Note: Descriptions are shown in the official language in which they were submitted.
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COMPOSITIONS AND METHODS RELATED TO ENGINEERED Fc-ANTIGEN BINDING DOMAIN
CONSTRUCTS TARGETED TO CD38
SUMMARY
CD38 is a type II transmembrane glycoprotein expressed at high density on
normal and
malignant plasmablasts and plasma cells and at low levels on certain lymphoid
and myeloid cells.
Darzalex (daratumumab) is an anti-CD38 cytolytic monoclonal antibody approved
for relapsed, refractory
multiple myeloma and for newly-diagnosed multiple myeloma.
SUMMARY OF THE DISCLOSURE
The present disclosure features compositions and methods for combining a CD38
binding domain
with at least two Fc domains to generate new therapeutics with unique
biological activity.
In some instances, the present disclosure contemplates combining a CD38
binding domain of a
known CD38 targeted single Fc-domain containing therapeutic, e.g., a known
therapeutic CD38 antibody,
with at least two Fc domains to generate a novel therapeutic with a biological
activity greater than that of
a known CD38 antibody. To generate such constructs, the disclosure provides
various methods for the
assembly of constructs having at least two, e.g., multiple, Fc domains, and to
control homodimerization
and heterodimerization of such, to assemble molecules of discrete size from a
limited number of
polypeptides. The properties of these constructs allow for the efficient
generation of substantially
homogenous pharmaceutical compositions. Such homogeneity in a pharmaceutical
composition is
desirable in order to ensure the safety, efficacy, uniformity, and reliability
of the pharmaceutical
composition.
In a first aspect, the disclosure features an Fc-antigen binding domain
construct including
enhanced effector function, where the Fc-antigen binding domain construct
includes a CD38 binding
domain and a first Fc domain joined to a second Fc domain by a linker, where
the Fc-antigen binding
domain construct has enhanced effector function in an antibody-dependent
cytotoxicity (ADCC) assay, an
antibody-dependent cellular phagocytosis (ADCP), and/or complement-dependent
cytotoxicity (CDC)
assay relative to a construct having a single Fc domain and the CD38 binding
domain.
In a second aspect, the disclosure features a composition including a
substantially homogenous
population of an Fc-antigen binding domain construct including a CD38 binding
domain and a first Fc
domain joined to a second Fc domain by a linker.
In a third aspect, the disclosure features an Fc-antigen binding domain
construct including a
CD38 binding domain and a first Fc domain joined to a second Fc domain by a
linker, where the Fc-
antigen binding domain construct includes a biological activity that is not
exhibited by a construct having a
single Fc domain and the CD38 binding domain.
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In a fourth aspect, the disclosure features a composition including a
substantially homogenous
population of an Fc-antigen binding domain construct including a) a first
polypeptide including i) a first Fc
domain monomer, ii) a second Fc domain monomer, and iii) a linker joining the
first Fc domain monomer
and the second Fc domain monomer; b) a second polypeptide including a third Fc
domain monomer; c) a
third polypeptide including a fourth Fc domain monomer; and d) a CD38 binding
domain joined to the first
polypeptide, second polypeptide, or third polypeptide; where the first Fc
domain monomer and the third
Fc domain monomer combine to form a first Fc domain and the second Fc domain
monomer and the
fourth Fc domain monomer combine to form a second Fc domain.
In some embodiments of the fourth aspect, the CD38 binding domain is joined to
the first
polypeptide and the second polypeptide or the third polypeptide, or to the
second polypeptide and the
third polypeptide, or the CD38binding domain is joined to the first
polypeptide, the second polypeptide,
and the third polypeptide.
In a fifth aspect, the disclosure features an Fc-antigen binding domain
construct including
enhanced effector function, where the Fc-antigen binding domain construct
includes: a) a first polypeptide
including i) a first Fc domain monomer, ii) a second Fc domain monomer, and
iii) a linker joining the first
Fc domain monomer and the second Fc domain monomer; b) a second polypeptide
including a third Fc
domain monomer; c) a third polypeptide including a fourth Fc domain monomer;
and d) a CD38 binding
domain joined to the first polypeptide, second polypeptide, or third
polypeptide; where the first Fc domain
monomer and the third Fc domain monomer combine to form a first Fc domain and
the second Fc domain
.. monomer and the fourth Fc domain monomer combine to form a second Fc
domain, and where the Fc-
antigen binding domain construct has enhanced effector function in an antibody-
dependent cytotoxicity
(ADCC) assay, an antibody-dependent cellular phagocytosis (ADCP), and/or
complement-dependent
cytotoxicity (CDC) assay relative to a construct having a single Fc domain and
the CD38 binding domain.
In some embodiments of the fifth aspect, the single Fc domain construct is an
antibody.
In a sixth aspect, the disclosure features an Fc-antigen binding domain
construct including: a) a
first polypeptide including i) a first Fc domain monomer, ii) a second Fc
domain monomer, and iii) a linker
joining the first Fc domain monomer and the second Fc domain monomer; b) a
second polypeptide
including a third Fc domain monomer; c) a third polypeptide including a fourth
Fc domain monomer; and
d) a CD38 binding domain joined to the first polypeptide, second polypeptide,
or third polypeptide;
where the first Fc domain monomer and the third Fc domain monomer combine to
form a first Fc domain
and the second Fc domain monomer and the fourth Fc domain monomer combine to
form a second Fc
domain, and where the Fc-antigen binding domain construct includes a
biological activity that is not
exhibited by a construct having a single Fc domain and the CD38 binding
domain.
In some embodiments of the sixth aspect, the biological activity is an Fc
receptor mediated
effector function, such as ADCC, ADCP and/or CDC activity (e.g., ADCC and ADCP
activity, ADCC and
CDC activity, ADCP and CDC activity, or ADCC, ADCP, and CDC activity).
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In a seventh aspect, the disclosure features an Fc-antigen binding domain
construct including: a)
a first polypeptide including: i) a first Fc domain monomer, ii) a second Fc
domain monomer, and iii) a
spacer joining the first Fc domain monomer and the second Fc domain monomer;
b) a second
polypeptide including a third Fc domain monomer; c) a third polypeptide
including a fourth Fc domain
monomer; and d) a CD38 binding domain joined to the first polypeptide, second
polypeptide, or third
polypeptide; where the first Fc domain monomer and the third Fc domain monomer
combine to form a
first Fc domain and the second Fc domain monomer and the fourth Fc domain
monomer combine to form
a second Fc domain.
In some embodiments of the fifth, sixth, and seventh aspects of the
disclosure, the CD38 binding
domain is joined to the first polypeptide and the second polypeptide or the
third polypeptide, or to the
second polypeptide and the third polypeptide, or the CD38 binding domain is
joined to the first
polypeptide, the second polypeptide, and the third polypeptide.
In some embodiments of the first, second, third and fourth aspects of the
disclosure, the CD38
binding domain is a Fab or the VH of a Fab.
In some embodiments of the fourth, fifth, sixth, and seventh aspects of the
disclosure, the binding
domain is part of the amino acid sequence of the first, second, or third
polypeptide, and, in some
embodiments, CD38 binding domain is a scFv.
In some embodiments of the fourth, fifth, sixth, and seventh aspects of the
disclosure, the CD38
binding domain includes a VH domain and a CH1 domain, and where the VH and CH1
domains are part of
the amino acid sequence of the first, second, or third polypeptide. In some
embodiments, the CD38
binding domain further includes a VL domain, where, in some embodiments the Fc-
antigen binding
domain construct includes a fourth polypeptide including the VL domain. In
some embodiments, the VH
domain includes a set of CDR-H1, CDR-H2 and CDR-H3 sequences set forth in
Table 1, the VH domain
includes CDR-H1, CDR-H2, and CDR-H3 of a VH domain including a sequence of an
antibody set forth in
Table 2, the VH domain includes CDR-H1, CDR-H2, and CDR-H3 of a VH sequence of
an antibody set
forth in Table 2, and the VH sequence, excluding the CDR-H1, CDR-H2, and CDR-
H3 sequence, is at
least 95% identical, at least 97% identical, at least 99% identical, or at
least 99.5% identical to the VH
sequence of an antibody set forth in Table 2, or the VH domain includes a VH
sequence of an antibody set
forth in Table 2.
In some embodiments of the fourth, fifth, sixth, and seventh aspects of the
disclosure, the CD38
binding domain includes a set of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and
CDR-L3 sequences
set forth in Table 1, CD38 binding domain includes CDR-H1, CDR-H2, CDR-H3, CDR-
L1, CDR-L2, and
CDR-L3 sequences from a set of a VH and a VL sequence of an antibody set forth
in Table 2, the CD38
binding domain includes a VH domain including CDR-H1, CDR-H2, and CDR-H3 of a
VH sequence of an
antibody set forth in Table 2, and a VL domain including CDR-L1, CDR-L2, and
CDR-L3 of a VL sequence
of an antibody set forth in Table 2, where the VH and the VL domain sequences,
excluding the CDR-H1,
CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 sequences, are at least 95%
identical, at least 97%
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identical, at least 99% identical, or at least 99.5% identical to the VH and
VL sequences of an antibody set
forth in Table 2, or CD38 binding domain includes a set of a VH and a VL
sequences of an antibody set
forth in Table 2.
In some embodiments of the fourth, fifth, sixth, and seventh aspects of the
disclosure, the Fc-
antigen binding domain construct, further includes an IgG CL antibody constant
domain and an IgG CH1
antibody constant domain, where the IgG CH1 antibody constant domain is
attached to the N-terminus of
the first polypeptide or the second polypeptide by way of a linker.
In some embodiments of the fourth, fifth, sixth, and seventh aspects of the
disclosure, the first Fc
domain monomer and the third Fc domain monomer include complementary
dimerization selectivity
modules that promote dimerization between the first Fc domain monomer and the
third Fc domain
monomer.
In some embodiments of the fourth, fifth, sixth, and seventh aspects of the
disclosure, the second
Fc domain monomer and the fourth Fc domain monomer include complementary
dimerization selectivity
modules that promote dimerization between the second Fc domain monomer and the
fourth Fc domain
monomer.
In some embodiments of the fourth, fifth, sixth, and seventh aspects of the
disclosure, the
dimerization selectivity modules include an engineered cavity into the CH3
domain of one of the Fc
domain monomers and an engineered protuberance into the CH3 domain of the
other of the Fc domain
monomers, where the engineered cavity and the engineered protuberance are
positioned to form a
protuberance-into-cavity pair of Fc domain monomers. In some embodiments, the
engineered
protuberance includes at least one modification selected from S354C, T366W,
T366Y, T394W, T394F,
and F405W, and the engineered cavity includes at least one modification
selected from Y349C, T366S,
L368A, Y407V, Y407T, Y407A, F405A, and T394S. In some embodiments, one of the
Fc domain
monomers includes Y407V and Y349C and the other of the Fc domain monomers
includes T366W and
S354C.
In some embodiments of the fourth, fifth, sixth, and seventh aspects of the
disclosure, the
dimerization selectivity modules include a negatively-charged amino acid into
the CH3 domain of one of
the domain monomers and a positively-charged amino acid into the CH3 domain of
the other of the Fc
domain monomers, where the negatively-charged amino acid and the positively-
charged amino acid are
positioned to promote formation of an Fc domain. In some embodiments, each of
the first Fc domain
monomer and third Fc domain monomer includes D399K and either K409D or K409E,
each of the first Fc
domain monomer and third Fc domain monomer includes K392D and D399K, each of
the first Fc domain
monomer and third Fc domain monomer includes E357K and K370E, each of the
first Fc domain
monomer and third Fc domain monomer includes D356K and K439D, each of the
first Fc domain
monomer and third Fc domain monomer includes K392E and D399K, each of the
first Fc domain
monomer and third Fc domain monomer includes E357K and K370D, each of the
first Fc domain
monomer and third Fc domain monomer includes D356K and K439E, each of the
second Fc domain
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monomer and fourth Fc domain monomer includes S354C and T366W and the third
and fourth
polypeptides each include Y349C, T366S, L368A, and Y407V, each of the third
and fourth polypeptides
includes S354C and T366W and the second Fc domain monomer and fourth Fc domain
monomer each
include Y349C, T366S, L368A, and Y407V, each of the second Fc domain monomer
and fourth Fc
domain monomer includes E357K or E357R and the third and fourth polypeptides
each include K370D or
K370E, each of the second Fc domain monomer and fourth Fc domain monomer
include K370D or
K370E and the third and fourth polypeptides each include E357K or 357R, each
of the second Fc domain
monomer and fourth Fc domain monomer include K409D or K409E and the third and
fourth polypeptides
each include D399K or D399R, or each of the second Fc domain monomer and
fourth Fc domain
monomer include D399K or D399R and the third and fourth polypeptides each
include K409D or K409E.
In some embodiments of the fourth, fifth, sixth, and seventh aspects of the
disclosure, the second
polypeptide and the third polypeptide have the same amino acid sequence.
In some embodiments of the fourth, fifth, sixth, and seventh aspects of the
disclosure, one or
more linker in the Fc-antigen binding domain construct is a bond.
In some embodiments of the fourth, fifth, sixth, and seventh aspects of the
disclosure, one or
more linker in the Fc-antigen binding domain construct is a spacer. In some
embodiments, the spacer
includes a polypeptide having the sequence GGGGGGGGGGGGGGGGGGGG, GGGGS, GGSG,
SGGG, GSGS, GSGSGS, GSGSGSGS, GSGSGSGSGS, GSGSGSGSGSGS, GGSGGS,
GGSGGSGGS, GGSGGSGGSGGS, GGSG, GGSG, GGSGGGSG,
GGSGGGSGGGSGGGGGSGGGGSGGGGSGGGGS, GENLYFQSGG, SACYCELS, RSIAT,
RPACKIPNDLKQKVMNH, GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG,
AAANSSIDLISVPVDSR, GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS,
GGGSGGGSGGGS, SGGGSGGGSGGGSGGGSGGG, GGSGGGSGGGSGGGSGGS, GGGG,
GGGGGGGG, GGGGGGGGGGGG, or GGGGGGGGGGGGGGGG. In some embodiments, the spacer
is a glycine spacer, for example, one consisting of 4 to 30, 8 to 30, or 12 to
30 glycine residues, such as a
spacer consisting of 20 glycine residues.
In some embodiments of the fourth, fifth, sixth, and seventh aspects of the
disclosure, the CD38
binding domain is joined to the Fc domain monomer by a linker. In some
embodiments, the linker is a
spacer.
