ID NO: 3 is at
position Y44. In some embodiments, the position of the structure of Formula
(X), Formula (XI), or
a mixture of Formula (X) and Formula (XI) in the amino acid sequence of the IL-
2 conjugate of
SEQ ID NO: 3 is at position V68. In some embodiments, the position of the
structure of Formula
(X), Formula (XI), or a mixture of Formula (X) and Formula (XI) in the amino
acid sequence of the
IL-2 conjugate of SEQ ID NO: 3 is at position L71.
[192] In some embodiments, the ratio of the amount of the structure of
Formula (X) to the
amount of the structure of Formula (XI) comprising the total amount of the IL-
2 conjugate is about
1:1. In some embodiments, the ratio of the amount of the structure of Formula
(X) to the amount of
the structure of Formula (XI) comprising the total amount of the IL-2
conjugate is greater than 1:1.
In some embodiments, the ratio of the amount of the structure of Formula (X)
to the amount of the
structure of Formula (XI) comprising the total amount of the IL-2 conjugate is
less than 1:1.
[193] In some embodiments, the IL-2 conjugate comprises the amino acid
sequence of SEQ ID
NO: 3 in which at least one amino acid residue in the IL-2 conjugate is
replaced by the structure of
Formula (X), Formula (XI), or a mixture of Formula (X) and Formula (XI),
wherein the amino acid
residue in SEQ ID NO: 3 that is replaced is selected from K34, F41, F43, K42,
E61, P64, R37, T40,
E67, Y44, V68, and L71, and wherein n is an integer from 100 to about 1150, or
from about 100 to
about 1100, or from about 100 to about 1000, or from about 100 to about 900,
or from about 100 to
about 750, or from about 100 to about 700, or from about 100 to about 600, or
from about 100 to
about 575, or from about 100 to about 500, or from about 100 to about 450, or
from about 100 to
about to about 350, or from about 100 to about 275, or from about 100 to about
230, or from about
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150 to about 475, or from about 150 to about 340, or from about 113 to about
340, or from about
450 to about 800, or from about 454 to about 796, or from about 454 to about
682, or from about
340 to about 795, or from about 341 to about 682, or from about 568 to about
909, or from about
227 to about 1500, or from about 225 to about 2280, or from about 460 to about
2160, or from
about 460 to about 2050, or from about 341 to about 1820, or from about 341 to
about 1710, or
from about 341 to about 1250, or from about 225 to about 1250, or from about
341 to about 1250,
or from about 341 to about 1136, or from about 341 to about 1023, or from
about 341 to about 910,
or from about 341 to about 796, or from about 341 to about 682, or from about
341 to about 568, or
from about 114 to about 1000, or from about 114 to about 950, or from about
114 to about 910, or
from about 114 to about 800, or from about 114 to about 690, or from about 114
to about 575. In
some embodiments, n in the compounds of Formula (VI) and (VII) is an integer
selected from 2, 5,
10, 11, 22, 23, 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681,
682, 794, 795, 796, 908,
909, 910, 1021, 1022, 1023, 1135, 1136, 1137, 1249, 1250, 1251, 1362, 1363,
1364, 1476, 1477,
1478, 1589, 1590, 1591, 1703, 1704, 1705, 1817, 1818, 1819, 1930, 1931, 1932,
2044, 2045, 2046,
2158, 2159, 2160, 2271, 2272, 2273, 2839, 2840, 2841, 2953, 2954, 2955, 3408,
3409, 3410, 3976,
3977, 3978, 4544, 4545, and 4546.
[194] In some embodiments, the IL-2 conjugate comprises the amino acid
sequence of SEQ ID
NO: 3 in which at least one amino acid residue in the IL-2 conjugate is
replaced by the structure of
Formula (X), Formula (XI), or a mixture of Formula (X) and Formula (XI),
wherein the amino acid
residue in in SEQ ID NO: 3 that is replaced is selected from F41, F43, K42,
E61, and P64, and
wherein n is an integer from about 450 to about 800, or from about 454 to
about 796, or from about
454 to about 682, or from about 568 to about 909. In some embodiments, n in
the compounds of
Formula (X) and Formula (XI) is an integer selected from 454, 455, 568, 569,
680, 681, 682, 794,
795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137, and 1249.
[195] In some embodiments, the IL-2 conjugate comprises the amino acid
sequence of SEQ ID
NO: 3 in which at least one amino acid residue in the IL-2 conjugate is
replaced by the structure of
Formula (X), Formula (XI), or a mixture of Formula (X) and Formula (XI),
wherein the amino acid
residue in SEQ ID NO: 3 that is replaced is selected from E61 and P64, and
wherein n is an integer
from about 450 to about 800, or from about 454 to about 796, or from about 454
to about 682, or
from about 568 to about 909. In some embodiments, n in the compounds of
Formula (X) and
Formula (X1) is an integer selected from 454, 455, 568, 569, 680, 681, 682,
794, 795, 796, 908, 909,
and 910.
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[196] In some embodiments, the IL-2 conjugate comprises the amino acid
sequence of SEQ ID
NO: 3 in which at least one amino acid residue in the IL-2 conjugate is
replaced by the structure of
Formula (X), Formula (XI), or a mixture of Formula (X) and Formula (XI),
wherein the amino acid
residue in SEQ ID NO: 3 that is replaced is E61, and wherein n is an integer
from about 450 to
about 800, or from about 454 to about 796, or from about 454 to about 682, or
from about 568 to
about 909. In some embodiments, n in the compounds of Formula (X) and Formula
(XI) is an
integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908,
909, and 910. In some
embodiments, n is from about 500 to about 1000. In some embodiments, n is from
about 550 to
about 800. In some embodiments, n is about 681.
[197] In some embodiments, the IL-2 conjugate comprises the amino acid
sequence of SEQ ID
NO: 3 in which at least one amino acid residue in the IL-2 conjugate is
replaced by the structure of
Formula (X), Formula (XI), or a mixture of Formula (X) and Formula (XI),
wherein the amino acid
residue in SEQ ID NO: 3 that is replaced is P64, and wherein n is an integer
from about 450 to
about 800, or from about 454 to about 796, or from about 454 to about 682, or
from about 568 to
about 909. In some embodiments, n in the compounds of Formula (X) and Formula
(XI) is an
integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908,
909, and 910. In some
embodiments, n is from about 500 to about 1000. In some embodiments, n is from
about 550 to
about 800. In some embodiments, n is about 681.
[198] In some embodiments, n in the structures of Formula (X) and Formula
(XI) is an integer
such that the molecular weight of the PEG moiety is in the range from about
1,000 Daltons about
200,000 Daltons, or from about 2,000 Daltons to about 150,000 Daltons, or from
about 3,000
Daltons to about 125,000 Daltons, or from about 4,000 Daltons to about 100,000
Daltons, or from
about 5,000 Daltons to about 100,000 Daltons, or from about 6,000 Daltons to
about 90,000
Daltons, or from about 7,000 Daltons to about 80,000 Daltons, or from about
8,000 Daltons to about
70,000 Daltons, or from about 5,000 Daltons to about 70,000 Daltons, or from
about 5,000 Daltons
to about 65,000 Daltons, or from about 5,000 Daltons to about 60,000 Daltons,
or from about 5,000
Daltons to about 50,000 Daltons, or from about 6,000 Daltons to about 50,000
Daltons, or from
about 7,000 Daltons to about 50,000 Daltons, or from about 7,000 Daltons to
about 45,000 Daltons,
or from about 7,000 Daltons to about 40,000 Daltons, or from about 8,000
Daltons to about 40,000
Daltons, or from about 8,500 Daltons to about 40,000 Daltons, or from about
8,500 Daltons to about
35,000 Daltons, or from about 9,000 Daltons to about 50,000 Daltons, or from
about 9,000 Daltons
to about 45,000 Daltons, or from about 9,000 Daltons to about 40,000 Daltons,
or from about 9,000
Daltons to about 35,000 Daltons, or from about 9,000 Daltons to about 30,000
Daltons, or from
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about 9,500 Daltons to about 35,000 Daltons, or from about 9,500 Daltons to
about 30,000 Daltons,
or from about 10,000 Daltons to about 50,000 Daltons, or from about 10,000
Daltons to about
45,000 Daltons, or from about 10,000 Daltons to about 40,000 Daltons, or from
about 10,000
Daltons to about 35,000 Daltons, or from about 10,000 Daltons to about 30,000
Daltons, or from
about 15,000 Daltons to about 50,000 Daltons, or from about 15,000 Daltons to
about 45,000
Daltons, or from about 15,000 Daltons to about 40,000 Daltons, or from about
15,000 Daltons to
about 35,000 Daltons, or from about 15,000 Daltons to about 30,000 Daltons, or
from about 20,000
Daltons to about 50,000 Daltons, or from about 20,000 Daltons to about 45,000
Daltons, or from
about 20,000 Daltons to about 40,000 Daltons, or from about 20,000 Daltons to
about 35,000
Daltons, or from about 20,000 Daltons to about 30,000 Daltons.
[199] In some embodiments, the IL-2 conjugate comprises the amino acid
sequence of SEQ ID
NO: 3 in which at least one amino acid residue in the IL-2 conjugate is
replaced by the structure of
Formula (X), Formula (XI), or a mixture of Formula (X) and Formula (XI),
wherein n is an integer
such that the molecular weight of the PEG moiety is about 5,000 Daltons, about
7,500 Daltons,
about 10,000 Daltons, about 15,000 Daltons, about 20,000 Daltons, about 25,000
Daltons, about
30,000 Daltons, about 35,000 Daltons, about 40,000 Daltons, about 45,000
Daltons, about 50,000
Daltons, about 60,000 Daltons, about 70,000 Daltons, about 80,000 Daltons,
about 90,000 Daltons,
about 100,000 Daltons, about 125,000 Daltons, about 150,000 Daltons, about
175,000 Daltons or
about 200,000 Daltons.
[200] In some embodiments, the IL-2 conjugate comprises the amino acid
sequence of SEQ ID
NO: 3 in which at least one amino acid residue in the IL-2 conjugate is
replaced by the structure of
Formula (X), Formula (XI), or a mixture of Formula (X) and Formula (XI),
wherein n is an integer
such that the molecular weight of the PEG moiety is about 5,000 Daltons, about
7,500 Daltons,
about 10,000 Daltons, about 15,000 Daltons, about 20,000 Daltons, about 25,000
Daltons, about
30,000 Daltons, about 35,000 Daltons, about 40,000 Daltons, about 45,000
Daltons, or about 50,000
Daltons.
[201] In some embodiments, the IL-2 conjugate comprises the amino acid
sequence of SEQ ID
NO: 3 in which at least one amino acid residue in the IL-2 conjugate is
replaced by the structure of
Formula (XII) or Formula (XIII), or a mixture of Formula (XII) and Formula
(XIII).
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'css5.'NH 0
N Arµy--)
N. 0
N
0
Formula (XII);
N" 0
CeY.- 0
Formula (XIII);
wherein:
n is an integer in the range from about 2 to about 5000;
q is 1,2, or 3; and
the wavy lines indicate covalent bonds to amino acid residues within SEQ ID
NO: 3 that are not
replaced.
[202] In some embodiments of Formula (XII) or Formula (XIII), or a mixture
of Formula (XII)
or Formula (XIII), q is 1. In some embodiments of Formula (XII) or Formula
(XIII), or a mixture of
Formula (XII) or Formula (XIII), q is 2. In some embodiments of Formula (XII)
or Formula (XIII),
or a mixture of Formula (XII) or Formula (XIII), q is 3.
[203] Here and throughout, the structure of Formula (XII) encompasses
pharmaceutically
acceptable salts, solvates, or hydrates thereof. Here and throughout, the
structure of Formula (XIII)
encompasses pharmaceutically acceptable salts, solvates, or hydrates thereof
In some
embodiments, the IL-2 conjugate is a pharmaceutically acceptable salt,
solvate, or hydrate.
[204] In some embodiments, q is 1 and the structures of Formula (XII) and
Formula (XIII) are
the structures of Formula (XIIa) and Formula (XIIIa):
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X NH 0
oNAOThS
0
Nõ
0CH3
=0
Formula (XIIa),
0
/n
0
0
0 /
Formula (XIIIa);
wherein:
n is an integer in the range from about 2 to about 5000; and
the wavy lines indicate covalent bonds to amino acid residues within SEQ ID
NO: 3 that are not
replaced.
[205] In some embodiments, the stereochemistry of the chiral center
within Formula (XII) and
Formula (XIII) is racemic, is enriched in (R), is enriched in (S), is
substantially (R), is substantially
(S), is (R) or is (S). In some embodiments, the stereochemistry of the chiral
center within Formula
(XII) and Formula (XIII) is racemic. In some embodiments, the stereochemistry
of the chiral center
within Formula (X11) and Formula (X111) is enriched in (R). In some
embodiments, the
stereochemistry of the chiral center within Formula (XII) and Formula (XIII)
is enriched in (S). In
some embodiments, the stereochemistry of the chiral center within Formula
(XII) and Formula
(XIII) is substantially (R). In some embodiments, the stereochemistry of the
chiral center within
Formula (XII) and Formula (XIII) is substantially (S). In some embodiments,
the stereochemistry
of the chiral center within Formula (XII) and Formula (XIII) is (R). In some
embodiments, the
stereochemistry of the chiral center within Formula (XII) and Formula (XIII)
is (S).
[206] In some embodiments, n in the compounds of Formula (XII) and
Formula (XIII) is in the
range from about 5 to about 4600, or from about 10 to about 4000, or from
about 20 to about 3000,
or from about 100 to about 3000, or from about 100 to about 2900, or from
about 150 to about
2900, or from about 125 to about 2900, or from about 100 to about 2500, or
from about 100 to
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about 2000, or from about 100 to about 1900, or from about 100 to about 1850,
or from about 100
to about 1750, or from about 100 to about 1650, or from about 100 to about
1500, or from about
100 to about 1400, or from about 100 to about 1300, or from about 100 to about
1250, or from
about 100 to about 1150, or from about 100 to about 1100, or from about 100 to
about 1000, or
from about 100 to about 900, or from about 100 to about 750, or from about 100
to about 700, or
from about 100 to about 600, or from about 100 to about 575, or from about 100
to about 500, or
from about 100 to about 450, or from about 100 to about to about 350, or from
about 100 to about
275, or from about 100 to about 230, or from about 150 to about 475, or from
about 150 to about
340, or from about 113 to about 340, or from about 450 to about 800, or from
about 454 to about
796, or from about 454 to about 682, or from about 340 to about 795, or from
about 341 to about
682, or from about 568 to about 909, or from about 227 to about 1500, or from
about 225 to about
2280, or from about 460 to about 2160, or from about 460 to about 2050, or
from about 341 to
about 1820, or from about 341 to about 1710, or from about 341 to about 1250,
or from about 225
to about 1250, or from about 341 to about 1250, or from about 341 to about
1136, or from about
341 to about 1023, or from about 341 to about 910, or from about 341 to about
796, or from about
341 to about 682, or from about 341 to about 568, or from about 114 to about
1000, or from about
114 to about 950, or from about 114 to about 910, or from about 114 to about
800, or from about
114 to about 690, or from about 114 to about 575. In some embodiments, n in
the compounds of
Formula (XII) and Formula (XIII) is an integer selected from 2, 5, 10, 11, 22,
23, 113, 114, 227,
228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909,
910, 1021, 1022, 1023,
1135, 1136, 1137, 1249, 1250, 1251, 1362, 1363, 1364, 1476, 1477, 1478, 1589,
1590, 1591, 1703,
1704, 1705, 1817, 1818, 1819, 1930, 1931, 1932, 2044, 2045, 2046, 2158, 2159,
2160, 2271, 2272,
2273, 2839, 2840, 2841, 2953, 2954, 2955, 3408, 3409, 3410, 3976, 3977, 3978,
4544, 4545, and
4546.
[207] In some embodiments, the position of the structure of Formula
(XII), Formula (XIII), or a
mixture of Formula (XII) and Formula (XIII) in the amino acid sequence of the
IL-2 conjugate is
selected from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68, and L71,
wherein the
position of the structure of Formula (XII), Formula (XIII), or a mixture of
Formula (XII) and
Formula (XIII) in the amino acid sequence of the IL-2 conjugate is in
reference to the positions in
SEQ ID NO: 3. In some embodiments, the position of the structure of Formula
(XII), Formula
(XIII), or a mixture of Formula (XII) and Formula (XIII) in the amino acid
sequence of the IL-2
conjugate of SEQ ID NO: 3 is selected from K34, F41, F43, 1(42, E61, P64, R37,
T40, E67, Y44,
V68, and L71. In some embodiments, the position of the structure of Formula
(XII), Formula
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(XIII), or a mixture of Formula (XII) and Formula (XIII) in the amino acid
sequence of the IL-2
conjugate of SEQ ID NO: 3 is at position K34. In some embodiments, the
position of the structure
of Formula (XII), Formula (XIII), or a mixture of Formula (XII) and Formula
(XIII) in the amino
acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is at position F41. In
some embodiments, the
position of the structure of Formula (XII), Formula (XIII), or a mixture of
Formula (XII) and
Formula (XIII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO:
3 is at position
F43. In some embodiments, the position of the structure of Formula (XII),
Formula (XIII), or a
mixture of Formula (XII) and Formula (XIII) in the amino acid sequence of the
IL-2 conjugate of
SEQ ID NO: 3 is at position K42. In some embodiments, the position of the
structure of Formula
(XII), Formula (XIII), or a mixture of Formula (XII) and Formula (XIII) in the
amino acid sequence
of the IL-2 conjugate of SEQ ID NO: 3 is at position E61. In some embodiments,
the position of
the structure of Formula (XII), Formula (XIII), or a mixture of Formula (XII)
and Formula (XIII) in
the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is at position
P64. In some
embodiments, the position of the structure of Formula (XII), Formula (XIII),
or a mixture of
Formula (XII) and Formula (XIII) in the amino acid sequence of the IL-2
conjugate of SEQ ID NO:
3 is at position R37. In some embodiments, the position of the structure of
Formula (XII), Formula
(XIII), or a mixture of Formula (XII) and Formula (XIII) in the amino acid
sequence of the IL-2
conjugate of SEQ ID NO: 3 is at position T40. In some embodiments, the
position of the structure
of Formula (XII), Formula (XIII), or a mixture of Formula (XII) and Formula
(XIII) in the amino
acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is at position E67. In
some embodiments, the
position of the structure of Formula (XII), Formula (XIII), or a mixture of
Formula (XII) and
Formula (XIII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO:
3 is at position
Y44. In some embodiments, the position of the structure of Formula (XII),
Formula (XIII), or a
mixture of Formula (XII) and Formula (XIII) in the amino acid sequence of the
IL-2 conjugate of
SEQ ID NO: 3 is at position V68. In some embodiments, the position of the
structure of Formula
(XII), Formula (XIII), or a mixture of Formula (XII) and Formula (XIII) in the
amino acid sequence
of the IL-2 conjugate of SEQ ID NO: 3 is at position L71.
[208]
In some embodiments, the ratio of the amount of the structure of Formula
(XII) to the
amount of the structure of Formula (XIII) comprising the total amount of the
IL-2 conjugate is
about 1:1. In some embodiments, the ratio of the amount of the structure of
Formula (XII) to the
amount of the structure of Formula (XIII) comprising the total amount of the
IL-2 conjugate is
greater than 1:1. In some embodiments, the ratio of the amount of the
structure of Formula (XII) to
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the amount of the structure of Formula (XIII) comprising the total amount of
the IL-2 conjugate is
less than 1:1.
[209] In some embodiments, the IL-2 conjugate comprises the amino acid
sequence of SEQ ID
NO: 3 in which at least one amino acid residue in the IL-2 conjugate is
replaced by the structure of
Formula (XII), Formula (XIII), or a mixture of Formula (XII) and Formula
(XIII), wherein the
amino acid residue in SEQ ID NO: 3 that is replaced is selected from K34, F41,
F43, K42, E61,
P64, R37, T40, E67, Y44, V68, and L71, and wherein n is an integer from 100 to
about 1150, or
from about 100 to about 1100, or from about 100 to about 1000, or from about
100 to about 900, or
from about 100 to about 750, or from about 100 to about 700, or from about 100
to about 600, or
from about 100 to about 575, or from about 100 to about 500, or from about 100
to about 450, or
from about 100 to about to about 350, or from about 100 to about 275, or from
about 100 to about
230, or from about 150 to about 475, or from about 150 to about 340, or from
about 113 to about
340, or from about 450 to about 800, or from about 454 to about 796, or from
about 454 to about
682, or from about 340 to about 795, or from about 341 to about 682, or from
about 568 to about
909, or from about 227 to about 1500, or from about 225 to about 2280, or from
about 460 to about
2160, or from about 460 to about 2050, or from about 341 to about 1820, or
from about 341 to
about 1710, or from about 341 to about 1250, or from about 225 to about 1250,
or from about 341
to about 1250, or from about 341 to about 1136, or from about 341 to about
1023, or from about
341 to about 910, or from about 341 to about 796, or from about 341 to about
682, or from about
341 to about 568, or from about 114 to about 1000, or from about 114 to about
950, or from about
114 to about 910, or from about 114 to about 800, or from about 114 to about
690, or from about
114 to about 575. In some embodiments, n in the compounds of Formula (XII) and
Formula (XIII)
is an integer selected from 2, 5, 10, 11, 22, 23, 113, 114, 227, 228, 340,
341, 454, 455, 568, 569,
680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136,
1137, 1249, 1250,
1251, 1362, 1363, 1364, 1476, 1477, 1478, 1589, 1590, 1591, 1703, 1704, 1705,
1817, 1818, 1819,
1930, 1931, 1932, 2044, 2045, 2046, 2158, 2159, 2160, 2271, 2272, 2273, 2839,
2840, 2841, 2953,
2954, 2955, 3408, 3409, 3410, 3976, 3977, 3978, 4544, 4545, and 4546.
[210] In some embodiments, the IL-2 conjugate comprises the amino acid
sequence of SEQ ID
NO: 3 in which at least one amino acid residue in the TL-2 conjugate is
replaced by the structure of
Formula (XII), Formula (XIII), or a mixture of Formula (XII) and Formula
(XIII), wherein the
amino acid residue in in SEQ ID NO: 3 that is replaced is selected from F41,
F43, K42, E61, and
P64, and wherein n is an integer from about 450 to about 800, or from about
454 to about 796, or
from about 454 to about 682, or from about 568 to about 909. In some
embodiments, n in the
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compounds of Formula (XII) and Formula (XIII) is an integer selected from 454,
455, 568, 569,
680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136,
1137, and 1249.
[211] In some embodiments, the IL-2 conjugate comprises the amino acid
sequence of SEQ ID
NO: 3 in which at least one amino acid residue in the IL-2 conjugate is
replaced by the structure of
Formula (XII), Formula (XIII), or a mixture of Formula (XII) and Formula
(XIII), wherein the
amino acid residue in SEQ ID NO: 3 that is replaced is selected from E61 and
P64, and wherein n is
an integer from about 450 to about 800, or from about 454 to about 796, or
from about 454 to about
682, or from about 568 to about 909. In some embodiments, n in the compounds
of Formula (XII)
and Formula (XIII) is an integer selected from 454, 455, 568, 569, 680, 681,
682, 794, 795, 796,
908, 909, and 910.
[212] In some embodiments, the IL-2 conjugate comprises the amino acid
sequence of SEQ ID
NO: 3 in which at least one amino acid residue in the IL-2 conjugate is
replaced by the structure of
Formula (XII), Formula (XIII), or a mixture of Formula (XII) and Formula
(XIII), wherein the
amino acid residue in SEQ ID NO: 3 that is replaced is E61, and wherein n is
an integer from about
450 to about 800, or from about 454 to about 796, or from about 454 to about
682, or from about
568 to about 909. In some embodiments, n in the compounds of Formula (XII) and
Formula (XIII)
is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796,
908, 909, and 910. In
some embodiments, n is from about 500 to about 1000. In some embodiments, n is
from about 550
to about 800. In some embodiments, n is about 681.
[213] In some embodiments, the IL-2 conjugate comprises the amino acid
sequence of SEQ ID
NO: 3 in which at least one amino acid residue in the IL-2 conjugate is
replaced by the structure of
Formula (XII), Formula (XIII), or a mixture of Foimula (XII) and Formula
(XIII), wherein the
amino acid residue in in SEQ ID NO: 3 that is replaced is P64, and wherein n
is an integer from
about 450 to about 800, or from about 454 to about 796, or from about 454 to
about 682, or from
about 568 to about 909. In some embodiments, n in the compounds of Formula
(XII) and Formula
(XIII) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794,
795, 796, 908, 909, and
910. In some embodiments, n is from about 500 to about 1000. In some
embodiments, n is from
about 550 to about 800. In some embodiments, n is about 681.
[214] In some embodiments, n in the structure of Formula (XII) or Formula
(XIII) is an integer
such that the molecular weight of the PEG moiety is in the range from about
1,000 Daltons about
200,000 Daltons, or from about 2,000 Daltons to about 150,000 Daltons, or from
about 3,000
Daltons to about 125,000 Daltons, or from about 4,000 Daltons to about 100,000
Daltons, or from
about 5,000 Daltons to about 100,000 Daltons, or from about 6,000 Daltons to
about 90,000
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Daltons, or from about 7,000 Daltons to about 80,000 Daltons, or from about
8,000 Daltons to about
70,000 Daltons, or from about 5,000 Daltons to about 70,000 Daltons, or from
about 5,000 Daltons
to about 65,000 Daltons, or from about 5,000 Daltons to about 60,000 Daltons,
or from about 5,000
Daltons to about 50,000 Daltons, or from about 6,000 Daltons to about 50,000
Daltons, or from
about 7,000 Daltons to about 50,000 Daltons, or from about 7,000 Daltons to
about 45,000 Daltons,
or from about 7,000 Daltons to about 40,000 Daltons, or from about 8,000
Daltons to about 40,000
Daltons, or from about 8,500 Daltons to about 40,000 Daltons, or from about
8,500 Daltons to about
35,000 Daltons, or from about 9,000 Daltons to about 50,000 Daltons, or from
about 9,000 Daltons
to about 45,000 Daltons, or from about 9,000 Daltons to about 40,000 Daltons,
or from about 9,000
Daltons to about 35,000 Daltons, or from about 9,000 Daltons to about 30,000
Daltons, or from
about 9,500 Daltons to about 35,000 Daltons, or from about 9,500 Daltons to
about 30,000 Daltons,
or from about 10,000 Daltons to about 50,000 Daltons, or from about 10,000
Daltons to about
45,000 Daltons, or from about 10,000 Daltons to about 40,000 Daltons, or from
about 10,000
Daltons to about 35,000 Daltons, or from about 10,000 Daltons to about 30,000
Daltons, or from
about 15,000 Daltons to about 50,000 Daltons, or from about 15,000 Daltons to
about 45,000
Daltons, or from about 15,000 Daltons to about 40,000 Daltons, or from about
15,000 Daltons to
about 35,000 Daltons, or from about 15,000 Daltons to about 30,000 Daltons, or
from about 20,000
Daltons to about 50,000 Daltons, or from about 20,000 Daltons to about 45,000
Daltons, or from
about 20,000 Daltons to about 40,000 Daltons, or from about 20,000 Daltons to
about 35,000
Daltons, or from about 20,000 Daltons to about 30,000 Daltons.
[215] In some embodiments, the IL-2 conjugate comprises the amino
acid sequence of SEQ ID
NO: 3 in which at least one amino acid residue in the IL-2 conjugate is
replaced by the structure of
Formula (XII), Formula (XIII), or a mixture of Formula (XII) and Formula
(XIII), wherein n is an
integer such that the molecular weight of the PEG moiety is about 5,000
Daltons, about 7,500
Daltons, about 10,000 Daltons, about 15,000 Daltons, about 20,000 Daltons,
about 25,000 Daltons,
about 30,000 Daltons, about 35,000 Daltons, about 40,000 Daltons, about 45,000
Daltons, about
50,000 Daltons, about 60,000 Daltons, about 70,000 Daltons, about 80,000
Daltons, about 90,000
Daltons, about 100,000 Daltons, about 125,000 Daltons, about 150,000 Daltons,
about 175,000
Daltons or about 200,000 Daltons In some embodiments, the 1L-2 conjugate
comprises the amino
acid sequence of SEQ ID NO: 3 in which at least one amino acid residue in the
IL-2 conjugate is
replaced by the structure of Formula (XII), Formula (XIII), or a mixture of
Formula (XII) and
Formula (XIII), wherein n is an integer such that the molecular weight of the
PEG moiety is about
5,000 Daltons, about 7,500 Daltons, about 10,000 Daltons, about 15,000
Daltons, about 20,000
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Daltons, about 25,000 Daltons, about 30,000 Daltons, about 35,000 Daltons,
about 40,000 Daltons,
about 45,000 Daltons, or about 50,000 Daltons.
[216] In some embodiments, the IL-2 conjugate comprises the amino
acid sequence of SEQ ID
NO: 3 in which the amino acid residue at E61 or P64 in the IL-2 conjugate is
replaced by the
structure of Formula (VIII) or Formula (IX), or a mixture of Formula (VIII)
and Formula (IX):
0
X *
Nõ 0
0
Formula (VIII);
NI: I 0
,CH3
n0
N 0
0
(IX),
wherein:
n is an integer such that the molecular weight of the PEG group is from about
15,000 Daltons to
about 60,000 Daltons;
q is 1,2, or 3; and
X has the structure:
0 cs-,.111
X-1 indicates the point of attachment to the preceding amino acid residue; and
X+1 indicates the point of attachment to the following amino acid residue.
1217] In some embodiments of Formula (VIII) or Formula (IX), or a
mixture of Formula (VIII)
or Formula (IX), q is 1. In some embodiments of Formula (VIII) or Formula
(IX), or a mixture of
Formula (VIII) or Formula (IX), q is 2. In some embodiments of Formula (VIII)
or Formula (IX),
or a mixture of Formula (VIII) or Formula (IX), q is 3.
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[218] Here and throughout, the structure of Formula (VIII) encompasses
pharmaceutically
acceptable salts, solvates, or hydrates thereof. Here and throughout, the
structure of Formula (IX)
encompasses pharmaceutically acceptable salts, solvates, or hydrates thereof.
In some
embodiments, the IL-2 conjugate is a pharmaceutically acceptable salt,
solvate, or hydrate.
[219] In some embodiments, q is 1 and the structures of Formula (VIII) and
Formula (IX) are the
structures of Formula (Villa) and Formula (IXa):
0
0
No I
CH3
/
0
Formula (Villa);
0
I
0CH3
xNyC)) 0
0
(IXa);
wherein:
n is an integer such that the molecular weight of the PEG group is from about
15,000 Daltons to
about 60,000 Daltons; and
X has the structure:
X-1
N H
0 csN
X-1 indicates the point of attachment to the preceding amino acid residue; and
X+1 indicates the point of attachment to the following amino acid residue.
[220] In some embodiments, the amino acid residue at E61 in the IL-2
conjugate is replaced by
the structure of Formula (VIII) or Formula (IX), or a mixture of Formula
(VIII) and Formula (IX),
and wherein n is an integer such that the molecular weight of the PEG group is
from about 20,000
Daltons to about 40,000 Daltons. In some embodiments, n is an integer such
that the molecular
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weight of the PEG group is from about 30,000 Daltons. In some embodiments, the
amino acid
residue at P64 in the IL-2 conjugate is replaced by the structure of Formula
(VIII) or Formula (IX),
or a mixture of Formula (VIII) and Formula (IX), and wherein n is an integer
such that the
molecular weight of the PEG group is from about 20,000 Daltons to about 40,000
Daltons. In some
embodiments, n is an integer such that the molecular weight of the PEG group
is from about 30,000
Daltons.
12211 In some embodiments, the IL-2 conjugate comprises the amino
acid sequence of SEQ ID
NO: 3 in which the amino acid residue at E61 or P64 in the IL-2 conjugate is
replaced by the
structure of Formula (VIII) or (IX), or a mixture of (VIII) and (IX):
0
*
1\1'õ 0
N 3
40 0
Formula (VIII);
411
NI: I 0
N N
C)40,C H3
x N 41 0
0
(IX),
wherein:
n is an integer such that the molecular weight of the PEG group is from about
15,000 Daltons to
about 60,000 Daltons;
q is 1,2, or 3; and
X has the structure:
ik,NH
0cs--
cs
-Eal
X-1 indicates the point of attachment to the preceding amino acid residue; and
X+1 indicates the point of attachment to the following amino acid residue.
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[222] In some embodiments, the amino acid residue at E61 in the IL-2
conjugate is replaced by
the structure of Formula (VIII) or Formula (IX), or a mixture of Formula
(VIII) and Formula (IX),
and wherein n is an integer such that the molecular weight of the PEG group is
from about 20,000
Daltons to about 40,000 Daltons. In some embodiments, n is an integer such
that the molecular
weight of the PEG group is from about 30,000 Daltons. In some embodiments, the
amino acid
residue at P64 in the IL-2 conjugate is replaced by the structure of Formula
(VIII) or Formula (IX),
or a mixture of Formula (VIII) and Formula (IX), and wherein n is an integer
such that the
molecular weight of the PEG group is from about 20,000 Daltons to about 40,000
Daltons. In some
embodiments, n is an integer such that the molecular weight of the PEG group
is from about 30,000
Daltons.
1223] In some embodiments, the IL-2 conjugate comprises the amino
acid sequence of SEQ ID
NO: 1, SEQ ID NO: 3, or SEQ ID NO: 4 in which at least one amino acid residue
in the IL-2
conjugate is replaced by a cysteine covalently bonded to a PEG group. In some
embodiments, the
PEG group has a molecular weight selected from 5kDa, 10kDa, 15kDa, 20kDa,
25kDa, 30kDa,
35kDa, 40kDa, 45kDa, 50kDa, and 60kDa. In some embodiments, the PEG group has
a molecular
weight of 5kDa. In some embodiments, the PEG group has a molecular weight of
10kDa. In some
embodiments, the PEG group has a molecular weight of 15kDa. In some
embodiments, the PEG
group has a molecular weight of 20kDa. In some embodiments, the PEG group has
a molecular
weight of 25kDa. In some embodiments, the PEG group has a molecular weight of
30kDa. In some
embodiments, the PEG group has a molecular weight of 35kDa. In some
embodiments, the PEG
group has a molecular weight of 40kDa. In some embodiments, the PEG group has
a molecular
weight of 45kDa. In some embodiments, the PEG group has a molecular weight of
50kDa. In some
embodiments, the PEG group has a molecular weight of 60kDa. In some
embodiments, the IL-2
conjugate comprises the amino acid sequence of SEQ ID NO: 3 and the at least
one amino acid
residue in the IL-2 conjugate that is replaced by a cysteine is selected from
K34, T36, R37, T40,
F41, K42, F43, Y44, E60, E61, E67, K63, P64, V68, L71, and Y106. In some
embodiments, the IL-
2 conjugate comprises the amino acid sequence of SEQ ID NO: 3 and the at least
one amino acid
residue in the IL-2 conjugate that is replaced by a cysteine is selected from
K34, T40, F41, K42,
Y44, E60, E61, E67, K63, P64, V68, and L71. In some embodiments, the IL-2
conjugate comprises
the amino acid sequence of SEQ ID NO: 4 and the at least one amino acid
residue in the IL-2
conjugate that is replaced by a cysteine is selected from K35, 137, R38, 141,
F42, K43, F44, Y45,
E61, E62, E68, 1(64, P65, V69, L72, and Y107
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[224] In some embodiments, the IL-2 conjugate comprises the amino acid
sequence of SEQ ID
NO: 3 in which at least one non-lysine residue is replaced by a lysine
comprising a linker and a
water-soluble polymer. In some embodiments, the water-soluble polymer is a PEG
group.
[225] In some embodiments, the IL-2 conjugate comprises a PEG group
covalently bonded via a
non-releasable linkage. In some embodiments, the IL-2 conjugate comprises a
non-releasable,
covalently bonded PEG group.