In some embodiments of the fourth, fifth, sixth, and seventh aspects of the
disclosure, at least
one of the Fc domains includes at least one amino acid modification at EU
position 1253. In some
embodiments, the each amino acid modification at position 1253 is
independently selected from 1253A,
1253C, 1253D, 1253E, 1253F, 1253G, 1253H, 12531, 1253K, 1253L, 1253M, 1253N,
1253P, 1253Q, 1253R,
1253S, 1253T, 1253V, 1253W, and 1253Y. In some embodiments, each amino acid
modification at position
1253 is 1253A.
In some embodiments of the fourth, fifth, sixth, and seventh aspects of the
disclosure, at least
one of the Fc domains includes at least one amino acid modification at EU
position R292. In some
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embodiments, each amino acid modification at position R292 is independently
selected from R292D,
R292E, R292L, R292P, R292Q, R292R, R292T, and R292Y. In some embodiments, each
amino acid
modification at position R292 is R292P.
In some embodiments of the fourth, fifth, sixth, and seventh aspects of the
disclosure, one or
more of the Fc domain monomers includes an IgG hinge domain, an IgG CH2
antibody constant domain,
and an IgG CH3 antibody constant domain. In some embodiments, each of the Fc
domain monomers
includes an IgG hinge domain, an IgG CH2 antibody constant domain, and an IgG
CH3 antibody constant
domain. In some embodiments, the IgG is of a subtype selected from the group
consisting of IgG1,
IgG2a, IgG2b, IgG3, and IgG4.
In some embodiments of the fourth, fifth, sixth, and seventh aspects of the
disclosure, the N-
terminal Asp in each of the fourth, fifth, sixth, and seventh polypeptides is
mutated to Gln.
In some embodiments of the fourth, fifth, sixth, and seventh aspects of the
disclosure, one or
more of the fourth, fifth, sixth, and seventh polypeptides lack a C-terminal
lysine. In some embodiments,
each of the fourth, fifth, sixth, and seventh polypeptides lacks a C-terminal
lysine.
In some embodiments of the fourth, fifth, sixth, and seventh aspects of the
disclosure, the Fc-
antigen binding domain construct further includes an albumin-binding peptide
joined to the N-terminus or
C-terminus of one or more of the polypeptides by a linker.
In an eighth aspect, the disclosure features a cell culture medium including a
population of Fc-
antigen binding domain constructs, where at least 50% of the Fc-antigen
binding domain constructs, on a
molar basis, are structurally identical, and where the Fc-antigen binding
domain constructs are present in
the culture medium at a concentration of at least 0.1 mg/L, 10 mg/L, 25 mg/L,
50 mg/L, 75 mg/L, or 100
mg/L.
In some embodiments of the eighth aspect of the disclosure, at least 75%%, at
least 85%, or at
least 95% of the Fc-antigen binding domain constructs, on a molar basis, are
structurally identical.
In a ninth aspect, the disclosure features a cell culture medium including a
population of Fc-
antigen binding domain constructs, where at least 50% of the Fc-antigen
binding domain constructs, on a
molar basis, include: a) a first polypeptide including i) a first Fc domain
monomer, ii) a second Fc domain
monomer, and iii) a linker joining the first Fc domain monomer and the second
Fc domain monomer; b) a
second polypeptide including a third Fc domain monomer; c) a third polypeptide
including a fourth Fc
domain monomer; and d) a CD38 binding domain joined to the first polypeptide,
second polypeptide, or
third polypeptide; where the first Fc domain monomer and the third Fc domain
monomer combine to form
a first Fc domain and the second Fc domain monomer and the fourth Fc domain
monomer combine to
form a second Fc domain.
In some embodiments of the ninth aspect of the disclosure at least 75%, at
least 85%, or at least
95% of the Fc-antigen binding domain constructs, on a molar basis, include the
first Fc domain, the
second Fc domain, and the CD38 binding domain.
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In a tenth aspect, the disclosure features a method of manufacturing an Fc-
antigen binding
domain construct, the method including: a) culturing a host cell expressing:
(1) a first polypeptide
including i) a first Fc domain monomer, ii) a second Fc domain monomer, and
iii) a linker joining the first
Fc domain monomer and the second Fc domain monomer; (2) a second polypeptide
including a third Fc
domain monomer; (3) a third polypeptide including a fourth Fc domain monomer;
and (4) a CD38 binding
domain; where the first Fc domain monomer and the third Fc domain monomer
combine to form a first Fc
domain and the second Fc domain monomer and the fourth Fc domain monomer
combine to form a
second Fc domain; where the CD38 binding domain is joined to the first
polypeptide, second polypeptide,
or third polypeptide, thereby forming an Fc-antigen binding domain construct;
and where at least 50% of
the Fc-antigen binding domain constructs in a cell culture supernatant, on a
molar basis, are structurally
identical, and b) purifying the Fc-antigen binding domain construct from the
cell culture supernatant.
In some embodiments of the ninth and tenth aspects of the disclosure, the CD38
binding domain
is joined to the first polypeptide and the second polypeptide or the third
polypeptide, or to the second
polypeptide and the third polypeptide, or the CD38 binding domain is joined to
the first polypeptide, the
second polypeptide, and the third polypeptide.
In some embodiments of the ninth and tenth aspects of the disclosure, the CD38
binding domain
is a Fab or a VH.
In some embodiments of the ninth and tenth aspects of the disclosure, the CD38
binding domain
is part of the amino acid sequence of the first, second, or third polypeptide,
and, in some embodiments,
the CD38 binding domain is a scFv.
In some embodiments of the ninth and tenth aspects of the disclosure, CD38
binding domain
includes a VH domain and a CH1 domain, and where the VH and CH1 domains are
part of the amino acid
sequence of the first, second, or third polypeptide. In some embodiments, the
CD38 binding domain
further includes a VL domain, where, in some embodiments the Fc-antigen
binding domain construct
includes a fourth polypeptide including the VL domain. In some embodiments,
the VH domain includes a
set of CDR-H1, CDR-H2 and CDR-H3 sequences set forth in Table 1, the VH domain
includes CDR-H1,
CDR-H2, and CDR-H3 of a VH domain including a sequence of an antibody set
forth in Table 2, the VH
domain includes CDR-H1, CDR-H2, and CDR-H3 of a VH sequence of an antibody set
forth in Table 2,
and the VH sequence, excluding the CDR-H1, CDR-H2, and CDR-H3 sequence, is at
least 95% identical,
at least 97% identical, at least 99% identical, or at least 99.5% identical to
the VH sequence of an
antibody set forth in Table 2, or the VH domain includes a VH sequence of an
antibody set forth in Table 2.
In some embodiments of the ninth and tenth aspects of the disclosure, the CD38
binding domain
includes a set of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 sequences
set forth in
Table 1, CD38 binding domain includes CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2,
and CDR-L3
sequences from a set of a VH and a VL sequences of an antibody set forth in
Table 2, CD38 binding
domain includes a VH domain including CDR-H1, CDR-H2, and CDR-H3 of a VH
sequence of an antibody
set forth in Table 2, and a VL domain including CDR-L1, CDR-L2, and CDR-L3 of
a VL sequence of an
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antibody set forth in Table 2, where the VH and the VL domain sequences,
excluding the CDR-H1, CDR-
H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 sequences, are at least 95% identical,
at least 97%
identical, at least 99% identical, or at least 99.5% identical to the VH and
VL sequences of an antibody set
forth in Table 2, or the CD38 binding domain includes a set of a VH and a VL
sequence of an antibody set
forth in Table 2.
In some embodiments of the ninth and tenth aspects of the disclosure, the Fc-
antigen binding
domain construct, further includes an IgG CL antibody constant domain and an
IgG CH1 antibody constant
domain, where the IgG CH1 antibody constant domain is attached to the N-
terminus of the first
polypeptide or the second polypeptide by way of a linker.
In some embodiments of the ninth and tenth aspects of the disclosure, the
first Fc domain
monomer and the third Fc domain monomer include complementary dimerization
selectivity modules that
promote dimerization between the first Fc domain monomer and the third Fc
domain monomer.
In some embodiments of the ninth and tenth aspects of the disclosure, the
second Fc domain
monomer and the fourth Fc domain monomer include complementary dimerization
selectivity modules
that promote dimerization between the second Fc domain monomer and the fourth
Fc domain monomer.
In some embodiments of the ninth and tenth aspects of the disclosure, the
dimerization selectivity
modules include an engineered cavity into the CH3 domain of one of the Fc
domain monomers and an
engineered protuberance into the CH3 domain of the other of the Fc domain
monomers, where the
engineered cavity and the engineered protuberance are positioned to form a
protuberance-into-cavity pair
of Fc domain monomers. In some embodiments, the engineered protuberance
includes at least one
modification selected from S354C, T366W, T366Y, T394W, T394F, and F405W, and
the engineered
cavity includes at least one modification selected from Y349C, T366S, L368A,
Y407V, Y407T, Y407A,
F405A, and T394S. In some embodiments, one of the Fc domain monomers includes
Y407V and Y349C
and the other of the Fc domain monomers includes T366W and S354C.
In some embodiments of the ninth and tenth aspects of the disclosure, the
dimerization selectivity
modules include a negatively-charged amino acid into the CH3 domain of one of
the domain monomers
and a positively-charged amino acid into the CH3 domain of the other of the Fc
domain monomers, where
the negatively-charged amino acid and the positively-charged amino acid are
positioned to promote
formation of an Fc domain. In some embodiments, each of the first Fc domain
monomer and third Fc
domain monomer includes D399K and either K409D or K409E, each of the first Fc
domain monomer and
third Fc domain monomer includes K392D and D399K, each of the first Fc domain
monomer and third Fc
domain monomer includes E357K and K370E, each of the first Fc domain monomer
and third Fc domain
monomer includes D356K and K439D, each of the first Fc domain monomer and
third Fc domain
monomer includes K392E and D399K, each of the first Fc domain monomer and
third Fc domain
monomer includes E357K and K370D, each of the first Fc domain monomer and
third Fc domain
monomer includes D356K and K439E, each of the second Fc domain monomer and
fourth Fc domain
monomer includes S354C and T366W and the third and fourth polypeptides each
include Y349C, T366S,
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L368A, and Y407V, each of the third and fourth polypeptides includes S354C and
T366W and the second
Fc domain monomer and fourth Fc domain monomer each include Y349C, T366S,
L368A, and Y407V,
each of the second Fc domain monomer and fourth Fc domain monomer includes
E357K or E357R and
the third and fourth polypeptides each include K370D or K370E, each of the
second Fc domain monomer
.. and fourth Fc domain monomer include K370D or K370E and the third and
fourth polypeptides each
include E357K or 357R, each of the second Fc domain monomer and fourth Fc
domain monomer include
K409D or K409E and the third and fourth polypeptides each include D399K or
D399R, or each of the
second Fc domain monomer and fourth Fc domain monomer include D399K or D399R
and the third and
fourth polypeptides each include K409D or K409E.
In some embodiments of the ninth and tenth aspects of the disclosure, the
second polypeptide
and the third polypeptide have the same amino acid sequence.
In some embodiments of the ninth and tenth aspects of the disclosure, one or
more linker in the
Fc-antigen binding domain construct is a bond.
In some embodiments of the ninth and tenth aspects of the disclosure, one or
more linker in the
.. Fc-antigen binding domain construct is a spacer. In some embodiments, the
spacer includes a
polypeptide having the sequence GGGGGGGGGGGGGGGGGGGG, GGGGS, GGSG, SGGG, GSGS,
GSGSGS, GSGSGSGS, GSGSGSGSGS, GSGSGSGSGSGS, GGSGGS, GGSGGSGGS,
GGSGGSGGSGGS, GGSG, GGSG, GGSGGGSG,
GGSGGGSGGGSGGGGGSGGGGSGGGGSGGGGS, GENLYFQSGG, SACYCELS, RSIAT,
RPACKIPNDLKQKVMNH, GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG,
AAANSSIDLISVPVDSR, GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS,
GGGSGGGSGGGS, SGGGSGGGSGGGSGGGSGGG, GGSGGGSGGGSGGGSGGS, GGGG,
GGGGGGGG, GGGGGGGGGGGG, or GGGGGGGGGGGGGGGG. In some embodiments, the spacer
is a glycine spacer, for example, one consisting of 4 to 30, 8 to 30, or 12 to
30 glycine residues, such as a
.. spacer consisting of 20 glycine residues.
In some embodiments of the ninth and tenth aspects of the disclosure, the CD38
binding domain
is joined to the Fc domain monomer by a linker. In some embodiments, the
linker is a spacer.
In some embodiments of the ninth and tenth aspects of the disclosure, at least
one of the Fc
domains includes at least one amino acid modification at position 1253. In
some embodiments, the each
.. amino acid modification at position 1253 is independently selected from
1253A, 1253C, 1253D, 1253E,
1253F, 1253G, 1253H, 12531, 1253K, 1253L, 1253M, 1253N, 1253P, 1253Q, 1253R,
1253S, 1253T, 1253V,
1253W, and 1253Y. In some embodiments, each amino acid modification at
position 1253 is 1253A.
In some embodiments of the ninth and tenth aspects of the disclosure, at least
one of the Fc
domains includes at least one amino acid modification at position R292. In
some embodiments, each
amino acid modification at position R292 is independently selected from R292D,
R292E, R292L, R292P,
R292Q, R292R, R292T, and R292Y. In some embodiments, each amino acid
modification at position
R292 is R292P.
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In some embodiments of the ninth and tenth aspects of the disclosure, one or
more of the Fc
domain monomers includes an IgG hinge domain, an IgG CH2 antibody constant
domain, and an IgG CH3
antibody constant domain. In some embodiments, each of the Fc domain monomers
includes an IgG
hinge domain, an IgG CH2 antibody constant domain, and an IgG CH3 antibody
constant domain. In
some embodiments, the IgG is of a subtype selected from the group consisting
of IgG1, IgG2a, IgG2b,
IgG3, and IgG4.
In some embodiments of the ninth and tenth aspects of the disclosure, the N-
terminal Asp in each
of the first, second, third, and fourth polypeptides is mutated to Gln.
In some embodiments of the ninth and tenth aspects of the disclosure, one or
more of the first,
second, third, and fourth polypeptides lack a C-terminal lysine. In some
embodiments, each of the first,
second, third, and fourth polypeptides lacks a C-terminal lysine.
In some embodiments of the ninth and tenth aspects of the disclosure, the Fc-
antigen binding
domain construct further includes an albumin-binding peptide joined to the N-
terminus or C-terminus of
one or more of the polypeptides by a linker.
In some embodiments of the eleventh aspect of the disclosure, the first Fc
domain monomer and
the third Fc domain monomer include complementary dimerization selectivity
modules that promote
dimerization between the first Fc domain monomer and the third Fc domain
monomer, where the second
Fc domain monomer and the fourth Fc domain monomer include complementary
dimerization selectivity
modules that promote dimerization between the second Fc domain monomer and the
fourth Fc domain
monomer, and where the second polypeptide and the third polypeptide have
different amino acid
sequences.
In some embodiments of the eleventh aspect of the disclosure, the first CD38
binding domain is
joined to the first polypeptide and the second CD38 binding domain is joined
to the second polypeptide
and the third polypeptide.
In some embodiments of the eleventh aspect of the disclosure, each of the
second Fc domain
monomer and the fourth Fc domain monomer includes E357K and K370D, and each of
the first Fc
domain monomer and the third Fc domain monomer includes K370D and E357K.
In some embodiments of the twelfth aspect of the disclosure, the first Fc
domain monomer and
the third Fc domain monomer include complementary dimerization selectivity
modules that promote
dimerization between the first Fc domain monomer and the third Fc domain
monomer, where the second
Fc domain monomer and the fourth Fc domain monomer include complementary
dimerization selectivity
modules that promote dimerization between the second Fc domain monomer and the
fourth Fc domain
monomer, and where the second polypeptide and the third polypeptide have
different amino acid
sequences.
In some embodiments of the twelfth aspect of the disclosure, each of the
second Fc domain
monomer and the fourth Fc domain monomer includes D399K and K409D, and each of
the first Fc
domain monomer and the third Fc domain monomer includes E357K and K370D.
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In some embodiments of the eleventh and twelfth aspects of the disclosure, the
first or CD38
binding domain is a Fab or a VH domain. In some embodiments of the eleventh
and twelfth aspects of the
disclosure, the first and second CD38 binding domain is a Fab. In some
embodiments of the ninth aspect
of the disclosure, the first, second, and third CD38 binding domain is a Fab
or a VH domain.
In some embodiments of the eleventh and twelfth aspects of the disclosure, the
first or second
CD38 binding domain is a scFv. In some embodiments of the eleventh and twelfth
aspects of the
disclosure, the first and second CD38 binding domain is a scFv. In some
embodiments of the ninth aspect
of the disclosure, the first, second, and third CD38 binding domain is a scFv.
In some embodiments of the eleventh aspect of the disclosure, the first or
second CD38 domain
.. includes a VH domain and a CH1 domain, and where the VH and CH1 domains are
part of the amino acid
sequence of the first, second, or third polypeptide. In some embodiments, the
CD38 binding domain
further includes a VL domain, where, in some embodiments the Fc-antigen
binding domain construct
includes a fourth polypeptide including the VL domain. In some embodiments,
the VH domain includes a
set of CDR-H1, CDR-H2 and CDR-H3 sequences set forth in Table 1, the VH domain
includes CDR-H1,
CDR-H2, and CDR-H3 of a VH domain including a sequence of an antibody set
forth in Table 2, the VH
domain includes CDR-H1, CDR-H2, and CDR-H3 of a VH sequence of an antibody set
forth in Table 2,
and the VH sequence, excluding the CDR-H1, CDR-H2, and CDR-H3 sequence, is at
least 95% identical,
at least 97% identical, at least 99% identical, or at least 99.5% identical to
the VH sequence of an
antibody set forth in Table 2, or the VH domain includes a VH sequence of an
antibody set forth in Table 2.
In some embodiments of the twelfth aspect of the disclosure, the first,
second, or third CD38
binding domain includes a VH domain and a CH1 domain, and where the VH and CH1
domains are part of
the amino acid sequence of the first, second, or third polypeptide. In some
embodiments, the CD38
binding domain further includes a VL domain, where, in some embodiments the Fc-
antigen binding
domain construct includes a fourth polypeptide including the VL domain. In
some embodiments, the VH
domain includes a set of CDR-H1, CDR-H2 and CDR-H3 sequences set forth in
Table 1, the VH domain
includes CDR-H1, CDR-H2, and CDR-H3 of a VH domain including a sequence of an
antibody set forth in
Table 2, the VH domain includes CDR-H1, CDR-H2, and CDR-H3 of a VH sequence of
an antibody set
forth in Table 2, and the VH sequence, excluding the CDR-H1, CDR-H2, and CDR-
H3 sequence, is at
least 95% identical, at least 97% identical, at least 99% identical, or at
least 99.5% identical to the VH
sequence of an antibody set forth in Table 2, or the VH domain includes a VH
sequence of an antibody set
forth in Table 2.
In some embodiments of the eleventh aspect of the disclosure, the first or
second CD38 binding
domain includes a set of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3
sequences set
forth in Table 1, the CD38 binding domain includes CDR-H1, CDR-H2, CDR-H3, CDR-
L1, CDR-L2, and
CDR-L3 sequences from a set of a VH and a VL sequence of an antibody set forth
in Table 2, the CD38
binding domain includes a VH domain including CDR-H1, CDR-H2, and CDR-H3 of a
VH sequence of an
antibody set forth in Table 2, and a VL domain including CDR-L1, CDR-L2, and
CDR-L3 of a VL
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sequences of an antibody set forth in Table 2, where the VH and the VL domain
sequences, excluding the
CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 sequences, are at least 95%
identical, at
least 97% identical, at least 99% identical, or at least 99.5% identical to
the VH and VL sequences of an
antibody set forth in Table 2, or the CD38 binding domain includes a set of a
VH and a VL sequence of an
antibody set forth in Table 2.
In some embodiments of the twelfth aspect of the disclosure, the first,
second, or third CD38
binding domain includes a set of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and
CDR-L3 sequences
set forth in Table 1, the CD38 binding domain includes CDR-H1, CDR-H2, CDR-H3,
CDR-L1, CDR-L2,
and CDR-L3 sequences from a set of a VH and a VL sequence of an antibody set
forth in Table 2, the
CD38 binding domain includes a VH domain including CDR-H1, CDR-H2, and CDR-H3
of a VH sequence
of an antibody set forth in Table 2, and a VL domain including CDR-L1, CDR-L2,
and CDR-L3 of a VL
sequence of an antibody set forth in Table 2, where the VH and the VL domain
sequences, excluding the
CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 sequences, are at least 95%
identical, at
least 97% identical, at least 99% identical, or at least 99.5% identical to
the VH and VL sequences of an
antibody set forth in Table 2, or the CD38 binding domain includes a set of a
VH and a VL sequences of
an antibody set forth in Table 2.
In some embodiments of the eleventh and twelfth aspects of the disclosure, the
Fc-antigen
binding domain construct, further includes an IgG CL antibody constant domain
and an IgG CH1 antibody
constant domain, where the IgG CH1 antibody constant domain is attached to the
N-terminus of the first
polypeptide or the second polypeptide by way of a linker.
In some embodiments of the eleventh and twelfth aspects of the disclosure, the
first Fc domain
monomer and the third Fc domain monomer include complementary dimerization
selectivity modules that
promote dimerization between the first Fc domain monomer and the third Fc
domain monomer.
In some embodiments of the eleventh and twelfth aspects of the disclosure, the
second Fc
domain monomer and the fourth Fc domain monomer include complementary
dimerization selectivity
modules that promote dimerization between the second Fc domain monomer and the
fourth Fc domain
monomer.
In some embodiments of the eleventh and twelfth aspects of the disclosure, the
dimerization
selectivity modules include an engineered cavity into the CH3 domain of one of
the Fc domain monomers
and an engineered protuberance into the CH3 domain of the other of the Fc
domain monomers, where the
engineered cavity and the engineered protuberance are positioned to form a
protuberance-into-cavity pair
of Fc domain monomers. In some embodiments, the engineered protuberance
includes at least one
modification selected from S354C, T366W, T366Y, T394W, T394F, and F405W, and
the engineered
cavity includes at least one modification selected from Y349C, T366S, L368A,
Y407V, Y407T, Y407A,
F405A, and T394S. In some embodiments, one of the Fc domain monomers includes
Y407V and Y349C
and the other of the Fc domain monomers includes T366W and S354C.
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In some embodiments of the eleventh and twelfth aspects of the disclosure, the
dimerization
selectivity modules include a negatively-charged amino acid into the CH3
domain of one of the domain
monomers and a positively-charged amino acid into the CH3 domain of the other
of the Fc domain
monomers, where the negatively-charged amino acid and the positively-charged
amino acid are
positioned to promote formation of an Fc domain. In some embodiments, each of
the first Fc domain
monomer and third Fc domain monomer includes D399K and either K409D or K409E,
each of the first Fc
domain monomer and third Fc domain monomer includes K392D and D399K, each of
the first Fc domain
monomer and third Fc domain monomer includes E357K and K370E, each of the
first Fc domain
monomer and third Fc domain monomer includes D356K and K439D, each of the
first Fc domain
monomer and third Fc domain monomer includes K392E and D399K, each of the
first Fc domain
monomer and third Fc domain monomer includes E357K and K370D, each of the
first Fc domain
monomer and third Fc domain monomer includes D356K and K439E, each of the
second Fc domain
monomer and fourth Fc domain monomer includes S354C and T366W and the third
and fourth
polypeptides each include Y349C, T366S, L368A, and Y407V, each of the third
and fourth polypeptides
includes S354C and T366W and the second Fc domain monomer and fourth Fc domain
monomer each
include Y349C, T366S, L368A, and Y407V, each of the second Fc domain monomer
and fourth Fc
domain monomer includes E357K or E357R and the third and fourth polypeptides
each include K370D or
K370E, each of the second Fc domain monomer and fourth Fc domain monomer
include K370D or
K370E and the third and fourth polypeptides each include E357K or 357R, each
of the second Fc domain
monomer and fourth Fc domain monomer include K409D or K409E and the third and
fourth polypeptides
each include D399K or D399R, or each of the second Fc domain monomer and
fourth Fc domain
monomer include D399K or D399R and the third and fourth polypeptides each
include K409D or K409E.
In some embodiments of the eleventh and twelfth aspects of the disclosure, one
or more linker in
the Fc-antigen binding domain construct is a bond.
In some embodiments of the eleventh and twelfth aspects of the disclosure, one
or more linker in
the Fc-antigen binding domain construct is a spacer. In some embodiments, the
spacer includes a
polypeptide having the sequence GGGGGGGGGGGGGGGGGGGG, GGGGS, GGSG, SGGG, GSGS,
GSGSGS, GSGSGSGS, GSGSGSGSGS, GSGSGSGSGSGS, GGSGGS, GGSGGSGGS,
GGSGGSGGSGGS, GGSG, GGSG, GGSGGGSG,
GGSGGGSGGGSGGGGGSGGGGSGGGGSGGGGS, GENLYFQSGG, SACYCELS, RSIAT,
RPACKIPNDLKQKVMNH, GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG,
AAANSSIDLISVPVDSR, GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS,
GGGSGGGSGGGS, SGGGSGGGSGGGSGGGSGGG, GGSGGGSGGGSGGGSGGS, GGGG,
GGGGGGGG, GGGGGGGGGGGG, or GGGGGGGGGGGGGGGG. In some embodiments, the spacer
is a glycine spacer, for example, one consisting of 4 to 30, 8 to 30, or 12 to
30 glycine residues, such as a
spacer consisting of 20 glycine residues.
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In some embodiments of the eleventh and twelfth aspects of the disclosure, one
or more of the
CD38 binding domains is joined to the Fc domain monomer by a linker. In some
embodiments, the linker
is a spacer.
In some embodiments of the eleventh and twelfth aspects of the disclosure, at
least one of the Fc
domains includes at least one amino acid modification at position 1253. In
some embodiments, the each
amino acid modification at position 1253 is independently selected from 1253A,
1253C, 1253D, 1253E,
1253F, 1253G, 1253H, 12531, 1253K, 1253L, 1253M, 1253N, 1253P, 1253Q, 1253R,
1253S, 1253T, 1253V,
1253W, and 1253Y. In some embodiments, each amino acid modification at
position 1253 is 1253A.
In some embodiments of the eleventh and twelfth aspects of the disclosure, at
least one of the Fc
domains includes at least one amino acid modification at position R292. In
some embodiments, each
amino acid modification at position R292 is independently selected from R292D,
R292E, R292L, R292P,
R292Q, R292R, R292T, and R292Y. In some embodiments, each amino acid
modification at position
R292 is R292P.
In some embodiments of the eleventh and twelfth aspects of the disclosure, one
or more of the Fc
domain monomers includes an IgG hinge domain, an IgG CH2 antibody constant
domain, and an IgG CH3
antibody constant domain. In some embodiments, each of the Fc domain monomers
includes an IgG
hinge domain, an IgG CH2 antibody constant domain, and an IgG CH3 antibody
constant domain. In
some embodiments, the IgG is of a subtype selected from the group consisting
of IgG1, IgG2a, IgG2b,
IgG3, and IgG4.
In some embodiments of the eleventh and twelfth aspects of the disclosure, the
N-terminal Asp in
each of the first, second, third, and fourth polypeptides is mutated to Gln.
In some embodiments of the eleventh and twelfth aspects of the disclosure, one
or more of the
first, second, third, and fourth polypeptides lack a C-terminal lysine. In
some embodiments, each of the
first, second, third, and fourth polypeptides lacks a C-terminal lysine.
In some embodiments of the eleventh and twelfth aspects of the disclosure, the
Fc-antigen
binding domain construct further includes an albumin-binding peptide joined to
the N-terminus or C-
terminus of one or more of the polypeptides by a linker.
In a thirteenth aspect, the disclosure features a composition including a
substantially
homogenous population of an Fc-antigen binding domain construct including: a)
a first polypeptide
including i) a first Fc domain monomer, ii) a second Fc domain monomer, and
iii) a first linker joining the
first Fc domain monomer and the second Fc domain monomer; and b) a second
polypeptide including i) a
third Fc domain monomer, ii) a fourth Fc domain monomer, and iv) a second
linker joining the third Fc
domain monomer and the fourth Fc domain monomer; and c) a third polypeptide
including a fifth Fc
domain monomer; d) a fourth polypeptide including an sixth Fc domain monomer;
and d) a CD38 binding
domain joined to the first polypeptide, second polypeptide, third polypeptide,
or fourth polypeptide; where
the first Fc domain monomer and the third Fc domain monomer combine to form a
first Fc domain and the
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second Fc domain monomer and the fifth Fc domain monomer combine to form a
second Fc domain, the
fourth Fc domain monomer and the sixth Fc domain monomer combine to form a
third Fc domain.
In some embodiments of the thirteenth aspect of the disclosure, each of the
first and third Fc
domain monomers includes a complementary dimerization selectivity module that
promote dimerization
between the first Fc domain monomer and the third Fc domain monomer, each of
the second and fifth Fc
domain monomers includes a complementary dimerization selectivity module that
promote dimerization
between the second Fc domain monomer and the fifth Fc domain monomer, and each
of the fourth and
sixth Fc domain monomers includes a complementary dimerization selectivity
module that promote
dimerization between the fourth Fc domain monomer and the sixth Fc domain
monomer.