[226] In some embodiments, the IL-2 conjugate comprises the amino acid
sequence of SEQ ID
NO: 3, wherein a non-lysine amino acid in the IL-2 conjugate is replaced by a
lysine residue, and
wherein the lysine residue comprises one or more water soluble polymers and a
covalent linker. In
some embodiments, the lysine residue is located in the region K34-Y106 of SEQ
ID NO: 3. In some
embodiments, the lysine residue is located at K34. In some embodiments, the
lysine residue is
located at F41. In some embodiments, the lysine residue is located at F43. In
some embodiments,
the lysine residue is located at K42. In some embodiments, the lysine residue
is located at E61. In
some embodiments, the lysine residue is located at P64. In some embodiments,
the lysine residue is
located at R37. In some embodiments, the lysine residue is located at T40. In
some embodiments,
the lysine residue is located at E67. In some embodiments, the lysine residue
is located at Y44. In
some embodiments, the lysine residue is located at V68. In some embodiments,
the lysine residue is
located at L71.
[227] In some embodiments, the IL-2 conjugate comprises the amino acid
sequence of SEQ ID
NO: 3, wherein a non-lysine amino acid in the amino acid sequence of the IL-2
conjugate is
replaced by an amino acid comprising: (a) a lysine; (b) a covalent linker; and
(3) and one or more
water-soluble polymers. In some embodiments, the one or more water-soluble
polymers comprises
a PEG group.
[228] In some embodiments, the IL-2 conjugate comprises the amino acid
sequence of SEQ ID
NO: 3 in which at least one amino acid residue in the IL-2 conjugate is
replaced by the structure of
Formula (XIV) or Formula (XV), or a mixture of Formula (XIV) and Formula (XV):
= s s
NH 0
N I 0
õ
'm N
0 H
Formula (XIV),
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N H3
Sr 0
m N
N 0
NH 0
Formula (XV);
wherein:
m is an integer from 0 to 20;
p is an integer from 0 to 20;
n is an integer in the range from about 2 to about 5000; and
the wavy lines indicate covalent bonds to amino acid residues within SEQ ID
NO: 3 that are not
replaced.
[229] Here and throughout, the structure of Formula (XIV)
encompasses pharmaceutically
acceptable salts, solvates, or hydrates thereof. Here and throughout, the
structure of Formula (XV)
encompasses pharmaceutically acceptable salts, solvates, or hydrates thereof.
In some
embodiments, the IL-2 conjugate is a pharmaceutically acceptable salt,
solvate, or hydrate.
[230] In some embodiments, the stereochemistry of the chiral center
within Formula (XIV) and
Formula (XV) is racemic, is enriched in (R), is enriched in (S), is
substantially (R), is substantially
(S), is (R) or is (S). In some embodiments, the stereochemistry of the chiral
center within Formula
(XIV) and Formula (XV) is racemic. In some embodiments, the stereochemistry of
the chiral center
within Formula (XIV) and Formula (XV) is enriched in (R). In some embodiments,
the
stereochemistry of the chiral center within Formula (XIV) and Formula (XV) is
enriched in (S). In
some embodiments, the stereochemistry of the chiral center within Formula
(XIV) and Formula
(XV) is substantially (R). In some embodiments, the stereochemistry of the
chiral center within
Formula (XIV) and Formula (XV) is substantially (S). In some embodiments, the
stereochemistry
of the chiral center within Formula (XIV) and Formula (XV) is (R). In some
embodiments, the
stereochemistry of the chiral center within Formula (XIV) and Formula (XV) is
(S).
[231] In some embodiments, the IL-2 conjugate comprises the amino
acid sequence of SEQ ID
NO: 3 in which m in the compounds of Formula (XIV) and Formula (XV) is from 0
to 20, or from 1
to 18, or from 1 to 16, or from 1 to 14, or from 1 to 12, or from 1 to 10, or
from 1 to 9, or from 1 to
8, or from 1 to 7, or from 1 to 6, or from 1 to 5, or from 1 to 4, or from 1
to 3, or from 1 to 2. In
some embodiments, m in the compounds of Formula (XIV) and Formula (XV) is L In
some
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embodiments, m in the compounds of Formula (XIV) and Formula (XV) is 2. In
some
embodiments, m in the compounds of Formula (XIV) and Formula (XV) is 3. In
some
embodiments, m in the compounds of Formula (XIV) and Formula (XV) is 4. In
some
embodiments, m in the compounds of Formula (XIV) and Formula (XV) is 5. In
some
embodiments, m in the compounds of Formula (XIV) and Formula (XV) is 6. In
some
embodiments, m in the compounds of Formula (XIV) and Formula (XV) is 7. In
some
embodiments, m in the compounds of Formula (XIV) and Formula (XV) is 8. In
some
embodiments, m in the compounds of Formula (XIV) and Formula (XV) is 9. In
some
embodiments, m in the compounds of Formula (XIV) and Formula (XV) is 10. In
some
embodiments, m in the compounds of Formula (XIV) and Formula (XV) is 11. In
some
embodiments, m in the compounds of Formula (XIV) and Formula (XV) is 12. In
some
embodiments, m in the compounds of Formula (XIV) and Formula (XV) is 13. In
some
embodiments, m in the compounds of Formula (XIV) and Formula (XV) is 14. In
some
embodiments, m in the compounds of Formula (XIV) and Formula (XV) is 15. In
some
embodiments, m in the compounds of Formula (XIV) and Formula (XV) is 16. In
some
embodiments, m in the compounds of Formula (XIV) and Formula (XV) is 17. In
some
embodiments, m in the compounds of Formula (XIV) and Formula (XV) is 18. In
some
embodiments, m in the compounds of Formula (XIV) and Formula (XV) is 19. In
some
embodiments, m in the compounds of Formula (XIV) and Formula (XV) is 20.
[232] In some embodiments, the IL-2 conjugate comprises the amino
acid sequence of SEQ ID
NO: 3 in which p in the compounds of Formula (XIV) and Formula (XV) is from 1
to 20, or from 1
to 18, or from 1 to 16, or from 1 to 14, or from 1 to 12, or from 1 to 10, or
from 1 to 9, or from 1 to
8, or from 1 to 7, or from 1 to 6, or from 1 to 5, or from 1 to 4, or from 1
to 3, or from 1 to 2. In
some embodiments, p in the compounds of Formula (XIV) and Formula (XV) is 1.
In some
embodiments, p in the compounds of Formula (XIV) and Formula (XV) is 2. In
some
embodiments, p in the compounds of Formula (XIV) and Formula (XV) is 3. In
some
embodiments, p in the compounds of Formula (XIV) and Formula (XV) is 4. In
some
embodiments, p in the compounds of Formula (XIV) and Formula (XV) is 5. In
some
embodiments, p in the compounds of Formula (XIV) and Formula (XV) is 6. In
some
embodiments, p in the compounds of Formula (XIV) and Formula (XV) is 7. In
some
embodiments, p in the compounds of Formula (XIV) and Formula (XV) is 8. In
some
embodiments, p in the compounds of Formula (XIV) and Formula (XV) is 9. In
some
embodiments, p in the compounds of Formula (XIV) and Formula (XV) is 10. In
some
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embodiments, pin the compounds of Formula (XIV) and Formula (XV) is 11. In
some
embodiments, p in the compounds of Formula (XIV) and Formula (XV) is 12. In
some
embodiments, p in the compounds of Formula (XIV) and Formula (XV) is 13. In
some
embodiments, p in the compounds of Formula (XIV) and Formula (XV) is 14. In
some
embodiments, p in the compounds of Formula (XIV) and Formula (XV) is 15. In
some
embodiments, m in the compounds of Formula (XIV) and Formula (XV) is 16. In
some
embodiments, pin the compounds of Formula (XIV) and Formula (XV) is 17. In
some
embodiments, p in the compounds of Formula (XIV) and Formula (XV) is 18. In
some
embodiments, p in the compounds of Formula (XIV) and Formula (XV) is 19. In
some
embodiments, p in the compounds of Formula (XIV) and Formula (XV) is 20.
12331 In some embodiments, the IL-2 conjugate comprises the amino
acid sequence of SEQ ID
NO: 3 in which n in the compounds of Formula (XIV) and Formula (XV) is in the
range from about
to about 4600, or from about 10 to about 4000, or from about 20 to about 3000,
or from about 100
to about 3000, or from about 100 to about 2900, or from about 150 to about
2900, or from about
125 to about 2900, or from about 100 to about 2500, or from about 100 to about
2000, or from
about 100 to about 1900, or from about 100 to about 1850, or from about 100 to
about 1750, or
from about 100 to about 1650, or from about 100 to about 1500, or from about
100 to about 1400,
or from about 100 to about 1300, or from about 100 to about 1250, or from
about 100 to about
1150, or from about 100 to about 1100, or from about 100 to about 1000, or
from about 100 to
about 900, or from about 100 to about 750, or from about 100 to about 700, or
from about 100 to
about 600, or from about 100 to about 575, or from about 100 to about 500, or
from about 100 to
about 450, or from about 100 to about to about 350, or from about 100 to about
275, or from about
100 to about 230, or from about 150 to about 475, or from about 150 to about
340, or from about
113 to about 340, or from about 450 to about 800, or from about 454 to about
796, or from about
454 to about 682, or from about 340 to about 795, or from about 341 to about
682, or from about
568 to about 909, or from about 227 to about 1500, or from about 225 to about
2280, or from about
460 to about 2160, or from about 460 to about 2050, or from about 341 to about
1820, or from
about 341 to about 1710, or from about 341 to about 1250, or from about 225 to
about 1250, or
from about 341 to about 1250, or from about 341 to about 1136, or from about
341 to about 1023,
or from about 341 to about 910, or from about 341 to about 796, or from about
341 to about 682, or
from about 341 to about 568, or from about 114 to about 1000, or from about
114 to about 950, or
from about 114 to about 910, or from about 114 to about 800, or from about 114
to about 690, or
from about 114 to about 575.
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[234] In some embodiments, the IL-2 conjugate comprises the amino
acid sequence of SEQ ID
NO: 3 in which m in the compounds of Formula (XIV) and Formula (XV) is an
integer from 1 to 6,
p is an integer from 1 to 6, and n is an integer selected from 113, 114, 227,
228, 340, 341, 454, 455,
568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135,
1136, and 1137. In
some embodiments of the compounds of Formula (XIV) and Formula (XV), m is an
integer from 2
to 6, p is an integer from 2 to 6, and n is an integer selected from 113, 114,
227, 228, 340, 341, 454,
455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023,
1135, 1136, and
1137. In some embodiments of the compounds of Formula (XIV) and Formula (XV),
m is an
integer from 2 to 4, p is an integer from 2 to 4, and n is an integer selected
from 113, 114, 227, 228,
340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910,
1021, 1022, 1023, 1135,
1136, and 1137. In some embodiments of the compounds of Formula (XIV) and
Formula (XV), m
is 1, p is 2, and n is an integer selected from 113, 114, 227, 228, 340, 341,
454, 455, 568, 569, 680,
681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and
1137. In some
embodiments of the compounds of Formula (XIV) and Formula (XV), m is 2, p is
2, and n is an
integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680,
681, 682, 794, 795,
796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some
embodiments of the
compounds of Formula (XIV) and Formula (XV), m is 3, p is 2, and n is an
integer selected from
113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795,
796, 908, 909, 910,
1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments of the compounds
of Formula
(XIV) and Formula (XV), m is 4, p is 2, and n is an integer selected from 113,
114, 227, 228, 340,
341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021,
1022, 1023, 1135,
1136, and 1137. In some embodiments of the compounds of Formula (XIV) and
Formula (XV), m
is 5, p is 2, and n is an integer selected from 113, 114, 227, 228, 340, 341,
454, 455, 568, 569, 680,
681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and
1137. In some
embodiments of the compounds of Formula (XIV) and Formula (XV), m is 6, p is
2, and n is an
integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680,
681, 682, 794, 795,
796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some
embodiments of the
compounds of Formula (XIV) and Formula (XV), m is 7, p is 2, and n is an
integer selected from
113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795,
796, 908, 909, 910,
1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments of the compounds
of Formula
(XIV) and Formula (XV), m is 8, p is 2, and n is an integer selected from 113,
114, 227, 228, 340,
341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021,
1022, 1023, 1135,
1136, and 1137. In some embodiments of the compounds of Formula (XIV) and
Formula (XV), m
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is 9, p is 2, and n is an integer selected from 113, 114, 227, 228, 340, 341,
454, 455, 568, 569, 680,
681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and
1137. In some
embodiments of the compounds of Formula (XIV) and Formula (XV), m is 10, p is
2, and n is an
integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680,
681, 682, 794, 795,
796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some
embodiments of the
compounds of Formula (XIV) and (XV), m is 11, p is 2, and n is an integer
selected from 113, 114,
227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908,
909, 910, 1021, 1022,
1023, 1135, 1136, and 1137. In some embodiments of the compounds of Formula
(XIV) and
Formula (XV), m is 11, p is 2, and n is an integer selected from 113, 114,
227, 228, 340, 341, 454,
455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023,
1135, 1136, and
1137. In some embodiments of the compounds of Formula (XIV) and Formula (XV),
m is 2, p is 2,
and n is an integer selected from 680, 681, 682, 794, 795, 796, 908, 909, 910,
1021, 1022, 1023,
1135, 1136, and 1137.
[235] In some embodiments, the IL-2 conjugate comprises the amino
acid sequence of SEQ ID
NO: 3 in which n in the compounds of Formula (XIV) and Formula (XV) is an
integer selected
from 2, 5, 10, 11, 22, 23, 113, 114, 227, 228, 340, 341, 454, 455, 568, 569,
680, 681, 682, 794, 795,
796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137, 1249, 1250, 1251,
1362, 1363, 1364,
1476, 1477, 1478, 1589, 1590, 1591, 1703, 1704, 1705, 1817, 1818, 1819, 1930,
1931, 1932, 2044,
2045, 2046, 2158, 2159, 2160, 2271, 2272, 2273, 2839, 2840, 2841, 2953, 2954,
2955, 3408, 3409,
3410, 3976, 3977, 3978, 4544, 4545, and 4546. In some embodiments, the
position of the structure
of Formula (XIV), Formula (XV), or a mixture of Formula (XIV) and Formula (XV)
in the amino
acid sequence of the IL-2 conjugate is selected from K34, F41, F43, K42, E61,
P64, R37, T40, E67,
Y44, V68, and L71, wherein the position of the structure of Formula (XIV),
Formula (XV), or a
mixture of Formula (XIV) and Formula (XV) in the amino acid sequence of the IL-
2 conjugate is in
reference to the positions in SEQ ID NO: 3. In some embodiments, the position
of the structure of
Formula (XIV), Formula (XV), or a mixture of Formula (XIV) and Formula (XV) in
the amino acid
sequence of the IL-2 conjugate of SEQ ID NO: 3 is selected from K34, F41, F43,
K42, E61, P64,
R37, T40, E67, Y44, V68, and L71. In some embodiments, the position of the
structure of Formula
(XIV), Formula (XV), or a mixture of Formula (XIV) and Formula (XV) in the
amino acid
sequence of the IL-2 conjugate of SEQ ID NO. 3 is at position K34. In some
embodiments, the
position of the structure of Formula (XIV), Formula (XV), or a mixture of
Formula (XIV) and
Formula (XV) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3
is at position
F41. In some embodiments, the position of the structure of Formula (XIV),
Formula (XV), or a
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mixture of Formula (XIV) and Formula (XV) in the amino acid sequence of the IL-
2 conjugate of
SEQ ID NO: 3 is at position F43. In some embodiments, the position of the
structure of Formula
(XIV), Formula (XV), or a mixture of Formula (XIV) and Formula (XV) in the
amino acid
sequence of the IL-2 conjugate of SEQ ID NO: 3 is at position K42. In some
embodiments, the
position of the structure of Formula (XIV), Formula (XV), or a mixture of
Formula (XIV) and
Formula (XV) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3
is at position
E61. In some embodiments, the position of the structure of Formula (XIV),
Formula (XV), or a
mixture of Formula (XIV) and Formula (XV) in the amino acid sequence of the IL-
2 conjugate of
SEQ ID NO: 3 is at position P64. In some embodiments, the position of the
structure of Formula
(XIV), Formula (XV), or a mixture of Formula (XIV) and Formula (XV) in the
amino acid
sequence of the IL-2 conjugate of SEQ ID NO: 3 is at position R37. In some
embodiments, the
position of the structure of Formula (XIV), Formula (XV), or a mixture of
Formula (XIV) and
Formula (XV) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3
is at position
T40. In some embodiments, the position of the structure of Formula (XIV),
Formula (XV), or a
mixture of Formula (XIV) and Formula (XV) in the amino acid sequence of the IL-
2 conjugate of
SEQ ID NO: 3 is at position E67. In some embodiments, the position of the
structure of Formula
(XIV), Formula (XV), or a mixture of Founula (XIV) and Formula (XV) in the
amino acid
sequence of the IL-2 conjugate of SEQ ID NO: 3 is at position Y44. In some
embodiments, the
position of the structure of Formula (XIV), Formula (XV), or a mixture of
Formula (XIV) and
Formula (XV) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3
is at position
V68. In some embodiments, the position of the structure of Formula (XIV),
Formula (XV), or a
mixture of Formula (XIV) and Formula (XV) in the amino acid sequence of the IL-
2 conjugate of
SEQ ID NO: 3 is at position L71. In some embodiments, the ratio of the amount
of the structure of
Formula (XIV) to the amount of the structure of Formula (XV) comprising the
total amount of the
IL-2 conjugate is about 1:1. In some embodiments, the ratio of the amount of
the structure of
Formula (XIV) to the amount of the structure of Formula (XV) comprising the
total amount of the
IL-2 conjugate is greater than 1:1. In some embodiments, the ratio of the
amount of the structure of
Formula (XIV) to the amount of the structure of Formula (XV) comprising the
total amount of the
IL-2 conjugate is less than 1.1
[236] In some embodiments, the IL-2 conjugate comprises the amino
acid sequence of SEQ ID
NO: 3 in which at least one amino acid residue in the IL-2 conjugate is
replaced by the structure of
Formula (XIV) or Formula (XV), or a mixture of Formula (XIV) and Formula (XV),
wherein the
amino acid residue in SEQ ID NO: 3 that is replaced is selected from K34, F41,
F43, K42, E61,
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P64, R37, T40, E67, Y44, V68, and L71, and wherein n is an integer from 100 to
about 1150, or
from about 100 to about 1100, or from about 100 to about 1000, or from about
100 to about 900, or
from about 100 to about 750, or from about 100 to about 700, or from about 100
to about 600, or
from about 100 to about 575, or from about 100 to about 500, or from about 100
to about 450, or
from about 100 to about to about 350, or from about 100 to about 275, or from
about 100 to about
230, or from about 150 to about 475, or from about 150 to about 340, or from
about 113 to about
340, or from about 450 to about 800, or from about 454 to about 796, or from
about 454 to about
682, or from about 340 to about 795, or from about 341 to about 682, or from
about 568 to about
909, or from about 227 to about 1500, or from about 225 to about 2280, or from
about 460 to about
2160, or from about 460 to about 2050, or from about 341 to about 1820, or
from about 341 to
about 1710, or from about 341 to about 1250, or from about 225 to about 1250,
or from about 341
to about 1250, or from about 341 to about 1136, or from about 341 to about
1023, or from about
341 to about 910, or from about 341 to about 796, or from about 341 to about
682, or from about
341 to about 568, or from about 114 to about 1000, or from about 114 to about
950, or from about
114 to about 910, or from about 114 to about 800, or from about 114 to about
690, or from about
114 to about 575. In some embodiments, n in the compounds of Formula Formula
(XIV) and
Formula (XV) is an integer selected from 2, 5, 10, 11, 22, 23, 113, 114, 227,
228, 340, 341, 454,
455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023,
1135, 1136, 1137,
1249, 1250, 1251, 1362, 1363, 1364, 1476, 1477, 1478, 1589, 1590, 1591, 1703,
1704, 1705, 1817,
1818, 1819, 1930, 1931, 1932, 2044, 2045, 2046, 2158, 2159, 2160, 2271, 2272,
2273, 2839, 2840,
2841, 2953, 2954, 2955, 3408, 3409, 3410, 3976, 3977, 3978, 4544, 4545, and
4546.
[237] In some embodiments, the IL-2 conjugate comprises the amino acid
sequence of SEQ ID
NO: 3 in which at least one amino acid residue in the IL-2 conjugate is
replaced by the structure of
Formula (XIV) or Formula (XV), or a mixture of Formula (XIV) and Formula (XV),
wherein the
amino acid residue in in SEQ ID NO: 3 that is replaced is selected from F41,
F43, K42, E61, and
P64, and wherein n is an integer from about 450 to about 800, or from about
454 to about 796, or
from about 454 to about 682, or from about 568 to about 909. In some
embodiments, n in the
compounds of Formula (XIV) and Formula (XV) is an integer selected from 454,
455, 568, 569,
680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136,
1137, and 1249
[238] In some embodiments, the IL-2 conjugate comprises the amino acid
sequence of SEQ ID
NO: 3 in which at least one amino acid residue in the 1L-2 conjugate is
replaced by the structure
Formula (XIV) or Formula (XV), or a mixture of Formula (XIV) and Formula (XV),
wherein the
amino acid residue in SEQ ID NO: 3 that is replaced is selected from E61 and
P64, and wherein n is
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an integer from about 450 to about 800, or from about 454 to about 796, or
from about 454 to about
682, or from about 568 to about 909. In some embodiments, n in the compounds
of Formula (XIV)
and Formula (XV) is an integer selected from 454, 455, 568, 569, 680, 681,
682, 794, 795, 796,
908, 909, and 910.
[239] In some embodiments, the IL-2 conjugate comprises the amino acid
sequence of SEQ ID
NO: 3 in which at least one amino acid residue in the IL-2 conjugate is
replaced by the structure of
Formula (XIV) or Formula (XV), or a mixture of Formula (XIV) and Formula (XV),
wherein the
amino acid residue in SEQ ID NO: 3 that is replaced is E61, and wherein n is
an integer from about
450 to about 800, or from about 454 to about 796, or from about 454 to about
682, or from about
568 to about 909. In some embodiments, n in the compounds of Formula (XIV) and
Formula (XV)
is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796,
908, 909, and 910. In
some embodiments, n is from about 500 to about 1000. In some embodiments, n is
from about 550
to about 800. In some embodiments, n is about 681.
[240] In some embodiments, the IL-2 conjugate comprises the amino acid
sequence of SEQ ID
NO: 3 in which at least one amino acid residue in the IL-2 conjugate is
replaced by the structure of
Formula (XIV) or Formula (XV), or a mixture of Formula (XIV) and Formula (XV),
wherein the
amino acid residue in in SEQ ID NO: 3 that is replaced is P64, and wherein n
is an integer from
about 450 to about 800, or from about 454 to about 796, or from about 454 to
about 682, or from
about 568 to about 909. In some embodiments, n in the compounds of Formula
(XIV) and Formula
(XV) is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795,
796, 908, 909, and
910. In some embodiments, n is from about 500 to about 1000. In some
embodiments, n is from
about 550 to about 800. In some embodiments, n is about 681.
[241] In some embodiments, the IL-2 conjugate comprises the amino acid
sequence of SEQ ID
NO: 3 in which at least one amino acid residue in the IL-2 conjugate is
replaced by the structure of
Formula (XIV) or Formula (XV), or a mixture of Formula (XIV) and Formula (XV),
wherein n is
an integer such that the molecular weight of the PEG moiety is in the range
from about 1,000
Daltons about 200,000 Daltons, or from about 2,000 Daltons to about 150,000
Daltons, or from
about 3,000 Daltons to about 125,000 Daltons, or from about 4,000 Daltons to
about 100,000
Daltons, or from about 5,000 Daltons to about 100,000 Daltons, or from about
6,000 Daltons to
about 90,000 Daltons, or from about 7,000 Daltons to about 80,000 Daltons, or
from about 8,000
Daltons to about 70,000 Daltons, or from about 5,000 Daltons to about 70,000
Daltons, or from
about 5,000 Daltons to about 65,000 Daltons, or from about 5,000 Daltons to
about 60,000 Daltons,
or from about 5,000 Daltons to about 50,000 Daltons, or from about 6,000
Daltons to about 50,000
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Daltons, or from about 7,000 Daltons to about 50,000 Daltons, or from about
7,000 Daltons to about
45,000 Daltons, or from about 7,000 Daltons to about 40,000 Daltons, or from
about 8,000 Daltons
to about 40,000 Daltons, or from about 8,500 Daltons to about 40,000 Daltons,
or from about 8,500
Daltons to about 35,000 Daltons, or from about 9,000 Daltons to about 50,000
Daltons, or from
about 9,000 Daltons to about 45,000 Daltons, or from about 9,000 Daltons to
about 40,000 Daltons,
or from about 9,000 Daltons to about 35,000 Daltons, or from about 9,000
Daltons to about 30,000
Daltons, or from about 9,500 Daltons to about 35,000 Daltons, or from about
9,500 Daltons to about
30,000 Daltons, or from about 10,000 Daltons to about 50,000 Daltons, or from
about 10,000
Daltons to about 45,000 Daltons, or from about 10,000 Daltons to about 40,000
Daltons, or from
about 10,000 Daltons to about 35,000 Daltons, or from about 10,000 Daltons to
about 30,000
Daltons, or from about 15,000 Daltons to about 50,000 Daltons, or from about
15,000 Daltons to
about 45,000 Daltons, or from about 15,000 Daltons to about 40,000 Daltons, or
from about 15,000
Daltons to about 35,000 Daltons, or from about 15,000 Daltons to about 30,000
Daltons, or from
about 20,000 Daltons to about 50,000 Daltons, or from about 20,000 Daltons to
about 45,000
Daltons, or from about 20,000 Daltons to about 40,000 Daltons, or from about
20,000 Daltons to
about 35,000 Daltons, or from about 20,000 Daltons to about 30,000 Daltons. In
some
embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID
NO: 3 in which at
least one amino acid residue in the IL-2 conjugate is replaced by the
structure of Formula (XIV) or
Formula (XV), or a mixture of Formula (XIV) and Formula (XV), wherein n is an
integer such that
the molecular weight of the PEG moiety is about 5,000 Daltons, about 7,500
Daltons, about 10,000
Daltons, about 15,000 Daltons, about 20,000 Daltons, about 25,000 Daltons,
about 30,000 Daltons,
about 35,000 Daltons, about 40,000 Daltons, about 45,000 Daltons, about 50,000
Daltons, about
60,000 Daltons, about 70,000 Daltons, about 80,000 Daltons, about 90,000
Daltons, about 100,000
Daltons, about 125,000 Daltons, about 150,000 Daltons, about 175,000 Daltons
or about 200,000
Daltons. In some embodiments, the IL-2 conjugate comprises the amino acid
sequence of SEQ ID
NO: 3 in which at least one amino acid residue in the IL-2 conjugate is
replaced by the structure of
Formula (XIV) or Formula (XV), or a mixture of Formula (XIV) and Formula (XV),
wherein n is
an integer such that the molecular weight of the PEG moiety is about 5,000
Daltons, about 7,500
Daltons, about 10,000 Daltons, about 15,000 Daltons, about 20,000 Daltons,
about 25,000 Daltons,
about 30,000 Daltons, about 35,000 Daltons, about 40,000 Daltons, about 45,000
Daltons, or about
50,000 Daltons.
1242] In some embodiments, the IL-2 conjugate comprises the amino
acid sequence of SEQ ID
NO: 3 in which at least one amino acid residue in the IL-2 conjugate is
replaced by the structure of
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Formula (XIV) or Formula (XV), or a mixture of Formula (XIV) and Formula (XV),
wherein the
amino acid residue in in SEQ ID NO: 3 that is replaced is selected from F41,
F43, K42, E61, and
P64, m is an integer from 1 to 6, p is an integer from 1 to 6, and n is an
integer from about 450 to
about 800, or from about 454 to about 796, or from about 454 to about 682, or
from about 568 to
about 909. In some embodiments of the compounds of Formula (XIV) and Formula
(XV), m is 2, p
is 2, and n is an integer selected from 454, 455, 568, 569, 680, 681, 682,
794, 795, 796, 908, 909,
910, 1021, 1022, 1023, 1135, 1136, 1137, and 1249.
[243] In some embodiments, the IL-2 conjugate comprises the amino
acid sequence of SEQ ID
NO: 3 in which at least one amino acid residue in the IL-2 conjugate is
replaced by the structure of
Formula (XIV) or Formula (XV), or a mixture of Formula (XIV) and Formula (XV),
wherein the
amino acid residue in SEQ ID NO: 3 that is replaced is selected from E61 and
P64, and wherein m
is an integer from 1 to 6, p is an integer from 1 to 6, and n is an integer
from about 450 to about
800, or from about 454 to about 796, or from about 454 to about 682, or from
about 568 to about
909. In some embodiments of the compounds of Formula (XIV) and Formula (XV), m
is 2, p is 2,
and n is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795,
796, 908, 909, and
910.
[244] In some embodiments, the IL-2 conjugate comprises the amino
acid sequence of SEQ ID
NO: 3 in which at least one amino acid residue in the IL-2 conjugate is
replaced by the structure of
Formula (XIV) or Formula (XV), or a mixture of Formula (XIV) and Formula (XV),
wherein the
amino acid residue in SEQ ID NO: 3 that is replaced is E61, and wherein m is
an integer from 1 to
6, p is an integer from 1 to 6, and n is an integer from about 450 to about
800, or from about 454 to
about 796, or from about 454 to about 682, or from about 568 to about 909. In
some embodiments
of the compounds of Formula (XIV) and Formula (XV), m is 2, p is 2, and n is
an integer selected
from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910. In
some embodiments, n
is from about 500 to about 1000. In some embodiments, n is from about 550 to
about 800. In some
embodiments, n is about 681.
[245] In some embodiments, the IL-2 conjugate comprises the amino
acid sequence of SEQ ID
NO: 3 in which at least one amino acid residue in the IL-2 conjugate is
replaced by the structure of
Formula (XIV) or Formula (XV), or a mixture of Formula (XIV) and Formula (XV),
wherein the
amino acid residue in SEQ ID NO. 3 that is replaced is P64, and wherein m is
an integer from 1 to
6, p is an integer from 1 to 6, and n is an integer from about 450 to about
800, or from about 454 to
about 796, or from about 454 to about 682, or from about 568 to about 909. In
some embodiments
of the compounds of Formula (XIV) and Formula (XV), m is 2, p is 2, and n is
an integer selected
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from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910. In
some embodiments, n
is from about 500 to about 1000. In some embodiments, n is from about 550 to
about 800. In some
embodiments, n is about 681.
[246] In some embodiments, the IL-2 conjugate comprises the amino
acid sequence of SEQ ID
NO: 3 in which at least one amino acid residue in the TL-2 conjugate is
replaced by the structure of
Formula (XVI) or Formula (XVII), or a mixture of Formula (XVI) and Formula
(XVII):
NH 0
OywN,A,e,,1
\-0
NõI H
CH3
0 m 0 n
Formula (XVI);
N: I
N \
N
/n m
0
Formula (XVII);
wherein:
m is an integer from 0 to 20;
n is an integer in the range from about 2 to about 5000; and
the wavy lines indicate covalent bonds to amino acid residues within SEQ ID
NO: 3 that are not
replaced.
[247] Here and throughout, the structure of Formula (XVI)
encompasses pharmaceutically
acceptable salts, solvates, or hydrates thereof. Here and throughout, the
structure of Formula
(XVII) encompasses pharmaceutically acceptable salts, solvates, or hydrates
thereof. In some
embodiments, the IL-2 conjugate is a pharmaceutically acceptable salt,
solvate, or hydrate.
[248] In some embodiments, the stereochemistry of the chital center
within Formula (XVI) and
Formula (XVII) is racemic, is enriched in (R), is enriched in (S), is
substantially (R), is substantially
(S), is (R) or is (S). In some embodiments, the stereochemistry of the chiral
center within Formula
(XVI) and Formula (XVII) is racemic. In some embodiments, the stereochemistry
of the chiral
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center within Formula (XVI) and Formula (XVII) is enriched in (R). In some
embodiments, the
stereochemistry of the chiral center within Formula (XVI) and Formula (XVII)
is enriched in (S).
In some embodiments, the stereochemistry of the chiral center within Formula
(XVI) and Formula
(XVII) is substantially (R). In some embodiments, the stereochemistry of the
chiral center within
Formula (XVI) and Formula (XVII) is substantially (S). In some embodiments,
the stereochemistry
of the chiral center within Formula (XVI) and Formula (XVII) is (R). In some
embodiments, the
stereochemistry of the chiral center within Formula (XVI) and Formula (XVII)
is (S).
1249] In some embodiments, the IL-2 conjugate comprises the amino
acid sequence of SEQ ID
NO: 3 in which m in the compounds of Formula (XVI) and Formula (XVII) is from
1 to 20, or from
1 to 18, or from 1 to 16, or from 1 to 14, or from 1 to 12, or from 1 to 10,
or from 1 to 9, or from 1
to 8, or from 1 to 7, or from 1 to 6, or from 1 to 5, or from 1 to 4, or from
1 to 3, or from 1 to 2. In
some embodiments, m in the compounds of Formula (XVI) and Formula (XVII) is 1.
In some
embodiments, m in the compounds of Formula (XVI) and Formula (XVII) is 2. In
some
embodiments, m in the compounds of Formula (XVI) and Formula (XVII) is 3. In
some
embodiments, m in the compounds of Formula (XVI) and Formula (XVII) is 4. In
some
embodiments, m in the compounds of Formula (XVI) and Formula (XVII) is 5. In
some
embodiments, m in the compounds of Formula (XVI) and Formula (XVII) is 6. In
some
embodiments, m in the compounds of Formula (XVI) and Formula (XVII) is 7. In
some
embodiments, m in the compounds of Formula (XVI) and Formula (XVII) is 8. In
some
embodiments, m in the compounds of Formula (XVI) and Formula (XVII) is 9. In
some
embodiments, m in the compounds of Formula (XVI) and Formula (XVII) is 10. In
some
embodiments, m in the compounds of Formula (XVI) and Formula (XVII) is 11. In
some
embodiments, m in the compounds of Formula (XVI) and Formula (XVII) is 12. In
some
embodiments, m in the compounds of Formula (XVI) and Formula (XVII) is 13. In
some
embodiments, m in the compounds of Formula (XVI) and Formula (XVII) is 14. In
some
embodiments, m in the compounds of Formula (XVI) and Formula (XVII) is 15. In
some
embodiments, m in the compounds of Formula (XVI) and Formula (XVII) is 16. In
some
embodiments, m in the compounds of Formula (XVI) and Formula (XVII) is 17. In
some
embodiments, m in the compounds of Formula (XVI) and Formula (XVII) is 18. In
some
embodiments, m in the compounds of Formula (XVI) and Formula (XVII) is 19. In
some
embodiments, m in the compounds of Formula (XVI) and Formula (XVII) is 20.
1250] In some embodiments, the IL-2 conjugate comprises the amino
acid sequence of SEQ ID
NO: 3 in which n in the compounds of Formula (XVI) and Formula (XVII) is in
the range from
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about 5 to about 4600, or from about 10 to about 4000, or from about 20 to
about 3000, or from
about 100 to about 3000, or from about 100 to about 2900, or from about 150 to
about 2900, or
from about 125 to about 2900, or from about 100 to about 2500, or from about
100 to about 2000,
or from about 100 to about 1900, or from about 100 to about 1850, or from
about 100 to about
1750, or from about 100 to about 1650, or from about 100 to about 1500, or
from about 100 to
about 1400, or from about 100 to about 1300, or from about 100 to about 1250,
or from about 100
to about 1150, or from about 100 to about 1100, or from about 100 to about
1000, or from about
100 to about 900, or from about 100 to about 750, or from about 100 to about
700, or from about
100 to about 600, or from about 100 to about 575, or from about 100 to about
500, or from about
100 to about 450, or from about 100 to about to about 350, or from about 100
to about 275, or from
about 100 to about 230, or from about 150 to about 475, or from about 150 to
about 340, or from
about 113 to about 340, or from about 450 to about 800, or from about 454 to
about 796, or from
about 454 to about 682, or from about 340 to about 795, or from about 341 to
about 682, or from
about 568 to about 909, or from about 227 to about 1500, or from about 225 to
about 2280, or from
about 460 to about 2160, or from about 460 to about 2050, or from about 341 to
about 1820, or
from about 341 to about 1710, or from about 341 to about 1250, or from about
225 to about 1250,
or from about 341 to about 1250, or from about 341 to about 1136, or from
about 341 to about
1023, or from about 341 to about 910, or from about 341 to about 796, or from
about 341 to about
682, or from about 341 to about 568, or from about 114 to about 1000, or from
about 114 to about
950, or from about 114 to about 910, or from about 114 to about 800, or from
about 114 to about
690, or from about 114 to about 575.