In an fourteenth aspect, the disclosure features a composition including a
substantially
homogenous population of an Fc-antigen binding domain construct including: a)
a first polypeptide
including i) a first Fc domain monomer, ii) a second Fc domain monomer, and
iii) a first linker joining the
first Fc domain monomer and the second Fc domain monomer; and b) a second
polypeptide including i) a
third Fc domain monomer, ii) a fourth Fc domain monomer, and iv) a second
linker joining the third Fc
domain monomer and the fourth Fc domain monomer; and c) a third polypeptide
including a fifth Fc
domain monomer; d) a fourth polypeptide including an sixth Fc domain monomer;
and e) a CD38 binding
domain joined to the first polypeptide, second polypeptide, third polypeptide,
or fourth polypeptide;
wherein the second Fc domain monomer and the fourth Fc domain monomer combine
to form a first Fc
domain and the first Fc domain monomer and the fifth Fc domain monomer combine
to form a second Fc
domain, the third Fc domain monomer and the sixth Fc domain monomer combine to
form a third Fc
domain.
In some embodiments of the fourteenth aspect of the disclosure, each of the
second and fourth
Fc domain monomers includes a complementary dimerization selectivity module
that promote
dimerization between the second Fc domain monomer and the fourth Fc domain
monomer, each of the
first and fifth Fc domain monomers includes a complementary dimerization
selectivity module that
promote dimerization between the first Fc domain monomer and the fifth Fc
domain monomer, and each
of the third and sixth Fc domain monomers includes a complementary
dimerization selectivity module that
promote dimerization between the third Fc domain monomer and the sixth Fc
domain monomer.
In a fifteenth aspect, the disclosure features a composition including a
substantially homogenous
population of an Fc-antigen binding domain construct including: a) a first
polypeptide including i) a first Fc
domain monomer, ii) a second Fc domain monomer, iii) a third Fc domain
monomer, iv) a first linker
joining the first Fc domain monomer and the second Fc domain monomer; and v) a
second linker joining
the second Fc domain monomer and the third Fc domain monomer; b) a second
polypeptide including i) a
fourth Fc domain monomer, ii) a fifth Fc domain monomer, iii) a sixth Fc
domain monomer, iv) a third
linker joining the fourth Fc domain monomer and the fifth Fc domain monomer;
and v) a fourth linker
joining the fifth Fc domain monomer and the sixth Fc domain monomer; c) a
third polypeptide including a
seventh Fc domain monomer; d) a fourth polypeptide including an eighth Fc
domain monomer; e) a fifth
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polypeptide including a ninth Fc domain monomer; 0 a sixth polypeptide
including a tenth Fc domain
monomer; and g) a CD38 binding domain joined to the first polypeptide, second
polypeptide, third
polypeptide, fourth polypeptide, fifth polypeptide, or sixth polypeptide;
where the second Fc domain
monomer and the fifth Fc domain monomer combine to form a first Fc domain and
the first Fc domain
monomer and the seventh Fc domain monomer combine to form a second Fc domain,
the fourth Fc
domain monomer and the eighth Fc domain monomer combine to form a third Fc
domain, the third Fc
domain monomer and the ninth Fc domain monomer combine to form a fourth Fc
domain, and the sixth
Fc domain monomer and the tenth Fc domain monomer combine to form a fifth Fc
domain.
In some embodiments of the fifteenth aspect of the disclosure, each of the
second and fifth Fc
domain monomers includes a complementary dimerization selectivity module that
promote dimerization
between the second Fc domain monomer and the fifth Fc domain monomer, each of
the first and seventh
Fc domain monomers includes a complementary dimerization selectivity module
that promote
dimerization between the first Fc domain monomer and the seventh Fc domain
monomer, each of the
fourth and eighth Fc domain monomers includes a complementary dimerization
selectivity module that
promote dimerization between the fourth Fc domain monomer and the eighth Fc
domain monomer, each
of the third and ninth Fc domain monomers includes a complementary
dimerization selectivity module that
promote dimerization between the third Fc domain monomer and the ninth Fc
domain monomer, and
each of the sixth and tenth Fc domain monomers includes a complementary
dimerization selectivity
module that promote dimerization between the sixth Fc domain monomer and the
tenth Fc domain
monomer.
In some embodiments of the thirteenth, fourteenth, and fifteenth aspects of
the disclosure, the
CD38 binding domain is a Fab or a VH domain
In some embodiments of the thirteenth, fourteenth, and fifteenth aspects of
the disclosure, the
CD38 binding domain is part of the amino acid sequence of one or more of the
polypeptides, and, in
some embodiments, the CD38 binding domain is a scFv.
In some embodiments of the thirteenth, fourteenth, and fifteenth aspects of
the disclosure, the
CD38 binding domain includes a VH domain and a CH1 domain, and where the VH
and CH1 domains are
part of the amino acid sequence of the first, second, or third polypeptide. In
some embodiments, the
CD38 binding domain further includes a VL domain, where, in some embodiments
the Fc-antigen binding
domain construct includes a fourth polypeptide including the VL domain. In
some embodiments, the VH
domain includes a set of CDR-H1, CDR-H2 and CDR-H3 sequences set forth in
Table 1, the VH domain
includes CDR-H1, CDR-H2, and CDR-H3 of a VH domain including a sequence of an
antibody set forth in
Table 2, the VH domain includes CDR-H1, CDR-H2, and CDR-H3 of a VH sequence of
an antibody set
forth in Table 2, and the VH sequence, excluding the CDR-H1, CDR-H2, and CDR-
H3 sequence, is at
least 95% identical, at least 97% identical, at least 99% identical, or at
least 99.5% identical to the VH
sequence of an antibody set forth in Table 2, or the VH domain includes a VH
sequence of an antibody set
forth in Table 2.
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In some embodiments of the thirteenth, fourteenth, and fifteenth aspects of
the disclosure, the
CD38 binding domain includes a set of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2,
and CDR-L3
sequences set forth in Table 1, the CD38 binding domain includes CDR-H1, CDR-
H2, CDR-H3, CDR-L1,
CDR-L2, and CDR-L3 sequences from a set of a VH and a VL sequences of an
antibody set forth in Table
2, the CD38 binding domain includes a VH domain including CDR-H1, CDR-H2, and
CDR-H3 of a VH
sequence of an antibody set forth in Table 2, and a VL domain including CDR-
L1, CDR-L2, and CDR-L3
of a VL sequence of an antibody set forth in Table 2, where the VH and the VL
domain sequences,
excluding the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 sequences,
are at least 95%
identical, at least 97% identical, at least 99% identical, or at least 99.5%
identical to the VH and VL
.. sequences of an antibody set forth in Table 2, or the CD38 binding domain
includes a set of a VH and a
VL sequences of an antibody set forth in Table 2.
In some embodiments of the thirteenth, fourteenth, and fifteenth aspects of
the disclosure, the Fc-
antigen binding domain construct, further includes an IgG CL antibody constant
domain and an IgG CH1
antibody constant domain, where the IgG CH1 antibody constant domain is
attached to the N-terminus of
the first polypeptide or the second polypeptide by way of a linker.
In some embodiments of the thirteenth, fourteenth, and fifteenth aspects of
the disclosure, the
dimerization selectivity modules include an engineered cavity into the CH3
domain of one of the Fc
domain monomers and an engineered protuberance into the CH3 domain of the
other of the Fc domain
monomers, where the engineered cavity and the engineered protuberance are
positioned to form a
protuberance-into-cavity pair of Fc domain monomers. In some embodiments, the
engineered
protuberance includes at least one modification selected from S354C, T366W,
T366Y, T394W, T394F,
and F405W, and the engineered cavity includes at least one modification
selected from Y349C, T366S,
L368A, Y407V, Y407T, Y407A, F405A, and T394S. In some embodiments, one of the
Fc domain
monomers includes Y407V and Y349C and the other of the Fc domain monomers
includes T366W and
S354C.
In some embodiments of the thirteenth, fourteenth, and fifteenth aspects of
the disclosure, the
dimerization selectivity modules include a negatively-charged amino acid into
the CH3 domain of one of
the domain monomers and a positively-charged amino acid into the CH3 domain of
the other of the Fc
domain monomers, where the negatively-charged amino acid and the positively-
charged amino acid are
positioned to promote formation of an Fc domain. In some embodiments, each of
the first Fc domain
monomer and third Fc domain monomer includes D399K and either K409D or K409E,
each of the first Fc
domain monomer and third Fc domain monomer includes K392D and D399K, each of
the first Fc domain
monomer and third Fc domain monomer includes E357K and K370E, each of the
first Fc domain
monomer and third Fc domain monomer includes D356K and K439D, each of the
first Fc domain
monomer and third Fc domain monomer includes K392E and D399K, each of the
first Fc domain
monomer and third Fc domain monomer includes E357K and K370D, each of the
first Fc domain
monomer and third Fc domain monomer includes D356K and K439E, each of the
second Fc domain
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monomer and fourth Fc domain monomer includes S354C and T366W and the third
and fourth
polypeptides each include Y349C, T366S, L368A, and Y407V, each of the third
and fourth polypeptides
includes S354C and T366W and the second Fc domain monomer and fourth Fc domain
monomer each
include Y349C, T366S, L368A, and Y407V, each of the second Fc domain monomer
and fourth Fc
domain monomer includes E357K or E357R and the third and fourth polypeptides
each include K370D or
K370E, each of the second Fc domain monomer and fourth Fc domain monomer
include K370D or
K370E and the third and fourth polypeptides each include E357K or 357R, each
of the second Fc domain
monomer and fourth Fc domain monomer include K409D or K409E and the third and
fourth polypeptides
each include D399K or D399R, or each of the second Fc domain monomer and
fourth Fc domain
monomer include D399K or D399R and the third and fourth polypeptides each
include K409D or K409E.
In some embodiments of the thirteenth, fourteenth, and fifteenth aspects of
the disclosure, one or
more linker in the Fc-antigen binding domain construct is a bond.
In some embodiments of the thirteenth, fourteenth, and fifteenth aspects of
the disclosure, one or
more linker in the Fc-antigen binding domain construct is a spacer. In some
embodiments, the spacer
includes a polypeptide having the sequence GGGGGGGGGGGGGGGGGGGG, GGGGS, GGSG,
SGGG, GSGS, GSGSGS, GSGSGSGS, GSGSGSGSGS, GSGSGSGSGSGS, GGSGGS,
GGSGGSGGS, GGSGGSGGSGGS, GGSG, GGSG, GGSGGGSG,
GGSGGGSGGGSGGGGGSGGGGSGGGGSGGGGS, GENLYFQSGG, SACYCELS, RSIAT,
RPACKIPNDLKQKVMNH, GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG,
AAANSSIDLISVPVDSR, GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS,
GGGSGGGSGGGS, SGGGSGGGSGGGSGGGSGGG, GGSGGGSGGGSGGGSGGS, GGGG,
GGGGGGGG, GGGGGGGGGGGG, or GGGGGGGGGGGGGGGG. In some embodiments, the spacer
is a glycine spacer, for example, one consisting of 4 to 30, 8 to 30, or 12 to
30 glycine residues, such as a
spacer consisting of 20 glycine residues.
In some embodiments of the thirteenth, fourteenth, and fifteenth aspects of
the disclosure, the
CD38 binding domain is joined to the Fc domain monomer by a linker. In some
embodiments, the linker
is a spacer.
In some embodiments of the thirteenth, fourteenth, and fifteenth aspects of
the disclosure, at
least one of the Fc domains includes at least one amino acid modification at
position 1253. In some
embodiments, the each amino acid modification at position 1253 is
independently selected from 1253A,
1253C, 1253D, 1253E, 1253F, 1253G, 1253H, 12531, 1253K, 1253L, 1253M, 1253N,
1253P, 1253Q, 1253R,
1253S, 1253T, 1253V, 1253W, and 1253Y. In some embodiments, each amino acid
modification at position
1253 is 1253A.
In some embodiments of the thirteenth, fourteenth, and fifteenth aspects of
the disclosure, at
least one of the Fc domains includes at least one amino acid modification at
position R292. In some
embodiments, each amino acid modification at position R292 is independently
selected from R292D,
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R292E, R292L, R292P, R292Q, R292R, R292T, and R292Y. In some embodiments, each
amino acid
modification at position R292 is R292P.
In some embodiments of the thirteenth, fourteenth, and fifteenth aspects of
the disclosure, one or
more of the Fc domain monomers includes an IgG hinge domain, an IgG CH2
antibody constant domain,
and an IgG CH3 antibody constant domain. In some embodiments, each of the Fc
domain monomers
includes an IgG hinge domain, an IgG CH2 antibody constant domain, and an IgG
CH3 antibody constant
domain. In some embodiments, the IgG is of a subtype selected from the group
consisting of IgG1,
IgG2a, IgG2b, IgG3, and IgG4.
In some embodiments of the thirteenth, fourteenth, and fifteenth aspects of
the disclosure, the N-
terminal Asp in each of the polypeptides is mutated to Gln.
In some embodiments of the thirteenth, fourteenth, and fifteenth aspects of
the disclosure, one or
more of the polypeptides lack a C-terminal lysine. In some embodiments, each
of the polypeptides lacks
a C-terminal lysine.
In some embodiments of the thirteenth, fourteenth, and fifteenth aspects of
the disclosure, the Fc-
antigen binding domain construct further includes an albumin-binding peptide
joined to the N-terminus or
C-terminus of one or more of the polypeptides by a linker.
In a sixteenth aspect, the disclosure features an Fc-antigen binding domain
construct including:
a) a first polypeptide including i) a first Fc domain monomer, ii) a second Fc
domain monomer, and iii) a
linker joining the first Fc domain monomer and the second Fc domain monomer;
b) a second polypeptide
including a third Fc domain monomer; c) a third polypeptide including a fourth
Fc domain monomer; and
d) a first CD38 binding domain joined to the first polypeptide; and e) a
second CD38 binding domain
joined to the second polypeptide and/or third polypeptide; where the first Fc
domain monomer and the
third Fc domain monomer combine to form a first Fc domain and the second Fc
domain monomer and the
fourth Fc domain monomer combine to form a second Fc domain, where the first
and the second CD38
binding domains bind different antigens, and where the Fc-antigen binding
domain construct has
enhanced effector function in an antibody-dependent cytotoxicity (ADCC) assay,
an antibody-dependent
cellular phagocytosis (ADCP) and/or complement-dependent cytotoxicity (CDC)
assay relative to a
construct having a single Fc domain and the CD38 binding domain.