[251] In some embodiments, the IL-2 conjugate comprises the amino
acid sequence of SEQ ID
NO: 3 in which m in the compounds of Formula (XVI) and Formula (XVII) is an
integer from 1 to
6, and n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455,
568, 569, 680, 681, 682,
794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some
embodiments of
the compounds of Formula (XVI) and Formula (XVII), m is an integer from 2 to
6, and n is an
integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680,
681, 682, 794, 795,
796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some
embodiments of the
compounds of Formula (XVI) and Formula (XVII), m is an integer from 2 to 4,
and n is an integer
selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682,
794, 795, 796, 908,
909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments of the
compounds of
Formula (XVI) and Formula (XVII), m is 1, and n is an integer selected from
113, 114, 227, 228,
340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910,
1021, 1022, 1023, 1135,
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1136, and 1137. In some embodiments of the compounds of Formula (XVI) and
Formula (XVII),
m is 2, and n is an integer selected from 113, 114, 227, 228, 340, 341, 454,
455, 568, 569, 680, 681,
682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In
some embodiments
of the compounds of Formula (XVI) and Formula (XVII), m is 3, and n is an
integer selected from
113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795,
796, 908, 909, 910,
1021, 1022, 1023, 1135, 1136, and 1137. In some embodiments of the compounds
of Formula
(XVI) and Formula (XVII), m is 4, and n is an integer selected from 113, 114,
227, 228, 340, 341,
454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022,
1023, 1135, 1136, and
1137. In some embodiments of the compounds of Formula (XVI) and Formula
(XVII), m is 5, and
n is an integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568,
569, 680, 681, 682, 794,
795, 796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some
embodiments of the
compounds of Formula (XVI) and Formula (XVII), m is 6, and n is an integer
selected from 113,
114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796,
908, 909, 910, 1021,
1022, 1023, 1135, 1136, and 1137. In some embodiments of the compounds of
Formula (XVI) and
Formula (XVII), m is 7, and n is an integer selected from 113, 114, 227, 228,
340, 341, 454, 455,
568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135,
1136, and 1137. In
some embodiments of the compounds of Formula (XVI) and Formula (XVII), m is 8,
and n is an
integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680,
681, 682, 794, 795,
796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some
embodiments of the
compounds of Formula (XVI) and Formula (XVII), m is 9, and n is an integer
selected from 113,
114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796,
908, 909, 910, 1021,
1022, 1023, 1135, 1136, and 1137. In some embodiments of the compounds of
Formula (XVI) and
Formula (XVII), m is 10, and n is an integer selected from 113, 114, 227, 228,
340, 341, 454, 455,
568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910, 1021, 1022, 1023, 1135,
1136, and 1137. In
some embodiments of the compounds of Formula (XVI) and Formula (XVII), m is
11, and n is an
integer selected from 113, 114, 227, 228, 340, 341, 454, 455, 568, 569, 680,
681, 682, 794, 795,
796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, and 1137. In some
embodiments of the
compounds of Formula (XVI) and Formula (XVII), m is 12, and n is an integer
selected from 113,
114, 227, 228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796,
908, 909, 910, 1021,
1022, 1023, 1135, 1136, and 1137. In some embodiments of the compounds of
Formula (XVI) and
Formula (XVII), m is 2, and n is an integer selected from 680, 681, 682, 794,
795, 796, 908, 909,
910, 1021, 1022, 1023, 1135, 1136, and 1137.
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[252] In some embodiments, the IL-2 conjugate comprises the amino
acid sequence of SEQ ID
NO: 3 in which n in the compounds of Formula (XVI) and Formula (XVII) is an
integer selected
from 2, 5, 10, 11, 22, 23, 113, 114, 227, 228, 340, 341, 454, 455, 568, 569,
680, 681, 682, 794, 795,
796, 908, 909, 910, 1021, 1022, 1023, 1135, 1136, 1137, 1249, 1250, 1251,
1362, 1363, 1364,
1476, 1477, 1478, 1589, 1590, 1591, 1703, 1704, 1705, 1817, 1818, 1819, 1930,
1931, 1932, 2044,
2045, 2046, 2158, 2159, 2160, 2271, 2272, 2273, 2839, 2840, 2841, 2953, 2954,
2955, 3408, 3409,
3410, 3976, 3977, 3978, 4544, 4545, and 4546. In some embodiments, the
position of the structure
of Formula (XVI), Formula (XVII), or a mixture of Formula (XVI) and Formula
(XVII) in the
amino acid sequence of the IL-2 conjugate is selected from K34, F41, F43, K42,
E61, P64, R37,
T40, E67, Y44, V68, and L71, wherein the position of the structure of Formula
(XVI), Formula
(XVII), or a mixture of Formula (XVI) and Formula (XVII) in the amino acid
sequence of the IL-2
conjugate is in reference to the positions in SEQ ID NO: 3. In some
embodiments, the position of
the structure of Formula (XVI), Formula (XVII), or a mixture of Formula (XVI)
and Formula
(XVII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is
selected from K34,
F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68, and L71. In some
embodiments, the position
of the structure of Formula (XVI), Formula (XVII), or a mixture of Formula
(XVI) and Formula
(XVII) in the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is at
position K34. In
some embodiments, the position of the structure of Formula (XVI), Formula
(XVII), or a mixture of
Formula (XVI) and Formula (XVII) in the amino acid sequence of the IL-2
conjugate of SEQ ID
NO: 3 is at position F41. In some embodiments, the position of the structure
of Formula (XVI),
Formula (XVII), or a mixture of Formula (XVI) and Formula (XVII) in the amino
acid sequence of
the IL-2 conjugate of SEQ ID NO: 3 is at position F43. In some embodiments,
the position of the
structure of Formula (XVI), Formula (XVII), or a mixture of Formula (XVI) and
Formula (XVII) in
the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is at position
K42. In some
embodiments, the position of the structure of Formula (XVI), Formula (XVII),
or a mixture of
Formula (XVI) and Formula (XVII) in the amino acid sequence of the IL-2
conjugate of SEQ ID
NO: 3 is at position E61. In some embodiments, the position of the structure
of Formula (XVI),
Formula (XVII), or a mixture of Formula (XVI) and Formula (XVII) in the amino
acid sequence of
the IL-2 conjugate of SEQ ID NO: 3 is at position P64. In some embodiments,
the position of the
structure of Formula (XVI), Fomiula (XVII), or a mixture of Formula (XVI) and
Formula (XVII) in
the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is at position
R37. In some
embodiments, the position of the structure of Formula (XVI), Formula (XVII),
or a mixture of
Formula (XVI) and Formula (XVII) in the amino acid sequence of the IL-2
conjugate of SEQ ID
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NO: 3 is at position T40. In some embodiments, the position of the structure
of Formula (XVI),
Formula (XVII), or a mixture of Formula (XVI) and Formula (XVII) in the amino
acid sequence of
the IL-2 conjugate of SEQ ID NO: 3 is at position E67. In some embodiments,
the position of the
structure of Formula (XVI), Formula (XVII), or a mixture of Formula (XVI) and
Formula (XVII) in
the amino acid sequence of the IL-2 conjugate of SEQ ID NO: 3 is at position
Y44. In some
embodiments, the position of the structure of Formula (XVI), Formula (XVII),
or a mixture of
Formula (XVI) and Formula (XVII) in the amino acid sequence of the IL-2
conjugate of SEQ ID
NO: 3 is at position V68. In some embodiments, the position of the structure
of Formula (XVI),
Formula (XVII), or a mixture of Formula (XVI) and Formula (XVII) in the amino
acid sequence of
the IL-2 conjugate of SEQ ID NO: 3 is at position L71. In some embodiments,
the ratio of the
amount of the structure of Formula (XVI) to the amount of the structure of
Formula (XVII)
comprising the total amount of the IL-2 conjugate is about 1:1. In some
embodiments, the ratio of
the amount of the structure of Formula (XVI) to the amount of the structure of
Formula (XVII)
comprising the total amount of the IL-2 conjugate is greater than 1:1. In some
embodiments, the
ratio of the amount of the structure of Formula (XVI) to the amount of the
structure of Formula
(XVII) comprising the total amount of the IL-2 conjugate is less than 1:1.
[253] In some embodiments, the IL-2 conjugate comprises the amino
acid sequence of SEQ ID
NO: 3 in which the at least one amino acid residue in the IL-2 conjugate is
replaced by the structure
of Formula (XVI), Formula (XVII), or a mixture of Formula (XVI) and Formula
(XVII) is selected
from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44, V68, and L71, and
wherein n is an
integer from 100 to about 1150, or from about 100 to about 1100, or from about
100 to about 1000,
or from about 100 to about 900, or from about 100 to about 750, or from about
100 to about 700, or
from about 100 to about 600, or from about 100 to about 575, or from about 100
to about 500, or
from about 100 to about 450, or from about 100 to about to about 350, or from
about 100 to about
275, or from about 100 to about 230, or from about 150 to about 475, or from
about 150 to about
340, or from about 113 to about 340, or from about 450 to about 800, or from
about 454 to about
796, or from about 454 to about 682, or from about 340 to about 795, or from
about 341 to about
682, or from about 568 to about 909, or from about 227 to about 1500, or from
about 225 to about
2280, or from about 460 to about 2160, or from about 460 to about 2050, or
from about 341 to
about 1820, or from about 341 to about 1710, or from about 341 to about 1250,
or from about 225
to about 1250, or from about 341 to about 1250, or from about 341 to about
1136, or from about
341 to about 1023, or from about 341 to about 910, or from about 341 to about
796, or from about
341 to about 682, or from about 341 to about 568, or from about 114 to about
1000, or from about
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114 to about 950, or from about 114 to about 910, or from about 114 to about
800, or from about
114 to about 690, or from about 114 to about 575. In some embodiments, n in
the compounds of
Formula (XVI) and Formula (XVII) is an integer selected from 2, 5, 10, 11, 22,
23, 113, 114, 227,
228, 340, 341, 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909,
910, 1021, 1022, 1023,
1135, 1136, 1137, 1249, 1250, 1251, 1362, 1363, 1364, 1476, 1477, 1478, 1589,
1590, 1591, 1703,
1704, 1705, 1817, 1818, 1819, 1930, 1931, 1932, 2044, 2045, 2046, 2158, 2159,
2160, 2271, 2272,
2273, 2839, 2840, 2841, 2953, 2954, 2955, 3408, 3409, 3410, 3976, 3977, 3978,
4544, 4545, and
4546.
[254] In some embodiments, the IL-2 conjugate comprises the amino acid
sequence of SEQ ID
NO: 3 in which the at least one amino acid residue in the IL-2 conjugate
replaced by the structure of
Formula (XVI), Formula (XVII), or a mixture of Formula (XVI) and Formula
(XVII), is selected
from F41, F43, K42, E61, and P64, and wherein n is an integer from about 450
to about 800, or
from about 454 to about 796, or from about 454 to about 682, or from about 568
to about 909. In
some embodiments, n in the compounds of Formula (XVI) and Formula (XVII) is an
integer
selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, 910,
1021, 1022, 1023,
1135, 1136, 1137, and 1249.
[255] In some embodiments, the IL-2 conjugate comprises the amino acid
sequence of SEQ ID
NO: 3 in which the at least one amino acid residue in the IL-2 conjugate
replaced by the structure of
Formula (XVI), Formula (XVII), or a mixture of Formula (XVI) and Formula
(XVII), is selected
from E61 and P64, and wherein n is an integer from about 450 to about 800, or
from about 454 to
about 796, or from about 454 to about 682, or from about 568 to about 909. In
some embodiments,
n in the compounds of Formula (XVI) and Formula (XVII) is an integer selected
from 454, 455,
568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910.
[256] In some embodiments, the IL-2 conjugate comprises the amino acid
sequence of SEQ ID
NO: 3 in which the at least one amino acid residue in the IL-2 conjugate
replaced by the structure of
Formula (XVI), Formula (XVII), or a mixture of Formula (XVI) and Formula
(XVII), that is
replaced is E61, and wherein n is an integer from about 450 to about 800, or
from about 454 to
about 796, or from about 454 to about 682, or from about 568 to about 909. In
some embodiments,
n in the compounds of Formula (XVI) and Formula (XVII) is an integer selected
from 454, 455,
568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910. In some
embodiments, n is from about
500 to about 1000. In some embodiments, n is from about 550 to about 800. In
some
embodiments, n is about 681.
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[257] In some embodiments, the IL-2 conjugate comprises the amino acid
sequence of SEQ ID
NO: 3 in which the at least one amino acid residue in the IL-2 conjugate is
replaced by the structure
of Formula (XVI), Formula (XVII), or a mixture of Formula (XVI) and Formula
(XVII), that is
replaced is P64, and wherein n is an integer from about 450 to about 800, or
from about 454 to
about 796, or from about 454 to about 682, or from about 568 to about 909. In
some embodiments,
n in the compounds of Formula (XVI) and Formula (XVII) is an integer selected
from 454, 455,
568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and 910. In some
embodiments, n is from about
500 to about 1000. In some embodiments, n is from about 550 to about 800. In
some
embodiments, n is about 681.
[258] In some embodiments, the IL-2 conjugate comprises the amino acid
sequence of SEQ ID
NO: 3 in which the at least one amino acid residue in the IL-2 conjugate
replaced by the structure of
Formula (XVI), Formula (XVII), or a mixture of Formula (XVI) and Formula
(XVII), wherein n is
an integer such that the molecular weight of the PEG moiety is in the range
from about 1,000
Daltons about 200,000 Daltons, or from about 2,000 Daltons to about 150,000
Daltons, or from
about 3,000 Daltons to about 125,000 Daltons, or from about 4,000 Daltons to
about 100,000
Daltons, or from about 5,000 Daltons to about 100,000 Daltons, or from about
6,000 Daltons to
about 90,000 Daltons, or from about 7,000 Daltons to about 80,000 Daltons, or
from about 8,000
Daltons to about 70,000 Daltons, or from about 5,000 Daltons to about 70,000
Daltons, or from
about 5,000 Daltons to about 65,000 Daltons, or from about 5,000 Daltons to
about 60,000 Daltons,
or from about 5,000 Daltons to about 50,000 Daltons, or from about 6,000
Daltons to about 50,000
Daltons, or from about 7,000 Daltons to about 50,000 Daltons, or from about
7,000 Daltons to about
45,000 Daltons, or from about 7,000 Daltons to about 40,000 Daltons, or from
about 8,000 Daltons
to about 40,000 Daltons, or from about 8,500 Daltons to about 40,000 Daltons,
or from about 8,500
Daltons to about 35,000 Daltons, or from about 9,000 Daltons to about 50,000
Daltons, or from
about 9,000 Daltons to about 45,000 Daltons, or from about 9,000 Daltons to
about 40,000 Daltons,
or from about 9,000 Daltons to about 35,000 Daltons, or from about 9,000
Daltons to about 30,000
Daltons, or from about 9,500 Daltons to about 35,000 Daltons, or from about
9,500 Daltons to about
30,000 Daltons, or from about 10,000 Daltons to about 50,000 Daltons, or from
about 10,000
Daltons to about 45,000 Daltons, or from about 10,000 Daltons to about 40,000
Daltons, or from
about 10,000 Daltons to about 35,000 Daltons, or from about 10,000 Daltons to
about 30,000
Daltons, or from about 15,000 Daltons to about 50,000 Daltons, or from about
15,000 Daltons to
about 45,000 Daltons, or from about 15,000 Daltons to about 40,000 Daltons, or
from about 15,000
Daltons to about 35,000 Daltons, or from about 15,000 Daltons to about 30,000
Daltons, or from
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about 20,000 Daltons to about 50,000 Daltons, or from about 20,000 Daltons to
about 45,000
Daltons, or from about 20,000 Daltons to about 40,000 Daltons, or from about
20,000 Daltons to
about 35,000 Daltons, or from about 20,000 Daltons to about 30,000 Daltons. In
some
embodiments, the IL-2 conjugate comprises the amino acid sequence of SEQ ID
NO: 3 in which at
least one amino acid residue in the IL-2 conjugate is replaced by the
structure of Formula (XVI),
Formula (XVII), or a mixture of Formula (XVI) and Formula (XVII), wherein n is
an integer such
that the molecular weight of the PEG moiety is about 5,000 Daltons, about
7,500 Daltons, about
10,000 Daltons, about 15,000 Daltons, about 20,000 Daltons, about 25,000
Daltons, about 30,000
Daltons, about 35,000 Daltons, about 40,000 Daltons, about 45,000 Daltons,
about 50,000 Daltons,
about 60,000 Daltons, about 70,000 Daltons, about 80,000 Daltons, about 90,000
Daltons, about
100,000 Daltons, about 125,000 Daltons, about 150,000 Daltons, about 175,000
Daltons or about
200,000 Daltons. In some embodiments, the IL-2 conjugate comprises the amino
acid sequence of
SEQ ID NO: 3 in which at least one amino acid residue in the IL-2 conjugate is
replaced by the
structure of Formula (XVI), Formula (XVII), or a mixture of Formula (XVI) and
Formula (XVII),
wherein n is an integer such that the molecular weight of the PEG moiety is
about 5,000 Daltons,
about 7,500 Daltons, about 10,000 Daltons, about 15,000 Daltons, about 20,000
Daltons, about
25,000 Daltons, about 30,000 Daltons, about 35,000 Daltons, about 40,000
Daltons, about 45,000
Daltons, or about 50,000 Daltons.
[259] In some embodiments, the IL-2 conjugate comprises the amino acid
sequence of SEQ ID
NO: 3 in which the at least one amino acid residue in the IL-2 conjugate is
replaced by the structure
of Formula (XVI), Formula (XVII), or a mixture of Formula (XVI) and Formula
(XVII), is selected
from F41, F43, K42, E61, and P64, m is an integer from 1 to 6, and n is an
integer from about 450
to about 800, or from about 454 to about 796, or from about 454 to about 682,
or from about 568 to
about 909. In some embodiments of the compounds of Formula (XVI) and Formula
(XVII), m is 2,
and n is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795,
796, 908, 909, 910,
1021, 1022, 1023, 1135, 1136, 1137, and 1249.
[260] In some embodiments, the IL-2 conjugate comprises the amino acid
sequence of SEQ ID
NO: 3 in which the at least one amino acid residue in the IL-2 conjugate
replaced by the structure of
Formula (XVI), Formula (XVII), or a mixture of Formula (XVI) and Formula
(XVII), is selected
from E61 and P64, and wherein m is an integer from 1 to 6, and n is an integer
from about 450 to
about 800, or from about 454 to about 796, or from about 454 to about 682, or
from about 568 to
about 909. In some embodiments of the compounds of Formula (XVI) and Formula
(XVII), m is 2,
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and n is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795,
796, 908, 909, and
910.
[261] In some embodiments, the IL-2 conjugate comprises the amino acid
sequence of SEQ ID
NO: 3 in which the at least one amino acid residue in the IL-2 conjugate
replaced by the structure of
Formula (XVI), Formula (XVII), or a mixture of Formula (XVI) and Formula
(XVII), that is
replaced is E61, and wherein m is an integer from 1 to 6, and n is an integer
from about 450 to
about 800, or from about 454 to about 796, or from about 454 to about 682, or
from about 568 to
about 909. In some embodiments of the compounds of Formula (XVI) and Formula
(XVII), m is 2,
and n is an integer selected from 454, 455, 568, 569, 680, 681, 682, 794, 795,
796, 908, 909, and
910. In some embodiments, n is from about 500 to about 1000. In some
embodiments, n is from
about 550 to about 800. In some embodiments, n is about 681.
[262] In some embodiments, the IL-2 conjugate comprises the amino acid
sequence of SEQ ID
NO: 3 in which the at least one amino acid residue in the IL-2 conjugate
replaced by the structure of
Formula (XVI), Formula (XVII), or a mixture of Formula (XVI) and Formula
(XVII), is P64, and
wherein m is an integer from 1 to 6, and n is an integer from about 450 to
about 800, or from about
454 to about 796, or from about 454 to about 682, or from about 568 to about
909. In some
embodiments of the compounds of Formula (XVI) and Formula (XVII), m is 2, and
n is an integer
selected from 454, 455, 568, 569, 680, 681, 682, 794, 795, 796, 908, 909, and
910. In some
embodiments, n is from about 500 to about 1000. In some embodiments, n is from
about 550 to
about 800. In some embodiments, n is about 681.
[263] In some embodiments, the IL-2 conjugate comprises SEQ ID NOs,: 1-98.
In some
embodiments, the IL-2 conjugate comprises SEQ ID NOs.: 15-29. In some
embodiments, the IL-2
conjugate comprises SEQ ID NOs.: 40-54. In some embodiments, the IL-2
conjugate comprises
SEQ ID NOs.: 55-69. In some embodiments, the IL-2 conjugate comprises SEQ ID
NOs.: 70-84. In
some embodiments, the IL-2 conjugate comprises a structure of Formula (I). In
some embodiments,
the IL-2 conjugate comprises a structure of Formula (II). In some embodiments,
the IL-2 conjugate
comprises a structure of Formula (III). In some embodiments, the IL-2
conjugate comprises a
structure of Formula (IV). In some embodiments, the IL-2 conjugate comprises a
structure of
Formula (V). In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 1.
In some
embodiments, the IL-2 conjugate comprises SEQ ID NO: 2. In some embodiments,
the IL-2
conjugate comprises SEQ ID NO: 3. In some embodiments, the 1L-2 conjugate
comprises SEQ ID
NO: 4. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 5. In some
embodiments,
the IL-2 conjugate comprises SEQ ID NO: 6. In some embodiments, the IL-2
conjugate comprises
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SEQ ID NO: 7. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 8.
In some
embodiments, the IL-2 conjugate comprises SEQ ID NO: 9. In some embodiments,
the IL-2
conjugate comprises SEQ ID NO: 10. In some embodiments, the IL-2 conjugate
comprises SEQ ID
NO: 11. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 12. In
some
embodiments, the IL-2 conjugate comprises SEQ ID NO: 13. In some embodiments,
the IL-2
conjugate comprises SEQ ID NO: 14. In some embodiments, the IL-2 conjugate
comprises SEQ ID
NO: 15. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 16. In
some
embodiments, the IL-2 conjugate comprises SEQ ID NO: 17. In some embodiments,
the IL-2
conjugate comprises SEQ ID NO: 18. In some embodiments, the IL-2 conjugate
comprises SEQ ID
NO: 19. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 20. In
some
embodiments, the IL-2 conjugate comprises SEQ ID NO: 21. In some embodiments,
the IL-2
conjugate comprises SEQ ID NO: 22. In some embodiments, the IL-2 conjugate
comprises SEQ ID
NO: 23. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 24. In
some
embodiments, the IL-2 conjugate comprises SEQ ID NO: 25. In some embodiments,
the IL-2
conjugate comprises SEQ ID NO: 26. In some embodiments, the IL-2 conjugate
comprises SEQ ID
NO: 27. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 28. In
some
embodiments, the IL-2 conjugate comprises SEQ ID NO: 24. In some embodiments,
the IL-2
conjugate comprises SEQ ID NO: 25. In some embodiments, the IL-2 conjugate
comprises SEQ ID
NO: 26. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 27. In
some
embodiments, the IL-2 conjugate comprises SEQ ID NO: 28. In some embodiments,
the IL-2
conjugate comprises SEQ ID NO: 29. In some embodiments, the IL-2 conjugate
comprises SEQ ID
NO: 30. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 31. In
some
embodiments, the IL-2 conjugate comprises SEQ ID NO: 32. In some embodiments,
the IL-2
conjugate comprises SEQ ID NO: 33. In some embodiments, the IL-2 conjugate
comprises SEQ ID
NO: 34. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 35. In
some
embodiments, the IL-2 conjugate comprises SEQ ID NO: 36. In some embodiments,
the IL-2
conjugate comprises SEQ ID NO: 37. In some embodiments, the IL-2 conjugate
comprises SEQ ID
NO: 38. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 39. In
some
embodiments, the IL-2 conjugate comprises SEQ ID NO: 40. In some embodiments,
the 1L-2
conjugate comprises SEQ ID NO: 41. In some embodiments, the IL-2 conjugate
comprises SEQ ID
NO: 42. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 43. In
some
embodiments, the IL-2 conjugate comprises SEQ ID NO: 44. In some embodiments,
the 1L-2
conjugate comprises SEQ ID NO: 45. In some embodiments, the IL-2 conjugate
comprises SEQ ID
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NO: 46. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 47. In
some
embodiments, the IL-2 conjugate comprises SEQ ID NO: 48. In some embodiments,
the IL-2
conjugate comprises SEQ ID NO: 49. In some embodiments, the IL-2 conjugate
comprises SEQ ID
NO: 50. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 51. In
some
embodiments, the IL-2 conjugate comprises SEQ ID NO: 52. In some embodiments,
the IL-2
conjugate comprises SEQ ID NO: 53. In some embodiments, the IL-2 conjugate
comprises SEQ ID
NO: 54. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 55. In
some
embodiments, the IL-2 conjugate comprises SEQ ID NO: 56. In some embodiments,
the IL-2
conjugate comprises SEQ ID NO: 57. In some embodiments, the IL-2 conjugate
comprises SEQ ID
NO: 58. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 59. In
some
embodiments, the IL-2 conjugate comprises SEQ ID NO: 60. In some embodiments,
the IL-2
conjugate comprises SEQ ID NO: 61. In some embodiments, the IL-2 conjugate
comprises SEQ ID
NO: 62. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 63. In
some
embodiments, the IL-2 conjugate comprises SEQ ID NO: 64. In some embodiments,
the IL-2
conjugate comprises SEQ ID NO: 65. In some embodiments, the IL-2 conjugate
comprises SEQ ID
NO: 66. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 67. In
some
embodiments, the IL-2 conjugate comprises SEQ ID NO: 68. In some embodiments,
the IL-2
conjugate comprises SEQ ID NO: 69. In some embodiments, the IL-2 conjugate
comprises SEQ ID
NO: 70. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 71. In
some
embodiments, the IL-2 conjugate comprises SEQ ID NO: 72. In some embodiments,
the IL-2
conjugate comprises SEQ ID NO: 73. In some embodiments, the IL-2 conjugate
comprises SEQ ID
NO: 74. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 75. In
some
embodiments, the IL-2 conjugate comprises SEQ ID NO: 76. In some embodiments,
the IL-2
conjugate comprises SEQ ID NO: 77. In some embodiments, the IL-2 conjugate
comprises SEQ ID
NO: 78. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 79. In
some
embodiments, the IL-2 conjugate comprises SEQ ID NO: 80. In some embodiments,
the IL-2
conjugate comprises SEQ ID NO: 81. In some embodiments, the IL-2 conjugate
comprises SEQ ID
NO: 82. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 83. In
some
embodiments, the IL-2 conjugate comprises SEQ ID NO: 84. In some embodiments,
the 1L-2
conjugate comprises SEQ ID NO: 85. In some embodiments, the IL-2 conjugate
comprises SEQ ID
NO: 86. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 87. In
some
embodiments, the IL-2 conjugate comprises SEQ ID NO: 88. In some embodiments,
the 1L-2
conjugate comprises SEQ ID NO: 89. In some embodiments, the IL-2 conjugate
comprises SEQ ID
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NO: 90. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 91. In
some
embodiments, the IL-2 conjugate comprises SEQ ID NO: 92. In some embodiments,
the IL-2
conjugate comprises SEQ ID NO: 93. In some embodiments, the IL-2 conjugate
comprises SEQ ID
NO: 94. In some embodiments, the IL-2 conjugate comprises SEQ ID NO: 95. In
some
embodiments, the IL-2 conjugate comprises SEQ ID NO: 96. In some embodiments,
the IL-2
conjugate comprises SEQ ID NO: 97. In some embodiments, the IL-2 conjugate
comprises SEQ ID
NO: 98.
[264] In some embodiments, the IL-2 conjugate comprises the amino acid
sequence of any one
of SEQ ID NOS: 86, 88, 90, 92, 94, 96, and 98. In any of these embodiments,
the structure of
Formula (I), or any variation thereof', such as Formula (II)-Formula (XV) or
any variation thereof, is
incorporated into the site comprising the unnatural amino acid.
[265] In some embodiments, the IL-2 conjugate is modified at an amino acid
position. In some
instances, the modification is to a natural amino acid. In some instances, the
modification is to an
unnatural amino acid. In some instances, described herein is an isolated and
modified IL-2
polypeptide that comprises at least one unnatural amino acid. In some cases,
the IL-2 polypeptide
comprises about 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to
any one of
SEQ ID NOS: 3 to 84.
[266] In some embodiments, the IL-2 conjugate further comprises an
additional mutation. In
some cases, the additional mutation is at an amino acid position selected from
K35, T37, R38, T41,
F42, K43, F44, Y45, E61, E62, E68, K64, P65, V69, L72, and Y107. In such
cases, the amino acid
is conjugated to an additional conjugating moiety for increase in serum half-
life, stability, or a
combination thereof. Alternatively, the amino acid is first mutated to a
natural amino acid such as
lysine, cysteine, histidine, arginine, aspartic acid, glutamic acid, serine,
threonine, or tyrosine; or to
an unnatural amino acid prior to binding to the additional conjugating moiety.
[267] In some cases, the PEG group is not limited to a particular
structure. In some cases, the
PEG is linear (e.g., an end capped, e.g., alkoxy PEG or a bifunctional PEG),
branched or multi-
armed (e.g., forked PEG or PEG attached to a polyol core), a dendritic (or
star) architecture, each
with or without one or more degradable linkages. Moreover, the internal
structure of the water-
soluble polymer can be organized in any number of different repeat patterns
and can be selected
from the group consisting of homopolymer, alternating copolymer, random
copolymer, block
copolymer, alternating tripolymer, random tripolymer, and block tripolymer.
[268] PEGs will typically comprise a number of (OCH2CH2) monomers [or
(CH2CH20)
monomers, depending on how the PEG is defined]. As used herein, the number of
repeating units is
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identified by the subscript "n" in "(OCH2CH2)n." Thus, the value of (n)
typically falls within one or
more of the following ranges: from 2 to about 3400, from about 100 to about
2300, from about 100
to about 2270, from about 136 to about 2050, from about 225 to about 1930,
from about 450 to
about 1930, from about 1200 to about 1930, from about 568 to about 2727, from
about 660 to about
2730, from about 795 to about 2730, from about 795 to about 2730, from about
909 to about 2730,
and from about 1,200 to about 1,900. For any given polymer in which the
molecular weight is
known, it is possible to determine the number of repeating units (i.e., "n")
by dividing the total
weight-average molecular weight of the polymer by the molecular weight of the
repeating
monomer.
[269] In some instances, the PEG is an end-capped polymer, that is, a
polymer having at least
one terminus capped with a relatively inert group, such as a lower C1-6 alkoxy
group, or a hydroxyl
group. When the polymer is PEG, for example, a methoxy-PEG (commonly referred
to as mPEG)
may be used, which is a linear form of PEG wherein one terminus of the polymer
is a methoxy (¨
OCH3) group, while the other terminus is a hydroxyl or other functional group
that can be
optionally chemically modified.
[270] In some embodiments, the PEG group comprising the IL-2 conjugates
disclosed herein is a
linear or branched PEG group. In some embodiments, the PEG group is a linear
PEG group. In
some embodiments, the PEG group is a branched PEG group. In some embodiments,
the PEG
group is a methoxy PEG group. In some embodiments, the PEG group is a linear
or branched
methoxy PEG group. In some embodiments, the PEG group is a linear methoxy PEG
group. In
some embodiments, the PEG group is a branched methoxy PEG group. In some
embodiments, the
PEG group is a linear or branched PEG group having an average molecular weight
of from about
100 Daltons to about 150,000 Daltons. Exemplary ranges include, for example,
weight-average
molecular weights in the range of greater than 5,000 Daltons to about 100,000
Daltons, in the range
of from about 6,000 Daltons to about 90,000 Daltons, in the range of from
about 10,000 Daltons to
about 85,000 Daltons, in the range of greater than 10,000 Daltons to about
85,000 Daltons, in the
range of from about 20,000 Daltons to about 85,000 Daltons, in the range of
from about 53,000
Daltons to about 85,000 Daltons, in the range of from about 25,000 Daltons to
about 120,000
Daltons, in the range of from about 29,000 Daltons to about 120,000 Daltons,
in the range of from
about 35,000 Daltons to about 120,000 Daltons, and in the range of from about
40,000 Daltons to
about 120,000 Daltons. Exemplary weight-average molecular weights for the PEG
group include
about 100 Daltons, about 200 Daltons, about 300 Daltons, about 400 Daltons,
about 500 Daltons,
about 600 Daltons, about 700 Daltons, about 750 Daltons, about 800 Daltons,
about 900 Daltons,
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about 1,000 Daltons, about 1,500 Daltons, about 2,000 Daltons, about 2,200
Daltons, about 2,500
Daltons, about 3,000 Daltons, about 4,000 Daltons, about 4,400 Daltons, about
4,500 Daltons, about
5,000 Daltons, about 5,500 Daltons, about 6,000 Daltons, about 7,000 Daltons,
about 7,500 Daltons,
about 8,000 Daltons, about 9,000 Daltons, about 10,000 Daltons, about 11,000
Daltons, about
12,000 Daltons, about 13,000 Daltons, about 14,000 Daltons, about 15,000
Daltons, about 20,000
Daltons, about 22,500 Daltons, about 25,000 Daltons, about 30,000 Daltons,
about 35,000 Daltons,
about 40,000 Daltons, about 45,000 Daltons, about 50,000 Daltons, about 55,000
Daltons, about
60,000 Daltons, about 65,000 Daltons, about 70,000 Daltons, about 75,000
Daltons, about 80,000
Daltons, about 90,000 Daltons, about 95,000 Daltons, and about 100,000
Daltons. In some
embodiments, the PEG group is a linear PEG group having an average molecular
weight as
disclosed above. In some embodiments, the PEG group is a branched PEG group
having an average
molecular weight as disclosed above. In some embodiments, the PEG group
comprising the IL-2
conjugates disclosed herein is a linear or branched PEG group having a defined
molecular weight
10%, or 15% or 20% or 25%. For example, included within the scope of the
present disclosure are
IL-2 conjugates comprising a PEG group having a molecular weight of 30,000 Da
+ 3000 Da, or
30,000 Da 4,500 Da, or 30,000 Da 6,000 Da.
[271] In some embodiments, the PEG group comprising the IL-2
conjugates disclosed herein is a
linear or branched PEG group having an average molecular weight of from about
5,000 Daltons to
about 60,000 Daltons. In some embodiments, the PEG group is a linear or
branched PEG group
having an average molecular weight of about 5,000 Daltons, about 5,500
Daltons, about 6,000
Daltons, about 7,000 Daltons, about 7,500 Daltons, about 8,000 Daltons, about
9,000 Daltons, about
10,000 Daltons, about 11,000 Daltons, about 12,000 Daltons, about 13,000
Daltons, about 14,000
Daltons, about 15,000 Daltons, about 20,000 Daltons, about 22,500 Daltons,
about 25,000 Daltons,
about 30,000 Daltons, about 35,000 Daltons, about 40,000 Daltons, about 45,000
Daltons, about
50,000 Daltons, about 55,000 Daltons, about 60,000 Daltons, about 65,000
Daltons, about 70,000
Daltons, about 75,000 Daltons, about 80,000 Daltons, about 90,000 Daltons,
about 95,000 Daltons,
and about 100,000 Daltons. In some embodiments, the PEG group is a linear or
branched PEG
group having an average molecular weight of about 5,000 Daltons, about 10,000
Daltons, about
20,000 Daltons, about 30,000 Daltons, about 50,000 Daltons, or about 60,000
Daltons In some
embodiments, the PEG group is a linear or branched PEG group having an average
molecular
weight of about 5,000 Daltons, about 30,000 Daltons, about 50,000 Daltons, or
about 60,000
Daltons. In some embodiments, the PEG group is a linear PEG group having an
average molecular
of about 5,000 Daltons, about 10,000 Daltons, about 20,000 Daltons, about
30,000 Daltons, about
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50,000 Daltons, or about 60,000 Daltons. In some embodiments, the PEG group is
a branched PEG
group having an average molecular weight of about 5,000 Daltons, about 10,000
Daltons, about
20,000 Daltons, about 30,000 Daltons, about 50,000 Daltons, or about 60,000
Daltons.