In a twenty sixth aspect, the disclosure features an Fc-antigen binding domain
construct
including: a) a first polypeptide including i) a first Fc domain monomer, ii)
a second Fc domain monomer,
and iii) a first linker joining the first Fc domain monomer and the second Fc
domain monomer; and b) a
second polypeptide including iv) a third Fc domain monomer, v) a fourth Fc
domain monomer, and vi) a
second linker joining the third Fc domain monomer and the fourth Fc domain
monomer; and c) a third
polypeptide including a fifth Fc domain monomer; d) a fourth polypeptide
including an sixth Fc domain
monomer; and d) a CD38 binding domain joined to the first polypeptide, second
polypeptide, third
polypeptide, or fourth polypeptide, where the first Fc domain monomer and the
third Fc domain monomer
combine to form a first Fc domain and the second Fc domain monomer and the
fifth Fc domain monomer
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combine to form a second Fc domain, the fourth Fc domain monomer and the sixth
Fc domain monomer
combine to form a third Fc domain, and where the Fc-antigen binding domain
construct has enhanced
effector function in an antibody-dependent cytotoxicity (ADCC) assay, an
antibody-dependent cellular
phagocytosis (ADCP) and/or complement-dependent cytotoxicity (CDC) assay
relative to a construct
having a single Fc domain and the CD38 binding domain.
In a twenty seventh aspect, the disclosure features a Fc-antigen binding
domain construct
including: a) a first polypeptide including i) a first Fc domain monomer, ii)
a second Fc domain monomer,
and iii) a first linker joining the first Fc domain monomer and the second Fc
domain monomer; and b) a
second polypeptide including iv) a third Fc domain monomer, v) a fourth Fc
domain monomer, and vi) a
second linker joining the third Fc domain monomer and the fourth Fc domain
monomer; and c) a third
polypeptide including a fifth Fc domain monomer; d) a fourth polypeptide
including an sixth Fc domain
monomer; and e) a CD38 binding domain joined to the first polypeptide, second
polypeptide, third
polypeptide, or fourth polypeptide; where the first Fc domain monomer and the
third Fc domain monomer
combine to form a first Fc domain and the second Fc domain monomer and the
fifth Fc domain monomer
combine to form a second Fc domain, the fourth Fc domain monomer and the sixth
Fc domain monomer
combine to form a third Fc domain, and where the Fc-antigen binding domain
construct includes a
biological activity that is not exhibited by a construct having a single Fc
domain and the CD38 binding
domain.
In a twenty eighth aspect, the disclosure features an Fc-antigen binding
domain construct
including: a) a first polypeptide including i) a first Fc domain monomer, ii)
a second Fc domain monomer,
and iii) a first spacer joining the first Fc domain monomer and the second Fc
domain monomer; and b) a
second polypeptide including iv) a third Fc domain monomer, v) a fourth Fc
domain monomer, and vi) a
second spacer joining the third Fc domain monomer and the fourth Fc domain
monomer; and c) a third
polypeptide including a fifth Fc domain monomer; d) a fourth polypeptide
including an sixth Fc domain
monomer; and e) a CD38 binding domain joined to the first polypeptide, second
polypeptide, third
polypeptide, or fourth polypeptide; where the first Fc domain monomer and the
third Fc domain monomer
combine to form a first Fc domain and the second Fc domain monomer and the
fifth Fc domain monomer
combine to form a second Fc domain, the fourth Fc domain monomer and the sixth
Fc domain monomer
combine to form a third Fc domain.
In a twenty ninth aspect, the disclosure features a cell culture medium
including a population of
Fc-antigen binding domain constructs, where at least 50% of the Fc-antigen
binding domain constructs,
on a molar basis, include: a) a first polypeptide including i) a first Fc
domain monomer, ii) a second Fc
domain monomer, and iii) a first linker joining the first Fc domain monomer
and the second Fc domain
monomer; and b) a second polypeptide including iv) a third Fc domain monomer,
v) a fourth Fc domain
monomer, and vi) a second linker joining the third Fc domain monomer and the
fourth Fc domain
monomer; and c) a third polypeptide including a fifth Fc domain monomer; d) a
fourth polypeptide
including an sixth Fc domain monomer; and e) a CD38 binding domain joined to
the first polypeptide,
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second polypeptide, third polypeptide, or fourth polypeptide; where the first
Fc domain monomer and the
third Fc domain monomer combine to form a first Fc domain and the second Fc
domain monomer and the
fifth Fc domain monomer combine to form a second Fc domain, the fourth Fc
domain monomer and the
sixth Fc domain monomer combine to form a third Fc domain.
In a thirtieth aspect, the disclosure features a method of manufacturing an Fc-
antigen binding
domain construct, the method including: a) culturing a host cell expressing:
(1) a first polypeptide
including i) a first Fc domain monomer, ii) a second Fc domain monomer, and
iii) a first linker joining the
first Fc domain monomer and the second Fc domain monomer; and (2) a second
polypeptide including iv)
a third Fc domain monomer, v) a fourth Fc domain monomer, and vi) a second
linker joining the third Fc
domain monomer and the fourth Fc domain monomer; and (3) a third polypeptide
including a fifth Fc
domain monomer; (4) a fourth polypeptide including an sixth Fc domain monomer;
and (5) a CD38
binding domain joined to the first polypeptide, second polypeptide, third
polypeptide, or fourth
polypeptide; where the first Fc domain monomer and the third Fc domain monomer
combine to form a
first Fc domain and the second Fc domain monomer and the fifth Fc domain
monomer combine to form a
second Fc domain, the fourth Fc domain monomer and the sixth Fc domain monomer
combine to form a
third Fc domain, and where at least 50% of the Fc-antigen binding domain
constructs in a cell culture
supernatant, on a molar basis, are structurally identical, and b) purifying
the Fc-antigen binding domain
construct from the cell culture supernatant.
In some embodiments of the twenty sixth, twenty seventh, twenty eighth, twenty
ninth, and
thirtieth aspect of the disclosure, each of the first and third Fc domain
monomers includes a
complementary dimerization selectivity module that promote dimerization
between the first Fc domain
monomer and the third Fc domain monomer, each of the second and fifth Fc
domain monomers includes
a complementary dimerization selectivity module that promote dimerization
between the second Fc
domain monomer and the fifth Fc domain monomer, and each of the fourth and
sixth Fc domain
monomers includes a complementary dimerization selectivity module that promote
dimerization between
the fourth Fc domain monomer and the sixth Fc domain monomer.
In some embodiments of all aspects of the disclosure, the Fc-antigen binding
domain construct
has reduced fucosylation. Thus, in some embodiments, less than 40%, 30%, 20%,
15%, 10% or 5% of
the Fc domain monomers in a composition comprising an Fc-antigen binding
domain construct are
fucosylated.
In some embodiments of all aspects of the disclosure, the Fc domain monomer
comprises the
amino acid sequence of FIG. 24A (SEQ ID NO: 43) with up to 10 (9, 8, 7, 6, 5,
4, 3, 2 or 1) single amino
acid changes in the CH3 domain.
In some embodiments of all aspects of the disclosure, the Fc domain monomer
comprises the
amino acid sequence of FIG. 24B (SEQ ID NO: 45) with up to 10 (9, 8, 7, 6, 5,
4, 3, 2 or 1) single amino
acid changes in the CH3 domain.
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In some embodiments of all aspects of the disclosure, the Fc domain monomer
comprises the
amino acid sequence of FIG. 24C (SEQ ID NO: 47) with up to 10 (9, 8, 7, 6, 5,
4, 3, 2 or 1) single amino
acid changes in the CH3 domain.
In some embodiments of all aspects of the disclosure, the Fc domain monomer
comprises the
amino acid sequence of FIG. 24D (SEQ ID NO: 42) with up to 10 (9, 8, 7, 6, 5,
4, 3, 2 or 1) single amino
acid changes in the CH3 domain.
In some embodiments of all aspects of the disclosure, for example, when the Fc
domain
monomer is at the carboxy-terminal end of a polypeptide, the Fc domain monomer
does not include
K447. In other embodiments, for example, when the Fc domain monomer is not at
the carboxy-terminal
end of a polypeptide, the Fc domain monomer includes K447.
In some embodiments of all aspects of the disclosure, for example, when the Fc
domain
monomer is amino terminal to a linker, the Fc domain monomer does not include
the portion of the hinge
from E216 to C220, inclusive, but does include the portion of the hinge from
D221 to L235, inclusive. In
other embodiments, for example, when the Fc domain monomer is carboxy-terminal
to a CH1 domain, the
Fc domain monomer includes the portion of the hinge from E216 to L235,
inclusive. In some
embodiments of all aspects of the disclosure, a hinge domain, for example a
hinge domain at the amino
terminus of a polypeptide, has an Asp to Gln mutation at EU position 221.
As noted above, the Fc-antigen binding domain constructs of the disclosure are
assembled from
polypeptides, including polypeptides comprising two or more IgG1 Fc domain
monomers, and such
polypeptides are an aspect of the present disclosure.
In a forty first aspect, the disclosure features a polypeptide comprising a
CD38 binding domain; a
linker; a first IgG1 Fc domain monomer comprising a hinge domain, a CH2 domain
and a CH3 domain; a
second linker; a second IgG1 Fc domain monomer comprising a hinge domain, a
CH2 domain and a CH3
domain; an optional third linker; and an optional third IgG1 Fc domain monomer
comprising a hinge
.. domain, a CH2 domain and a CH3 domain, wherein at least one Fc domain
monomer comprises
mutations forming an engineered protuberance.
In various embodiments of the forty first aspect: the CD38 binding domain
comprises an antibody
heavy chain variable domain; the CD38 binding domain comprises an antibody
light chain variable
domain; the first IgG1 Fc domain monomer comprises two or four reverse charge
mutations and the
second IgG1 Fc domain monomer comprises mutations forming an engineered
protuberance; the first
IgG1 Fc domain monomer comprises mutations forming an engineered protuberance
and the second
IgG1 Fc domain monomer comprises two or four reverse charge mutations; both
the first IgG1 Fc domain
monomer and the second IgG constant domain monomer comprise mutations forming
an engineered
protuberance; the polypeptide comprises a third linker and a third IgG1 Fc
domain monomer wherein the
first IgG1 Fc domain monomer, the second IgG1 Fc domain monomer and the third
IgG1 Fc domain
monomer each comprise mutations forming an engineered protuberance; the
polypeptide comprises a
third linker and a third IgG1 Fc domain monomer wherein both the first IgG1 Fc
domain monomer and the
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second IgG1 Fc domain monomer each comprise mutations forming an engineered
protuberance and the
third IgG1 Fc domain monomer comprises two or four reverse charge mutations;
the polypeptide
comprises a third linker and third IgG1 Fc domain monomer wherein both the
first IgG1 Fc domain
monomer and the third IgG1 Fc domain monomer each comprise mutations forming
an engineered
protuberance and the second IgG1 domain monomer comprises two or four reverse
charge mutations;
the polypeptide comprises a third linker and a third IgG1 Fc domain monomer
wherein both the second
IgG1 Fc domain monomer and the third IgG1 Fc domain monomer each comprise
mutations forming an
engineered protuberance and the first IgG1 domain monomer comprises two or
four reverse charge
mutations.