[272] In some embodiments, the PEG group comprising the IL-2
conjugates disclosed herein is a
linear methoxy PEG group having an average molecular weight of from about
5,000 Daltons to
about 60,000 Daltons. In some embodiments, the PEG group is a linear methoxy
PEG group having
an average molecular weight of about 5,000 Daltons, about 5,500 Daltons, about
6,000 Daltons,
about 7,000 Daltons, about 7,500 Daltons, about 8,000 Daltons, about 9,000
Daltons, about 10,000
Daltons, about 11,000 Daltons, about 12,000 Daltons, about 13,000 Daltons,
about 14,000 Daltons,
about 15,000 Daltons, about 20,000 Daltons, about 22,500 Daltons, about 25,000
Daltons, about
30,000 Daltons, about 35,000 Daltons, about 40,000 Daltons, about 45,000
Daltons, about 50,000
Daltons, about 55,000 Daltons, about 60,000 Daltons, about 65,000 Daltons,
about 70,000 Daltons,
about 75,000 Daltons, about 80,000 Daltons, about 90,000 Daltons, about 95,000
Daltons, and
about 100,000 Daltons. In some embodiments, the PEG group is a linear methoxy
PEG group
having an average molecular weight of about 5,000 Daltons, about 10,000
Daltons, about 20,000
Daltons, about 30,000 Daltons, about 50,000 Daltons, or about 60,000 Daltons.
In some
embodiments, the PEG group is a linear methoxy PEG group having an average
molecular weight
of about 5,000 Daltons, about 30,000 Daltons, about 50,000 Daltons, or about
60,000 Daltons. In
some embodiments, the PEG group is a linear methoxy PEG group having an
average molecular of
about 5,000 Daltons, about 10,000 Daltons, about 20,000 Daltons, about 30,000
Daltons, about
50,000 Daltons, or about 60,000 Daltons. In some embodiments, the PEG group is
a linear methoxy
PEG group having an average molecular of about 5,000 Daltons. In some
embodiments, the PEG
group is a linear methoxy PEG group having an average molecular of about
10,000 Daltons. In
some embodiments, the PEG group is a linear methoxy PEG group having an
average molecular of
about 20,000 Daltons. In some embodiments, the PEG group is a linear methoxy
PEG group having
an average molecular of about 30,000 Daltons. In some embodiments, the PEG
group is a linear
methoxy PEG group having an average molecular of about 50,000 Daltons. In some
embodiments,
the PEG group is a linear methoxy PEG group having an average molecular of
about 60,000
Daltons In some embodiments, the PEG group comprising the IL-2 conjugates
disclosed herein is
a linear methoxy PEG group having a defined molecular weight 10%, or 15% or
20% or 25%.
For example, included within the scope of the present disclosure are 1L-2
conjugates comprising a
linear methoxy PEG group having a molecular weight of 30,000 Da 1 3000 Da, or
30,000 Da I
4,500 Da, or 30,000 Da 6,000 Da,
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[273] In some embodiments, the PEG group comprising the IL-2
conjugates disclosed herein is a
branched methoxy PEG group having an average molecular weight of from about
5,000 Daltons to
about 60,000 Daltons. In some embodiments, the PEG group is a branched methoxy
PEG group
having an average molecular weight of about 5,000 Daltons, about 5,500
Daltons, about 6,000
Daltons, about 7,000 Daltons, about 7,500 Daltons, about 8,000 Daltons, about
9,000 Daltons, about
10,000 Daltons, about 11,000 Daltons, about 12,000 Daltons, about 13,000
Daltons, about 14,000
Daltons, about 15,000 Daltons, about 20,000 Daltons, about 22,500 Daltons,
about 25,000 Daltons,
about 30,000 Daltons, about 35,000 Daltons, about 40,000 Daltons, about 45,000
Daltons, about
50,000 Daltons, about 55,000 Daltons, about 60,000 Daltons, about 65,000
Daltons, about 70,000
Daltons, about 75,000 Daltons, about 80,000 Daltons, about 90,000 Daltons,
about 95,000 Daltons,
and about 100,000 Daltons. In some embodiments, the PEG group is a branched
methoxy PEG
group having an average molecular weight of about 5,000 Daltons, about 10,000
Daltons, about
20,000 Daltons, about 30,000 Daltons, about 50,000 Daltons, or about 60,000
Daltons. In some
embodiments, the PEG group is a branched methoxy PEG group having an average
molecular
weight of about 5,000 Daltons, about 30,000 Daltons, about 50,000 Daltons, or
about 60,000
Daltons. In some embodiments, the PEG group is a branched methoxy PEG group
having an
average molecular of about 5,000 Daltons, about 10,000 Daltons, about 20,000
Daltons, about
30,000 Daltons, about 50,000 Daltons, or about 60,000 Daltons. In some
embodiments, the PEG
group is a branched methoxy PEG group having an average molecular of about
5,000 Daltons,
about 10,000 Daltons, about 20,000 Daltons, about 30,000 Daltons, about 50,000
Daltons, or about
60,000 Daltons. In some embodiments, the PEG group is a branched methoxy PEG
group having
an average molecular of about 5,000 Daltons. In some embodiments, the PEG
group is a branched
methoxy PEG group having an average molecular of about 10,000 Daltons. In some
embodiments,
the PEG group is a branched methoxy PEG group having an average molecular of
about 20,000
Daltons. In some embodiments, the PEG group is a branched methoxy PEG group
having an
average molecular of about 30,000 Daltons. In some embodiments, the PEG group
is a branched
methoxy PEG group having an average molecular of about 50,000 Daltons. In some
embodiments,
the PEG group is a branched methoxy PEG group having an average molecular of
about 60,000
Daltons In some embodiments, the PEG group comprising the IL-2 conjugates
disclosed herein is
a branched methoxy PEG group having a defined molecular weight 10%, or 15%
or 20% or 25%.
For example, included within the scope of the present disclosure are IL-2
conjugates comprising a
branched methoxy PEG group having a molecular weight of 30,000 Da 1 3000 Da,
or 30,000 Da I
4,500 Da, or 30,000 Da 6,000 Da,
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[274] In some embodiments, exemplary PEG groups include, but are not
limited to, linear or
branched discrete PEG (dPEG) from Quanta Biodesign, Ltd; linear, branched, or
forked PEGs from
Nektar Therapeutics; and Y-shaped PEG derivatives from JenKem Technology.
[275] In any of the embodiments or variations of Formula (I) described
herein and
pharmaceutical compositions comprising the same, average molecular weight
encompasses both
weight average molecular weight and number average molecular weight; in other
words, for
example, both a 30 kDa number average molecular weight and a 30 kDa weight
average molecular
weight qualify as a 30 kDa molecular weight. In some embodiments, the average
molecular weight
is weight average molecular weight. In other embodiments, the average
molecular weight is
number average molecular weight. It is understood that in the methods provided
herein,
administering an IL-2 conjugate as described herein to a subject comprises
administering more than
a single molecule of IL-2 conjugate; as such, use of the term "average" to
describe the molecular
weight of the PEG group refers to the average molecular weight of the PEG
groups of the IL-2
conjugate molecules in a dose administered to the subject.
Conjugation Chemistry
[276] Various conjugation reactions are used to conjugate linkers,
conjugation moieties, and
unnatural amino acids incorporated into the IL-2 polypeptides described
herein. Such conjugation
reactions are often compatible with aqueous conditions, such as
"bioorthogonal" reactions. In some
embodiments, conjugation reactions are mediated by chemical reagents such as
catalysts, light, or
reactive chemical groups found on linkers, conjugation moieties, or unnatural
amino acids. In some
embodiments, conjugation reactions are mediated by enzymes. In some
embodiments, a conjugation
reaction used herein is described in Gong, Y., Pan, L. Tett. Lett. 2015, 56,
2123, the disclosure of
which is herein incorporated by reference. In some embodiments, a conjugation
reaction used herein
is described in Chen, X.; Wu. Y-W. Org. Biomol. Chem. 2016, 14, 5417, the
disclosure of which is
herein incorporated by reference.
[277] In some variation, the IL-2 conjugates described herein can be
prepared by a conjugation
reaction comprising a 1,3-dipolar cycloaddition reaction. In some embodiments,
the 1,3-dipolar
cycloaddition reaction comprises reaction of an azide and a phosphine ("Click"
reaction). In some
embodiments, the conjugation reaction is catalyzed by copper. In some
embodiments, a conjugation
reaction described herein results in cytokine peptide comprising a linker or
conjugation moiety
attached via a triazole. In some embodiments, a conjugation reaction described
herein comprises
reaction of an azide with a strained olefin In some embodiments, a conjugation
reaction described
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herein comprises reaction of an azide with a strained alkyne. In some
embodiments, a conjugation
reaction described herein comprises reaction of an azide with a cycloalkyne,
for example DBCO.
[278] In some embodiments described herein, a conjugation reaction
described herein comprises
the reaction outlined in Scheme 1, wherein Xis the position in the IL-2
conjugate comprising an
unnatural amino acid, such as in any one of SEQ ID NOS: 5, 6, 7, 8, 9, 30, 31,
32, 33, and 34.
Scheme 1.
Reactive G Position X-1 Conjugating Moiety
roup
Reactive
Sidechain., ,NH Group
Position X-1
0 frrr Conjugating Moiety -Sidechain
NH
Position X+1
Position X+1
[279] In some embodiments, the conjugating moiety comprises a water soluble
polymer. In some
embodiments, a reactive group comprises an alkyne or azide.
[280] In some embodiments, a conjugation reaction described herein
comprises the reaction
outlined in Scheme 2, wherein X is the position in the IL-2 conjugate
comprising an unnatural
amino acid, such as in any one of SEQ ID NOS: 5, 6, 7, 8, 9, 30, 31, 32, 33,
and 34.
Scheme 2.
Position X-1 -Conjugating Moiety
s
N3-SidechainT,NH Conjugating M9iety-1[
Position X-1
Click
N
Sidechain,y NH
0 cos Reaction
Position X+1
O/
Position X+1
[281] In some embodiments, a conjugation reaction described herein
comprises the reaction
outlined in Scheme 3, wherein X is the position in the IL-2 conjugate
comprising an unnatural
amino acid, such as in any one of SEQ ID NOS: 5, 6, 7, 8, 9, 30, 31, 32, 33,
and 34.
Scheme 3.
Position X-1
Position X-1
N3-Conjugating Moiety
Sidechpin NH Nii ____________
Sidechainji\JH
Click N
1.,';00,ss' Position X+1 Reaction Conjugating Moiety
Position X+1
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[282] In some embodiments described herein, a conjugation reaction
described herein comprises
the reaction outlined in Scheme 4, wherein X is the position in the IL-2
conjugate comprising an
unnatural amino acid, such as in any one of SEQ ID NOS: 5, 6, 7, 8, 9, 30, 31,
32, 33, and 34.
Scheme 4.
Position X-1
___________________________________________________________ Conjugating Moiety
N3
0 Click
Position X+1 Reaction
Position X-1
Conjugating Moiety _______________________ µ:1\1
,
___________________________________________________ y
0
0 rsss
Position X+1
[283] In some embodiments, a conjugation reaction described herein
comprises a cycloaddition
reaction between an azide moiety, such as that contained in a protein
containing an amino acid
residue derived from N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK), and a
strained cycloalkyne,
such as that derived from DBCO, which is a chemical moiety comprising a
dibenzocyclooctyne
group. PEG groups comprising a DBCO moiety are commercially available or may
be prepared by
methods know to those of ordinary skill in the art. An exemplary reaction is
shown in Schemes 5
and 6.
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Scheme 5.
.....
to Position X-1
Posin "X"
H
1L-2 variant protein -== N3' - -sr. '' '' r
0 ..i,
--..... -... ...
0 '--i-
e
Position X+1
õ.
...7-7.
iy \\
0 0
C
..,'
,-..
aid<
Reaction - - n H K .)1
inPEG-DBCO . ...
(.,
...;.)
,,:s.,
,..<,
1; ..................................................... N
0 0<.. ,..^ .%;
. .
..0õ-^ --`= .0 ='`. ='` A A >'1" \ N
I, n H II µN
1
/ \
t.,... .e,..
b
.=::".,
.:^ . .................................................. f....
o-- =---r
+
..,
r----,
,...,,/ µ ,
,õ i /
µ
..,.;.-,..-..,.. . ....................................
.(... )....-N N
=
H ..,...,
- .---..
'1-- . .N, .. ....0,N
.. õNH
0 0 ::'µ. 7., 0I
i
1, (7
N.1.......2i.
........:1
1L-2 Azic_PES variant proteins
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Scheme 6.
Position X-1
Position "X"
Cytokine variant protein N3 N N H
0
0 /
Position X+1
0=
H3COo1LN
ii
Click in
II
Reaction 0
mPEG-DBCO
0
0
N
NH
0
0 /
0 '1\1
n H
0
0
Of
Cytokine Azk_L1_PEG variant proteins
[284] Conjugation reactions such as a click reaction described
herein may generate a single
regi oi somer, or a mixture of regioisomers. In some instances, the ratio of
regioisomers is about 1:1.
In some instances, the ratio of regioisomers is about 2:1. In some instances,
the ratio of
regioisomers is about 1.5:1. In some instances, the ratio of regioisomers is
about 1.2:1. In some
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instances, the ratio of regioisomers is about LI: L In some instances the
ratio of regioisomers is
greater than 1:1.
IL-2 Polypeptide Production
[285] In some instances, the IL-2 conjugates described herein, either
containing a natural amino
acid mutation or an unnatural amino acid mutation, are generated recombinantly
or are synthesized
chemically. In some instances, IL-2 conjugates described herein are generated
recombinantly, for
example, either by a host cell system, or in a cell-free system. In any of the
embodiments or
variations described herein, the amino acid may be an L-amino acid or a D-
amino acid. In some
embodiments, the amino acid is an L-amino acid. In other embodiments, the
amino acid is a D-
amino acid.
[286] In some instances, IL-2 conjugates are generated recombinantly
through a host cell
system. In some cases, the host cell is a eukaryotic cell (e.g., mammalian
cell, insect cells, yeast
cells or plant cell) or a prokaryotic cell (e.g., gram-positive bacterium or a
gram-negative
bacterium). In some cases, a eukaryotic host cell is a mammalian host cell. In
some cases, a
mammalian host cell is a stable cell line, or a cell line that has
incorporated a genetic material of
interest into its own genome and has the capability to express the product of
the genetic material
after many generations of cell division. In other cases, a mammalian host cell
is a transient cell line,
or a cell line that has not incorporated a genetic material of interest into
its own genome and does
not have the capability to express the product of the genetic material after
many generations of cell
division.
[287] Exemplary mammalian host cells include 293T cell line, 293A cell
line, 293FT cell line,
293F cells, 293 H cells, A549 cells, MDCK cells, CHO DG44 cells, CHO-S cells,
CHO-Kl cells,
Expi293FTM cells, Flp-InTm T-RExTm 293 cell line, Flp-InTm-293 cell line, Flp-
InTm-3T3 cell line,
Flp-InTm-BHK cell line, Flp-InTm-CHO cell line, Flp-InTm-CV-1 cell line, Flp-
InTm-Jurkat cell line,
FreeStyleTM 293-F cells, FreeStyleTM CHO-S cells, GripTiteTm 293 MSR cell
line, GS-CHO cell
line, HepaRGTM cells, T-RExTm Jurkat cell line, Per.C6 cells, T-RExTm-293 cell
line, T_RExTm_
CHO cell line, and T-RExTm-HeLa cell line.
[288] In some embodiments, a eukaryotic host cell is an insect host cell.
Exemplary insect host
cell include Drosophila S2 cells, Sf9 cells, Sf21 cells, High FiveTM cells,
and expresSF+R cells.
[289] In some embodiments, a eukaryotic host cell is a yeast host cell.
Exemplary yeast host
cells include Pichia pastoris (K. phaffii) yeast strains such as GS115, KM71H,
SMD1168,
SMD1168H, and X-33, and Saccharomyces cerevisiae yeast strain such as INVSc1.
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[290] In some embodiments, a eukaryotic host cell is a plant host cell. In
some instances, the
plant cells comprise a cell from algae. Exemplary plant cell lines include
strains from
Chlamydomonas reinhardtii 137c, or Synechococcus elongatus PPC 7942.
[291] In some embodiments, a host cell is a prokaryotic host cell.
Exemplary prokaryotic host
cells include BL21, MachlTM, DH1OBTM, TOP10, DH5a, DH10BacTm, OmniMaxTm,
MegaXTM,
DH12STM, INV110, TOP1OF', INVaF, TOP10/P3, ccdB Survival, PIRL PIR2, Stb12Tm,
Stbl3TM, or
Stbl4TM.
[292] In some instances, suitable polynucleic acid molecules or vectors for
the production of an
IL-2 polypeptide described herein include any suitable vectors derived from
either a eukaryotic or
prokaryotic source. Exemplary polynucleic acid molecules or vectors include
vectors from bacteria
(e.g., E. colt), insects, yeast (e.g., Pichia pastoris, K. phaffii), algae, or
mammalian source. Bacterial
vectors include, for example, pACYC177, pASK75, pBAD vector series, pBADM
vector series,
pET vector series, pETM vector series, pGEX vector series, pHAT, pHAT2, pMal-
c2, pMal-p2,
pQE vector series, pRSET A, pRSET B, pRSET C, pTrcHis2 series, pZA31-Luc,
pZE21-MCS-1,
pFLAG ATS, pFLAG CTS, pFLAG MAC, pFLAG Shift-12c, pTAC-MAT-1, pFLAG CTC, or
pTAC-MAT-2.
[293] Insect vectors include, for example, pFastBacl, pFastBac DUAL,
pFastBac ET, pFastBac
HTa, pFastBac HTb, pFastBac HTc, pFastBac M30a, pFastBact M30b, pFastBac,
M30c, pVL1392,
pVL1393, pVL1393 M10, pVL1393 M11, pVL1393 M12, FLAG vectors such as pPolh-
FLAG1 or
pPolh-MAT 2, or MAT vectors such as pPolh-MAT1, or pPolh-MAT2.
[294] Yeast vectors include, for example, Gateway pDESTTm 14 vector,
Gateway pDESTrm 15
vector, Gateway pDEST- 17 vector, Gateway pDEST- 24 vector, Gateway pYES-
DEST52
vector, pBAD-DEST49 Gateway destination vector, pA0815 Pichia vector, pFLD1
Pichi pastoris
(K phaffii) vector, pGAPZA, B, & C Pichia pastoris (K phaffii) vector,
pPIC3.5K Pichia vector,
pPIC6 A, B, & C Pichia vector, pPIC9K Pichia vector, pTEF1/Zeo, pYES2 yeast
vector,
pYES2/CT yeast vector, pYES2/NT A, B, & C yeast vector, or pYES3/CT yeast
vector.
[295] Algae vectors include, for example, pChlamy-4 vector or MCS vector.
[296] Mammalian vectors include, for example, transient expression vectors
or stable expression
vectors. Exemplary mammalian transient expression vectors include p3xFLAG-CMV
8, pFLAG-
Myc-CMV 19, pFLAG-Myc-CMV 23, pFLAG-CMV 2, pFLAG-CMV 6a,b,c, pFLAG-CMV 5.1,
pFLAG-CMV 5a,b,c, p3xFLAG-CMV 7.1, pFLAG-CMV 20, p3xFLAG-Myc-CMV 24, pCMV-
FLAG-MAT1, pCMV-FLAG-MAT2, pBICEP-CMV 3, or pBICEP-CMV 4. Exemplary
mammalian stable expression vectors include pFLAG-CMV 3, p3xFLAG-CMV 9,
p3xFLAG-CMV
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13, pFLAG-Myc-CMV 21, p3xFLAG-Myc-CMV 25, pFLAG-CMV 4, p3xFLAG-CMV 10,
p3xFLAG-CMV 14, pFLAG-Myc-CMV 22, p3xFLAG-Myc-CMV 26, pBICEP-CMV 1, or
pBICEP-CMV 2.
[297] In some instances, a cell-free system is used for the production of
an IL-2 polypeptide
described herein. In some cases, a cell-free system comprises a mixture of
cytoplasmic and/or
nuclear components from a cell and is suitable for in vitro nucleic acid
synthesis. In some instances,
a cell-free system utilizes prokaryotic cell components. In other instances, a
cell-free system utilizes
eukaryotic cell components. Nucleic acid synthesis is obtained in a cell-free
system based on, for
example, Drosophila cell, Xenopus egg, Archaea, or HeLa cells. Exemplary cell-
free systems
include E. coli S30 Extract system, E. coli T7 S30 system, or PURExpresse,
XpressCF, and
XpressCF+.
[298] Cell-free translation systems variously comprise components such as
plasmids, mRNA,
DNA, tRNAs, synthetases, release factors, ribosomes, chaperone proteins,
translation initiation and
elongation factors, natural and/or unnatural amino acids, and/or other
components used for protein
expression. Such components are optionally modified to improve yields,
increase synthesis rate,
increase protein product fidelity, or incorporate unnatural amino acids. In
some embodiments,
cytokines described herein are synthesized using cell-free translation systems
described in US
8,778,631; US 2017/0283469; US 2018/0051065; US 2014/0315245; or US 8,778,631.
In some
embodiments, cell-free translation systems comprise modified release factors,
or even removal of
one or more release factors from the system. In some embodiments, cell-free
translation systems
comprise a reduced protease concentration. In some embodiments, cell-free
translation systems
comprise modified tRNAs with re-assigned codons used to code for unnatural
amino acids. In some
embodiments, the synthetases described herein for the incorporation of
unnatural amino acids are
used in cell-free translation systems. In some embodiments, tRNAs are pre-
loaded with unnatural
amino acids using enzymatic or chemical methods before being added to a cell-
free translation
system. In some embodiments, components for a cell-free translation system are
obtained from
modified organisms, such as modified bacteria, yeast, or other organism.
[299] In some embodiments, an IL-2 polypeptide is generated as a circularly
permuted form,
either via an expression host system or through a cell-free system.
Production of IL-2 Polypeptide Comprising an Unnatural Amino Acid
[300] An orthogonal or expanded genetic code can be used in the present
disclosure, in which
one or more specific codons present in the nucleic acid sequence of an IL-2
polypeptide are
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allocated to encode the unnatural amino acid so that it can be genetically
incorporated into the IL-2
by using an orthogonal tRNA synthetase/tRNA pair. The orthogonal tRNA
synthetase/tRNA pair is
capable of charging a tRNA with an unnatural amino acid and is capable of
incorporating that
unnatural amino acid into the polypeptide chain in response to the codon.
[301] In some instances, the codon is the codon amber, ochre, opal or a
quadruplet codon. In
some cases, the codon corresponds to the orthogonal tRNA which will be used to
carry the
unnatural amino acid In some cases, the codon is amber. In other cases, the
codon is an orthogonal
codon.
[302] In some instances, the codon is a quadruplet codon, which can be
decoded by an
orthogonal ribosome ribo-Ql. In some cases, the quadnwlet codon is as
illustrated in Neumann, et
al., "Encoding multiple unnatural amino acids via evolution of a quadruplet-
decoding ribosome,"
Nature, 464(7287): 441-444 (2010), the disclosure of which is herein
incorporated by reference.
[303] In some instances, a codon used in the present disclosure is a
recoded codon, e.g., a
synonymous codon or a rare codon that is replaced with alternative codon. In
some cases, the
recoded codon is as described in Napolitano, et al., "Emergent rules for codon
choice elucidated by
editing rare arginine codons in Escherichia coli," PNAS, 113(38): E5588-5597
(2016), the
disclosure of which is herein incorporated by reference. In some cases, the
recoded codon is as
described in Ostrov et al., "Design, synthesis, and testing toward a 57-codon
genome," Science
353(6301): 819-822 (2016), the disclosure of which is herein incorporated by
reference.
[304] In some instances, unnatural nucleic acids are utilized leading to
incorporation of one or
more unnatural amino acids into the IL-2 Exemplary unnatural nucleic acids
include, but are not
limited to, uracil-5-yl, hypoxanthin-9-y1 (I), 2-aminoadenin-9-yl, 5-
methylcytosine (5-me-C), 5-
hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and
other alkyl
derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of
adenine and guanine, 2-
thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-
propynyl uracil and
cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-
thiouracil, 8-halo, 8-amino,
8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and
guanines, 5-halo particularly 5-
bromo, 5-trifiuoromethyl and other 5-substituted uracils and cytosines, 7-
methylguanine and 7-
methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-
deazaadenine and 3-
deazaguanine and 3-deazaadenine. Certain unnatural nucleic acids, such as 5-
substituted
pyrimidines, 6-azapyrimidines and N-2 substituted purines, N-6 substituted
purines, 0-6 substituted
purines, 2-aminopropyladenine, 5-propynyluracil, 5-propynylcytosine, 5-
methylcytosine, those that
increase the stability of duplex formation, universal nucleic acids,
hydrophobic nucleic acids,
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promiscuous nucleic acids, size-expanded nucleic acids, fluorinated nucleic
acids, 5-substituted
pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines,
including 2-
aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine
(5-me-C), 5-
hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl,
other alkyl derivatives
of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and
guanine, 2-thiouracil, 2-
thiothymine and 2-thiocytosine, 5-halouracil, 5-halocytosine, 5-propynyl (-CC-
CH3) uracil, 5-
propynyl cytosine, other alkynyl derivatives of pyrimidine nucleic acids, 6-
azo uracil, 6-azo
cytosine, 6-azo thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-
amino, 8-thiol, 8-thioalkyl,
8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly
5-bromo, 5-
trifluoromethyl, other 5-substituted uracils and cytosines, 7-methylguanine, 7-
methyladenine, 2-F-
adenine, 2-amino-adenine, 8-azaguanine, 8-azaadenine, 7-deazaguanine, 7-
deazaadenine, 3-
deazaguanine, 3-deazaadenine, tricyclic pyrimidines, phenoxazine cytidine(
[5,4-b][1,4]benzoxazin-
2(3H)-one), phenothiazine cytidine (1H- pyrimido[5,4-b][1,4]benzothiazin-2(3H)-
one), G-clamps,
phenoxazine cytidine (e.g. 9- (2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-
2(3H)-one),
carbazole cytidine (2H-pyrimido[4,5- b]indo1-2-one), pyridoindole cytidine (H-
pyrido[3',2':4,5]pyrrolo[2,3-d]pyrimidin-2-one), those in which the purine or
pyrimidine base is
replaced with other heterocycles, 7-deaza-adenine, 7-deazaguanosine, 2-
aminopyridine, 2-pyridone,
, azacytosine, 5-bromocytosine, bromouracil, 5-chlorocytosine, chlorinated
cytosine, cyclocytosine,
cytosine arabinoside, 5-fluorocytosine, fluoropyrimidine, fluorouracil, 5,6-
dihydrocytosine, 5-
iodocytosine, hydroxyurea, iodouracil, 5-nitrocytosine, 5- bromouracil, 5-
chlorouracil, 5-
fluorouracil, and 5-iodouracil, 2-amino-adenine, 6-thio-guanine, 2-thio-
thymine, 4-thio-thymine, 5-
propynyl-uracil, 4-thio-uracil, N4-ethylcytosine, 7-deazaguanine, 7-deaza-8-
azaguanine, 5-
hydroxycytosine, 2'-deoxyuridine, 2-amino-2'-deoxyadenosine, and those
described in U.S. Patent
Nos. 3,687,808; 4,845,205; 4,910,300; 4,948,882; 5,093,232; 5,130,302;
5,134,066; 5,175,273;
5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711;
5,552,540;
5,587,469; 5,594,121; 5,596,091; 5,614,617; 5,645,985; 5,681,941; 5,750,692;
5,763,588;
5,830,653 and 6,005,096; WO 99/62923; Kandimalla et al., (2001) Bioorg. Med.
Chem. 9:807-813;
The Concise Encyclopedia of Polymer Science and Engineering, Kroschwitz, J.I.,
Ed., John Wiley
& Sons, 1990, 858- 859; Englisch et al , Angewandte Chemie, International
Edition, 1991, 30, 613;
and Sanghvi, Chapter 15, Antisense Research and Applications, Crooke and
Lebleu Eds., CRC
Press, 1993, 273-288. Additional base modifications can be found, for example,
in U.S. Pat. No.
3,687,808; Englisch et al., Angewandte Chemie, International Edition, 1991,
30, 613; and Sanghvi,
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Chapter 15, Antisense Research and Applications, pages 289-302, Crooke and
Lebleu ed., CRC
Press, 1993; the disclosure of each of which is herein incorporated by
reference.
[305] Unnatural nucleic acids comprising various heterocyclic bases and
various sugar moieties
(and sugar analogs) are available in the art, and the nucleic acids in some
cases include one or
several heterocyclic bases other than the principal five base components of
naturally-occurring
nucleic acids. For example, the heterocyclic base includes, in some cases,
uracil-5-yl, cytosin-5-yl,
adenin-7-yl, adenin-8-yl, guanin-7-yl, guanin-8-yl, 4- aminopyrrolo [2.3-di
pyrimidin-5-yl, 2-
amino-4-oxopyrolo [2, 3-dl pyrimidin-5-yl, 2- amino-4-oxopyrrolo pyrimidin-
3-y1 groups,
where the purines are attached to the sugar moiety of the nucleic acid via the
9-position, the
pyrimidines via the 1 -position, the pyrrolopyrimidines via the 7-position and
the
pyrazolopyrimidines via the 1-position.
[306] In some embodiments, nucleotide analogs are also modified at the
phosphate moiety.
Modified phosphate moieties include, but are not limited to, those with
modification at the linkage
between two nucleotides and contains, for example, a phosphorothioate, chiral
phosphorothioate,
phosphorodithioate, phosphotriester, aminoalkylphosphotriester, methyl and
other alkyl
phosphonates including 3'-alkylene phosphonate and chiral phosphonates,
phosphinates,
phosphoramidates including 3'-amino phosphoramidate and
aminoalkylphosphoramidates,
thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters,
and
boranophosphates. It is understood that these phosphate or modified phosphate
linkage between two
nucleotides are through a 3'-5' linkage or a 2'-5' linkage, and the linkage
contains inverted polarity
such as 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts and free
acid forms are also
included. Numerous United States patents teach how to make and use nucleotides
containing
modified phosphates and include but are not limited to, 3,687,808; 4,469,863;
4,476,301; 5,023,243;
5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;
5,399,676;
5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821;
5,541,306;
5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050; the disclosure of
each of which is
herein incorporated by reference.
[307] In some embodiments, unnatural nucleic acids include 2',3'-dideoxy-
2',3'-didehydro-
nucleosides (PCT/11S2002/006460), 5'-substituted DNA and RNA derivatives
(PCT/US2011/033961; Saha et al., J. Org Chem., 1995, 60, 788-789; Wang et al.,
Bioorganic &
Medicinal Chemistry Letters, 1999, 9, 885-890; Mikhailov et al., Nucleosides &
Nucleotides, 1991,
10(1-3), 339-343; Leonid et al., 1995, 14(3-5), 901-905; Eppacher et al.,
Helvetica Chimica Acta,
2004, 87, 3004-3020; PCT/JP2000/004720; PCT/JP2003/002342; PCT/JP2004/013216;
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PCT/JP2005/020435; PCT/JP2006/315479; PCT/JP2006/324484; PCT/JP2009/056718;
PCT/JP2010/067560), or 5'-substituted monomers made as the monophosphate with
modified bases
(Wang et al., Nucleosides Nucleotides & Nucleic Acids, 2004, 23 (1 & 2), 317-
337); the disclosure
of each of which is herein incorporated by reference.
[308] In some embodiments, unnatural nucleic acids include
modifications at the 5'-position and
the 2'-position of the sugar ring (PCT/US94/02993), such as 5'-C112-
substituted 2'-0-protected
nucleosides (Wu et al., Helvetica Chimica Acta, 2000, 83, 1127-1143 and Wu et
al., Bioconjugate
Chem. 1999, 10, 921-924, the disclosure of which is herein incorporated by
reference). In some
cases, unnatural nucleic acids include amide linked nucleoside dimers have
been prepared for
incorporation into oligonucleotides wherein the 3' linked nucleoside in the
dimer (5' to 3')
comprises a 2'-OCH3 and a 5'-(S)-CH3 (Mesmaeker et al., Synlett, 1997, 1287-
1290). Unnatural
nucleic acids can include 2'-substituted 5'-CH2 (or 0) modified nucleosides
(PCT/US92/01020).
Unnatural nucleic acids can include 5'-methylenephosphonate DNA and RNA
monomers, and
dimers (Bohringer et al., Tet. Lett., 1993, 34, 2723-2726; Collingwood et al.,
Synlett, 1995, 7, 703-
705; and Hutter et al., Helvetica Chimica Acta, 2002, 85, 2777-2806).
Unnatural nucleic acids can
include 5'-phosphonate monomers having a 2'-substitution (US2006/0074035) and
other modified
5'-phosphonate monomers (W01997/35869). Unnatural nucleic acids can include 5'-
modified
methylenephosphonate monomers (EP614907 and EP629633). Unnatural nucleic acids
can include
analogs of 5' or 6'-phosphonate ribonucleosides comprising a hydroxyl group at
the 5' and/or 6'-
position (Chen et al., Phosphorus, Sulfur and Silicon, 2002, 777, 1783-1786;
Jung et al., Bioorg.
Med. Chem., 2000, 8, 2501-2509; Gallier et al., Eur. J. Org. Chem., 2007, 925-
933; and Hampton et
al., J. Med. Chem., 1976, 19(8), 1029-1033). Unnatural nucleic acids can
include 5'-phosphonate
deoxyribonucleoside monomers and dimers having a 5'-phosphate group (Nawrot et
al.,
Oligonucleotides, 2006, 16(1), 68-82). Unnatural nucleic acids can include
nucleosides having a 6'-
phosphonate group wherein the 5' or/and 6'-position is unsubstituted or
substituted with a thio-tert-
butyl group (SC(CH3)3) (and analogs thereof); a methyleneamino group (CH2NH2)
(and analogs
thereof) or a cyano group (CN) (and analogs thereof) (Fairhurst et al.,
Synlett, 2001, 4, 467-472;
Kappler et al., J. Med. Chem., 1986, 29, 1030-1038; Kappler et al., J. Med.
Chem., 1982, 25, 1179-
1184; Vrudhula et al., J. Med. Chem., 1987, 30, 888-894; Hampton et al., J
Med. Chem., 1976, 19,
1371-1377; Geze et al., J. Am. Chem. Soc, 1983, 105(26), 7638-7640; and
Hampton et al., J. Am.
Chem. Soc, 1973, 95(13), 4404-4414). The disclosure of each reference listed
in this paragraph is
herein incorporated by reference.
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[309] In some embodiments, unnatural nucleic acids also include
modifications of the sugar
moiety. In some cases, nucleic acids contain one or more nucleosides wherein
the sugar group has
been modified. Such sugar modified nucleosides may impart enhanced nuclease
stability, increased
binding affinity, or some other beneficial biological property. In certain
embodiments, nucleic acids
comprise a chemically modified ribofuranose ring moiety. Examples of
chemically modified
ribofuranose rings include, without limitation, addition of substituent groups
(including 5' and/or 2'
substituent groups; bridging of two ring atoms to form bicyclic nucleic acids
(BNA); replacement of
the ribosyl ring oxygen atom with S, N(R), or C(R1)(R2) (R = H, Ci-C12 alkyl
or a protecting
group); and combinations thereof Examples of chemically modified sugars can be
found in
W02008/101157, US2005/0130923, and W02007/134181, the disclosure of each of
which is
herein incorporated by reference.
[310] In some instances, a modified nucleic acid comprises modified sugars
or sugar analogs.
Thus, in addition to ribose and deoxyribose, the sugar moiety can be pentose,
deoxypentose, hexose,
deoxyhexose, glucose, arabinose, xylose, lyxose, or a sugar "analog"
cyclopentyl group. The sugar
can be in a pyranosyl or furanosyl form. The sugar moiety may be the
furanoside of ribose,
deoxyribose, arabinose or 2'-0-alkylribose, and the sugar can be attached to
the respective
heterocyclic bases either in [alpha] or [beta] anomeric configuration. Sugar
modifications include,
but are not limited to, 2'-alkoxy-RNA analogs, 2'-amino-RNA analogs, 2'-fluoro-
DNA, and 2'-
alkoxy- or amino-RNA/DNA chimeras. For example, a sugar modification may
include 2'-0-
methyl-uridine or 2'-0-methyl-cytidine. Sugar modifications include 2'-0-alkyl-
substituted
deoxyribonucleosides and 2'-0-ethyleneglycol like ribonucleosides. The
preparation of these sugars
or sugar analogs and the respective "nucleosides" wherein such sugars or
analogs are attached to a
heterocyclic base (nucleic acid base) is known. Sugar modifications may also
be made and
combined with other modifications.
[311] Modifications to the sugar moiety include natural modifications of
the ribose and deoxy
ribose as well as unnatural modifications. Sugar modifications include, but
are not limited to, the
following modifications at the 2' position: OH; F; 0-, S-, or N-alkyl; 0-, S-,
or N-alkenyl; 0-, S- or
N-alkynyl; or 0-alkyl-0-alkyl, wherein the alkyl, alkenyl and alkynyl may be
substituted or
unsubstituted CI to Cm, alkyl or C2 to C 10 alkenyl and alkynyl 2' sugar
modifications also include
but are not limited to -0[(CH2)nO]m CH3, -0(CH2)nOCH3, -0(CH2)nNH2, -
0(CH2),CH3, -
0(CH2)nONH2, and -0(CH2)11ONRCH2)n CH3)]2, where n and m are from I to about
10.
[312] Other modifications at the 2' position include but are not limited
to: CI_ to Cm lower alkyl,
substituted lower alkyl, alkaryl, aralkyl, 0-alkaryl, 0-aralkyl, SH, SCH3,
OCN, Cl, Br, CN, CF3,
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OCF3, SOCH3, SO2 CH3, 0NO2, NO2, N3, NH2, heterocycloalkyl,
heterocycloalkaryl,
aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a
reporter group, an
intercalator, a group for improving the pharmacokinetic properties of an
oligonucleotide, or a group
for improving the pharmacodynamic properties of an oligonucleotide, and other
sub stituents having
similar properties. Similar modifications may also be made at other positions
on the sugar,
particularly the 3' position of the sugar on the 3' terminal nucleotide or in
2'-5' linked
oligonucleotides and the 5' position of the 5' terminal nucleotide. Modified
sugars also include
those that contain modifications at the bridging ring oxygen, such as CH2 and
S. Nucleotide sugar
analogs may also have sugar mimetics such as cyclobutyl moieties in place of
the pentofuranosyl
sugar. There are numerous United States patents that teach the preparation of
such modified sugar
structures and which detail and describe a range of base modifications, such
as U.S. Patent Nos.
4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786;
5,514,785;
5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053;
5,639,873;
5,646,265; 5,658,873; 5,670,633; 4,845,205; 5,130,302; 5,134,066; 5,175,273;
5,367,066;
5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711, 5,552,540;
5,587,469,
5,594,121, 5,596,091; 5,614,617; 5,681,941; and 5,700,920, the disclosure of
each of which is
herein incorporated by reference in its entirety.
[313] Examples of nucleic acids having modified sugar moieties include,
without limitation,
nucleic acids comprising 5'-vinyl, 5'-methyl (R or S), 4'-S, 2'-F, 2'-OCH3,
and 2'-0(CH2)20CH3
sub stituent groups. The sub stituent at the 2' position can also be selected
from allyl, amino, azido,
thio, 0-allyl, 0-(Ci-Cio alkyl), OCF3, 0(CH2)2SCH3, 0(CH2)2-0-N(Rin)(Rn), and
0-C112-C(=0)-
N(Rm)(Rn), where each Rm and Rn is, independently, H or substituted or
unsubstituted Ci-Cio alkyl.
[314] In certain embodiments, nucleic acids described herein include one or
more bicyclic
nucleic acids. In certain such embodiments, the bicyclic nucleic acid
comprises a bridge between
the 4' and the 2' ribosyl ring atoms. In certain embodiments, nucleic acids
provided herein include
one or more bicyclic nucleic acids wherein the bridge comprises a 4' to 2'
bicyclic nucleic acid.
Examples of such 4' to 2' bicyclic nucleic acids include, but are not limited
to, one of the Formulae:
4'-(CH2)-0-2' (LNA); 4'-(CH2)-S-2'; 4'-(CH2)2-0-2' (ENA); 4'-CH(CH3)-0-2' and
4'-
CH(CH2OCH3)-0-2', and analogs thereof (see, U.S. Patent No. 7,399,845); 4'-
C(CH3)(CH3)-0-
2' and analogs thereof, (see W02009/006478, W02008/150729, US2004/0171570,
U.S. Patent No.
7,427,672, Chattopadhyaya et al., J. Org. Chem., 209, 74, 118-134, and
W02008/154401). Also
see, for example: Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et
al., Tetrahedron,
1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl. Acad, Sci. U. S. A., 2000,
97, 5633-5638;
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Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Singh et al., J.
Org. Chem., 1998, 63,
10035-10039; Srivastava et al., J. Am. Chem. Soc., 2007, 129(26) 8362-8379;
Elayadi et al., Curr.
Opinion Invens. Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol, 2001, 8,
1-7; Oram et al.,
Curr. Opinion Mol. Ther., 2001, 3, 239-243; U.S. Patent Nos. 4,849,513;
5,015,733; 5,118,800;
5,118,802; 7,053,207; 6,268,490; 6,770,748; 6,794,499; 7,034,133, 6,525,191;
6,670,461, and
7,399,845; International Publication Nos. W02004/106356, W01994/14226,
W02005/021570,
W02007/090071, and W02007/134181; U.S. Patent Publication Nos. U
S2004/0171570,
US2007/0287831, and US2008/0039618; U.S. Provisional Application Nos.
60/989,574,
61/026,995, 61/026,998, 61/056,564, 61/086,231, 61/097,787, and 61/099,844;
and International
Applications Nos PCT/US2008/064591, PCT US2008/066154, PCT US2008/068922, and
PCT/DK98/00393. The disclosure of each reference listed in this paragraph is
herein incorporated
by reference.
[315] In certain embodiments, nucleic acids comprise linked nucleic acids.
Nucleic acids can be
linked together using any inter nucleic acid linkage. The two main classes of
inter nucleic acid
linking groups are defined by the presence or absence of a phosphorus atom.
Representative
phosphorus containing inter nucleic acid linkages include, but are not limited
to, phosphodiesters,
phosphotriesters, methylphosphonates, phosphoramidate, and phosphorothioates
(P=S).
Representative non-phosphorus containing inter nucleic acid linking groups
include, but are not
limited to, methylenemethylimino (-CH2-N(CH3)-0-CH2-), thiodiester (-0-C(0)-S-
),
thionocarbamate (-0-C(0)(NH)-5-); siloxane (-0-Si(H)2-0-); and N,N*-
dimethylhydrazine (-CH2-
N(CH3)-N(CH3)). In certain embodiments, inter nucleic acids linkages having a
chiral atom can be
prepared as a racemic mixture, as separate enantiomers, e.g, alkylphosphonates
and
phosphorothioates. Unnatural nucleic acids can contain a single modification.
Unnatural nucleic
acids can contain multiple modifications within one of the moieties or between
different moieties.
[316] Backbone phosphate modifications to nucleic acid include, but are not
limited to, methyl
phosphonate, phosphorothioate, phosphoramidate (bridging or non-bridging),
phosphotriester,
phosphorodithioate, phosphodithioate, and boranophosphate, and may be used in
any combination.
Other non- phosphate linkages may also be used.
[317] In some embodiments, backbone modifications (e.g., methylphosphonate,
phosphorothioate, phosphoroamidate and phosphorodithioate internucleotide
linkages) can confer
immunomodulatory activity on the modified nucleic acid and/or enhance their
stability in vivo.
[318] In some instances, a phosphorous derivative (or modified phosphate
group) is attached to
the sugar or sugar analog moiety in and can be a monophosphate, diphosphate,
triphosphate,
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alkylphosphonate, phosphorothioate, phosphorodithioate, phosphoramidate or the
like. Exemplary
polynucleotides containing modified phosphate linkages or non-phosphate
linkages can be found in
Peyrottes et al., 1996, Nucleic Acids Res. 24: 1841-1848; Chaturvedi et al.,
1996, Nucleic Acids
Res. 24:2318-2323; Schultz et al., (1996) Nucleic Acids Res. 24:2966-2973;
Matteucci, 1997,
"Oligonucleotide Analogs: an Overview" in Oligonucleotides as Therapeutic
Agents, (Chadwick
and Cardew, ed.) John Wiley and Sons, New York, NY; Zon, 1993,
"Oligonucleoside
Phosphorothioates" in Protocols for Oligonucleotides and Analogs, Synthesis
and Properties,
Humana Press, pp. 165-190; Miller et al., 1971, JACS 93:6657-6665; Jager et
al., 1988, Biochem.
27:7247-7246; Nelson et al., 1997, JOC 62:7278-7287; U.S. Patent No.
5,453,496; and Micklefield,
2001, Curr. Med. Chem. 8: 1157-1179; the disclosure of each of which is herein
incorporated by
reference.
[319] In some cases, backbone modification comprises replacing the
phosphodiester linkage
with an alternative moiety such as an anionic, neutral or cationic group.
Examples of such
modifications include: anionic internucleoside linkage; N3' to P5'
phosphoramidate modification;
boranophosphate DNA; prooligonucleotides; neutral internucleoside linkages
such as
methylphosphonates; amide linked DNA; methylene(methylimino) linkages;
formacetal and
thioformacetal linkages; backbones containing sulfonyl groups; morpholino
oligos; peptide nucleic
acids (PNA); and positively charged deoxyribonucleic guanidine (DNG) oligos
(Micklefield, 2001,
Current Medicinal Chemistry 8: 1157-1179). A modified nucleic acid may
comprise a chimeric or
mixed backbone comprising one or more modifications, e.g. a combination of
phosphate linkages
such as a combination of phosphodiester and phosphorothioate linkages.
[320] Substitutes for the phosphate include, for example, short chain alkyl
or cycloalkyl
internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl
intemucleoside linkages, or one
or more short chain heteroatomic or heterocyclic internucleoside linkages.
These include those
having morpholino linkages (formed in part from the sugar portion of a
nucleoside); siloxane
backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and
thioformacetyl backbones;
methylene formacetyl and thioformacetyl backbones; alkene containing
backbones; sulfamate
backbones; methyleneimino and methylenehydrazino backbones; sulfonate and
sulfonamide
backbones; amide backbones; and others having mixed N, 0, S and CH2 component
parts.
Numerous United States patents disclose how to make and use these types of
phosphate
replacements and include but are not limited to U.S. Patent Nos. 5,034,506;
5,166,315; 5,185,444;
5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257;
5,466,677;
5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289;
5,602,240;
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5,608,046; 5,6.10,289; 5,6.18,704; 5,623,070; 5,663,3.12; 5,633,360;
5,677,437; and 5,677,439. It is
also understood in a nucleotide substitute that both the sugar and the
phosphate moieties of the
nucleotide can be replaced, by for example an amide type linkage
(aminoethylglycine) (PNA).
United States Patent Nos. 5,539,082; 5,714,331; and 5,719,262 teach how to
make and use PNA
molecules, each of which is herein incorporated by reference. See also Nielsen
et al., Science, 1991,
254, 1497-1500. It is also possible to link other types of molecules
(conjugates) to nucleotides or
nucleotide analogs to enhance for example, cellular uptake. Conjugates can be
chemically linked to
the nucleotide or nucleotide analogs. Such conjugates include but are not
limited to lipid moieties
such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA,
1989, 86, 6553-6556),
cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a
thioether, e.g.,
hexyl-S-tritylthiol (Manoharan et al., Ann. KY. Acad. Sci., 1992, 660, 306-
309; Manoharan et al.,
Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et
al., Nucl. Acids
Res., 1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or undecyl
residues (Saison-
Behmoaras et al., EM50J, 1991, 10, 1111-1118; Kabanov et al., FEBS Lett.,
1990, 259, 327-330;
Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g., di-
hexadecyl-rac-glycerol or
triethylammoniuml-di-O-hexadecyl-rac-glycero-S-H-phosphonate (Manoharan et
al., Tetrahedron
Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-
3783), a polyamine or a
polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995,
14, 969-973), or
adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-
3654), a palmityl
moiety (Mishra et al., Biochem. Biophys. Acta, 1995, 1264, 229-237), or an
octadecylamine or
hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp.
Ther., 1996, 277,
923-937). Numerous United States patents teach the preparation of such
conjugates and include, but
are not limited to U.S. Patent Nos. 4,828,979; 4,948,882; 5,218,105;
5,525,465; 5,541,313;
5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584; 5,109,124;
5,118,802;
5,138,045; 5,414,077; 5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044;
4,605,735;
4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582;
4,958,013;
5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136; 5,245,022;
5,254,469;
5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723;
5,416,203,
5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142;
5,585,481;
5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941. The
disclosure of each
reference listed in this paragraph is herein incorporated by reference.
1321] In some cases, the unnatural nucleic acids further form
unnatural base pairs. Exemplary
unnatural nucleotides capable of forming an unnatural DNA or RNA base pair
(UBP) under
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conditions in vivo includes, but is not limited to, TAT I, dTAT1, 5FM, d5FM,
TPT3, dTPT3,
5SICS, d5SICS, NaM, dNaM, CNMO, dCNMO, and combinations thereof. In some
embodiments,
unnatural nucleotides include:
CN
N S N S 111111 C) N S
C)
-r-
(d)TAT1 , (d)TPT3 , (d)NaM , (d)5FM
, (d)5SICS , and (d)CNMO
Exemplary unnatural base pairs include: (d)TPT3-(d)NaM; (d)5SICS-(d)NaM;
(d)CNMO-(d)TAT1;
(d)NaM-(d)TAT1; (d)CNMO-(d)TPT3; and (d)5FM-(d)TAT1.
13221 Other examples of unnatural nucleotides capable of forming
unnatural UBPs that may be
used to prepare the IL-2 conjugates disclosed herein may be found in Dien et
al., J Am Chem Soc.,
2018, 140:16115-16123; Feldman et al., J Am Chem Soc, 2017, 139:11427-11433;
Ledbetter et al.,
J Am Chem Soc., 2018, 140:758-765; Dhami et al., Nucleic Acids Res. 2014,
42:10235-10244;
Malyshev et al., Nature, 2014, 509:385-388; Betz et al., J Am Chem Soc., 2013,
135:18637-18643;
Lavergne et al., J Am Chem Soc. 2013, 135:5408-5419; and Malyshev et al. Proc
Natl Acad Sci
USA, 2012, 109:12005-12010; the disclosure of each of which is herein
incorporated by reference.
In some embodiments, unnatural nucleotides include:
,novv. 01
01 I
N s
0 i
d5SICS dNAM 01
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.AAA,
o oI
o
0
5SICS NAM OH 0.;,53s
0 0H
[323] In some embodiments, the unnatural nucleotides that may be used to
prepare the IL-2
conjugates disclosed herein may be derived from a compound of the Formula
R2
NS
wherein R2 is selected from hydrogen, alkyl, alkenyl, alkynyl, methoxy,
methanethiol,
methaneseleno, halogen, cyano, and azido; and
the wavy line indicates a bond to a ribosyl or 2'-deoxyribosyl, wherein the 5'-
hydroxy
group of the ribosyl or 2'-deoxyribosyl moiety is in free form, is connected
to a monophosphate,
diphosphate, triphosphate, a-thiotriphosphate, 13-thiotriphosphate, or y-
thiotriphosphate group, or is
included in an RNA or a DNA or in an RNA analog or a DNA analog
[324] In some embodiments, the unnatural nucleotides that may be used to
prepare the IL-2
conjugates disclosed herein may be derived from a compound of the Formula
F2
N=-X
R2.
X-
R2 X, N
-4-
wherein.
each X is independently carbon or nitrogen;
R2 is absent when X is nitrogen, and is present when X is carbon and is
independently
hydrogen, alkyl, alkenyl, alkynyl, methoxy, methanethiol, methaneseleno,
halogen, cyano, or azide;
Y is sulfur, oxygen, selenium, or secondary amine;
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E is oxygen, sulfur, or selenium; and
the wavy line indicates a point of bonding to a ribosyl, deoxyribosyl, or
dideoxyribosyl
moiety or an analog thereof, wherein the ribosyl, deoxyribosyl, or
dideoxyribosyl moiety or analog
thereof is in free form, is connected to a mono-phosphate, diphosphate,
triphosphate, ct-
thiotriphosphate, II-thiotriphosphate, or 7-thiotriphosphate group, or is
included in an RNA or a
DNA or in an RNA analog or a DNA analog.
[325] In some embodiments, each X is carbon. In some embodiments, at least
one X is carbon.
In some embodiments, one X is carbon. In some embodiments, at least two X are
carbon. In some
embodiments, two X are carbon. In some embodiments, at least one X is
nitrogen. In some
embodiments, one X is nitrogen. In some embodiments, at least two X are
nitrogen. In some
embodiments, two X are nitrogen.
[326] In some embodiments, Y is sulfur. In some embodiments, Y is oxygen.
In some
embodiments, Y is selenium. In some embodiments, Y is a secondary amine.
[327] In some embodiments, E is sulfur. In some embodiments, E is oxygen.
In some
embodiments, E is selenium.
[328] In some embodiments, R2 is present when X is carbon. In some
embodiments, R2 is absent
when X is nitrogen. In some embodiments, each R2, where present, is hydrogen.
In some
embodiments, R2 is alkyl, such as methyl, ethyl, or propyl. In some
embodiments, R2 is alkenyl,
such as -CH2=CH2. In some embodiments, R2 is alkynyl, such as ethynyl. In some
embodiments, R2
is methoxy. In some embodiments, R2 is methanethiol. In some embodiments, R2
is methaneseleno.
In some embodiments, R2 is halogen, such as chloro, bromo, or fluoro. In some
embodiments, R2 is
cyano. In some embodiments, R2 is azide.
[329] In some embodiments, E is sulfur, Y is sulfur, and each X is
independently carbon or
nitrogen. In some embodiments, E is sulfur, Y is sulfur, and each X is carbon.
[330] In some embodiments, the unnatural nucleotides that may be used to
prepare the IL-2
(IL
two
..H,
OCH3
HO HO
0 0
conjugates disclosed herein may be derived from OH OH OH
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CH3 CH3
N.------\- N-------:\
1 1 ,µ,./, S Aµi S
N S N S I I
HO HO HO ,, N-NS HO
0
OH OH OH OH OH OH
,
CN CN
l
F 40 F
HO 0 HO u HO 0 HO
0 0 0 o0
OH OH OH OH OH OH
i ,
; 8 ,i,
..., .-.. ,---, . ......
11 i
=,, .--4..,., ...-;,.,
'N' 'S 'N' 'S
HO,µ HO. i
,
:
01-1 6H and
[331]
In some embodiments, the unnatural nucleotides that may be used to prepare
the IL-2
ilt
igr
0 0 0 OCH3
HO-P-O-P-O-P-0 0
OH OH 01-I
conjugates disclosed herein include OH OH ,
CH3
411.6
41, 1
0 0 0 0cH3 0 0 0 N S
II II II il II II
HO-PI-0-PI-0-PI-0 0 HOPOPOPO
OH OH OH OH OH OH (7)
OH OH
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CH3
Nr---\
1 S
9 9 9 N S 0 0 0
I I I I I 1 (L/
N S
HO-P-O-P-O-P-0- HO P OP OPO
OH OH OH I I I 0
OH OH OH
OH OH , OH OH ,
N---:"\-
-)s'=-:-/, S F
O 0 0 I 0 0 0
II I 1 II II II II 0 cp.,'
HO-P-O-P-O-P-0- I\IS HO-P-O-P-0-P-0
I I 1 0 I I 1 0
OH OH OH OH OH OH
OH OH OH
CN
F
O 0 0 0 0 0
I I II II IIP (:).." I I I I I I ..-
HO-P-O-P-O-P-0 HO-P-O-P-O-P-0 0
I I 1 0 1 I 1 0
OH OH OH OH OH OH
OH OH OH
7 ,
CN
R
O 0 0
II II II ... 0 0 0 N S
HO-P-O-P-O-P-0 o II ii ii
HO-P-O-P-O-P-0-
I I 1 0
OH OH OH OH OH OH
OH OH OH and
,
Zs
1 ,
0 0 0
II II II
HOPOPOPO
()
I _______________________
OH OH OH
OH , or salts thereof
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[332] In some embodiments, the unnatural nucleotides that may be used to
prepare the IL-2
N=\
is
is
HO HO
(/)
conjugates disclosed herein may be derived from OH OH and OH
[333] In some embodiments, the unnatural nucleotides that may be used to
prepare the IL-2
0 0 0
11 11 11
HO¨P-0 ¨P¨O¨P-0-
1 1 1
OH OH OH
conjugates disclosed herein include OH OH and
\
0 0 0
11 11 11
HO ¨P¨O¨P¨O¨P-0 ¨ N S
1 1
OH OH OH
OH
[334] In some embodiments, an unnatural base pair generates an unnatural
amino acid as
described in Dumas et al., "Designing logical codon reassignment ¨ Expanding
the chemistry in
biology," Chemical Science, 6: 50-69 (2015).
[335] In some embodiments, the unnatural amino acid is incorporated into
the IL-2 polypeptide
by a synthetic codon comprising an unnatural nucleic acid. In some instances,
the unnatural amino
acid is incorporated into the IL-2 by an orthogonal, modified synthetase/tRNA
pair. Such
orthogonal pairs comprise an unnatural synthetase that is capable of charging
the unnatural tRNA
with the unnatural amino acid, while minimizing charging of a) other
endogenous amino acids onto
the unnatural tRNA and b) unnatural amino acids onto other endogenous tRNAs.
Such orthogonal
pairs comprise tRNAs that are capable of being charged by the unnatural
synthetase, while avoiding
being charged with a) other endogenous amino acids by endogenous synthetases.
In some
embodiments, such pairs are identified from various organisms, such as
bacteria, yeast, Archaea, or
human sources. In some embodiments, an orthogonal synthetase/tRNA pair
comprises components
from a single organism. In some embodiments, an orthogonal synthetase/tRNA
pair comprises
components from two different organisms. In some embodiments, an orthogonal
synthetase/tRNA
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pair comprising components that prior to modification, promote translation of
two different amino
acids. In some embodiments, an orthogonal synthetase is a modified alanine
synthetase. In some
embodiments, an orthogonal synthetase is a modified arginine synthetase. In
some embodiments, an
orthogonal synthetase is a modified asparagine synthetase. In some
embodiments, an orthogonal
synthetase is a modified aspartic acid synthetase. In some embodiments, an
orthogonal synthetase is
a modified cysteine synthetase. In some embodiments, an orthogonal synthetase
is a modified
glutamine synthetase. In some embodiments, an orthogonal synthetase is a
modified glutamic acid
synthetase. In some embodiments, an orthogonal synthetase is a modified
alanine glycine. In some
embodiments, an orthogonal synthetase is a modified histidine synthetase. In
some embodiments, an
orthogonal synthetase is a modified leucine synthetase. In some embodiments,
an orthogonal
synthetase is a modified isoleucine synthetase. In some embodiments, an
orthogonal synthetase is a
modified lysine synthetase. In some embodiments, an orthogonal synthetase is a
modified
methionine synthetase. In some embodiments, an orthogonal synthetase is a
modified phenylalanine
synthetase. In some embodiments, an orthogonal synthetase is a modified
proline synthetase. In
some embodiments, an orthogonal synthetase is a modified scrim synthetase. In
some
embodiments, an orthogonal synthetase is a modified threonine synthetase. In
some embodiments,
an orthogonal synthetase is a modified tryptophan synthetase. In some
embodiments, an orthogonal
synthetase is a modified tyrosine synthetase. In some embodiments, an
orthogonal synthetase is a
modified valine synthetase. In some embodiments, an orthogonal synthetase is a
modified
phosphoserine synthetase. In some embodiments, an orthogonal tRNA is a
modified alanine tRNA.
In some embodiments, an orthogonal tRNA is a modified arginine tRNA. In some
embodiments, an
orthogonal tRNA is a modified asparagine tRNA. In some embodiments, an
orthogonal tRNA is a
modified aspartic acid tRNA. In some embodiments, an orthogonal tRNA is a
modified cysteine
tRNA. In some embodiments, an orthogonal tRNA is a modified glutamine tRNA. In
some
embodiments, an orthogonal tRNA is a modified glutamic acid tRNA. In some
embodiments, an
orthogonal tRNA is a modified alanine glycine. In some embodiments, an
orthogonal tRNA is a
modified histidine tRNA. In some embodiments, an orthogonal tRNA is a modified
leucine tRNA.
In some embodiments, an orthogonal tRNA is a modified isoleucine tRNA. In some
embodiments,
an orthogonal tRNA is a modified lysine tRNA. In some embodiments, an
orthogonal tRNA is a
modified methionine tRNA. In some embodiments, an orthogonal tRNA is a
modified
phenylalanine tRNA. In some embodiments, an orthogonal tRNA is a modified
proline tRNA. In
some embodiments, an orthogonal tRNA is a modified serine tRNA. In some
embodiments, an
orthogonal tRNA is a modified threonine tRNA. In some embodiments, an
orthogonal tRNA is a
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modified tryptophan tRNA. In some embodiments, an orthogonal tRNA is a
modified tyrosine
tRNA. In some embodiments, an orthogonal tRNA is a modified valine tRNA. In
some
embodiments, an orthogonal tRNA is a modified phosphoserine tRNA.
[336] In some embodiments, the unnatural amino acid is incorporated into
the IL-2 polypeptide
by an aminoacyl (aaRS or RS)-tRNA synthetase-tRNA pair. Exemplary aaRS-tRNA
pairs include,
but are not limited to, Methanococcus jannaschii (Mj-Tyr) aaRS/tRNA pairs, E.
colt TyrRS (Ec-
Tyr)1 11. stearothermophihts tRNAcuA pairs, E. coil LeuRS (Ec-Leu)IB.
stearothermophilus
tRNAcuA pairs, and pyrrolysyl-tRNA pairs. In some instances, the unnatural
amino acid is
incorporated into the IL-2 polypeptide by a Mi-TyrRS/tRNA pair. Exemplary UAAs
that can be
incorporated by a Mj-TyrRS/tRNA pair include, but are not limited to, para-
substituted
phenylalanine derivatives such as p-aminophenylalanine and p-
methoyphenylalanine; meta-
substituted tyrosine derivatives such as 3-aminotyrosine, 3-nitrotyrosine, 3,4-
dihydroxyphenylalanine, and 3-iodotyrosine; phenylselenocysteine; p-
boronophenylalanine; and o-
nitrobenzyltyrosine.
[337] In some instances, the unnatural amino acid is incorporated into the
IL-2 polypeptide by a
Ec-Tyr/tRNAcuA or a Ec-Leu/tRNAcuA pair. Exemplary UAAs that can be
incorporated by a Ec-
Tyr/tRNAcuA or a Ec-Leu/tRNAcuA pair include, but are not limited to,
phenylalanine derivatives
containing benzophenone, ketone, iodide, or azide substituents; O-
propargyltyrosine; a-
aminocaprylic acid, 0-methyl tyrosine, 0-nitrobenzyl cysteine; and 3-
(naphthalene-2-ylamino)-2-
amino-propanoic acid.
[338] In some instances, the unnatural amino acid is incorporated into the
IL-2 polypeptide by a
pyrrolysyl-tRNA pair. In some cases, the Py1RS is obtained from an
archaebacterial, e.g., from a
methanogenic archaebacterial. In some cases, the Py1RS is obtained from
Methanosarcina barkeri,
Methanosarcina tnazei, or Methanosarcina acetivorans. Exemplary UAAs that can
be incorporated
by a pyrrolysyl-tRNA pair include, but are not limited to, amide and carbamate
substituted lysines
such as 2-amino-6-((R)-tetrahydrofuran-2-carboxamido)hexanoic acid, N-E-o-
prolyl-L-lysine, and
N-c-cyclopentyloxycarbonyl-L-lysine; N-E-Acryloyl-L-lysine; N-r-[(1-(6-
nitrobenzo[d][1,3]dioxol-
5-y1)ethoxy)carbony1FL-lysine; and N-c-(1-methylcyclopro-2-
enecarboxamido)lysine. In some
embodiments, the IL-2 conjugates disclosed herein may be prepared by use of M.
mazei tRNA
which is selectively charged with a non-natural amino acid such as N6-((2-
azidoethoxy)-carbony1)-
L-lysine (AzK) by the M. barkeri pyrrolysyl-tRNA synthetase (Mb Py1RS). Other
methods are
known to those of ordinary skill in the art, such as those disclosed in Zhang
et al., Nature 2017,
551(7682): 644-647, the disclosure of which is herein incorporated by
reference.
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[339] In some instances, an unnatural amino acid is incorporated into the
IL-2 polypeptide by a
synthetase disclosed in US 9,988,619 and US 9,938,516, the disclosure of each
of which is herein
incorporated by reference.
[340] The host cell into which the constructs or vectors disclosed herein
are introduced is
cultured or maintained in a suitable medium such that the tRNA, the tRNA
synthetase and the
protein of interest are produced. The medium also comprises the unnatural
amino acid(s) such that
the protein of interest incorporates the unnatural amino acid(s). In some
embodiments, a nucleoside
triphosphate transporter (NTT) from bacteria, plant, or algae is also present
in the host cell. In some
embodiments, the IL-2 conjugates disclosed herein are prepared by use of a
host cell that expresses
a NTT. In some embodiments, the nucleotide nucleoside triphosphate transporter
used in the host
cell may be selected from TpNTT1, TpNTT2, TpNTT3, TpNTT4, TpNTT5, TpNTT6,
TpNTT7,
TpNTT8 (T. pseudonana), PtNTT1, PtNTT2, PtNTT3, PtNTT4, PtNTT5, PtNTT6 (P.
tricornutum),
GsNTT (Galdieria sulphuraria), AtNTT1, AtNTT2 (Arabidopsis thaliana), CtNTT1,
CtNTT2
(Chlamydia trachomatis), PamNTT1, PamNTT2 (Protochlamydia amoebophila), CeNTT
(Caedibacter caryophilus), RpNTT1 (Rickettsia prowazekii). In some
embodiments, the NTT is
selected from PtNTT1, PtNTT2, PtNTT3, PtNTT4, PtNTT5, and PtNTT6. In some
embodiments,
the NTT is PtNTT1. In some embodiments, the NTT is PtNTT2. In some
embodiments, the NTT
is PtNTT3. In some embodiments, the NTT is PtNTT4. In some embodiments, the
NTT is
PtNTT5. In some embodiments, the NTT is PtNTT6. Other NTTs that may be used
are disclosed
in Zhang et al., Nature 2017, 551(7682): 644-647; Malyshev et al. Nature 2014
(509(7500), 385-
388; and Zhang et al. Proc Natl Acad Sci USA, 2017, 114:1317-1322; the
disclosure of each of
which is herein incorporated by reference.
[341] The orthogonal tRNA synthetase/tRNA pair charges a tRNA with an
unnatural amino acid
and incorporates the unnatural amino acid into the polypeptide chain in
response to the codon.
Exemplary aaRS-tRNA pairs include, but are not limited to, Methanococcus
jamaschii (11/1j-Tyr)
aaRS/tRNA pairs, E. colt TyrRS (Ec-Tyr)IB. stearotherniophilus tRNAcuA pairs,
E. colt LeuRS
(Ec-Leu)IB. stearothermophilus tRNAcuA pairs, and pyrrolysyl-tRNA pairs. Other
aaRS-tRNA
pairs that may be used according to the present disclosure include those
derived from M. mazei
those described in Feldman et al., J Am Chem Soc., 2018 140:1447-1454; and
Zhang et al. Proc
Natl Acad Sci USA, 2017, 114:1317-1322; the disclosure of each of which is
herein incorporated
by reference.
[342] In some embodiments are provided methods of preparing the IL-2
conjugates disclosed
herein in a cellular system that expresses a NTT and a tRNA synthetase. In
some embodiments
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described herein, the NTT is selected from PtNTT1, PtNTT2, PtNTT3, PtNTT4,
PtNTT5, and
PtNTT6, and the tRNA synthetase is selected from Methanococcus jannaschii, E.
colt TyrRS (Ec-
Tyr)1B. stearothermophilus, and M. mazei. In some embodiments, the NTT is
PtNTT1 and the
tRNA synthetase is derived from Methanococcus jannaschii, E. colt TyrRS (Ec-
Tyr)1B.
stearothermophilus, or M. mazei. In some embodiments, the NTT is PtNTT2 and
the tRNA
synthetase is derived from Methanococcus jannaschii, E. colt TyrRS (Ec-Tyr)IB.
stearothermophilus, or M. mazei. In some embodiments, the NTT is PtNTT3 and
the tRNA
synthetase is derived from Methanococcus jannaschii, E. coil TyrRS (Ec-Tyr)IB.
stearothermophilus, or M. mazei. In some embodiments, the NTT is PtNTT3 and
the tRNA
synthetase is derived from Methanococcus jannaschii, E. coil TyrRS (Ec-Tyr)IB.
stearothermophihts, or M. mazei. In some embodiments, the NTT is PtNTT4 and
the tRNA
synthetase is derived from Methanococcus jannaschii, E. colt TyrRS (Ec-Tyr)IB.
stearothermophilus, or M. mazei. In some embodiments, the NTT is PtNTT5 and
the tRNA
synthetase is derived from Methanococcus jannaschii, E. colt TyrRS (Ec-Tyr)IB.
stearolhermophilus, or M. mazei. In some embodiments, the NTT is PtNTT6 and
the tRNA
synthetase is derived from Methanococcus jannaschii, E. coil TyrRS (Ec-Tyr)IB.
stearothermophilus, or M mazei.