In various embodiments of the forty first aspect: the IgG1 Fc domain monomers
comprising
mutations forming an engineered protuberance further comprise one, two or
three reverse charge
mutations; the mutations forming an engineered protuberance and the reverse
charge mutations are in
the CH3 domain; the mutations are within the sequence from EU Numbering
position G341 to EU
Numbering position K447, inclusive; the mutations are single amino acid
changes; the second linker and
the optional third linker comprise or consist of an amino acid sequence
selected from the group consisting
of: GGGGGGGGGGGGGGGGGGGG, GGGGS, GGSG, SGGG, GSGS, GSGSGS, GSGSGSGS,
GSGSGSGSGS, GSGSGSGSGSGS, GGSGGS, GGSGGSGGS, GGSGGSGGSGGS, GGSG, GGSG,
GGSGGGSG, GGSGGGSGGGSGGGGGSGGGGSGGGGSGGGGS, GENLYFQSGG, SACYCELS,
RSIAT, RPACKIPNDLKQKVMNH, GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG,
AAANSSIDLISVPVDSR, GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS,
GGGSGGGSGGGS, SGGGSGGGSGGGSGGGSGGG, GGSGGGSGGGSGGGSGGS, GGGG,
GGGGGGGG, GGGGGGGGGGGG and GGGGGGGGGGGGGGGG; the second linker and the
optional
third linker is a glycine spacer; the second linker and the optional third
linker independently consist of 4 to
30, 4 to 20, 8 to 30, 8 to 20, 12 to 20 or 12 to 30 glycine residues; the
second linker and the optional third
linker consist of 20 glycine residues; at least one of the Fc domain monomers
comprises a single amino
acid mutation at EU Numbering position 1253 each amino acid mutation at EU
Numbering position 1253 is
independently selected from the group consisting of 1253A, 1253C, 1253D,
1253E, 1253F, 1253G, 1253H,
12531, 1253K, 1253L, 1253M, 1253N, 1253P, 1253Q, 1253R, 1253S, 1253T, 1253V,
1253W, and 1253Y; each
amino acid mutation at position 1253 is 1253A; at least one of the Fc domain
monomers comprises a
single amino acid mutation at EU Numbering position R292; each amino acid
mutation at EU Numbering
position R292 is independently selected from the group consisting of R292D,
R292E, R292L, R292P,
R292Q, R292R, R292T, and R292Y; each amino acid mutation at position R292 is
R292P; each Fc
domain monomer independently comprises or consists of an amino acid sequence
selected from the
group consisting of EPKSCDKTHTCPPCPAPELL and DKTHTCPPCPAPELL; the hinge
portion of the
second Fc domain monomer and the third Fc domain monomer have the amino acid
sequence
DKTHTCPPCPAPELL; the hinge portion of the first Fc domain monomer has the
amino acid sequence
EPKSCDKTHTCPPCPAPEL; the hinge portion of the first Fc domain monomer has the
amino acid
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sequence EPKSCDKTHTCPPCPAPEL and the hinge portion of the second Fc domain
monomer and the
third Fc domain monomer have the amino acid sequence DKTHTCPPCPAPELL; the CH2
domains of
each Fc domain monomer independently comprise the amino acid sequence:
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK with no more than two single amino acid
deletions
or substitutions; the CH2 domains of each Fc domain monomer are identical and
comprise the amino acid
sequence:
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK with no more than two single amino acid
deletions
or substitutions; the CH2 domains of each Fc domain monomer are identical and
comprise the amino acid
sequence:
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK with no more than two single amino acid
substitutions; the CH2 domains of each Fc domain monomer are identical and
comprise the amino acid
sequence:
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK; the CH3 domains of each Fc domain
monomer
independently comprise the amino acid sequence:
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK
LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG with no more than 10 single amino acid
substitutions; the CH3 domains of each Fc domain monomer independently
comprise the amino acid
sequence:
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK
LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG with no more than 8 single amino acid
substitutions; the CH3 domains of each Fc domain monomer independently
comprise the amino acid
sequence:
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK
LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG with no more than 6 single amino acid
substitutions; wherein the CH3 domains of each Fc domain monomer independently
comprise the amino
acid sequence:
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK
LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG with no more than 5 single amino acid
substitutions; the single amino acid substitutions are selected from the group
consisting of: T366Y,
T366W, T394W, T394Y, F405W, F405A, Y407A, S354C, Y349T, T394F, K409D, K409E,
K392D, K392E,
K370D, K370E, D399K, D399R, E357K, E357R, D356K, and D356R; each of the Fc
domain monomers
independently comprises the amino acid sequence of any of SEQ ID NOs:42, 43,
45, and 47 having up to
10 single amino acid substitutions; up to 6 of the single amino acid
substitutions are reverse charge
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mutations in the CH3 domain or are mutations forming an engineered
protuberance; the single amino
acid substitutions are within the sequence from EU Numbering position G341 to
EU Numbering position
K447, inclusive; at least one of the mutations forming an engineered
protuberance is selected from the
group consisting of T366Y, T366W, T394W, T394Y, F405Wõ S354C, Y349T, and
T394F; the two or four
.. reverse charge mutations are selected from: K409D, K409E, K392D. K392E,
K370D, K370E, D399K,
D399R, E357K, E357R, D356K, and D356R; the CD38 binding domain is a scFv; the
CD38 binding
domain comprises a VH domain and a CH1 domain; the CD38 binding domain further
comprises a VL
domain; the VH domain comprises a set of CDR-H1, CDR-H2 and CDR-H3 sequences
set forth in Table
1; the VH domain comprises CDR-H1, CDR-H2, and CDR-H3 of a VH domain
comprising a sequence of
.. an antibody set forth in Table 2; the VH domain comprises CDR-H1, CDR-H2,
and CDR-H3 of a VH
sequence of an antibody set forth in Table 2, and the VH sequence, excluding
the CDR-H1, CDR-H2, and
CDR-H3 sequence, is at least 95% or 98% identical to the VH sequence of an
antibody set forth in Table
2; the VH domain comprises a VH sequence of an antibody set forth in Table 2;
the CD38 binding domain
comprises a set of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3
sequences set forth in
Table 1; the CD38 binding domain comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-
L2, and CDR-
L3 sequences from a set of a VH and a VL sequence of an antibody set forth in
Table 2; the CD38
binding domain comprises a VH domain comprising CDR-H1, CDR-H2, and CDR-H3 of
a VH sequence of
an antibody set forth in Table 2, and a VL domain comprising CDR-L1, CDR-L2,
and CDR-L3 of a VL
sequence of an antibody set forth in Table 2, wherein the VH and the VL domain
sequences, excluding
the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 sequences, are at least
95% or 98%
identical to the VH and VL sequences of an antibody set forth in Table 2; the
CD38 binding domain
comprises a set of a VH and a VL sequence of an antibody set forth in Table 2;
CD38 binding domain
comprises an IgG CL antibody constant domain and an IgG CH1 antibody constant
domain; the CD38
binding domain comprises a VH domain and CH1 domain and can bind to a
polypeptide comprising a VL
domain and a CL domain to form a Fab.
Also described is a polypeptide complex comprising two copies of the
polypeptide of described
above joined by disulfide bonds between cysteine residues within the hinge of
first or second IgG1 Fc
domain monomers.
Also described is a polypeptide complex comprising a polypeptide described
above
joined to a second polypeptide comprising and IgG1 Fc domain monomer
comprising a hinge domain, a
CH2 domain and a CH3 domain, wherein the polypeptide and the second
polypeptide are joined by
disulfide bonds between cysteine residues within the hinge domain of the
first, second or third IgG1 Fc
domain monomer of the polypeptide and the hinge domain of the second
polypeptide.
In various embodiments of the complexes: the second polypeptide monomer
comprises
mutations forming an engineered cavity; the mutations forming the engineered
cavity are selected from
the group consisting of: Y407T, Y407A, F405A, T394S, T394W/Y407A, T366W/T394S,
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T366S/L368A/Y407V/Y349C, S364H/F405A; the second polypeptide comprises the
amino acid sequence
of any of SEQ ID NOs: 42, 43, 45, and 47 having up to 10 single amino acid
substitutions.
In a forty second aspect, the disclosure features: a polypeptide comprising:
aCD38 binding
domain; a linker; a first IgG1 Fc domain monomer comprising a hinge domain, a
CH2 domain and a CH3
domain; a second linker; a second IgG1 Fc domain monomer comprising a hinge
domain, a CH2 domain
and a CH3 domain; an optional third linker; and an optional third IgG1 Fc
domain monomer comprising a
hinge domain, a CH2 domain and a CH3 domain, wherein at least one Fc domain
monomer comprises
one, two or three reverse charge amino acid mutations.
In various embodiments of the forty second aspect: the CD38 binding domain
comprises an
antibody heavy chain variable domain; the CD38 binding domain comprises an
antibody light chain
variable domain; the first IgG1 Fc domain monomer comprises a set of two
reverse charge mutations
selected from those in Tables 4A and 4B or a set of four reverse charge
mutation selected from those in
Tables 4A and 4B and the second IgG1 Fc domain monomer comprises one, two or
three reverse charge
amino acid mutations selected from Tables 4A and 4B; the first IgG1 Fc domain
monomer comprises one,
two or three reverse charge amino acid mutations selected from Tables 4A and
4B and the second IgG1
Fc domain monomer comprises a set of two reverse charge mutations selected
from those in Tables 4a
and 4b or a set of four reverse charge mutation selected from those in Tables
4A and 4B; both the first
IgG1 Fc domain monomer and the second IgG constant domain monomer comprise
one, two or three
reverse charge amino acid mutations selected from Tables 4A and 4B; the
polypeptide further comprises
a third linker and a third IgG1 Fc domain monomer wherein the first IgG1 Fc
domain monomer, the
second IgG1 Fc domain monomer and the third IgG1 Fc domain monomer each
comprise one, two or
three reverse charge amino acid mutations selected from Tables 4A and 4B; the
polypeptide further
comprises a third linker and a third IgG1 Fc domain monomer wherein both the
first IgG1 Fc domain
monomer and the second IgG1 Fc domain monomer each comprise one, two or three
reverse charge
amino acid mutations selected from Tables 4A and 4B and the third IgG1 Fc
domain monomer comprises
a set of two reverse charge mutations selected from those in Tables 4A and 4B
or a set of four reverse
charge mutation selected from those in Tables 4A and 4B; the polypeptide
further comprises a third linker
and third IgG1 Fc domain monomer wherein both the first IgG1 Fc domain monomer
and the third IgG1
Fc domain monomer each comprise one, two or three reverse charge amino acid
mutations selected from
Tables 4A and 4B and the second IgG1 domain monomer comprises a set of two
reverse charge
mutations selected from those in Tables 4A and 4B or a set of four reverse
charge mutation selected from
those in Tables 4A and 4B; the polypeptide further comprises a third linker
and a third IgG1 Fc domain
monomer wherein both the second IgG1 Fc domain monomer and the third IgG1 Fc
domain monomer
each comprise one, two or three reverse charge amino acid mutations selected
from Tables 4A and 4B
and the first IgG1 domain monomer comprises a set of two reverse charge
mutations selected from those
in Tables 4A and 4B or a set of four reverse charge mutation selected from
those in Tables 4A and 4B;
the IgG1 Fc domain monomers comprising one, two or three reverse charge amino
acid mutations
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selected from Tables 4A and 4B have identical CH3 domains; one, two or three
reverse charge amino
acid mutations selected from Tables 4A and 4B are in the CH3 domain; the
mutations are within the
sequence from EU Numbering position G341 to EU Numbering position K447,
inclusive; the mutations
are each single amino acid changes; the mutations are within the sequence from
EU Numbering position
G341 to EU Numbering position K446, inclusive; the mutations are single amino
acid changes; the
second linker and the optional third linker comprise or consist of an amino
acid sequence selected from
the group consisting of: GGGGGGGGGGGGGGGGGGGG, GGGGS, GGSG, SGGG, GSGS,
GSGSGS,
GSGSGSGS, GSGSGSGSGS, GSGSGSGSGSGS, GGSGGS, GGSGGSGGS, GGSGGSGGSGGS,
GGSG, GGSG, GGSGGGSG, GGSGGGSGGGSGGGGGSGGGGSGGGGSGGGGS, GENLYFQSGG,
SACYCELS, RSIAT, RPACKIPNDLKQKVMNH,
GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG, AAANSSIDLISVPVDSR,
GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS, GGGSGGGSGGGS,
SGGGSGGGSGGGSGGGSGGG, GGSGGGSGGGSGGGSGGS, GGGG, GGGGGGGG,
GGGGGGGGGGGG and GGGGGGGGGGGGGGGG; the second linker and the optional third
linker is a
glycine spacer; the second linker and the optional third linker independently
consist of 4 to 30, 4 to 20, 8
to 30, 8 to 20, 12 to 20 or 12 to 30 glycine residues; the second linker and
the optional third linker consist
of 20 glycine residues; at least one of the Fc domain monomers comprises a
single amino acid mutation
at EU Numbering position 1253 each amino acid mutation at EU Numbering
position 1253 is independently
selected from the group consisting of 1253A, 1253C, 1253D, 1253E, 1253F,
1253G, 1253H, 12531, 1253K,
I253L, 1253M, 1253N, 1253P, 1253Q, 1253R, 1253S, 1253T, 1253V, 1253W, and
1253Y; each amino acid
mutation at position 1253 is 1253A; at least one of the Fc domain monomers
comprises a single amino
acid mutation at EU Numbering position R292; each amino acid mutation at EU
Numbering position R292
is independently selected from the group consisting of R292D, R292E, R292L,
R292P, R292Q, R292R,
R292T, and R292Y; each amino acid mutation at position R292 is R292P; each Fc
domain monomer
independently comprises or consists of an amino acid sequence selected from
the group consisting of
EPKSCDKTHTCPPCPAPELL and DKTHTCPPCPAPELL; the hinge portion of the second Fc
domain
monomer and the third Fc domain monomer have the amino acid sequence
DKTHTCPPCPAPELL; the
hinge portion of the first Fc domain monomer has the amino acid sequence
EPKSCDKTHTCPPCPAPEL;
the hinge portion of the first Fc domain monomer has the amino acid sequence
EPKSCDKTHTCPPCPAPEL and the hinge portion of the second Fc domain monomer and
the third Fc
domain monomer have the amino acid sequence DKTHTCPPCPAPELL; the CH2 domains
of each Fc
domain monomer independently comprise the amino acid sequence:
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK with no more than two single amino acid
deletions
or substitutions; the CH2 domains of each Fc domain monomer are identical and
comprise the amino acid
sequence:
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
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VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK with no more than two single amino acid
deletions
or substitutions; the CH2 domains of each Fc domain monomer are identical and
comprise the amino acid
sequence:
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
.. VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK with no more than two single amino
acid
substitutions; the CH2 domains of each Fc domain monomer are identical and
comprise the amino acid
sequence:
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK; the CH3 domains of each Fc domain
monomer
.. independently comprise the amino acid sequence:
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK
LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG with no more than 10 single amino acid
substitutions; the CH3 domains of each Fc domain monomer independently
comprise the amino acid
sequence:
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK
LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG with no more than 8 single amino acid
substitutions; the CH3 domains of each Fc domain monomer independently
comprise the amino acid
sequence:
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK
.. LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG with no more than 6 single amino acid
substitutions; wherein the CH3 domains of each Fc domain monomer independently
comprise the amino
acid sequence:
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK
LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG with no more than 5 single amino acid
.. substitutions; the single amino acid substitutions are selected from the
group consisting of: T366Y,
T366W, T394W, T394Y, F405W, F405A, Y407A, S354C, Y349T, T394F, K409D, K409E,
K392D, K392E,
K370D, K370E, D399K, D399R, E357K, E357R, D356K, and D356R; each of the Fc
domain monomers
independently comprises the amino acid sequence of any of SEQ ID NOs:42, 43,
45, and 47 having up to
10 single amino acid substitutions; up to 6 of the single amino acid
substitutions are reverse charge
.. mutations in the CH3 domain or are mutations forming an engineered
protuberance; the single amino
acid substitutions are within the sequence from EU Numbering position G341 to
EU Numbering position
K447, inclusive; at least one of the mutations forming an engineered
protuberance is selected from the
group consisting of T366Y, T366W, T394W, T394Y, F405W, 5354C, Y349T, and
T394F; the two or four
reverse charge mutations are selected from: K409D, K409E, K392D. K392E, K370D,
K370E, D399K,
.. D399R, E357K, E357R, D356K, and D356R; the CD38 binding domain is a scFv;
CD38 binding domain
comprises a VH domain and a CH1 domain; the CD38 binding domain further
comprises a VL domain;
the VH domain comprises a set of CDR-H1, CDR-H2 and CDR-H3 sequences set forth
in Table 1; the VH
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domain comprises CDR-H1, CDR-H2, and CDR-H3 of a VH domain comprising a
sequence of an
antibody set forth in Table 2; the VH domain comprises CDR-H1, CDR-H2, and CDR-
H3 of a VH
sequence of an antibody set forth in Table 2, and the VH sequence, excluding
the CDR-H1, CDR-H2, and
CDR-H3 sequence, is at least 95% or 98% identical to the VH sequence of an
antibody set forth in Table
2; the VH domain comprises a VH sequence of an antibody set forth in Table 2;
the CD38 binding domain
comprises a set of CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3
sequences set forth in
Table 1; the CD38 binding domain comprises CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-
L2, and CDR-
L3 sequences from a set of a VH and a VL sequence of an antibody set forth in
Table 2; the CD38
binding domain comprises a VH domain comprising CDR-H1, CDR-H2, and CDR-H3 of
a VH sequence of
.. an antibody set forth in Table 2, and a VL domain comprising CDR-L1, CDR-
L2, and CDR-L3 of a VL
sequence of an antibody set forth in Table 2, wherein the VH and the VL domain
sequences, excluding
the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 sequences, are at least
95% or 98%
identical to the VH and VL sequences of an antibody set forth in Table 2; the
CD38 binding domain
comprises a set of a VH and a VL sequence of an antibody set forth in Table 2;
the CD38 binding domain
comprises an IgG CL antibody constant domain and an IgG CH1 antibody constant
domain; the CD38
binding domain comprises a VH domain and CH1 domain and can bind to a
polypeptide comprising a VL
domain and a CL domain to form a Fab.