[343] In some embodiments, the IL-2 conjugates disclosed herein may
be prepared in a cell,
such as E. colt, comprising (a) nucleotide triphosphate transporter PtNTT2
(including a truncated
variant in which the first 65 amino acid residues of the full-length protein
are deleted), (b) a plasmid
comprising a double-stranded oligonucleotide that encodes an IL-2 variant
having a desired amino
acid sequence and that contains a unnatural base pair comprising a first
unnatural nucleotide and a
second unnatural nucleotide to provide a codon at the desired position at
which an unnatural amino
acid, such as N6-((2-azidoethoxy)-carbonyl)-L-lysine (AzK), will be
incorporated, (c) a plasmid
encoding a tRNA derived from M mazei and which comprises an unnatural
nucleotide to provide a
recognized anticodon (to the codon of the IL-2 variant) in place of its native
sequence, and (d) a
plasmid encoding a M. barkeri derived pyrrolysyl-tRNA synthetase (Mb Py1RS),
which may be the
same plasmid that encodes the tRNA or a different plasmid. In some
embodiments, the cell is
further supplemented with deoxyribo triphosphates comprising one or more
unnatural bases. In
some embodiments, the cell is further supplemented with ribo triphosphates
comprising one or more
unnatural bases. In some embodiments, the cells is further supplemented with
one or more
unnatural amino acids, such as N6((2-azidoethoxy)-carbony1)-L-lysine (AzK). In
some
embodiments, the double-stranded oligonucleotide that encodes the amino acid
sequence of the
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desired IL-2 variant contains a codon AXC at, for example, position 34, 37,
40, 41, 42, 43, 44, 61,
64, 68, or 71 of the sequence that encodes the protein having SEQ ID NO: 3, or
at position 35, 38,
41, 42, 43, 45, 62, 65, 69, or 72 of the sequence that encodes the protein
having SEQ ID NO: 4,
wherein X is an unnatural nucleotide. In some embodiments, the cell further
comprises a plasmid,
which may be the protein expression plasmid or another plasmid, that encodes
an orthogonal tRNA
gene from M mazei that comprises an AXC-matching anticodon GYT in place of its
native
sequence, wherein Y is an unnatural nucleotide that is complementary and may
be the same or
different as the unnatural nucleotide in the codon. In some embodiments, the
unnatural nucleotide
in the codon is different than and complimentary to the unnatural nucleotide
in the anti-codon. In
some embodiments, the unnatural nucleotide in the codon is the same as the
unnatural nucleotide in
the anti-codon. In some embodiments, the first and second unnatural
nucleotides comprising the
unnatural base pair in the double-stranded oligonucleotide may be derived from
CH3
Odah,
OCH3 N S
HO HO N S HO F
0
0 0
OH OH OH OH
CN $
N 'S
HO 0 HO,
0 I
I
\-01
OH ,and 6H
. In some embodiments, the first and second unnatural
nucleotides comprising the unnatural base pair in the double-stranded
oligonucleotide may be
0õ,
HO HO
0 0 -õ
\-1
derived from OH and 6H . In some
embodiments, the
triphosphates of the first and second unnatural nucleotides include,
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CH3
0101
iiir 1
O 0 0 õ. OCH3 0 0 0 N
S
HO-P-O-P-O-P-0 HO-P-0-P-O-P-0-
I I I 0
OH OH OH OH OH OH
OH OH and
,
ZS
1
O 0 0 ''N"---S
HO-P-O-P-O-P-0
I I I
OH OH OH
OH , or salts thereof In some embodiments,
the triphosphates of
IL
RP'
0 0 0
OCH3
ii ii II
HO-P-O-P-O-P-0
1 1 1 0
OH OH OH
the first and second unnatural nucleotides include, OH
,and
S
..,
1
O 0 0 N S
II II II
HO-P-O-P-O-P-0-
1 1 1 0
OH OH OH
OH
, or salts thereof In some embodiments, the mRNA derived
the double-stranded oligonucleotide comprising a first unnatural nucleotide
and a second unnatural
nucleotide may comprise a codon comprising an unnatural nucleotide derived
from
CH3
411, fr.õ
.... ,..s
isp-
..... .....;õ.=
OCH3
HO HO,, HO,,
0
k".---,-
OH OH , OH OH , and OH OH
. In some embodiments, the
M mazei tRNA may comprise an anti-codon comprising an unnatural nucleotide
that recognizes the
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codon comprising the unnatural nucleotide of the mRNA. The anti-codon in the
M. mazei tRNA
CH3
RP'
OCH3
N S
HO HO
0 V12_
may comprise an unnatural nucleotide derived from OH OH OH OH
ON $
F 401II I
HO NS HO 0 HO 0 HO.,
0 0
OH OH OH OH OH OH , and OH 6H
In some
11101) 00H3
HO
0
embodiments, the mRNA comprises an unnatural nucleotide derived from OH OH
In some embodiments, the mRNA comprises an unnatural nucleotide derived from
CH3
N S
HO
Ic_04
OH OH
. In some embodiments, the mRNA comprises an unnatural nucleotide
derived
HO
from OH OH . In some embodiments, the mRNA comprises an
unnatural nucleotide
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F
HO 0
0
derived from OH OH
. In some embodiments, the mRNA comprises an unnatural
c N
HO 0
0
nucleotide derived from OH OH
. In some embodiments, the mRNA comprises an
j .5
"1µ1" S
HO.,
unnatural nucleotide derived from OH OH . In some embodiments,
the tRNA comprises
1.-11 OC H3
HO
0
an unnatural nucleotide derived from OH OH
In some embodiments, the tRNA
CH3
N S
HO
comprises an unnatural nucleotide derived from OH OH
. In some embodiments, the
Ls
HO
tRNA comprises an unnatural nucleotide derived from OH OH
. In some embodiments,
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F
HO 0
0
the tRNA comprises an unnatural nucleotide derived from OH OH . In
some
ON
HO
0
0
embodiments, the tRNA comprises an unnatural nucleotide derived from OH OH
. In
,
,.S
h,
.
HO,
1
some embodiments, the tRNA comprises an unnatural nucleotide derived from
6H at-4
In some embodiments, the mRNA comprises an unnatural nucleotide derived from
1110Irdi
OCH3
HO
0
OH OH and the tRNA comprises an unnatural nucleotide
derived from
N
OH 6H . In some embodiments, the mRNA comprises an unnatural
nucleotide derived
tHO
S
s
0
from OH OH and the tRNA comprises an unnatural
nucleotide derived from
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OCH3
HO
0
OH OH In some embodiments, the mRNA comprises an
unnatural nucleotide
Oidik
OCH3
HO
0
derived from OH OH
and the tRNA comprises an unnatural nucleotide derived from
HOõ NS
OH OH . In some embodiments, the mRNA comprises an unnatural
nucleotide derived
CL/
N S
from OH OH and the tRNA comprises an unnatural
nucleotide derived from
Oral
lir OCH3
HO
0
OH OH
. The host cell is cultured in a medium containing appropriate
nutrients, and
is supplemented with (a) the triphosphates of the deoxyribo nucleosides
comprising one or more
unnatural bases that are necessary for replication of the plasmid(s) encoding
the cytokine gene
harboring the codon, (b) the triphosphates of the ribo nucleosides comprising
one or more unnatural
bases necessary for transcription of (i) the mRNA corresponding to the coding
sequence of the
cytokine and containing the codon comprising one or more unnatural bases, and
(ii) the tRNA
containing the anticodon comprising one or more unnatural bases, and (c) the
unnatural amino
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acid(s) to be incorporated in to the polypeptide sequence of the cytokine of
interest. The host cells
are then maintained under conditions which permit expression of the protein of
interest.
[344] The resulting AzK-containing protein that is expressed may be
purified by methods known
to those of ordinary skill in the art and may then be allowed to react with an
alkyne, such as DBCO
comprising a PEG chain having a desired average molecular weight as disclosed
herein, under
conditions known to those of ordinary skill in the art, to afford the IL-2
conjugates disclosed herein.
Other methods are known to those of ordinary skill in the art, such as those
disclosed in Zhang et
al., Nature 2017, 551(7682): 644-647; WO 2015157555; WO 2015021432; WO
2016115168; WO
2017106767; WO 2017223528; WO 2019014262; WO 2019014267; WO 2019028419; and
W02019/028425; the disclosure of each of which is herein incorporated by
reference.
[345] The resulting protein comprising the one or more unnatural amino
acids, Azk for example,
that is expressed may be purified by methods known to those of ordinary skill
in the art and may
then be allowed to react with an alkyne, such as DBCO comprising a PEG chain
having a desired
average molecular weight as disclosed herein, under conditions known to those
of ordinary skill in
the art, to afford the IL-2 conjugates disclosed herein. Other methods are
known to those of
ordinary skill in the art, such as those disclosed in Zhang et al., Nature
2017, 551(7682): 644-647;
WO 2015157555; WO 2015021432; WO 2016115168; WO 2017106767; WO 2017223528; WO
2019014262; WO 2019014267; WO 2019028419; and W02019/028425; the disclosure of
which is
herein incorporated by reference.
[346] Alternatively, IL-2 polypeptides comprising an unnatural amino
acid(s) are prepared by
introducing the nucleic acid constructs described herein comprising the tRNA
and aminoacyl tRNA
synthetase and comprising a nucleic acid sequence of interest with one or more
in-frame orthogonal
(stop) codons into a host cell. The host cell is cultured in a medium
containing appropriate nutrients,
is supplemented with (a) the triphosphates of the deoxyribo nucleosides
comprising one or more
unnatural bases required for replication of the plasmid(s) encoding the
cytokine gene harboring the
new codon and anticodon, (b) the triphosphates of the ribo nucleosides
required for transcription of
the mRNA corresponding to (i) the cytokine sequence containing the codon, and
(ii) the orthogonal
tRNA containing the anticodon, and (c) the unnatural amino acid(s). The host
cells are then
maintained under conditions which permit expression of the protein of interest
The unnatural
amino acid(s) is incorporated into the polypeptide chain in response to the
unnatural codon. For
example, one or more unnatural amino acids are incorporated into the IL-2
polypeptide.
Alternatively, two or more unnatural amino acids may be incorporated into the
IL-2 polypeptide at
two or more sites in the protein.
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[347] Once the IL-2 polypeptide incorporating the unnatural amino acid(s)
has been produced in
the host cell it can be extracted therefrom by a variety of techniques known
in the art, including
enzymatic, chemical and/or osmotic lysis and physical disruption. The IL-2
polypeptide can be
purified by standard techniques known in the art such as preparative ion
exchange chromatography,
hydrophobic chromatography, affinity chromatography, or any other suitable
technique known to
those of ordinary skill in the art.
[348] Suitable host cells may include bacterial cells (e g , E. coli,
BL21(DE3)), but most suitably
host cells are eukaryotic cells, for example insect cells (e.g. Drosophila
such as Drosophila
melanogaster), yeast cells, nematodes (e.g. C. elegans), mice (e.g. Mus
muscu/us), or mammalian
cells (such as Chinese hamster ovary cells (CHO) or COS cells, human 293T
cells, HeLa cells, NIH
3T3 cells, and mouse erythroleukemia (MEL) cells) or human cells or other
eukaryotic cells. Other
suitable host cells are known to those skilled in the art. Suitably, the host
cell is a mammalian cell -
such as a human cell or an insect cell. In some embodiments, the suitable host
cells comprise E.
colt.
[349] Other suitable host cells which may be used generally in the
embodiments of the invention
are those mentioned in the examples section. Vector DNA can be introduced into
host cells via
conventional transformation or transfection techniques. As used herein, the
terms "transformation"
and "transfection" are intended to refer to a variety of well-recognized
techniques for introducing a
foreign nucleic acid molecule (e.g., DNA) into a host cell, including calcium
phosphate or calcium
chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or
electroporation.
Suitable methods for transforming or transfecting host cells are well known in
the art.
[350] When creating cell lines, it is generally preferred that stable cell
lines are prepared. For
stable transfection of mammalian cells for example, it is known that,
depending upon the expression
vector and transfection technique used, only a small fraction of cells may
integrate the foreign DNA
into their genome. In order to identify and select these integrants, a gene
that encodes a selectable
marker (for example, for resistance to antibiotics) is generally introduced
into the host cells along
with the gene of interest. Preferred selectable markers include those that
confer resistance to drugs,
such as G418, hygromycin, or methotrexate. Nucleic acid molecules encoding a
selectable marker
can be introduced into a host cell on the same vector or can be introduced on
a separate vector.
Cells stably transfected with the introduced nucleic acid molecule can be
identified by drug
selection (for example, cells that have incorporated the selectable marker
gene will survive, while
the other cells die).
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[351] In one embodiment, the constructs described herein are integrated
into the genome of the
host cell. An advantage of stable integration is that the uniformity between
individual cells or clones
is achieved. Another advantage is that selection of the best producers may be
carried out.
Accordingly, it is desirable to create stable cell lines. In another
embodiment, the constructs
described herein are transfected into a host cell. An advantage of
transfecting the constructs into the
host cell is that protein yields may be maximized. In one aspect, there is
described a cell comprising
the nucleic acid construct or the vector described herein.
Anti-EGFR Antibodies
[352] The methods of treating cancer described herein include
administration of an anti-EGFR
antibody in combination with the IL-2 conjugates described herein.
[353] In some embodiments, the anti-EGFR antibody is an inhibitory
antibody. In some
embodiments, the anti-EGFR inhibitor antibody is selected from cetuximab
(Erbitux), panitumumab
(Vectibix), necitumumab (Portrazza), JNJ-61186372 (Amivantamab), IMC-C225, ABX-
EGF,
ICR62, and EMD 55900. In some embodiments, the anti-EGFR inhibitor antibody is
cetuximab
(Erbitux) In some embodiments, the anti-EGFR inhibitor antibody is panitumumab
(Vectibix) In
some embodiments, the anti-EGFR inhibitor antibody is necitumumab (Portrazza).
In some
embodiments, the anti-EGFR inhibitor antibody is JNJ-61186372 (Amivantamab).
In some
embodiments, the anti-EGFR inhibitor antibody is IMC-C225. In some
embodiments, the anti-
EGFR inhibitor antibodies is ABX-EGF. In some embodiments, the anti-EGFR
inhibitor antibody
is ICR62. In some embodiments, the anti-EGFR inhibitor antibody is EMD 55900.x
Methods of Treatment
[354] In one aspect, provided herein are methods of treating cancer in a
subject in need thereof,
comprising administering to the subject an effective amount of (a) an IL-2
conjugate as described
herein, and (b) an anti-EGFR antibody.
[355] Also provided is an IL-2 conjugate as described herein for use in a
method disclosed
herein of treating cancer in a subject in need thereof.
13561 In a further aspect, provided is use of an IL-2 conjugate as
described herein for the
manufacture of a medicament for a method disclosed herein of treating cancer
in a subject in need
thereof.
Cancer types
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[357] In some embodiments, the cancer is selected from renal cell carcinoma
(RCC), non-small
cell lung cancer (NSCLC), head and neck squamous cell cancer (HNSCC),
classical Hodgkin
lymphoma (cHL), primary mediastinal large B-cell lymphoma (PMBCL), urothelial
carcinoma,
microsatellite unstable cancer, microsatellite stable cancer, gastric cancer,
colon cancer, colorectal
cancer (CRC), cervical cancer, hepatocellular carcinoma (HCC), Merkel cell
carcinoma (MCC),
melanoma, small cell lung cancer (SCLC), esophageal, esophageal squamous cell
carcinoma
(ESCC), glioblastoma, mesothelioma, breast cancer, triple-negative breast
cancer, prostate cancer,
castrate-resistant prostate cancer, metastatic castrate-resistant prostate
cancer, or metastatic castrate-
resistant prostate cancer having DNA damage response (DDR) defects, bladder
cancer, ovarian
cancer, tumors of moderate to low mutational burden, cutaneous squamous cell
carcinoma (CSCC),
squamous cell skin cancer (SCSC), tumors of low- to non-expressing PD-L1,
tumors disseminated
systemically to the liver and CNS beyond their primary anatomic originating
site, and diffuse large
B-cell lymphoma.
[358] In some embodiments, the cancer in the subject is selected from renal
cell carcinoma
(RCC), non-small cell lung cancer (NSCLC), urothelial carcinoma, melanoma,
Merkel cell
carcinoma (MCC), and head and neck squamous cell cancer (HNSCC). In some
embodiments, the
cancer is renal cell carcinoma (RCC). In some embodiments, the cancer is non-
small cell lung
cancer (NSCLC). In some embodiments, the cancer is urothelial carcinoma. In
some embodiments,
the cancer is melanoma. In some embodiments, the cancer is Merkel cell
carcinoma (MCC). In
some embodiments, the cancer is head and neck squamous cell cancer (HNSCC).
[359] In some embodiments, the cancer is in the form of a solid tumor. In
some embodiments,
the cancer is an advanced or metastatic solid tumor. In some embodiments, the
cancer is in the form
of a liquid tumor.
Administration
[360] In some embodiments, the response is a complete response, a partial
response or stable
disease. In some embodiments, the IL-2 conjugate is administered to the
subject by intravenous,
subcutaneous, intramuscular, intracerebral, intranasal, intra-arterial, intra-
articular, intradermal,
intravitreal, intraosseous infusion, intraperitoneal, or intrathecal
administration. In some
embodiments, the IL-2 conjugate is administered to the subject by intravenous,
subcutaneous, or
intramuscular administration. In some embodiments, the IL-2 conjugate is
administered to the
subject by intravenous administration. In some embodiments, the IL-2 conjugate
is administered to
the subject by subcutaneous administration. In some embodiments, the IL-2
conjugate is
administered to the subject by intramuscular administration.
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[361] In some embodiments, the duration of the treatment is up to 24
months, such as 1 month, 2
months, 3 months, 6 months, 9 months, 12 months, 15 months, 18 months, 21
months or 24 months.
In some embodiments, the duration of treatment is further extended by up to
another 24 months.
[362] In some embodiments, the IL-2 conjugate is administered to the
subject prior to the
administration to the subject of the anti-EGFR antibody. In some embodiments,
the anti-EGFR
antibody is administered to the subject prior to the administration to the
subject of the IL-2
conjugate. In some embodiments, the IL-2 conjugate and the anti-EGFR antibody
are
simultaneously administered to the subject. In some embodiments, the IL-2
conjugate is
administered to the subject separately from the administration of the anti-
EGFR antibody. In some
embodiments, the IL-2 conjugate and the anti-EGFR antibody are administered
sequentially to the
subject. In some embodiments, the IL-2 conjugate and the anti-EGFR antibody
are administered to
the subject on the same day. In some embodiments, the IL-2 conjugate and the
anti-EGFR antibody
are administered to the subject on different days.
[363] In some embodiments, an effective amount of the IL-2 conjugate is
administered to a
subject in need thereof once per week, once every two weeks, once every three
weeks, once every 4
weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, once every
8 weeks, once
every 9 weeks, once every 10 weeks, once every 11 weeks, once every 12 weeks,
once every 13
weeks, once every 14 weeks, once every 15 weeks, once every 16 weeks, once
every 17 weeks,
once every 18 weeks, once every 19 weeks, once every 20 weeks, once every 21
weeks, once every
22 weeks, once every 23 weeks, once every 24 weeks, once every 25 weeks, once
every 26 weeks,
once every 27 weeks, or once every 28 weeks. In some embodiments, an effective
amount of the
IL-2 conjugate is administered to a subject in need thereof once per week. In
some embodiments,
an effective amount of the IL-2 conjugate is administered to a subject in need
thereof once every
two weeks. In some embodiments, an effective amount of the IL-2 conjugate is
administered to a
subject in need thereof once every three weeks. In some embodiments, an
effective amount of the
IL-2 conjugate is administered to a subject in need thereof once every 4
weeks. In some
embodiments, an effective amount of the IL-2 conjugate is administered to a
subject in need thereof
once every 5 weeks. In some embodiments, an effective amount of the IL-2
conjugate is
administered to a subject in need thereof once every 6 weeks. In some
embodiments, an effective
amount of the IL-2 conjugate is administered to a subject in need thereof once
every 7 weeks. In
some embodiments, an effective amount of the IL-2 conjugate is administered to
a subject in need
thereof once every 8 weeks. In some embodiments, an effective amount of the IL-
2 conjugate is
administered to a subject in need thereof once every 9 weeks. In some
embodiments, an effective
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amount of the IL-2 conjugate is administered to a subject in need thereof once
every 10 weeks. In
some embodiments, an effective amount of the IL-2 conjugate is administered to
a subject in need
thereof once every 11 weeks. In some embodiments, an effective amount of the
IL-2 conjugate is
administered to a subject in need thereof once every 12 weeks. In some
embodiments, an effective
amount of the IL-2 conjugate is administered to a subject in need thereof once
every 13 weeks. In
some embodiments, an effective amount of the IL-2 conjugate is administered to
a subject in need
thereof once every 14 weeks. In some embodiments, an effective amount of the
IL-2 conjugate is
administered to a subject in need thereof once every 15 weeks. In some
embodiments, an effective
amount of the IL-2 conjugate is administered to a subject in need thereof once
every 16 weeks. In
some embodiments, an effective amount of the IL-2 conjugate is administered to
a subject in need
thereof once every 17 weeks. In some embodiments, an effective amount of the
IL-2 conjugate is
administered to a subject in need thereof once every 18 weeks. In some
embodiments, an effective
amount of the IL-2 conjugate is administered to a subject in need thereof once
every 19 weeks. In
some embodiments, an effective amount of the IL-2 conjugate is administered to
a subject in need
thereof once every 20 weeks. In some embodiments, an effective amount of the
IL-2 conjugate is
administered to a subject in need thereof once every 21 weeks. In some
embodiments, an effective
amount of the IL-2 conjugate is administered to a subject in need thereof once
every 22 weeks. In
some embodiments, an effective amount of the IL-2 conjugate is administered to
a subject in need
thereof once every 23 weeks. In some embodiments, an effective amount of the
IL-2 conjugate is
administered to a subject in need thereof once every 24 weeks. In some
embodiments, an effective
amount of the IL-2 conjugate is administered about once every 7, 8, 9, 10, 11,
12, 13, 14, 15, 16, 17,
18, 19, 20, or 21 days. In some embodiments, an effective amount of the IL-2
conjugate is
administered about once every 14, 15, 16, 17, 18, 19, 20, or 21 days.
[364] In some embodiments, the amount of a given agent that corresponds to
such an amount
varies depending upon factors such as the particular compound, the severity of
the disease, the
identity (e.g., weight) of the subject or host in need of treatment, but
nevertheless is routinely
determined in a manner known in the art according to the particular
circumstances surrounding the
case, including, e.g., the specific agent being administered, the route of
administration, and the
subject or host being treated In some instances, the desired dose is
conveniently presented in a
single dose or as divided doses administered simultaneously (or over a short
period of time) or at
appropriate intervals, for example as two, three, four or more sub-doses per
day.
[365] In some embodiments, the IL-2 conjugate is administered at a dose
from about 8 pg/kg to
24 fig/kg, In some embodiments, the IL-2 conjugate is administered at a dose
of about 8 pg/kg. In
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some embodiments, the IL-2 conjugate is administered at a dose of about 16
ig/kg. In some
embodiments, the IL-2 conjugate is administered at a dose of about 24 itig/kg.
In any of these
embodiments, the IL-2 conjugate is administered at a dose as described herein
every 3 weeks.
[366] In some embodiments, an anti-EGFR antibody may be administered
at a dose and using a
dosing regimen that has been determined to be safe and efficacious for that
antibody alone or in
combination with an IL-2 conjugate. In some embodiments, an anti-EGFR antibody
is administered
by intravenous infusion. In some embodiments, cetuximab (or another anti-EGFR
antibody) is
administered to a subject in need thereof once per week, once every two weeks,
once every three
weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every
7 weeks, once
every 8 weeks, once every 9 weeks, once every 10 weeks, once every 11 weeks,
once every 12
weeks, once every 13 weeks, once every 14 weeks, once every 15 weeks, once
every 16 weeks,
once every 17 weeks, once every 18 weeks, once every 19 weeks, once every 20
weeks, once every
21 weeks, once every 22 weeks, once every 23 weeks, once every 24 weeks, once
every 25 weeks,
once every 26 weeks, once every 27 weeks, or once every 28 weeks. In some
embodiments,
cetuximab (or another anti-EGFR antibody) is administered to a subject in need
thereof once per
week. In some embodiments, cetuximab (or another anti-EGFR antibody) is
administered to a
subject in need thereof once every two weeks. In some embodiments, cetuximab
(or another anti-
EGFR antibody) is administered to a subject in need thereof once every three
weeks. In some
embodiments, cetuximab (or another anti-EGFR antibody) is administered to a
subject in need
thereof once every 4 weeks. In some embodiments, cetuximab (or another anti-
EGFR antibody) is
administered to a subject in need thereof once every 5 weeks. In some
embodiments, cetuximab (or
another anti-EGFR antibody) is administered to a subject in need thereof once
every 6 weeks. In
some embodiments, cetuximab (or another anti-EGFR antibody) is administered to
a subject in need
thereof once every 7 weeks. In some embodiments, cetuximab (or another anti-
EGFR antibody) is
administered to a subject in need thereof once every 8 weeks. In some
embodiments, cetuximab (or
another anti-EGFR antibody) is administered to a subject in need thereof once
every 9 weeks. In
some embodiments, cetuximab (or another anti-EGFR antibody) is administered to
a subject in need
thereof once every 10 weeks. In some embodiments, cetuximab (or another anti-
EGFR antibody) is
administered to a subject in need thereof once every 11 weeks In some
embodiments, cetuximab
(or another anti-EGFR antibody) is administered to a subject in need thereof
once every 12 weeks.
In some embodiments, cetuximab (or another anti-EGFR antibody) is administered
to a subject in
need thereof once every 13 weeks. In some embodiments, cetuximab (or another
anti-EGFR
antibody) is administered to a subject in need thereof once every 14 weeks. In
some embodiments,
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cetuximab (or another anti-EGFR antibody) is administered to a subject in need
thereof once every
15 weeks. In some embodiments, cetuximab (or another anti-EGFR antibody) is
administered to a
subject in need thereof once every 16 weeks. In some embodiments, cetuximab
(or another anti-
EGFR antibody) is administered to a subject in need thereof once every 17
weeks. In some
embodiments, cetuximab (or another anti-EGFR antibody) is administered to a
subject in need
thereof once every 18 weeks. In some embodiments, cetuximab (or another anti-
EGFR antibody) is
administered to a subject in need thereof once every 19 weeks. In some
embodiments, cetuximab
(or another anti-EGFR antibody) is administered to a subject in need thereof
once every 20 weeks.
In some embodiments, cetuximab (or another anti-EGFR antibody) is administered
to a subject in
need thereof once every 21 weeks. In some embodiments, cetuximab (or another
anti-EGFR
antibody) is administered to a subject in need thereof once every 22 weeks. In
some embodiments,
cetuximab (or another anti-EGFR antibody) is administered to a subject in need
thereof once every
23 weeks. In some embodiments, cetuximab (or another anti-EGFR antibody) is
administered to a
subject in need thereof once every 24 weeks. In some embodiments, cetuximab
(or another anti-
EGFR antibody) is administered about once every 7, 8, 9, 10, 11, 12, 13, 14,
15, 16, 17, 18, 19, 20,
or 21 days.
[367]
In some embodiments, the anti-EGFR antibody is cetuximab. In some
embodiments,
cetuximab is administered at a loading dose from about 100 mg/m2 to about 500
mg/m2 by
intravenous infusion. In any of the embodiments described herein, the loading
dose of cetuximab is
mg/m2 of the subject's body surface area. In some embodiments, cetuximab is
administered at a
loading dose of about 100 mg/m2 by intravenous infusion. In some embodiments,
cetuximab is
administered at a loading dose of about 150 mg/m2 by intravenous infusion. In
some embodiments,
cetuximab is administered at a loading dose of about 200 mg/m2 by intravenous
infusion. In some
embodiments, cetuximab is administered at a loading dose of about 250 mg/m2 by
intravenous
infusion. In some embodiments, cetuximab is administered at a loading dose of
about 300 mg/m2
by intravenous infusion. In some embodiments, cetuximab is administered at a
loading dose of
about 350 mg/m2 by intravenous infusion. In some embodiments, cetuximab is
administered at a
loading dose of about 400 mg/m2 by intravenous infusion. In some embodiments,
cetuximab is
administered at a loading dose of about 450 mg/m2 by intravenous infusion In
some embodiments,
cetuximab is administered at a loading dose of about 500 mg/m2 by intravenous
infusion. In some
embodiments, the initial dose of cetuximab is administered at a loading dose
of about 400 mg/m2 by
intravenous infusion, and all subsequent doses of cetuximab are administered
at a loading dose of
about 250 mg/m2 by intravenous infusion. In any of these embodiments,
cetuximab is infused over
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about 30-240 minutes. In some embodiments, cetuximab is infused over about 30
minutes. In some
embodiments, cetuximab is infused over about 60 minutes. In some embodiments,
cetuximab is
infused over about 90 minutes. In some embodiments, cetuximab is infused over
about 120 minutes.
In some embodiments, cetuximab is infused over about 150 minutes. In some
embodiments,
cetuximab is infused over about 180 minutes. In some embodiments, cetuximab is
infused over
about 210 minutes. In some embodiments, cetuximab is infused over about 240
minutes. In any of
these embodiments, cetixumab is administered at an infusion rate of about 1
mg/min to about 10
mg/min, such as 1 mg/min, 2 mg-/min, 3 mg/min, 4 mg/min, 5 mg/min, 6 mg/min, 7
mg/min, 8
mg/min, 9 mg/min, or 10 mg/min. In some embodiments, the first dose of
cetuximab is
administered at a higher loading dose than the dose of subsequent doses of
cetuximab. In some
embodiments, the infusion time of the first dose of cetuximab is longer than
the infusion time of
subsequent doses of cetuximab. In some embodiments, cetuximab is administered
at a dose as
described herein every 3 weeks. In some embodiments, cetuximab is administered
at a dose as
described herein every 2 weeks. In some embodiments, cetuximab is administered
at a dose as
described herein every week.
Additional agents/premedication
[368] In some embodiments, any of the methods described herein further
comprises
administering an antihistamine. In some embodiments, the antihistamine is
cetirizine. In some
embodiments, the antihistamine is promethazine. In some embodiments, the
antihistamine is
dexchlorpheniramine. In some embodiments, the antihistamine is
diphenhydramine. In some
embodiments, diphenhydramine is administered intravenously at a dose from
about 25 to 50 mg.
[369] In some embodiments, any of the methods described herein further
comprises
administering an analgesic, such as acetaminophen. In some embodiments,
acetaminophen is
administered orally at a dose from about 650 to 1000 mg.
[370] In some embodiments, any of the methods described herein further
comprises
administering a serotonin 5-H13 receptor antagonist. In some embodiments, the
serotonin 5-
HT3 receptor antagonist is granisetron. In some embodiments, the serotonin 5-
HT3 receptor
antagonist is dolasetron. In some embodiments, the serotonin 5-HT 3 receptor
antagonist is
tropisetron In some embodiments, the serotonin 5-HT3 receptor antagonist is
palonosetron In
some embodiments, the serotonin 5-HT3 receptor antagonist is ondansetron. In
some embodiments,
ondansetron is administered intravenously at a dose from about 8 mg to 0.15
mg/kg.
[371] In some embodiments, any of the methods described herein further
comprises
administering an antihistamine (such as cetirizine, promethazine,
dexchlorpheniramine, or
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diphenhydramine), an analgesic (such as acetaminophen), and/or a serotonin 5-
HT3 receptor
antagonist (such as granisetron, dolasetron, tropisetron, palonosetron, or
ondansetron). In some
embodiments, the method further comprises administering an antihistamine (such
as cetirizine,
promethazine, dexchlorpheniramine, or diphenhydramine) and an analgesic (such
as
acetaminophen). In some embodiments, the method further comprising
administering an
antihistamine (such as cetirizine, promethazine, dexchlorpheniramine, or
diphenhydramine) and a
serotonin 5-HT 3 receptor antagonist (such as granisetron, dolasetron,
tropisetron, palonosetron, or
ondansetron). In some embodiments, the method further comprising administering
an analgesic
(such as acetaminophen) and a serotonin 5-HT3 receptor antagonist (such as
granisetron, dolasetron,
tropisetron, palonosetron, or ondansetron). In some embodiments, any of the
methods described
herein further comprises administering an antihistamine (such as cetirizine,
promethazine,
dexchlorpheniramine, or diphenhydramine), an analgesic (such as
acetaminophen), and a serotonin
5-HT3 receptor antagonist (such as granisetron, dolasetron, tropisetron,
palonosetron, or
ondansetron).
[372] In some embodiments, any of the methods described herein further
comprises
administering a premedication, for example to prevent or reduce the acute
effect of infusion-
associated reactions (IAR) or flu-like symptoms. In some embodiments, the
premedication is
administered prior to administering the IL-2 conjugate and/or the anti-EGFR
antibody (such as
cetuximab). In some embodiments, the premedication is administered prior to
administering the IL-
2 conjugate. In some embodiments, the premedication is administered prior to
administering the
anti-EGFR antibody (such as cetuximab). In some embodiments, the premedication
is administered
prior to administering the IL-2 conjugate and the anti-EGFR antibody (such as
cetuximab).
[373] In some embodiments, the premedication for the IL-2 conjugate is
different from the
premedication for the anti-EGFR antibody (such as cetuximab). In some
embodiments, the
premedication for the IL-2 conjugate is the same as the premedication for the
anti-EGFR antibody
(such as cetuximab). In some instances where the premedication for the IL-2
conjugate and the
anti-EGFR antibody (such as cetuximab) is the same, only a single dose of
premedication is
administered. In other instances where the premedication for the IL-2
conjugate and the anti-EGFR
antibody (such as cetuximab) is the same, multiple doses of premedication are
administered In
some embodiments, the premedication is administered for all doses administered
of the IL-2
conjugate. In some embodiments, the premedication is administered for the
first 1, 2, 3, 4, 5, 6, 7,
8, 9, or 10 doses of the IL-2 conjugate and not for any subsequent doses of
the IL-2 conjugate. In
some embodiments, the premedication is administered for the first 4 doses of
the IL-2 conjugate and
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not for any subsequent doses of the IL-2 conjugate. In some embodiments, the
premedication is
administered for all doses administered of the anti-EGFR antibody (such as
cetuximab). In some
embodiments, the premedication is administered for the first 1, 2, 3, 4, 5, 6,
7, 8, 9, or 10 doses of
the anti-EGFR antibody (such as cetuximab) and not for any subsequent doses of
the anti-EGFR
antibody. In some embodiments, the premedication is administered for the first
dose of the anti-
EGFR antibody (such as cetuximab) and not for any subsequent doses of the anti-
EGFR antibody.
[374] In some embodiments, any of the methods described herein further
comprises
administering premedication prior to administering the IL-2 conjugate. In some
embodiments, the
IL-2 conjugate premedication is an antihistamine, such as cetirizine,
promethazine,
dexchlorpheniramine, or diphenhydramine. In some embodiments, the
antihistamine is
diphenhydramine. In some embodiments, diphenhydramine is administered
intravenously at a dose
from about 25 to 50 mg. In some embodiments, the IL-2 conjugate premedication
is a serotonin 5-
HT3 receptor antagonist (such as granisetron, dolasetron, tropisetron,
palonosetron, or ondansetron).
In some embodiments, the serotonin 5-HT3 receptor antagonist is ondansetron.
In some
embodiments, ondansetron is administered intravenously at a dose from about 8
mg to 0.15 mg/kg.
In some embodiments, the IL-2 conjugate premedication is an analgesic (such as
acetaminophen).
In some embodiments, acetaminophen is administered orally at a dose from about
650 to 1000 mg.
[375] In some embodiments, any of the methods described herein further
comprises
administering premedication prior to administering the anti-EGFR antibody
(such as cetuximab). In
some embodiments, the anti-EGFR antibody premedication is an antihistamine,
such as cetirizine,
promethazine, dexchlorpheniramine, or diphenhydramine In some embodiments, the
antihistamine
is diphenhydramine. In some embodiments, diphenhydramine is administered
intravenously at a
dose from about 25 to 50 mg. In some embodiments, the cetuximab premedication
is a serotonin 5-
HT3 receptor antagonist (such as granisetron, dolasetron, tropisetron,
palonosetron, or ondansetron).
In some embodiments, the serotonin 5-HT3 receptor antagonist is ondansetron.
In some
embodiments, ondansetron is administered intravenously at a dose from about 8
mg to 0.15 mg/kg.
In some embodiments, the anti-EGFR antibody premedication is an analgesic
(such as
acetaminophen). In some embodiments, acetaminophen is administered orally at a
dose from about
650 to 1000 mg
[376] In some embodiments, any of the methods described herein further
comprises
administering a first dose of premedication prior to administering the IL-2
conjugate and a second
dose of premedication prior to administering the anti-EGFR antibody (such as
cetuximab). In some
embodiments, the premedication for the IL-2 conjugate is the same as the
premedication for the
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anti-EGFR antibody (such as cetuximab). In some embodiments, the premedication
for the IL-2
conjugate is different from the premedication for the anti-EGFR antibody (such
as cetuximab). In
some embodiments, the premedication is an antihistamine, such as cetirizine,
promethazine,
dexchlorpheniramine, or diphenhydramine. In some embodiments, the
antihistamine is
diphenhydramine In some embodiments, diphenhydramine is administered
intravenously at a dose
from about 25 to 50 mg. In some embodiments, the premedication is a serotonin
5-HT3 receptor
antagonist (such as granisetron, dolasetron, tropisetron, pal onosetron, or
ondansetron). In some
embodiments, the serotonin 5-HT3 receptor antagonist is ondansetron. In some
embodiments,
ondansetron is administered intravenously at a dose from about 8 mg to 0.15
mg/kg. In some
embodiments, the premedication is an analgesic (such as acetaminophen). In
some embodiments,
acetaminophen is administered orally at a dose from about 650 to 1000 mg. In
some embodiments,
the premedication comprises an antihistamine and a serotonin 5-HT3 receptor
antagonist. In some
embodiments, the premedication comprises an antihistamine and an analgesic. In
some
embodiments, the premedication comprises a serotonin 5-HT3 receptor antagonist
and an analgesic.
In some embodiments, the premedication comprises an antihistamine, a serotonin
5-HT3 receptor
antagonist, and an analgesic. In some instances where the premedication for
the IL-2 conjugate and
the anti-EGFR antibody (such as cetuximab) is the same (such as
diphenhydramine), only a single
dose of premedication is administered. In other instances where the
premedication for the IL-2
conjugate and the anti-EGFR antibody (such as cetuximab) is the same, multiple
doses of
premedication are administered.
[377] In some embodiments of the methods described herein, the
dosing sequence is as follows:
(i) premedication for the anti-EGFR antibody (such as cetuximab); (ii) the
anti-EGFR antibody
(such as cetuximab); (iii) premedication for the IL-2 conjugate; and (iv) the
IL-2 conjugate. In
some variations where the premedication for the anti-EGFR antibody (such as
cetuximab) is the
same as the premedication for the IL-2 conjugate (such as diphenhydramine),
administering the
premedication for the IL-2 conjugate may be omitted. In some embodiments, the
dosing sequence
is as follows: (i) premedication for the IL-2 conjugate; (ii) the IL-2
conjugate; (iii) premedication
for the anti-EGFR antibody (such as cetuximab); and (iv) the anti-EGFR
antibody (such as
cetuximab) In some variations where the premedication for the anti-EGFR
antibody (such as
cetuximab) is the same as the premedication for the IL-2 conjugate (such as
diphenhydramine),
administering the premedication for the anti-EGFR antibody may be omitted.
Subject
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[378] In some embodiments, administration of the effective amount of the IL-
2 conjugate and an
anti-EGFR antibody is to an adult. In some embodiments, the adult is a male.
In other
embodiments, the adult is a female. In some embodiments, the adult is at least
age 20, 25, 30, 35,
40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, or 95 years of age. In some
embodiments, administration
of the effective amount of the IL-2 conjugate and an anti-EGFR antibody is to
an infant, child, or
adolescent. In some embodiments, the subject is at least 1 month, 2 months, 3
months, 6 months, 9
months or 12 months of age. In some embodiments, the subject is at least 1, 2,
3, 4, 5, 6, 7, 8,9, 10,
11, 12, 13, 14, 15, 16, 17, 18, or 19 years of age.
[379] In some embodiments, the subject has measurable disease (i.e.,
cancer) as determined by
RECIST v1.1. In some embodiments, the subject has been determined to have
Eastern Cooperative
Oncology Group (ECOG) performance status of 0 or 1. In some embodiments, the
subject has
adequate cardiovascular, hematological, liver, and renal function, as
determined by a physician. In
some embodiments, the subject has been determined (e.g., by a physician) to
have a life expectancy
greater than or equal to 12 weeks.
[380] In some embodiments, the subject has adequate cardiovascular,
hematological, liver, and
renal function.
[381] In some embodiments, the subject has histologically or cytologically
confirmed diagnosis
of advanced and/or metastatic solid tumors with at least one tumor lesion with
location accessible to
safely biopsy per clinical judgment (i.e., as determined by a physician). In
some embodiments, the
subject has had prior anti-cancer therapy before administration of the first
treatment dose. In some
embodiments, treatment related toxicity of the prior anti-cancer therapy has
been resolved to an
appropriate level.
[382] In some embodiments, the subject is a female of childbearing
potential and is using a
medically-accepted method of birth control during the treatment and for at
least 3 months after the
last treatment dose is administered. In some embodiments, the subject is a pre-
menopausal female
who has tested negative for pregnancy (by a serum pregnancy test) within 7
days prior to
administration of the first treatment dose. In some embodiments, the subject
is a female less than
12 months after menopause who has tested negative for pregnancy (by a serum
pregnancy test)
within 7 days prior to administration of the first treatment dose
[383] In some embodiments, the subject is a male who is not surgically
sterile and who is using a
medically-accepted method of birth control during the treatment and for at
least 3 months after the
last dose is administered. In some embodiments, the male is not donating or
banking sperm during
the treatment period and for at least 3 months after administration of the
last treatment dose.
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[384] In some embodiments, the subject has not received radiotherapy within
14 days of
administration of the first treatment dose. In some embodiments, the subject
has not received
palliative radiation or stereotactic radiosurgery within 7 days of
administration of the first treatment
dose.
[385] In some embodiments, the subject has not been treated with systemic
anti-cancer therapy
or an investigational anti-cancer agent within 2 weeks of administration of
the first treatment dose.
In some embodiments, the subject has not been treated with immunotherapy or
tyrosine kinase
inhibitor therapy within 4 weeks of administration of the first treatment
dose.
[386] In some embodiments, the subject has not had major surgery within 30
days of
administration of the first treatment dose. In some embodiments, the subject
has had major surgery
more than 30 days prior to administration of the first treatment dose and has
recovered to at least
Grade 1 from any adverse effects associated with the procedure. In some
embodiments, the subject
does not anticipate the need for major surgery during the course of treatment.
[387] In some embodiments, the subject has not had active autoimmune
disease requiring
systemic treatment within 3 months prior to administration of the first
treatment dose. In some
embodiments, the subject has not had a documented history of clinically severe
autoimmune disease
that requires systemic steroids or immunosuppressive agents prior to
administration of the first
treatment dose.
[388] In some embodiments, the subject does not have primary central
nervous system (CNS)
disease or leptomeningeal disease. In some embodiments, the subject has known
CNS metastases
but has received appropriate treatment and is asymptomatic, without evidence
of radiological
progression for at least 8 weeks prior to administration of the first
treatment dose, and has had no
requirement for steroids or enzyme inducing anticonvulsants within 14 days
prior to administration
of the first treatment dose.
[389] In some embodiments, the subject has not had abnormal pulmonary
function, including
pneumonitis, active pneumonitis, interstitial lung disease requiring the use
of steroids, idiopathic
pulmonary fibrosis, confirmed pleural effiision, and severe dyspnea at rest or
requiring
supplementary oxygen therapy, within 6 months of administration of the first
treatment dose.
[390] In some embodiments, the subject has not taken parenteral antibiotics
within 14 days of
administration of the first treatment dose.
[391] In some embodiments, the subject does not have a history of allogenic
or solid organ
transplant. In some embodiments, the subject does not have human
immunodeficiency virus (HIV)
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infection or active infection with hepatitis C. In some embodiments, the
subject does not have
uncontrolled hepatitis B virus (HBV) infection.
[392] In some embodiments, the subject has had no clinically significant
bleeding (e.g.,
gastrointestinal bleeding, intracranial hemorrhage) within 2 weeks prior to
administration of the first
treatment dose. In some embodiments, the subject has not had a prior diagnosis
of deep vein
thrombosis or pulmonary embolism within 3 months of administration of the
first treatment dose.
[393] In some embodiments, the subject has not had a severe or unstable
cardiac condition (such
as congestive heart failure (New York Heart Association Class III or IV),
cardiac bypass surgery or
coronary artery stent placement, angioplasty, cardiac ejection fraction below
the lower limit of
normal, unstable angina, medically uncontrolled hypertension (e.g. >160 mm Hg
systolic or >100
mm Hg diastolic), uncontrolled cardiac arrhythmia requiring medication (>
grade 2, according to
NCI CTCAE v5.0), or myocardial infarction) within 6 months prior to
administration of the first
treatment dose.
[394] In some embodiments, the subject has no history of non-
pharmacologically induced
prolonged corrected QT interval determined using Fridericia's formula (QTcF) >
450 milliseconds
(msec) in males or > 470 msec in females.
[395] In some embodiments, the subject has no known hypersensitivity or
contraindications to
any of the IL-2 conjugates disclosed herein, PEG, pegylated drugs, or anti-
EGFR antibody, such as,
for example, cetuximab.
[396] In some embodiments, the subject does not have an active second
malignancy. In some
embodiments, the subject does not have a history of a previous malignancy. In
some embodiments,
the subject has had a non-melanomatous skin cancer or cervical cancer that has
been curatively
surgically resected prior to administration of the first treatment dose.
[397] In some embodiments, the subject does not have any serious medical
condition (including
pre-existing autoimmune disease or inflammatory disorder), laboratory
abnormality, psychiatric
condition, or any other significant or unstable concurrent medical illness
that would preclude
treatment or would make treatment inappropriate.
[398] In some embodiments, the subject is not pregnant or breastfeeding. In
some embodiments,
the subject is not expecting to conceive or father children during the course
of the treatment and
following up to 3 months after administration of the final treatment dose.
[399] In some embodiments, the subject is not receiving a concurrent
therapy with any
investigational agent, vaccine, or device during the course of treatment. In
some embodiments, the
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subject is receiving concurrent therapy with an investigational agent,
vaccine, or device during the
course of treatment after physician approval.
Effects of Administration
[400] In some embodiments, following administration of the IL-2 conjugate
and an anti-EGFR
antibody, the subject experiences a response as measured by the Immune-related
Response
Evaluation Criteria in Solid Tumors (iRECIST). In some embodiments, following
administration of
the IL-2 conjugate and an anti-EGFR antibody, the subject experiences an
Objective Response Rate
(ORR) according to RECIST version 1.1. In some embodiments, following
administration of the
IL-2 conjugate and an anti-EGFR antibody, the subject experiences Duration of
Response (DOR)
according to RECIST versions 1.1. In some embodiments, following
administration of the IL-2
conjugate and an anti-EGFR antibody, the subject experiences Progression-Free
Survival (PFS)
according to RECIST version 1.1. In some embodiments, following administration
of the IL-2
conjugate and an anti-EGFR antibody, the subject experiences Overall Survival
according to
RECIST version 1.1. In some embodiments, following administration of the IL-2
conjugate and an
anti-EGFR antibody, the subject experiences Time to Response (TTR) according
to RECIST
version 1.1. In some embodiments, following administration of the IL-2
conjugate and an anti-
EGFR antibody, the subject experiences Disease Control Rate (DCR) according to
RECIST version
1.1. In any of these embodiments, the subject's experience is based on a
physician's review of a
radiographic image taken of the subject.
[401] In some embodiments, treatment is discontinued based on a physician's
review of a
radiographic image taken of the subject.
[402] In some embodiments, treatment is discontinued based on a physician's
review of
immunophenotyping of peripheral blood at various timepoints. In some
embodiments, treatment is
discontinued based on a physician's review of immunophenotyping of tumor
samples at various
timepoints. In some embodiments, treatment is discontinued based on a
physician's review of the
presence of antibodies to any of the IL-2 conjugates disclosed herein at
various timepoints. In some
embodiments, treatment is discontinued based on a physician's review of the
plasma concentration
of any of the IL-2 conjugates disclosed herein at various timepoints. In any
of these embodiments,
the physican's review is based on an appropriate assay of the relevant
parameters
[403] In some embodiments, administration of the effective amount of the IL-
2 conjugate and an
anti-EGFR antibody to the subject does not cause vascular leak syndrome in the
subject. In some
embodiments, administration of the effective amount of the IL-2 conjugate and
an anti-EGFR
antibody to the subject does not cause Grade 2, Grade 3, or Grade 4 vascular
leak syndrome in the
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subject. In some embodiments, administration of the effective amount of the IL-
2 conjugate and an
anti-EGFR antibody to the subject does not cause Grade 2 vascular leak
syndrome in the subject. In
some embodiments, administration of the effective amount of the IL-2 conjugate
and an anti-EGFR
antibody to the subject does not cause Grade 3 vascular leak syndrome in the
subject. In some
embodiments, administration of the effective amount of the IL-2 conjugate and
an anti -EGFR
antibody to the subject does not cause Grade 4 vascular leak syndrome in the
subject.
[404] In some embodiments, administration of the effective amount of the IL-
2 conjugate and an
anti-EGFR antibody to the subject does not cause loss of vascular tone in the
subject.
[405] In some embodiments, administration of the effective amount of the IL-
2 conjugate and an
anti-EGFR antibody to the subject does not cause extravasation of plasma
proteins and fluid into the
extravascular space in the subject.
[406] In some embodiments, administration of the effective amount of the IL-
2 conjugate and an
anti-EGFR antibody to the subject does not cause hypotension and reduced organ
perfusion in the
subj ect.
[407] In some embodiments, administration of the effective amount of the IL-
2 conjugate and an
anti-EGFR antibody to the subject does not cause impaired neutrophil function
in the subject. In
some embodiments, administration of the effective amount of the IL-2 conjugate
and an anti-EGFR
antibody to the subject does not cause reduced chemotaxis in the subject.
[408] In some embodiments, administration of the effective amount of the IL-
2 conjugate and an
anti-EGFR antibody to the subject is not associated with an increased risk of
disseminated infection
in the subject. In some embodiments, the disseminated infection is sepsis or
bacterial endocarditis.
In some embodiments, the disseminated infection is sepsis. In some
embodiments, the disseminated
infection is bacterial endocarditis. In some embodiments, the subject is
treated for any preexisting
bacterial infections prior to administration of the IL-2 conjugate and an anti-
EGFR antibody. In
some embodiments, the subject is treated with an antibacterial agent selected
from oxacillin,
nafcillin, ciprofloxacin, and vancomycin prior to administration of the IL-2
conjugate and an anti-
EGFR antibody.
[409] In some embodiments, administration of the effective amount of the IL-
2 conjugate and an
anti-EGFR antibody to the subject does not exacerbate a pre-existing or
initial presentation of an
autoimmune disease or an inflammatory disorder in the subject. In some
embodiments, the
administration of the effective amount of the 1L-2 conjugate and an anti-EGFR
antibody to the
subject does not exacerbate a pre-existing or initial presentation of an
autoimmune disease in the
subject. In some embodiments, the administration of the effective amount of
the IL-2 conjugate and
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an anti-EGFR antibody to the subject does not exacerbate a pre-existing or
initial presentation of an
inflammatory disorder in the subject. In some embodiments, the autoimmune
disease or
inflammatory disorder in the subject is selected from Crohn's disease,
scleroderma, thyroiditis,
inflammatory arthritis, diabetes mellitus, oculo-bulbar myasthenia gravis,
crescentic IgA
glomerulonephritis, cholecystitis, cerebral vasculitis, Stevens-Johnson
syndrome and bullous
pemphigoid. In some embodiments, the autoimmune disease or inflammatory
disorder in the subject
is Crohn's disease. In some embodiments, the autoimmune disease or
inflammatory disorder in the
subject is scleroderma. In some embodiments, the autoimmune disease or
inflammatory disorder in
the subject is thyroiditis. In some embodiments, the autoimmune disease or
inflammatory disorder
in the subject is inflammatory arthritis. In some embodiments, the autoimmune
disease or
inflammatory disorder in the subject is diabetes mellitus. In some
embodiments, the autoimmune
disease or inflammatory disorder in the subject is oculo-bulbar myasthenia
gravis. In some
embodiments, the autoimmune disease or inflammatory disorder in the subject is
crescentic IgA
glomerulonephritis. In some embodiments, the autoimmune disease or
inflammatory disorder in the
subject is cholecystitis. In some embodiments, the autoimmune disease or
inflammatory disorder in
the subject is cerebral vasculitis. In some embodiments, the autoimmune
disease or inflammatory
disorder in the subject is Stevens-Johnson syndrome. In some embodiments, the
autoimmune
disease or inflammatory disorder in the subject is bullous pemphigoid.
[410] In some embodiments, administration of the effective amount of the IL-
2 conjugate and an
anti-EGFR antibody to the subject does not cause changes in mental status,
speech difficulties,
cortical blindness, limb or gait ataxia, hallucinations, agitation,
obtundation, or coma in the subject.
In some embodiments, administration of the effective amount of the IL-2
conjugate and an anti-
EGFR antibody to the subject does not cause seizures in the subject. In some
embodiments,
administration of the effective amount of the IL-2 conjugate and an anti-EGFR
antibody to the
subject is not contraindicated in subjects having a known seizure disorder.
[411] In some embodiments, administration of the effective amount of the IL-
2 conjugate and an
anti-EGFR antibody to the subject does not cause capillary leak syndrome in
the subject. In some
embodiments, administration of the effective amount of the IL-2 conjugate and
an anti-EGFR
antibody to the subject does not cause Grade 2, Grade 3, or Grade 4 capillary
leak syndrome in the
subject. In some embodiments, administration of the effective amount of the IL-
2 conjugate and an
anti-EGFR antibody to the subject does not cause Grade 2 capillary leak
syndrome in the subject. In
some embodiments, administration of the effective amount of the IL-2 conjugate
and an anti-EGFR
antibody to the subject does not cause Grade 3 capillary leak syndrome in the
subject. In some
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embodiments, administration of the effective amount of the IL-2 conjugate and
an anti-EGFR
antibody to the subject does not cause Grade 4 capillary leak syndrome in the
subject.
[412] In some embodiments, administration of the effective amount of the IL-
2 conjugate and an
anti-EGFR antibody to the subject does not cause a drop in mean arterial blood
pressure in the
subject following administration of the IL-2 conjugate to the subject. In some
embodiments,
administration of the effective amount of the IL-2 conjugate and an anti-EGFR
antibody to the
subject does cause hypotension in the subject. In some embodiments,
administration of the effective
amount of the IL-2 conjugate and an anti-EGFR antibody to the subject does not
cause the subject
to experience a systolic blood pressure below 90 mm Hg or a 20 mm Hg drop from
baseline systolic
pressure
[413] In some embodiments, administration of the effective amount of the IL-
2 conjugate and an
anti-EGFR antibody to the subject does not cause edema or impairment of kidney
or liver function
in the subject.
[414] In some embodiments, administration of the effective amount of the IL-
2 conjugate and an
anti-EGFR antibody to the subject does not cause eosinophilia in the subject.
In some
embodiments, administration of the effective amount of the IL-2 conjugate and
an anti-EGFR
antibody to the subject does not cause the eosinophil count in the peripheral
blood of the subject to
exceed 500 per [tL. In some embodiments, administration of the effective
amount of the IL-2
conjugate and an anti-EGFR antibody to the subject does not cause the
eosinophil count in the
peripheral blood of the subject to exceed 500 L to 1500 per uL. In some
embodiments,
administration of the effective amount of the IL-2 conjugate and an anti-EGFR
antibody to the
subject does not cause the eosinophil count in the peripheral blood of the
subject to exceed 1500 per
p,L to 5000 per [iL. In some embodiments, administration of the effective
amount of the IL-2
conjugate and an anti-EGFR antibody to the subject does not cause the
eosinophil count in the
peripheral blood of the subject to exceed 5000 per uL. In some embodiments,
administration of the
effective amount of the IL-2 conjugate and an anti-EGFR antibody to the
subject is not
contraindicated in subjects on an existing regimen of psychotropic drugs.
[415] In some embodiments, administration of the effective amount of the IL-
2 conjugate and
an anti-EGFR antibody to the subject is not contraindicated in subjects on an
existing regimen of
nephrotoxic, myelotoxic, cardiotoxic, or hepatotoxic drugs. In some
embodiments, administration of
the effective amount of the IL-2 conjugate and an anti-EGFR antibody the
subject is not
contraindicated in subjects on an existing regimen of aminoglycosides,
cytotoxic chemotherapy,
doxorubicin, methotrexate, or asparaginase. In some embodiments,
administration of the effective
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amount of the IL-2 conjugate and an anti-EGFR antibody to the subject is not
contraindicated in
subjects receiving combination regimens containing antineoplastic agents. In
some embodiments,
the antineoplastic agent is selected from dacarbazine, cis-platinum, tamoxifen
and interferon-alpha.
[416] In some embodiments, administration of the effective amount of the IL-
2 conjugate and an
anti -EGFR antibody to the subject does not cause one or more Grade 4 adverse
events in the subject
following administration of the IL-2 conjugate to the subject. In some
embodiments, Grade 4
adverse events are selected from hypothermia; shock; bradycardia; ventricular
extrasystol es;
myocardial ischemia; syncope; hemorrhage; atrial arrhythmia; phlebitis; AV
block second degree;
endocarditis; pericardial effusion; peripheral gangrene; thrombosis; coronary
artery disorder;
stomatitis; nausea and vomiting; liver function tests abnormal;
gastrointestinal hemorrhage;
hematemesis; bloody diarrhea; gastrointestinal disorder; intestinal
perforation; pancreatitis; anemia;
leukopenia; leukocytosis; hypocalcemia; alkaline phosphatase increase; blood
urea nitrogen (BUN)
increase; hypeniricemia; non-protein nitrogen (NPN) increase; respiratory
acidosis; somnolence;
agitation; neuropathy; paranoid reaction; convulsion; grand mal convulsion;
delirium; asthma, lung
edema, hyperventilation; hypoxia; hemoptysis; hypoventilation; pneumothorax;
mydriasis; pupillary
disorder; kidney function abnormal; kidney failure; and acute tubular
necrosis. In some
embodiments, administration of the effective amount of the IL-2 conjugate and
an anti-EGFR
antibody to a group of subjects does not cause one or more Grade 4 adverse
events in greater than
1% of the subjects following administration of the IL-2 conjugate to the
subjects. In some
embodiments, Grade 4 adverse events are selected from hypothermia; shock;
bradycardia;
ventricular extrasystoles; myocardial ischemia; syncope; hemorrhage; atrial
arrhythmia; phlebitis;
AV block second degree; endocarditis; pericardial effusion; peripheral
gangrene; thrombosis;
coronary artery disorder; stomatitis; nausea and vomiting; liver function
tests abnormal;
gastrointestinal hemorrhage; hematemesis; bloody diarrhea; gastrointestinal
disorder; intestinal
perforation; pancreatitis; anemia; leukopenia; leukocytosis; hypocalcemia;
alkaline phosphatase
increase; blood urea nitrogen (BUN) increase; hyperuricemia; non-protein
nitrogen (NPN) increase;
respiratory acidosis; somnolence; agitation; neuropathy; paranoid reaction;
convulsion; grand mal
convulsion; delirium; asthma, lung edema; hyperventilation; hypoxia;
hemoptysis; hypoventilation;
pneumothorax; mydriasis; pupillary disorder; kidney function abnormal; kidney
failure; and acute
tubular necrosis.
[417] In some embodiments, administration of the effective amount of the IL-
2 conjugate and an
anti-EGFR antibody to a group of subjects does not cause one or more adverse
events in greater
than 1% of the subjects following administration of the IL-2 conjugate to the
subjects, wherein the
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one or more adverse events is selected from duodenal ulceration; bowel
necrosis; myocarditis;
supraventricular tachycardia; permanent or transient blindness secondary to
optic neuritis; transient
ischemic attacks; meningitis; cerebral edema; pericarditis; allergic
interstitial nephritis; and tracheo-
esophageal fistula.
[418] In some embodiments, administration of the effective amount of the IL-
2 conjugate and an
anti-EGFR antibody to a group of subjects does not cause one or more adverse
events in greater
than 1% of the subjects following administration, wherein the one or more
adverse events is
selected from malignant hyperthermia; cardiac arrest; myocardial infarction;
pulmonary emboli;
stroke; intestinal perforation; liver or renal failure; severe depression
leading to suicide; pulmonary
edema; respiratory arrest; respiratory failure.
[419] In some embodiments, administration of the IL-2 conjugate and an anti-
EGFR antibody to
the subject increases the number of peripheral CD8+ T and NK cells in the
subject without
increasing the number of peripheral CD4+ regulatory T cells in the subject. In
some embodiments,
administration of the IL-2 conjugate and an anti-EGFR antibody to the subject
increases the number
of peripheral CD8+ T and NK cells in the subject without increasing the number
of peripheral
eosinophils in the subject. In some embodiments, administration of the IL-2
conjugate and an anti-
EGFR antibody to the subject increases the number of peripheral CD8+ T and NK
cells in the
subject without increasing the number of intratumoral CD8+ T and NK cells in
the subject without
increasing the number of intratumoral CD4+ regulatory T cells in the subject.
[420] In some embodiments, administration of the effective amount of the IL-
2 conjugate and an
anti-EGFR antibody to the subject does not require the availability of an
intensive care facility or
skilled specialists in cardiopulmonary or intensive care medicine. In some
embodiments,
administration of the effective amount of the IL-2 conjugate to the subject
does not require the
availability of an intensive care facility or skilled specialists in
cardiopulmonary or intensive care
medicine. In some embodiments, administration of the effective amount of the
IL-2 conjugate to the
subject does not require the availability of an intensive care facility. In
some embodiments,
administration of the effective amount of the IL-2 conjugate to the subject
does not require the
availability of skilled specialists in cardiopulmonary or intensive care
medicine.
[421] In some embodiments, administration of the IL-2 conjugate and the
anti-EGFR antibody
combination therapy improves an ADCC response to a cancer, for example, by
improving the
ADCC function of the anti-EGFR antibody. In some embodiments, administration
of the IL-2
conjugate and the anti-EGFR antibody combination therapy expands innate and
adaptive immune
cells. In some embodiments, administration of the IL-2 conjugate and the anti-
EGFR antibody
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combination therapy promotes immune activation within the tumor
microenvironment. In some
embodiments, administration of the IL-2 conjugate and the anti-EGFR antibody
results in a
synergistic improvement in the anti-cancer activity of the combination of the
two agents when
compared to the anti-cancer activity of either agent alone. In some
embodiments, administration of
the IL-2 conjugate increases the number and amount of activation of NK cells,
which potentiates the
ADCC triggered by the anti-EGFR antibody.
Additional Agents
[422] In some embodiments, the methods further comprise administering to
the subject a
therapeutically effective amount of one or more chemotherapeutic agents, in
addition to an anti-
EGFR antibody. In some embodiments, the one or more chemotherapeutic agents
comprises one or
more platinum-based chemotherapeutic agents. In some embodiments, the one or
more
chemotherapeutic agents comprises carboplatin and pemetrexed. In some
embodiments, the one or
more chemotherapeutic agents comprises carboplatin and nab-paclitaxel. In some
embodiments, the
one or more chemotherapeutic agents comprises carboplatin and docetaxel. In
some embodiments,
the cancer in the subject is non-small cell lung cancer (NSCLC).
Kits/Article of Manufacture
[423] Disclosed herein, in certain embodiments, are kits and articles of
manufacture for use with
one or more methods and compositions described herein. Such kits include a
carrier, package, or
container that is compartmentalized to receive one or more containers such as
vials, tubes, and the
like, each of the container(s) comprising one of the separate elements to be
used in a method
described herein. Suitable containers include, for example, bottles, vials,
syringes, and test tubes. In
one embodiment, the containers are formed from a variety of materials such as
glass or plastic. The
kit comprises an IL-2 conjugate and an anti-EGFR antibody.
[424] A kit typically includes labels listing contents and/or instructions
for use, and package
inserts with instructions for use. A set of instructions will also typically
be included.
[425] In one embodiment, a label is on or associated with the container. In
one embodiment, a
label is on a container when letters, numbers or other characters forming the
label are attached,
molded or etched into the container itself, a label is associated with a
container when it is present
within a receptacle or carrier that also holds the container, e.g., as a
package insert. In one
embodiment, a label is used to indicate that the contents are to be used for a
specific therapeutic
application. The label also indicates directions for use of the contents, such
as in the methods
described herein.
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[426] In certain embodiments, the pharmaceutical compositions are presented
in a pack or
dispenser device which contains one or more unit dosage forms containing a
compound provided
herein. The pack, for example, contains metal or plastic foil, such as a
blister pack. In one
embodiment, the pack or dispenser device is accompanied by instructions for
administration. In one
embodiment, the pack or dispenser is also accompanied with a notice associated
with the container
in form prescribed by a governmental agency regulating the manufacture, use,
or sale of
pharmaceuticals, which notice is reflective of approval by the agency of the
form of the drug for
human or veterinary administration. Such notice, for example, is the labeling
approved by the U.S.
Food and Drug Administration for drugs, or the approved product insert. In one
embodiment,
compositions containing a compound provided herein formulated in a compatible
pharmaceutical
carrier are also prepared, placed in an appropriate container, and labeled for
treatment of an
indicated condition.
Exemplary Embodiments
[427] The present disclosure is further described by the following
embodiments. The features of
each of the embodiments are combinable with any of the other embodiments where
appropriate and
practical.
[428] Embodiment Pl. A method of treating a cancer in a subject in need
thereof, comprising
administering to the subject a therapeutically effective amount of (a) an IL-2
conjugate, and (b) an
anti-EGFR antibody, wherein the IL-2 conjugate comprises the amino acid
sequence of SEQ ID
NO: 3 in which at least one amino acid residue in the IL-2 conjugate is
replaced by the structure of
Formula (I):
X N
0
Formula (I);
wherein:
Z is CH2 and Y is 0 0 =
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Y is CH2 and Z is 0 0
0
Z is CH2 and Y is 0 ;or
0
= Y is CH2 and Z is 0
W is a PEG group having an average molecular weight selected from 5kDa,
101(Da, 15kDa, 20kDa,
25kDa, 30kDa, 35kDa, 40kDa, 45kDa, 50kDa, and 60kDa; and
X has the structure:
o/ _________ -
x+1
=
X-1 indicates the point of attachment to the preceding amino acid residue; and
X+1 indicates the point of attachment to the following amino acid residue;
wherein the position of the structure of Formula (I) in the amino acid
sequence of the IL-2
conjugate is selected from K34, F41, F43, K42, E61, P64, R37, T40, E67, Y44,
V68, and L71,
or a pharmaceutically acceptable salt, solvate, or hydrate thereof
[429] Embodiment P2. The method of embodiment P1, wherein in the IL-
2 conjugate Z is CH2
=itr" 0
and Y is 0
[430] Embodiment P3. The method of embodiment Pl, wherein in the IL-2
conjugate Y is CH2
J+Isi 0
and Z is 0
[431] Embodiment P4. The method of any one of embodiments P1-3, wherein in
the IL-2
conjugate the PEG group has an average molecular weight of 25 kDa, 30kDa, or
35 kDa.
[432] Embodiment P5. The method of embodiment 4, wherein in the IL-2
conjugate the PEG
group has an average molecular weight of 30kDa.
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[433] Embodiment P6. The method of any one of embodiments P1-5, wherein the
position of the
structure of Formula (I) in the amino acid sequence of the IL-2 conjugate is
P64.
[434] Embodiment P7. The method of embodiment P1, wherein the structure of
Formula (I) has
the structure of Formula (XII) or Formula (XIII), or is a mixture of the
structures of Formula (XII)
and Formula (XIII) :
NH 0
ON)-Lo
0
Nõ= I
N
0 /n
Formula (XII);
=
0
14: I
N N ,Tr. 0 =
0
0 / 0
(XIII);
wherein:
n is an integer in the range from about 2 to about 5000; and
the wavy lines indicate covalent bonds to amino acid residues within SEQ ID
NO: 3 that are not
replaced,
or a pharmaceutically acceptable salt, solvate, or hydrate thereof.
[435] Embodiment P8. The method of embodiment P7, wherein in the IL-2
conjugate n is an
integer such that -(OCH2CH2)n-OCH3 has a molecular weight of about 25 l(Da, 30
lcDa, or 35 lcDa.
[436] Embodiment P9. The method of embodiment P8, wherein in the IL-2
conjugate n is an
integer such that -(OCH2CH2)n-OCH3 has a molecular weight of about 30 l(Da.
[437] Embodiment P10. The method of any one of embodiments P7-9, wherein
the position of
the structure of Formula (XII) or Formula (XIII) in the amino acid sequence of
the IL-2 conjugate is
P64.
[438] Embodiment P11. A method of treating a cancer in a subject in need
thereof, comprising
administering to the subject a therapeutically effective amount of (a) an IL-2
conjugate, and (b) an
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anti-EGFR antibody, wherein the IL-2 conjugate comprises the amino acid
sequence of SEQ ID
NO: 50, wherein [AzK Li PEG30kD] has the structure of Formula (IV) or Formula
(V), or is a
_ _
mixture of the structures of Formula (IV) and Formula (V):
N 0
X
I I 0
0 N.
N
0
Formula (IV);
0
X y0 0w
0
Formula (V);
wherein:
W is a PEG group having an average molecular weight selected from 51(Da,
10kDa, 15kDa, 20Wa,
25kDa, 30kDa, 35kDa, 40kDa, 45kDa, 50kDa, and 60kDa;
X has the structure:
Ell
0s5-
X-1 indicates the point of attachment to the preceding amino acid residue; and
X+1 indicates the point of attachment to the following amino acid residue;
or a pharmaceutically acceptable salt, solvate, or hydrate thereof.
[439] Embodiment P12. The method according to embodiment P11, wherein W is
a PEG group
having an average molecular weight selected from 25kDa, 30kDa, or 35kDa.
[440] Embodiment P13. The method according to embodiment P12, wherein W is
a PEG group
having an average molecular weight of 30kDa.
[441] Embodiment P14. The method according to any one of embodiments P1-13,
wherein the
anti-EGFR antibody is cetuximab.
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[442] Embodiment P15. A method of treating a cancer in a subject in need
thereof, comprising
administering to the subject a therapeutically effective amount of (a) an IL-2
conjugate, and (b)
cetuximab, wherein the IL-2 conjugate comprises the amino acid sequence of SEQ
ID NO: 50,
wherein [AzK Ll PEG30kD] has the structure of Formula (XII) or Formula (XIII),
or is a mixture
of the structures of Formula (XII) and Formula (XIII):
NH 0
ON)-Lo
0
Nõ= I
,CH3
in
0
Formula (XII);
=
0
NI: I
)c,N N ,TrOj =
0
0
0 /
(XIII);
wherein:
n is an integer such that -(OCH2CH2)n-OCH3 has a molecular weight of about 30
kDa; and
the wavy lines indicate covalent bonds to amino acid residues within SEQ ID
NO: 50 that are not
replaced,
or a pharmaceutically acceptable salt, solvate, or hydrate thereof.
[443] Embodiment P16. The method according to any one of embodiments P1-15,
wherein the
cancer is selected from renal cell carcinoma (RCC), non-small cell lung cancer
(NSCLC), head and
neck squamous cell cancer (HNSCC), classical Hodgkin lymphoma (cHL), primary
mediastinal
large B-cell lymphoma (PMBCL), urothelial carcinoma, microsatellite unstable
cancer,
microsatellite stable cancer, gastric cancer, colon cancer, colorectal cancer
(CRC), cervical cancer,
hepatocellular carcinoma (HCC), Merkel cell carcinoma (MCC), melanoma, small
cell lung cancer
(SCLC), esophageal, esophageal squamous cell carcinoma (ESCC), glioblastoma,
mesothelioma,
breast cancer, triple-negative breast cancer, prostate cancer, castrate-
resistant prostate cancer,
metastatic castrate-resistant prostate cancer, or metastatic castrate-
resistant prostate cancer having
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DNA damage response (DDR) defects, bladder cancer, ovarian cancer, tumors of
moderate to low
mutational burden, cutaneous squamous cell carcinoma (CSCC), squamous cell
skin cancer
(SCSC), tumors of low- to non-expressing PD-L1, tumors disseminated
systemically to the liver and
CNS beyond their primary anatomic originating site, and diffuse large B-cell
lymphoma.
[444] Embodiment P17. The method according to any one of embodiments P1-16,
wherein the
IL-2 conjugate is administered to the subject once per week, once every two
weeks, once every
three weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once
every 7 weeks, or
once every 8 weeks.
[445] Embodiment P18. The method according to any one of embodiments P1-17,
wherein the
IL-2 conjugate is administered to a subject by intravenous administration.
EXAMPLES
[446] These examples are provided for illustrative purposes only and not to
limit the scope of the
claims provided herein.
Example 1. Preparation of the IL-2 conjugate IL-2_P653AzK_L1_PEG30kM-1.
[447] IL-2 employed for bioconjugation was expressed as inclusion bodies in
E. coli using
methods disclosed herein, using: (a) an expression plasmid encoding (i) the
protein with the desired
amino acid sequence, which gene contains a first unnatural base pair to
provide a codon at the
desired position at which the unnatural amino acid N6((2-azidoethoxy)-
carbony1)-L-lysine (AzK)
was incorporated and (ii) a tRNA derived from M. mazei Pyl, which gene
comprises a second
unnatural nucleotide to provide a matching anticodon in place of its native
sequence; (b) a plasmid
encoding a M. barkeri derived pyrrolysyl4RNA synthetase (Mb Py1RS), (c) AzK,
and (d)
truncated variant of nucleotide triphosphate transporter PtNTT2 in which the
first 65 amino acid
residues of the full-length protein were deleted. The double-stranded
oligonucleotide that encodes
the amino acid sequence of the IL-2 variant contained a codon AXC as codon 64
of the sequence
that encodes the protein having SEQ ID NO: 3 in which P64 is replaced with an
unnatural amino
acid described herein. The plasmid encoding an orthogonal tRNA gene from M
mazei comprised
an AXC-matching anticodon GYT in place of its native sequence, wherein Y is an
unnatural
nucleotide as disclosed herein. X and Y were selected from unnatural
nucleotides dTPT3 and
dNaM as disclosed herein. The expressed protein was extracted from inclusion
bodies and re-
folded using standard procedures before site-specifically pegylating the AzK-
containing IL-2
product using DBCO-mediated copper-free click chemistry to attach stable,
covalent mPEG
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moieties (methoxy, linear PEG group having an average molecular weight of
30kDa) to the AzK (as
outlined in Scheme 6 above).