Also described is a polypeptide complex comprising two copies of any of the
polypeptides
described above joined by disulfide bonds between cysteine residues within the
hinge of first or second
.. IgG1 Fc domain monomers.
Also described is a polypeptide complex comprising a polypeptide described
above joined to a
second polypeptide comprising and IgG1 Fc domain monomer comprising a hinge
domain, a CH2 domain
and a CH3 domain, wherein the polypeptide and the second polypeptide are
joined by disulfide bonds
between cysteine residues within the hinge domain of the first, second or
third IgG1 Fc domain monomer
of the polypeptide and the hinge domain of the second polypeptide. In various
embodiments: the second
polypeptide monomer comprises one, two or three reverse charge mutations; the
second polypeptide
monomer comprises one, two or three reverse charge mutations selected from
Tables 4A and 4B and are
complementary to the one, two or three reverse charge mutations selected
Tables 4A and 4B in the
polypeptide; the second polypeptide comprises the amino acid sequence of any
of SEQ ID NOs: 42, 43,
45, and 47 having up to 10 single amino acid substitutions.
In a forty third aspect, the disclosure features a polypeptide comprising: a
first IgG1 Fc domain
monomer comprising a hinge domain, a CH2 domain and a CH3 domain; a second
linker; a second IgG1
Fc domain monomer comprising a hinge domain, a CH2 domain and a CH3 domain; an
optional third
linker; and an optional third IgG1 Fc domain monomer comprising a hinge
domain, a CH2 domain and a
CH3 domain, wherein at least one Fc domain monomer comprises mutations forming
an engineered
protuberance.
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In various embodiments of the forty third aspect: the polypeptide further
comprises: an antibody
heavy chain variable domain and CH1 domain amino terminal to the first IgG1
monomer or an scFv
amino terminal to the first IgG1 monomer; the first IgG1 Fc domain monomer
comprises two or four
reverse charge mutations and the second IgG1 Fc domain monomer comprises
mutations forming an
engineered protuberance; the first IgG1 Fc domain monomer comprises mutations
forming an engineered
protuberance and the second IgG1 Fc domain monomer comprises two or four
reverse charge mutations;
both the first IgG1 Fc domain monomer and the second IgG constant domain
monomer comprise
mutations forming an engineered protuberance; the polypeptide comprises a
third linker and a third IgG1
Fc domain monomer wherein the first IgG1 Fc domain monomer, the second IgG1 Fc
domain monomer
and the third IgG1 Fc domain monomer each comprise mutations forming an
engineered protuberance;
the polypeptide comprises a third linker and a third IgG1 Fc domain monomer
wherein both the first IgG1
Fc domain monomer and the second IgG1 Fc domain monomer each comprise
mutations forming an
engineered protuberance and the third IgG1 Fc domain monomer comprises two or
four reverse charge
mutations; the polypeptide comprises a third linker and third IgG1 Fc domain
monomer wherein both the
first IgG1 Fc domain monomer and the third IgG1 Fc domain monomer each
comprise mutations forming
an engineered protuberance and the second IgG1 domain monomer comprises two or
four reverse
charge mutations; the polypeptide comprises a third linker and a third IgG1 Fc
domain monomer wherein
both the second IgG1 Fc domain monomer and the third IgG1 Fc domain monomer
each comprise
mutations forming an engineered protuberance and the first IgG1 domain monomer
comprises two or four
reverse charge mutations.
In various embodiments of the forty third aspect: the IgG1 Fc domain monomers
comprising
mutations forming an engineered protuberance further comprise one, two or
three reverse charge
mutations;
the mutations forming an engineered protuberance and the reverse charge
mutations are in the CH3
domain; the mutations are within the sequence from EU Numbering position G341
to EU Numbering
position K447, inclusive; the mutations are single amino acid changes; the
second linker and the optional
third linker comprise or consist of an amino acid sequence selected from the
group consisting of:
GGGGGGGGGGGGGGGGGGGG, GGGGS, GGSG, SGGG, GSGS, GSGSGS, GSGSGSGS,
GSGSGSGSGS, GSGSGSGSGSGS, GGSGGS, GGSGGSGGS, GGSGGSGGSGGS, GGSG, GGSG,
GGSGGGSG, GGSGGGSGGGSGGGGGSGGGGSGGGGSGGGGS, GENLYFQSGG, SACYCELS,
RSIAT, RPACKIPNDLKQKVMNH, GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG,
AAANSSIDLISVPVDSR, GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS,
GGGSGGGSGGGS, SGGGSGGGSGGGSGGGSGGG, GGSGGGSGGGSGGGSGGS, GGGG,
GGGGGGGG, GGGGGGGGGGGG and GGGGGGGGGGGGGGGG; the second linker and the
optional
third linker is a glycine spacer; the second linker and the optional third
linker independently consist of 4 to
30, 4 to 20, 8 to 30, 8 to 20, 12 to 20 or 12 to 30 glycine residues; the
second linker and the optional third
linker consist of 20 glycine residues; at least one of the Fc domain monomers
comprises a single amino
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acid mutation at EU Numbering position 1253 each amino acid mutation at EU
Numbering position 1253 is
independently selected from the group consisting of 1253A, 1253C, 1253D,
1253E, 1253F, 1253G, 1253H,
12531, 1253K, 1253L, 1253M, 1253N, 1253P, 1253Q, 1253R, 1253S, 1253T, 1253V,
1253W, and 1253Y; each
amino acid mutation at position 1253 is 1253A; at least one of the Fc domain
monomers comprises a
single amino acid mutation at EU Numbering position R292; each amino acid
mutation at EU Numbering
position R292 is independently selected from the group consisting of R292D,
R292E, R292L, R292P,
R292Q, R292R, R292T, and R292Y; each amino acid mutation at position R292 is
R292P; each Fc
domain monomer independently comprises or consists of an amino acid sequence
selected from the
group consisting of EPKSCDKTHTCPPCPAPELL and DKTHTCPPCPAPELL; the hinge
portion of the
second Fc domain monomer and the third Fc domain monomer have the amino acid
sequence
DKTHTCPPCPAPELL; the hinge portion of the first Fc domain monomer has the
amino acid sequence
EPKSCDKTHTCPPCPAPEL; the hinge portion of the first Fc domain monomer has the
amino acid
sequence EPKSCDKTHTCPPCPAPEL and the hinge portion of the second Fc domain
monomer and the
third Fc domain monomer have the amino acid sequence DKTHTCPPCPAPELL; the CH2
domains of
.. each Fc domain monomer independently comprise the amino acid sequence:
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK with no more than two single amino acid
deletions
or substitutions; the CH2 domains of each Fc domain monomer are identical and
comprise the amino acid
sequence:
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK with no more than two single amino acid
deletions
or substitutions; the CH2 domains of each Fc domain monomer are identical and
comprise the amino acid
sequence:
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK with no more than two single amino acid
substitutions; the CH2 domains of each Fc domain monomer are identical and
comprise the amino acid
sequence:
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK; the CH3 domains of each Fc domain
monomer
independently comprise the amino acid sequence:
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK
LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG with no more than 10 single amino acid
substitutions; the CH3 domains of each Fc domain monomer independently
comprise the amino acid
sequence:
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK
LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG with no more than 8 single amino acid
substitutions; the CH3 domains of each Fc domain monomer independently
comprise the amino acid
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sequence:
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK
LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG with no more than 6 single amino acid
substitutions; wherein the CH3 domains of each Fc domain monomer independently
comprise the amino
acid sequence:
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK
LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG with no more than 5 single amino acid
substitutions; the single amino acid substitutions are selected from the group
consisting of: T366Y,
T366W, T394W, T394Y, F405W, F405A, Y407A, S354C, Y349T, T394F, K409D, K409E,
K392D, K392E,
K370D, K370E, D399K, D399R, E357K, E357R, D356K, and D356R; each of the Fc
domain monomers
independently comprises the amino acid sequence of any of SEQ ID NOs:42, 43,
45, and 47 having up to
10 single amino acid substitutions; up to 6 of the single amino acid
substitutions are reverse charge
mutations in the CH3 domain or are mutations forming an engineered
protuberance; the single amino
acid substitutions are within the sequence from EU Numbering position G341 to
EU Numbering position
K447, inclusive; at least one of the mutations forming an engineered
protuberance is selected from the
group consisting of T366Y, T366W, T394W, T394Y, F405W, 5354C, Y349T, and
T394F; the two or four
reverse charge mutations are selected from: K409D, K409E, K392D. K392E, K370D,
K370E, D399K,
D399R, E357K, E357R, D356K, and D356R.
In a forty fourth aspect the disclosure features a polypeptide comprising: a
first IgG1 Fc domain
monomer comprising a hinge domain, a CH2 domain and a CH3 domain; a second
linker; a second IgG1
Fc domain monomer comprising a hinge domain, a CH2 domain and a CH3 domain; an
optional third
linker; and an optional third IgG1 Fc domain monomer comprising a hinge
domain, a CH2 domain and a
CH3 domain, wherein at least one Fc domain monomer comprises one, two or three
reverse charge
amino acid mutations.
In various embodiments of the forty fourth aspect: the polypeptide further
comprises an antibody
heavy chain variable domain and CH1 domain amino terminal to the first IgG1 Fc
domain monomer or
scFv amino terminal to the first IgG1 Fc domain monomer; the first IgG1 Fc
domain monomer comprises
a set of two reverse charge mutations selected from those in Tables 4A and 4B
or a set of four reverse
charge mutation selected from those in Tables 4A and 4B and the second IgG1 Fc
domain monomer
comprises one, two or three reverse charge amino acid mutations selected from
Tables 4A and 4B; the
first IgG1 Fc domain monomer comprises one, two or three reverse charge amino
acid mutations selected
from Tables 4A and 4B and the second IgG1 Fc domain monomer comprises a set of
two reverse charge
mutations selected from those in Tables 4a and 4b or a set of four reverse
charge mutation selected from
those in Tables 4A and 4B; both the first IgG1 Fc domain monomer and the
second IgG constant domain
monomer comprise one, two or three reverse charge amino acid mutations
selected from Tables 4A and
4B; the polypeptide further comprises a third linker and a third IgG1 Fc
domain monomer wherein the first
IgG1 Fc domain monomer, the second IgG1 Fc domain monomer and the third IgG1
Fc domain monomer
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each comprise one, two or three reverse charge amino acid mutations selected
from Tables 4A and 4B;
the polypeptide further comprises a third linker and a third IgG1 Fc domain
monomer wherein both the
first IgG1 Fc domain monomer and the second IgG1 Fc domain monomer each
comprise one, two or
three reverse charge amino acid mutations selected from Tables 4A and 4B and
the third IgG1 Fc domain
monomer comprises a set of two reverse charge mutations selected from those in
Tables 4A and 4B or a
set of four reverse charge mutation selected from those in Tables 4A and 4B;
the polypeptide further
comprises a third linker and third IgG1 Fc domain monomer wherein both the
first IgG1 Fc domain
monomer and the third IgG1 Fc domain monomer each comprise one, two or three
reverse charge amino
acid mutations selected from Tables 4A and 4B and the second IgG1 domain
monomer comprises a set
of two reverse charge mutations selected from those in Tables 4A and 4B or a
set of four reverse charge
mutation selected from those in Tables 4A and 4B; the polypeptide further
comprises a third linker and a
third IgG1 Fc domain monomer wherein both the second IgG1 Fc domain monomer
and the third IgG1 Fc
domain monomer each comprise one, two or three reverse charge amino acid
mutations selected from
Tables 4A and 4B and the first IgG1 domain monomer comprises a set of two
reverse charge mutations
selected from those in Tables 4A and 4B or a set of four reverse charge
mutation selected from those in
Tables 4A and 4BB; the IgG1 Fc domain monomers comprising one, two or three
reverse charge amino
acid mutations selected from Tables 4A and 4B have identical CH3 domains; one,
two or three reverse
charge amino acid mutations selected from Tables 4A and 4B are in the CH3
domain; the mutations are
within the sequence from EU Numbering position G341 to EU Numbering position
K447, inclusive; the
mutations are each single amino acid changes; the mutations are within the
sequence from EU
Numbering position G341 to EU Numbering position K446, inclusive; the
mutations are single amino acid
changes; the second linker and the optional third linker comprise or consist
of an amino acid sequence
selected from the group consisting of: GGGGGGGGGGGGGGGGGGGG, GGGGS, GGSG,
SGGG,
GSGS, GSGSGS, GSGSGSGS, GSGSGSGSGS, GSGSGSGSGSGS, GGSGGS, GGSGGSGGS,
GGSGGSGGSGGS, GGSG, GGSG, GGSGGGSG,
GGSGGGSGGGSGGGGGSGGGGSGGGGSGGGGS, GENLYFQSGG, SACYCELS, RSIAT,
RPACKIPNDLKQKVMNH, GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG,
AAANSSIDLISVPVDSR, GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS,
GGGSGGGSGGGS, SGGGSGGGSGGGSGGGSGGG, GGSGGGSGGGSGGGSGGS, GGGG,
GGGGGGGG, GGGGGGGGGGGG and GGGGGGGGGGGGGGGG; the second linker and the
optional
third linker is a glycine spacer; the second linker and the optional third
linker independently consist of 4 to
30, 4 to 20, 8 to 30, 8 to 20, 12 to 20 or 12 to 30 glycine residues; the
second linker and the optional third
linker consist of 20 glycine residues; at least one of the Fc domain monomers
comprises a single amino
acid mutation at EU Numbering position 1253 each amino acid mutation at EU
Numbering position 1253 is
independently selected from the group consisting of 1253A, 1253C, 1253D,
1253E, 1253F, 1253G, 1253H,
12531, 1253K, 1253L, 1253M, 1253N, 1253P, 1253Q, 1253R, 1253S, 1253T, 1253V,
1253W, and 1253Y; each
amino acid mutation at position 1253 is 1253A; at least one of the Fc domain
monomers comprises a
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single amino acid mutation at EU Numbering position R292; each amino acid
mutation at EU Numbering
position R292 is independently selected from the group consisting of R292D,
R292E, R292L, R292P,
R292Q, R292R, R292T, and R292Y; each amino acid mutation at position R292 is
R292P; each Fc
domain monomer independently comprises or consists of an amino acid sequence
selected from the
.. group consisting of EPKSCDKTHTCPPCPAPELL and DKTHTCPPCPAPELL; the hinge
portion of the
second Fc domain monomer and the third Fc domain monomer have the amino acid
sequence
DKTHTCPPCPAPELL; the hinge portion of the first Fc domain monomer has the
amino acid sequence
EPKSCDKTHTCPPCPAPEL; the hinge portion of the first Fc domain monomer has the
amino acid
sequence EPKSCDKTHTCPPCPAPEL and the hinge portion of the second Fc domain
monomer and the
third Fc domain monomer have the amino acid sequence DKTHTCPPCPAPELL; the CH2
domains of
each Fc domain monomer independently comprise the amino acid sequence:
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK with no more than two single amino acid
deletions
or substitutions; the CH2 domains of each Fc domain monomer are identical and
comprise the amino acid
sequence:
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK with no more than two single amino acid
deletions
or substitutions; the CH2 domains of each Fc domain monomer are identical and
comprise the amino acid
sequence:
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK with no more than two single amino acid
substitutions; the CH2 domains of each Fc domain monomer are identical and
comprise the amino acid
sequence:
GGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVS
VLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK; the CH3 domains of each Fc domain
monomer
independently comprise the amino acid sequence:
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK
LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG with no more than 10 single amino acid
substitutions; the CH3 domains of each Fc domain monomer independently
comprise the amino acid
sequence:
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK
LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG with no more than 8 single amino acid
substitutions; the CH3 domains of each Fc domain monomer independently
comprise the amino acid
sequence:
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK
LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG with no more than 6 single amino acid
substitutions; wherein the CH3 domains of each Fc domain monomer independently
comprise the amino
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acid sequence:
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSK
LTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG with no more than 5 single amino acid
substitutions; the single amino acid substitutions are selected from the group
consisting of: T366Y,
T366W, T394W, T394Y, F405W, F405A, Y407A, S354C, Y349T, T394F, K409D, K409E,
K392D, K392E,
K370D, K370E, D399K, D399R, E357K, E357R, D356K, and D356R; each of the Fc
domain monomers
independently comprises the amino acid sequence of any of SEQ ID NOs:42, 43,
45, and 47 having up to
single amino acid substitutions; up to 6 of the single amino acid
substitutions are reverse charge
mutations in the CH3 domain or are mutations forming an engineered
protuberance; the single amino
10 acid substitutions are within the sequence from EU Numbering position
G341 to EU Numbering position
K447, inclusive; the VH domain or scFv comprises a set of CDR-H1, CDR-H2 and
CDR-H3 sequences
set forth in Table 1; the VH domain or scFv comprises CDR-H1, CDR-H2, and CDR-
H3 of a VH domain
comprising a sequence of an antibody set forth in Table 2; the VH domain or
scFv comprises CDR-H1,
CDR-H2, and CDR-H3 of a VH sequence of an antibody set forth in Table 2, and
the VH sequence,
excluding the CDR-H1, CDR-H2, and CDR-H3 sequence, is at least 95% or 98%
identical to the VH
sequence of an antibody set forth in Table 2; the VH domain or scFv comprises
a VH sequence of an
antibody set forth in Table 2; the VH domain or scFv comprises a set of CDR-
H1, CDR-H2, CDR-H3,
CDR-L1, CDR-L2, and CDR-L3 sequences set forth in Table 1; the VH domain or
scFv comprises CDR-
H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 sequences from a set of a VH
and a VL sequence
of an antibody set forth in Table 2; the VH domain or scFv main comprises a VH
domain comprising CDR-
H1, CDR-H2, and CDR-H3 of a VH sequence of an antibody set forth in Table 2,
and a VL domain
comprising CDR-L1, CDR-L2, and CDR-L3 of a VL sequence of an antibody set
forth in Table 2, wherein
the VH and the VL domain sequences, excluding the CDR-H1, CDR-H2, CDR-H3, CDR-
L1, CDR-L2, and
CDR-L3 sequences, are at least 95% or 98% identical to the VH and VL sequences
of an antibody set
forth in Table 2; the VH domain or scFv comprises a set of a VH and a VL
sequence of an antibody set
forth in Table 2.