[448] The IL-2 conjugate "IL-2 P65 [AzK Ll PEG3003]-1" comprises SEQ ID NO:
50 in
which the proline at position 64 is replaced by AzK Li PEG30kD, wherein AzK Li
PEG30kD is
defined as a structure of Formula (IV) or Formula (V), or a mixture of Formula
(IV) and Formula
(V), and a 30 kDa, linear mPEG chain. The IL-2 conjugate "IL-2 P65 [AzK Ll
PEG301X0]-1" is
also defined as the compound comprising SEQ ID NO: 3 in which the proline
residue at position 64
(P64) is replaced by the structure of Formula (VIII) or Formula (IX), or a
mixture of Formula (VIII)
and Formula (IX), wherein n is an integer such that the molecular weight of
the PEG group is about
30 kDa. The IL-2 conjugate "IL-2 P65[AzK Ll PEG301(D]-1" is also defined as
the compound
comprising SEQ ID NO: 3 in which the proline residue at position 64 (P64) is
replaced by the
structure of Formula (XII) or Formula (XIII), or a mixture of Formula (XII)
and Formula (XIII),
wherein n is an integer such that the molecular weight of the PEG group is
about 30 kDa.
Example 2. Antibody Dependent Cellular Cytotoxicity (ADCC) assays using IL-
2 _ P65 _ [AzK_ Ll _PEG301(D1-1 and cetuximab.
[449] The effect of IL-2 P65 [AzK Ll PEG30k_D]-1 on ADCC function of
cetuximab is
examined using a calcein-acetyoxymethyl (Calcein-AM; Invitrogen) release
assay.
[450] Materials.
[451] The following cell lines are used: A431 (EGFR high expression cell
line), human PBMCs
are obtained from healthy donors, and enriched using EasySep Human NK Cell
Enrichment Kit
(Stemcell).
[452] The following reagents are used: calcein-acetyoxymethyl (Calcein-AM;
Invitrogen),
Probenecid (Invitrogen), ultra low IgG fetal bovine serum (Thermofisher), and
human isotype IgG1
antibody (Biolegend).
[453] Procedure.
[454] Human primary NK cells are negatively selected from PBMC using a
RoboSepTM instrument according to manufacturer recommended protocols. Purified
NK cells are
cultured with IL-2 P65 [AzK Ll PEG30kD]-1 at varying concentrations (0.1
ug/mL, 0.01 pg/mL,
0.001 ttg/mL, and 0 ug/mL) in RPMI 1640 media supplemented with 1% low IgG FBS
for 18 hours
at 37 C in a humidified incubator with 5% CO2. These cultured cells are used
as effector cells.
Human EGFR positive cancer cell line (A431) is labeled with calcein-AM for 30
min (50 p,g diluted
in 25 [it DMSO to prepare a stock solution, then 10 [IL of calcein stock
solution is added to 4 mL
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RPMI 1640 + 1% low IgG FBS + 1% probenecid for the staining of 4 x 106 cells),
then washed and
plated onto 96-well round bottom plates at a density of 5 x 10 cells/well.
Cetuximab and isotype
human IgG1 antibody are added at various concentrations (from 101..ig/mL to 1
pg/mL) for 30 min
to allow opsonization before adding NK cells. The NK cells activated with IL-
2 P65 [AzK Ll PEG30kD]-1 are collected and added as effector cells at an E:T
ratio of 3:1
(6 x 104 NK cells for 2>< 104 target cells). The plates are then incubated for
1 hour at 37 C in a
humidified incubator with 5% CO2, and 90 uL of supernatants are harvested and
transferred into
opaque 96-well microplates for analysis using fluorometry on an Envision 2104
plate reader
(excitation: 492 nm; emission: 515 nm).
[455] For maximal release, the cells are lysed with 2% Triton X-100. The
fluorescence value of
the culture medium background is subtracted from that of the experimental
release (A), the target
cell spontaneous release (B), and the target cell maximal release (C).
[456] The cytotoxicity and ADCC percentages for each plate (in duplicate)
are calculated using
the following formulas:
Cytotoxicity (%) = (A ¨ B)/(C ¨ B) x 100
ADCC (%)= Cytotoxicity (%, with antibody) ¨ Cytotoxicity (%, without antibody)
For each experiment, measurements are conducted in triplicate using three
replicate wells. Each
experiment is repeated at least 3 times. The half-maximal effective
concentration (EC50) values are
calculated by fitting the data points to a 4-parameter equation using GraphPad
Prism 5 (GraphPad
Software, Inc., San Diego, CA)
[457] Results.
[458] Without activation by IL-2 P65 [AzK Ll PEG30k1D]-1, cetuximab-treated
human NK
cells exhibit cytotoxicity against EGFR expressed cancer cell lines (A431 and
A549), but not
against EGFR null expression cells (NCI-H69). After activation by IL-
2 P65 [AzK Ll PEG301(1)]-1, cetuximab-treated human NK cells exhibit an
enhanced
cytotoxicity against EGFR expressed cancer cell lines (A431 and A549).
Example 3. In vitro study of IL-2 conjugate and cetuximab (PBMC ADCC Assay).
[459] A study was performed to investigate the effects of antibody
dependent cellular
cytotoxicity (ADCC) by the IL-2 conjugate of Example 1 in combination with
cetuximab using a
co-culture of human PBMCs with calcein-labeled cancer cell lines (CAL27 and
A431).
CAL27 Cells.
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[460] Reagents.
[461] Bioassay buffer: 1% ultra low IgG FBS added to phenol-red-free RPMI.
Complete assay
buffer: 450 p1 probenecid added to 45 mL bioassay buffer with final probenecid
concentration of
77 pg/mL. Calcein-acetoxymethyl ester (Calcein-AM): 50 pg in 25 pt DMSO.
Calcein-AM
staining buffer: 10 lit Calcein-AM added to 4 mL complete assay buffer (final
Calcein-AM
concentration of 5 pg/mL). Triton-X-100 lysis buffer' 20 ittL Triton-X-100
added to 4 mL complete
assay buffer (final concentration of 0.5%).
[462] Procedure.
[463] On Day 1, a 6-point, 1 in 5 dilution series (in PBS) of the IL-2
conjugate was prepared.
The IL-2 conjugate concentrations were 2, 0.4, 0.08, 0.016, 0.0032, and 0
tg/mL. PBMCs were
collected by centrifugation at 200 x g for 5 minutes and resuspended in phenol
red-free RPMI +
10% ultra-low IgG at 20 million cells/mL. Appropriate volumes of these PBMCs
were transferred
to 6 sections of a multi-well reservoir to which a range of the IL-2 conjugate
dilutions was added.
PBMCs were mixed well with the IL-2 conjugate by pipetting up and down and 50
pt were
transferred into round-bottomed 96 well plates using a multi-channel pipette
(final PBMC number
per well was 1 million). Six empty wells were reserved for controls to be
added the following day.
The plates were incubated overnight in a humidified incubator at 37 C in the
presence of 5% carbon
dioxide.
[464] On Day 2, CAL27 cells (EGFR-expressing oral epithelial squamous cell
carcinoma cell
line) were harvested using TrypLE express dissociation buffer and collected by
centrifugation at
200 x g for 5 minutes. Cells were counted and 5 million cells were resuspended
in 4 mL calcein-
AM staining buffer and incubated for 30 minutes at 37 C in the presence of 5%
carbon dioxide.
Cells were then collected and washed twice in complete assay buffer by
centrifugation at 200 x g
for 5 minutes. Cells were counted and resuspended at 0.4 million cells/mL for
a final target cell
number of 20,000/well.
[465] Cetuximab antibody (Eli Lilly & Co.) was diluted to a working
concentration of 3X (3,
0.3, 0.03, 0.003 i.tg/mL) for final assay concentrations of 1, 0.1, 0.001,
0.0001 pg/mL. The isotype
control (hIgGl, Biolegend) was diluted to 3 pg/mL for a final concentration of
1 tg/mL in complete
assay buffer. Equal volumes of stained CAL27 cells at 0.4 million cells/mL
were mixed with
antibody dilutions or isotype control and incubated for 30 minutes at 4 C to
allow antibody to bind.
Following incubation, 1001AL of antibody-CAL27 cell mixture were added to the
96 well plates
containing 50 a, of the I1-2 conjugate treated PBMCs from Day 1.
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[466] Control wells without PBMCs but with 50 uL calcein-AM stained CAL27
cells treated
with complete assay buffer (background signal) or stained CAL27 with 501AL
Triton-X-100
treatment (for maximum signal following cell lysis), both made up to 150 uL
final volume with
complete assay buffer were prepared in triplicate. The plates were centrifuged
for 1 minute at 200 x
g, and then incubated for 60 minutes at 37 C in the presence of 5% carbon
dioxide. After
incubation, the plates were again briefly centrifuged before transferring 90
pt of supernatant into
fresh black, clear-bottomed plates, and the fluorescence signal was read on an
Envision 2104 plate
reader (excitation: 492 nm; emission: 515 nm).
[467] The cytotoxicity was calculated using the following formula:
Cytotoxicity (%) = (A ¨ B)/(C ¨ B) x 100
where A is the fluorescence value for treated cells; B is the background from
target cells alone; and
C is the maximum release valued obtained from Triton-X-100 treatment.
[468] The data represent the % cytotoxicity of the IL-2 conjugate treated
human PBMCs on
target cancer cells in the presence of cetuximab. The mean percentage from the
technical replicates
was converted to a proportion. The analysis was conducted using a two-way
generalized linear
mixed model (GLMM), with factors for the IL-2 conjugate, cetuximab and their
interaction, with
random donor effects, treating proportion as a pseudo-binomial variable. It
was followed by a post-
hoc test (with Dunnett-Hsu adjustment) to compare the 1L-2 conjugate treated
groups to the control
group. Statistical analyses were performed using SAS (1) version 9.4 software.
A probability less
than 5% (p<0.05) was considered as significant.
[469] Results.
[470] At cetuximab dose levels of 1, 0.1, and 0,01 mg/mL, the IL-2
conjugate enhanced ADCC
function of cetuximab against EGFR expressing CAL27 cells (p<0.05) at
concentrations of 0.08, 0.4
and 2 mg/mL (FIGS. 1A-C). At a cetuximab dose level of 0.001mg/mL, the 1L-2
conjugate
enhanced ADCC function of cetuximab against EGFR expressing CAL27 cells
(p<0.05) at
concentrations of 0.4 and 2 mg/mL. FIG. 2A further shows the enhanced ADCC
function of
cetuximab against EGFR expressing CAL27 cells (PBMC to CAL27 ratio 50:1).
[471] The tests of fixed effects from the GLMM model indicate that the
factors IL-2 conjugate,
cetuximab and their interaction have a significant effect on the cytotoxicity,
i.e., the differences
between IL-2 conjugate groups vary significantly for the different cetuximab
concentrations. The
pairwise comparisons indicated a significant difference between the IL-2
conjugate 2 mg/mL group
versus the control group (p=0.0001) and between the IL-2 conjugate 0.4 mg/mL
group versus the
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control group (p=0.0001) at a cetuximab concentration of 0.001 mg/mL. The
pairwise comparisons
also indicated a significant difference between the IL-2 conjugate 2 mg/mL
group versus the control
group (p<0.0001), between the IL-2 conjugate 0.4 mg/mL group versus the
control group
(p<0.0001), and between the IL-2 conjugate 0.08 mg/mL group versus the control
group (p=0.0003)
at a cetuximab concentration of 0.01 mg/mL. In addition, the pairwise
comparisons indicated a
significant difference between the IL-2 conjugate 2 mg/mL group versus the
control group
(p<0.0001), between the IL-2 conjugate 0.4 mg/mL group versus the control
group (p<0.0001), and
between the IL-2 conjugate 0.08 mg/mL group versus the control group
(p<0.0001) at a cetuximab
concentration of 0.1 mg/mL. Lastly, the pairwise comparisons indicated a
significant difference
between the IL-2 conjugate 2 mg/mL group versus the control group (p<0.0001),
between the IL-2
conjugate 0.4 mg/mL group versus the control group (p<0.0001), and between the
IL-2 conjugate
0.08 mg/mL group versus the control group (p<0.0001) at a cetuximab
concentration of 1 mg/mL.
[472] The data demonstrate that the IL-2 conjugate enhanced ADCC function
of cetuximab
against EGFR expressing CAL27 cancer cells. No significant differences were
observed using the
IL-2 conjugate in combination with the isotype control.
A431 Cells.
[473] Studies were performed using EGFR expressing A431 cells (epidermoid
carcinoma)
following the procedure outlined above for CAL27 cells. FIG. 2B shows the
enhanced ADCC
function of cetuximab against EGFR expressing A431 cells (PBMC to A431 ratio
50:1). The data
demonstrate that the IL-2 conjugate enhanced ADCC function of cetuximab
against EGFR
expressing A431 cancer cells.
Example 4. ADCC assay using an engineered cell line NK-92.CD16 V as effector
cells.
[474] The effect of IL-2 P65 [AzK LI PEG30k1)]-1 on ADCC function of
cetuximab was
examined using a calcein-acetyoxymethyl (Calcein-AM; Invitrogen) release
assay.
[475] Materials.
[476] NK-92.CD16 V (high affinity variant) (Conkwest Inc., San Diego, CA)
was used as the
effector cell line. The following cell lines were used as target cells: CAL27,
A431, DLD-1, and
FaDu.
[477] The following reagents were used: cetuximab antibody (Eli Lilly &
Co.); human isotype
IgG1 antibody (Biolegend); calcein-acetyoxymethyl (Calcein-AM; Invitrogen
C3100MP), and
probenecid (Invitrogen; P36400). The bioassay medium was phenol red-free RPMI
with 1% ultra
low IgG fetal bovine serum, supplemented with 1% probenecid for complete assay
medium.
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MyeloCult H5100 (Stemcell Cat# 05150) supplemented with IL-2 (100 U/mL) and
hydrocortisone
(Sigma H6909; 10 mL at 50 04) was used for the NK-92.CD16 V cell culture.
[478] Procedure.
[479] IL-2 supplement was withdrawn from the NK-92.CD16 V cell culture,
which was then
incubated overnight prior to starting the assay. The next day, cells were
plated in 96-well round-
bottom plates (60,000 cells were plated for a 3:1 ratio of effector to target
cells) in the presence of
IL-2 P65 [AzK Ll PEG30k1:30.1-1 at varying concentrations (0.1 g/mL, 0.01
g/mL, 0.001
ps/mL, and 0 ps/mL) in phenol red-free RPMI 1640 media supplemented with 1%
low IgG FBS
for 18 hours at 37 C in a humidified incubator with 5% CO2. These cells are
used as the effector
cells. The following day, human EGFR positive cancer cell lines (A431, DLD-1,
FaDu, or CAL27)
were labeled with calcein-AM for 30 min (50 idg diluted in 25 1.11. DMSO to
prepare a stock
solution, then 10 L of calcein stock solution was added to 4 mL RPMI 1640
containing 1% low
IgG FBS and 1% probenecid for the staining of 5 x 106 cells) and then washed.
Cells were divided
into several labeled tubes for incubation with varying concentrations of
cetuximab or isotype
control. Cetuximab and isotype human IgG1 antibody were added at 3X
concentrations (for final
assay concentrations from 10 pg/mL to 1 pg/mL), and the labeled target cells
and antibody were
mixed and allowed to sit for 30 min to allow opsonization. After this
incubation, target cells
(20,000) and antibody were added on top of NK-92.CD16 V cells in 100 L. The
plate was
centrifuged briefly for 1 minute at 1100 rpm before incubating at 37 C and 5%
CO2 for 1 hour.
Following incubation, the plates were again briefly centrifuged as before, and
90 L of supernatant
was transferred from each well to black plates with clear bottom without
disturbing the cells. The
fluorescence signal was read using Envision 2104 (excitation: 492 nm;
emission: 515 nm).
[480] For maximal release, the cells were lysed with 2% Triton X-100. The
fluorescence value
of the culture medium background was subtracted from that of the experimental
release (A), the
target cell spontaneous release (B), and the target cell maximal release (C).
[481] The cytotoxicity and ADCC percentages for each plate (in duplicate)
were calculated
using the following formulas:
Cytotoxicity (%) = (A ¨ B)/(C ¨ B) x 100
ADCC (%)= Cytotaxi city (%, with antibody) ¨ Cytotoxicity (%, without
antibody)
For each experiment, measurements were conducted in triplicate using three
replicate wells. Each
experiment is repeated at least 3 times. The half-maximal effective
concentration (EC50) values are
calculated by fitting the data points to a 4-parameter equation using GraphPad
Prism 5 (GraphPad
Software, Inc., San Diego, CA).
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[482] Results.
[483] Cytotoxicity data using the NK92 cell line ADCC assay is shown in
FIGS. 3A-D for
EGFR expressing A431 (epidermoid carcinoma) (NK92 to A431 ratio 3:1), DLD-1
(adenocarcinoma, colorectal) (NK92 to DLD-1 ratio 3:1), FaDu (epithelial
squamous cell
carcinoma) (NK92 to FaDu ratio 3:1), and CAL27 (epithelial squamous cell
carcinoma) (NK92 to
CAL27 ratio 3:1) cells, respectively. The data demonstrate that the IL-2
conjugate enhanced ADCC
function of cetuximab against EGFR expressing cancer cells.
Example 5. Clinical study of combination therapy using an IL-2 conjugate and
cetuximab.
[484] Overview. Monotherapy using the IL-2 conjugate of Example 1 has been
demonstrated to
promote a peripheral increase in the number of NK cells, which are important
effector cells
mediating antibody-dependent cellular cytotoxicity (ADCC) for IgG1 antibodies
such as cetuximab.
[485] A Phase 1/2, open-label, multi-center study assessing the clinical
benefit of the IL-2
conjugate described in Example 1 in combination with cetuximab for the
treatment of participants
with advanced or metastatic solid tumors was undertaken.
[486] Participants received the IL-2 conjugate (16 or 24 pig/kg dose) by IV
infusion once every 3
weeks. Here and throughout discussion of this cohort, drug mass per kg subject
(e.g., 16 ug/kg)
refers to IL-2 mass exclusive of PEG and linker mass. Cetuximab was given on
Cycle 1 Day 1 as
an initial loading dose of 400 mg/m2 infused over 120 minutes (maximum
infusion rate 10 mg/min),
followed by 250 mg/m2 infused over 60 minutes (maximum infusion rate 10
mg/min) for all
subsequent doses starting with the Cycle 1 Day 8 administration. Cetuximab was
given on days 1,
8, and 15 of each 21 day cycle. The infusion time of the IL-2 conjugate was
about 30 minutes each.
For each cycle of treatment, prior to administering the IL-2 conjugate, all
participants received IL-2
conjugate premedication to prevent or reduce the acute effect of infusion-
associated reactions (TAR)
or flu-like symptoms, 30 to 60 minutes prior to infusion of the IL-2
conjugate. The IL-2 conjugate
premedication was as follows: anti-pyretic, orally, and anti-histamine (H1
blocker). Antiemetics
were provided at the discretion of the supervising physician. Prior to
administration of the first dose
of cetuximab, all participants were pre-medicated with diphenhydramine (about
25 to 50 mg,
intravenous). Premedication for subsequent doses of cetuximab was optional
based on the
supervising physician's assessment. When the IL-2 conjugate and cetuximab were
given on the
same day, participants who received diphenhydramine as cetuximab premedication
may have
skipped the diphenhydramine as the IL-2 conjugate premedication. The dosing
sequence was as
follows: (i) premedication for cetuximab (30-60 min. prior to the start of
cetuximab infusion); (ii)
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cetuximab; (iii) premedication for the IL-2 conjugate (administered 30-60 min.
prior to the start of
the IL-2 conjugate infusion); and (iv) IL-2 conjugate. Treatment was repeated
for up to a total of 35
cycles or for a duration up to 735 days.
[487] The following biomarkers serve as surrogate predictors of safety
and/or efficacy:
Eosinophilia (elevated peripheral eosinophil count) Cell surrogate marker for
IL-2-induced
proliferation of cells (eosinophils) linked to vascular leak syndrome (VLS);
Interleukin 5 (IL-5): Cytokine surrogate marker for IL-2 induced activation of
type 2 innate
lymphoid cells and release of this chemoattractant that leads to eosinophilia
and potentially VLS;
Interleukin 6 (IL-6): Cytokine surrogate marker for IL-2 induced cytokine
release syndrome
(CRS); and
Interferon y (IFN- y): Cytokine surrogate marker for IL-2 induced activation
of CD8+ cytotoxic T
lymphocytes.
[488] The following biomarkers serve as surrogate predictors of anti-tumor
immune activity:
Peripheral CD8+ Effector Cells: Marker for IL-2-induced proliferation of these
target cells in the
periphery that upon infiltration become a surrogate marker of inducing a
potentially latent
therapeutic response;
Peripheral CD8+ Memory Cells: Marker for IL-2-induced proliferation of these
target cells in the
periphery that upon infiltration become a surrogate marker of inducing a
potentially durable latent
therapeutic and maintenance of the memory population;
Peripheral NK Cells: Marker for IL-2-induced proliferation of these target
cells in the periphery
that upon infiltration become a surrogate marker of inducing a potentially
rapid therapeutic
response; and
Peripheral CD4+ Regulatory Cells: Marker for IL-2-induced proliferation of
these target cells in
the periphery that upon infiltration become a surrogate marker of inducing an
immunosuppressive
TME and offsetting of an effector-based therapeutic effect.
First Cohort Using 16 pig/kg Dose of IL-2 Conjugate
[489] Results. The 5 subjects included two human males and 3 females with a
median age of 69
years (ranging from 65-72 years). All subjects had an Eastern Cooperative
Oncology Group
(ECOG) performance status of 0 or 1, and had received 1 to 4 prior lines of
systemic therapies. The
cancers were anal cancer (1 subject), colon adenocarcinoma (1 subject),
adrenocortical cancer (1
subject), squamous cell carcinoma of the lung (1 subject), and small
intestinal cancer (1 subject).
All five subjects had metastatic disease.
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[490] The subjects received the IL-2 conjugate (16 pg/kg) and cetuximab
combination treatment
for 2-7 cycles (2-7 doses of the IL-2 conjugate). Two subjects, one with anal
cancer and one with
metastatic adrenocortical carcinoma, showed progressive disease (PD) following
2 cycles of
combination treatment, leading to discontinuation of the IL-2 conjugate and
cetuximab combination
treatment. One subject with colon cancer showed disease progression after 5
cycles Two subjects
are ongoing: one at 3 cycles with squamous cell carcinoma of the lung and one
at 7 cycles with
small intestinal carcinoma.
[491] Peripheral CD8+ Tar cell counts were measured (FIGS. 4A-B). Prolonged
CD8+
expansion over baseline (e.g., greater than or equal to 2-fold change) was
observed at 3 weeks after
the previous dose in some subjects.
[492] Peripheral NK cell counts are shown in FIGS. 5A-B. An increase in NK
cell count was
observed in each subject. Subjects generally showed elevated NK cell counts
over baseline at 8
days and 3 weeks after the previous dose.
[493] Peripheral CD4+ Treg counts are shown in FIGS. 6A-B.
[494] Eosinophil counts were measured (FIGS. 7A-B). The measured values did
not exceed a
four-fold increase and were consistently below the range of 2328-15958
eosinophilsniL in patients
with IL-2 induced eosinophilia as reported in Pisani et al., Blood 1991 Sep
15;78(6):1538-44.
Lymphocyte counts were also measured (FIGS. 8A-B).
[495] Summary of Results and Discussion. All subjects tested had post-dose
peripheral
expansion of CD8+ T effector (Teff) cells, NK cells, and CD4+ Treg cells.
[496] An adverse event (AE) was any untoward medical occurrence in a
clinical investigation
subject administered a pharmaceutical product, regardless of causal
attribution. Dose-limiting
toxicities were defined as an AE occurring within Day 1 through Day 29
(inclusive) 1 day of a
treatment cycle that was not clearly or incontrovertibly solely related to an
extraneous cause and
that met at least one of the following criteria:
= Grade 3 neutropenia (absolute neutrophil count < 1000/mm3 > 500/mm3)
lasting > 7 days, or
Grade 4 neutropenia of any duration
= Grade 3+ febrile neutropenia
= Grade 4+ thrombocytopeni a (platelet count < 25,000/mm3)
= Grade 3+ thrombocytopenia (platelet count < 50,000-25,000/mm3) lasting >
5 days, or
associated with clinically significant bleeding or requiring platelet
transfusion
= Failure to meet recovery criteria of an absolute neutrophil count of at
least 1,000 cells/me
and a platelet count of at least 75,000 cells/me within 10 days
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= Any other grade 4+ hematologic toxicity lasting? 5 days
= Grade 3+ ALT or AST in combination with a bilirubin > 2 times ULN with no
evidence of
cholestasis or another cause such as viral infection or other drugs (i.e. Hy's
law)
= Grade 3 infusion-related reaction that occurs with premedication; Grade 4
infusion-related
reaction
= Grade 3 Vascular Leak Syndrome defined as hypotension associated with
fluid retention and
pulmonary edema
= Grade 3+ anaphylaxis
= Grade 3+ hypotension
= Grade 3+ AE that does not resolve to grade <2 within 7 days of starting
accepted standard
of care medical management
= Grade 3+ cytokine release syndrome
The following exceptions applied to non-hematologic AEs:
= Grade 3 fatigue, nausea, vomiting, or diarrhea that resolves to grade < 2
with optimal
medical management in < 3 days
= Grade 3 fever (as defined by > 40 C for < 24 hours)
= Grade 3 infusion-related reaction that occurs without premedication;
subsequent doses
should use premedication and if reaction recurs then it will be a DLT
= Grade 3 arthralgia or rash that resolves to grade < 2 within 7 days of
starting accepted
standard of care medical management (e.g., systemic corticosteroid therapy)
If a subject had grade 1 or 2 ALT or AST elevation at baseline considered
secondhand to liver
metastases, a grade 3 elevation must also be? 3 times baseline and last > 7
days.
[497] Serious AEs were defined as any AE that results in any of the
following outcomes: death;
life-threatening AE; inpatient hospitalization or prolongation of an existing
hospitalization; a
persistent or significant incapacity or substantial disruption of the ability
to conduct normal life
functions; or a congenital anomaly/birth defect. Important medical events that
may not result in
death, be life-threatening, or require hospitalization may be considered
serious when, based upon
appropriate medical judgment, they may jeopardize the subject and may require
medical or surgical
intervention to prevent one of the outcomes listed above Examples of such
medical events include
allergic bronchospasm requiring intensive treatment in an emergency room or at
home, blood
dyscrasias or convulsions that do not result in inpatient hospitalization, or
the development of drug
dependency or drug abuse.
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[498] There were no meaningful elevations in IL-5. There was no cumulative
toxicity. There
was no end organ toxicity. There was no QTc prolongation or other cardiac
toxicity. Overall, the
IL-2 conjugate was considered well-tolerated.
[499] Four of the 5 subjects had at least one treatment-emergent AE (TEAE).
Most of the
TEAEs were Grade 1-2, one subject had at least one Grade 3, and one subject at
least one Grade 4
TEAE. Four subjects had treatment related AEs. These included: one Grade 1
infusion reaction;
one Grade 1 nausea; one Grade 1 fatigue; one Grade 2 diarrhea; and one Grade 4
lymphocyte count
decrease. Two subjects had 3 unrelated SAEs: one dysphagia and spinal cord
compression; and one
pleural effusion. The TEAEs did not result in any drug discontinuations, no
dose reductions, no
DLTs, and no anaphylaxis or CRS. The treatment-related AEs resolved with
accepted standard of
care. TEAEs are detailed in Table 2, and treatment-related adverse events are
summarized in Table
3.
Table 2. Treatment Emergent Adverse Events: n = 5.
System Organ Class Grade 1
Grade 2 Grade 3 Grade 4 Grade 5
General disorders and
2/5 (40%)
0/5 (0%) 0/5 (0%) 0/5 (0%) 0/5 (0%)
administration site conditions
Gastrointestinal disorders 2/5 (40%) 0/5 (0%) 1/5 (20%) 0/5 (0%)
0/5 (0%)
Investigations 0/5 (0%)
0/5 (0%) 0/5 (0%) 1/5(20%) 0/5 (0%)
Infections and infestations 0/5 (0%) 1/5 (20%) 0/5 (0%) 0/5 (0%)
0/5 (0%)
Injury, Procedural
2/5 (40%)
0/5 (0%) 0/5 (0%) 0/5 (0%) 0/5 (0%)
Complications
Nervous system disorders 0/5 (0%) 0/5 (0%) 2/5(40%) 0/5 (0%)
0/5 (0%)
Skin and subcutaneous tissue
3/5 (60%) 0/5 (0%) 0/5 (0%) 0/5 (0%)
0/5 (0%)
disorders
Blood and lymphatic system
1/5 (20%) 0/5 (0%) 0/5 (0%) 0/5 (0%)
0/5 (0%)
disorders
Eye Disorders 1/5 (20%) 0/5 (0%) 0/5 (0%) 0/5 (0%)
0/5 (0%)
Metabolism and nutrition
1/5 (20%) 0/5 (0%) 0/5 (0%) 0/5 (0%)
0/5 (0%)
disorders
Respiratory, thoracic and
0/5 (0%) 1/5 (20%) 0/5 (0%) 0/5 (0%)
0/5 (0%)
mediastinal disorders
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System Organ Class Grade 1
Grade 2 Grade 3 Grade 4 Grade 5
Endocrine Disorders 0/5 (0%) 1/5 (20%) 0/5 (0%) 0/5 (0%)
0/5 (0%)
Mu s cul o skel etal and
1/5 (20%) 1/5 (20%) 0/5 (0%) 0/5 (0%)
0/5 (0%)
connective tissue disorders
Psychiatric disorders 1/5 (20%) 0/5 (0%) 0/5 (0%) 0/5 (0%)
0/5 (0%)
Reproductive and Breast
1/5 (20%) 0/5 (0%) 0/5 (0%) 0/5 (0%)
0/5 (0%)
Disorders
Renal and Urinary Disorders 1/5 (20%) 0/5 (0%) 0/5 (0%) 0/5 (0%)
0/5 (0%)
Table 3. Treatment Related Adverse Events: n = 5.
System Organ Class Grade 1
Grade 2 Grade 3 Grade 4 Grade 5
Endocrine Disorders 0/5 (0%) 1/5 (20%) 0/5 (0%) 0/5 (0%)
0/5 (0%)
General disorders and
2/5 (40%)
0/5 (0%) 0/5 (0%) 0/5 (0%) 0/5 (0%)
administration site conditions
Gastrointestinal disorders 1/5 (20%) 1/5(205%) 0/5 (0%) 0/5 (0%)
0/5 (0%)
Investigations 0/5 (0%)
0/5 (0%) 0/5 (0%) 1/5 (20%) 0/5 (0%)
Injury, Procedural
1/5 (20%) 0/5 (0%) 0/5 (0%) 0/5 (0%)
0/5 (0%)
Complications
Nervous system disorders 0/5 (0%) 0/5 (0%) 1/5(20%) 0/5 (0%)
0/5 (0%)
Second Cohort Using 24 lag/kg Dose of IL-2 Conjugate
15001 Results. The 3 subjects were human males with a median age of
71 years (ranging from
65-75 years). All subjects had an Eastern Cooperative Oncology Group (ECOG)
performance
status of 1. One subject had received 1 prior line of therapy, and a second
subject had received 4
prior lines of therapy. The cancers were gastric cancer (1 subject), head and
neck squa.mous cell
carcinoma (HNSCC) (1 subject), and colon cancer (1 subject). All of the
subjects had metastatic
disease. The subjects received the IL-2 conjugate (24 m.g/kg) and cetuximab
combination treatment
for 1 cycle (1 dose of the IL-2 conjugate). One subject showed progressive
disease (PD) following
the first cycle of combination treatment, preventing administration of a
further treatment dose of the
IL-2 conjugate and cetuximab combination treatment.
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[501] Two of the subjects experienced at least one TEAE. One of the
subjects experienced at
least one Grade 3 treatment-related AE, which was Grade 3 chills. None of the
subjects had related
SAEs. No DLTs were observed and no drug discontinuations resulted from the
TEAEs. TEAEs are
detailed in Table 4, and treatment-related adverse events are summarized in
Table 5.
Table 4. Treatment Emergent Adverse Events: n = 3.
System Organ Class Grade 1
Grade 2 Grade 3 Grade 4 Grade 5
Gastrointestinal disorders 0/3 (0%) 1/3 (33.3%)
0/3 (0%) 0/3 (0%) 0/3 (0%)
General disorders and
1/3 (33.3%) 0/3 (0%) 1/3 (33.3%) 0/3 (0%) 0/3
(0%)
administration conditions
Infections and infestations 0/3 (0%) 2/3 (66.7%)
0/3 (0%) 0/3 (0%) 0/3 (0%)
Metabolism and nutrition
0/3 (0%) 2/3 (66.7%) 0/3 (0%) 0/3 (0%) 0/3 (0%)
disorders
Musculoskeletal and
1/3 (33.3%) 0/3 (0%) 0/3 (0%) 0/3 (0%) 0/3
(0%)
connective tissue disorders
Nervous system disorders 0/3 (0%) 1/3 (33.3%)
0/3 (0%) 0/3 (0%) 0/3 (0%)
Renal and urinary disorders 1/3 (33.3%) 0/3 (0%) 0/3 (0%) 0/3 (0%) 0/3
(0%)
Respiratory and mediastinal 0/3 (0%) 1/3 (33.3%) 0/3 (0%) 0/3 (0%)
0/3 (0%)
disorders
Skin and subcutaneous
1/3 (33.3%) 1/3 (33.3%) 0/3 (0%) 0/3 (0%) 0/3 (0%)
tissue disorders
Table 5. Treatment Related Adverse Events: n = 3.
System Organ Class Grade 1
Grade 2 Grade 3 Grade 4 Grade 5
Gastrointestinal disorders 0/3 (0%) 1/3 (33,3%)
0/3 (0%) 0/3 (0%) 0/3 (0%)
General disorders and
administration conditions 1/3 (33.3%) 0/3 (0%)
1/3 (33.3%) 0/3 (0%) 0/3 (0%)
Infections and infestations 0/3 (0%) 2/3 (66.7%)
0/3 (0%) 0/3 (0%) 0/3 (0%)
Metabolism and nutrition
0/3 (0%) 2/3 (66.7%) 0/3 (0%) 0/3 (0%) 0/3 (0%)
disorders
Nervous system disorders 0/3 (0%) 1/3 (33.3%)
0/3 (0%) 0/3 (0%) 0/3 (0%)
Respiratory and mediastinal
disorders 0/3 (0%) 1/3 (33.3%)
0/3 (0%) 0/3 (0%) 0/3 (0%)
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System Organ Class Grade 1 Grade 2
Grade 3 Grade 4 Grade 5
Skin and subcutaneous
0/3 (0%) 1/3 (33.3%)
0/3 (0%) 0/3 (0%) 0/3 (0%)
tissue disorders
15021 Although the foregoing invention has been described in some
detail by way of illustration
and example for purposes of clarity of understanding, the descriptions and
examples should not be
construed as limiting the scope of the invention. Numerous variations,
changes, and substitutions
will now occur to those skilled in the art without departing from the
invention. It should be
understood that various alternatives to the embodiments of the invention
described herein may be
employed in practicing the invention. It is intended that the following claims
define the scope of
the invention and that methods and structures within the scope of these claims
and their equivalents
be covered thereby. The disclosures of all patent and scientific literature
cited herein are expressly
incorporated herein in their entirety by reference. To the extent any material
incorporated herein by
reference is inconsistent with the express content of this disclosure, the
express content controls.
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