Also describes is a nucleic acid molecule encoding any of the forgoing
polypeptides of the forty
first, forty second, forty third and forty fourth aspects.
Also described is: an expression vector that includes a nucleic acid encoding
any of the forgoing
polypeptide; host cells containing the nucleic acids or expression vectors;
host cells further containing a
nucleic acid molecule encoding a polypeptide comprising an antibody VL domain
(e.g., a nucleic acid
molecule encoding a polypeptide comprising an antibody VL domain and an
antibody CL domain); a host
cell further containing a nucleic acid molecule encoding a polypeptide
comprising an antibody VL domain
and an antibody CL domain; a host cells further containing a nucleic acid
molecule encoding a
polypeptide comprising an IgG1 Fc domain monomer having no more than 10 single
amino acid
mutations; a host cell further containing a nucleic acid molecule encoding a
polypeptide comprising IgG1
Fc domain monomer having no more than 10 single amino acid mutations. In
various embodiments: the
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IgG1 Fc domain monomer comprises the amino acid sequence of any of SEQ ID Nos;
42, 43, 45 and 47
having no more than 10, 8, 6 or 4 single amino acid mutations in the CH3
domain.
Also described is a pharmaceutical composition comprising any of the
polypeptide or polypeptide
complexes described herein. In various embodiments less than 40%, 30%, 20%,
10%, 5%, 2% of the
polypeptides have at least one fucose.
The polypeptides of the of forty first, forty second, forty third and forty
fourth aspects of the
disclosure are useful as components of the various Fc-antigen binding domain
constructs described
herein. Thus, the polypeptides of any of the first through fortieth aspects,
e.g., those can comprise a
CD38 binding domain, can comprise or consist of the polypeptides of any of
forty first, forty second, forty
third and forty fourth aspects of the disclosure.
Other useful polypeptides for use in all aspects of the disclosure include
polypeptides comprising
an Fc domain monomer (e.g., comprising or consisting of the amino acid
sequence of any of SEQ ID Nos:
42, 43, 45 and 47 with no more than 8, 6, 5, 4, or 3 single amino acid
substitutions) having one, two or
three mutations forming a cavity (e.g., selected from: Y407T Y407A, F405A,
T3945, T394W:Y407T,
T3945:Y407A, T366W:T3945, F405T, T3665:1_368A:Y407V:Y349C, 5364H:F405A). These
polypeptides
can optionally include one, two or three reverse charge mutations from Tables
4A and 4B.
Also described herein is an Fc-antigen binding domain construct comprising:
a) a first polypeptide comprising:
i) a first Fc domain monomer,
ii) a second Fc domain monomer
iii) a first CD38 heavy chain binding domain, and
iv) a linker joining the first and second Fc domain monomers;
b) a second polypeptide comprising:
i) a third Fc domain monomer,
ii) a fourth Fc domain monomer
iii) a second CD38 heavy chain binding domain and
iv) a linker joining the third and fourth Fc domain monomers;
c) a third polypeptide comprising a fifth Fc domain monomer;
d) a fourth polypeptide comprising a sixth Fc domain monomer;
e) a fifth polypeptide comprising a first CD38 light chain binding domain; and
0 a sixth polypeptide comprising a second CD38 light chain binding domain;
wherein the first and third Fc domain monomers together form a first Fc
domain, the second and
fifth Fc domain monomers together form a second Fc domain, the fourth and
sixth Fc monomers together
form a third Fc domain, the first CD38 heavy chain binding domain and first
CD38 light chain binding
domain together form a first Fab; and the second CD38 heavy chain binding
domain and second CD38
light chain binding domain together form a second Fab.
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In various embodiments: the first and second polypeptides are identical in
sequence; the third
and fourth polypeptides are identical in sequence; the fifth and sixth
polypeptides are identical in
sequence; the first and second polypeptides are identical in sequence, the
third and fourth polypeptides
are identical in sequence, and the fifth and sixth polypeptides are identical
in sequence; the CH3 domain
of each of the Fc domain monomers includes up to 8, 7, 6, 5, 4, 3, 2 or 1
single amino acid substitutions;
the CH3 domain of each of the Fc domain monomers includes up to 8, 7, 6, 5, 4,
3, 2 or 1 single amino
acid substitutions compared to the amino acid sequence of human IgG; each of
the Fc domain monomers
independently comprises the amino acid sequence of any of SEQ ID NOs:42, 43,
45, and 47 having up to
10, 8, 7, 6, 5, 4, 3, 2 or 1 single amino acid substitutions; the single amino
acids substitutions are only in
the CH3 domain; the first and third Fc domain monomers comprise up to 8, 7, 6,
5, 4, 3, 2 or 1 single
amino acid substitutions that promote homodimerization between the first and
third Fc domain monomers;
the second and fifth Fc domain monomers comprise up to 8, 7, 6, 5, 4, 3, 2 or
1 single amino acid
substitutions that promote heterodimerization between the second and fifth Fc
domain monomers and the
fourth and sixth Fc domain monomers comprise up to 8, 7, 6, 5, 4, 3, 2 or 1
single amino acid
substitutions that promote heterodimerization between the fourth and sixth Fc
domain monomers; the
substitutions that promote homodimerization are selected from substitutions in
Table 4A and 4B; and
the substitutions that promote heterodimerization are selected from
substitutions in Table 3.
Also described is an Fc-antigen binding domain construct comprising:
a) a first polypeptide comprising:
i) a first Fc domain monomer,
ii) a second Fc domain monomer
iii) a first CD38 heavy chain binding domain, and
iv) a linker joining the first and second Fc domain monomers;
b) a second polypeptide comprising:
i) a third Fc domain monomer,
ii) a fourth Fc domain monomer
iii) a second CD38 heavy chain binding domain and
iv) a linker joining the third and fourth Fc domain monomers;
c) a third polypeptide comprising a fifth Fc domain monomer and a first CD38
light chain binding
domain; and
d) a fourth polypeptide comprising a sixth Fc domain monomer and a second CD38
light chain
binding domain;
wherein the first and third Fc domain monomers together form a first Fc
domain, the second and
fifth Fc domain monomers together form a second Fc domain, the fourth and
sixth Fc monomers together
form a third Fc domain, the first CD38 heavy chain binding domain and first
CD38 light chain binding
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domain together form a first Fab; and the second CD38 heavy chain binding
domain and second CD38
light chain binding domain together form a second Fab.
Also described is an Fc-antigen binding domain construct, comprising:
a) a first polypeptide comprising:
i) a first Fc domain monomer,
ii) a second Fc domain monomer
iii) a first CD38 heavy chain binding domain ,and
iv) a linker joining the first and second Fc domain monomers;
b) a second polypeptide comprising:
i) a third Fc domain monomer,
ii) a fourth Fc domain monomer
iii) a second CD38 heavy chain binding domain and
iv) a linker joining the third and fourth Fc domain monomers;
c) a third polypeptide comprising a fifth Fc domain monomer;
d) a fourth polypeptide comprising a sixth Fc domain monomer;
e) a fifth polypeptide comprising a first CD38 light chain binding domain; and
0 a sixth polypeptide comprising a second CD38 light chain binding domain;
wherein the first and fifth Fc domain monomers together form a first Fc
domain, the third and sixth
Fc domain monomers together form an second Fc domain, the second and fourth Fc
monomers together
form a third Fc domain, the first CD38 heavy chain binding domain and first
CD38 light chain binding
domain together form a first Fab; and the second CD38 heavy chain binding
domain and second CD38
light chain binding domain together form a second F
In various embodiments: the first and second polypeptides are identical in
sequence; third and
fourth polypeptides are identical in sequence; the fifth and sixth
polypeptides are identical in sequence;
the first and second polypeptides are identical in sequence, the third and
fourth polypeptides are identical
in sequence, and the fifth and sixth polypeptides are identical in sequence;
the CH3 domain of each of
the Fc domain monomers includes up to 8, 7, 6, 5, 4, 3, 2 or 1 single amino
acid substitutions; the CH3
domain of each of the Fc domain monomers includes up to 8, 7, 6, 5, 4, 3, 2 or
1 single amino acid
substitutions compared to the amino acid sequence of human IgG1; each of the
Fc domain monomers
independently comprises the amino acid sequence of any of SEQ ID NOs:42, 43,
45, and 47 having up to
10, 8, 7, 6, 5, 4, 3, 2 or 1 single amino acid substitutions; the single amino
acids substitutions are only in
the CH3 domain; the second and fourth Fc domain monomers comprise up to 8, 7,
6, 5, 4, 3, 2 or 1 single
amino acid substitutions that promote homodimerization between the second and
fourth Fc domain
monomers; the first and fifth Fc domain monomers comprise up to 8, 7, 6, 5, 4,
3, 2 or 1 single amino acid
substitutions that promote heterodimerization between the first and fifth Fc
domain monomers and the
third and sixth Fc domain monomers comprise up to 8, 7, 6, 5, 4, 3, 2 or 1
single amino acid substitutions
that promote heterodimerization between the fourth and sixth Fc domain
monomers; the substitutions that
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promote homodimerization are selected from substitutions in Table 4A and 4B;
and the substitutions that
promote heterodimerization are selected from substitutions in Table 3.
Also described in an Fc-antigen binding domain construct, comprising:
a) a first polypeptide comprising:
i) a first Fc domain monomer,
ii) a second Fc domain monomer,
iii) a third Fc domain monomer,
iv) a first CD38 heavy chain binding domain,
v) a linker joining the first and he second Fc domain monomers, and
vi) a linker joining the second and third Fc domain monomers;
b) a second polypeptide comprising:
i) a fourth Fc domain monomer,
ii) a fifth Fc domain monomer,
iii) a sixth Fc domain monomer,
iv) a second CD38 heavy chain binding domain,
v) a linker joining the fourth and fifth Fc domain monomers, and
vi) a linker joining the fifth and sixth Fc domain monomers;
c) a third polypeptide comprising a seventh Fc domain monomer;
d) a fourth polypeptide comprising an eighth Fc domain monomer;
e) a fifth polypeptide comprising ninth Fc domain monomer;
0 a sixth polypeptide comprising a tenth Fc domain monomer;
g) a seventh polypeptide comprising a first CD38 light chain binding domain;
and
h) an eighth polypeptide comprising a second CD38 light chain binding domain;
wherein the first and seventh Fc domain monomers together form a first Fc
domain, the fourth
and eighth Fc domain monomers together form an second Fc domain, the second
and fifth Fc monomer
together form a third Fc domain, the third and ninth Fc domain monomers
together form a fourth Fc
domain, the sixth and tenth Fc monomers together form a fifth Fc domain, the
first CD38 heavy chain
binding domain and first CD38 light chain binding domain together form a first
Fab; and the second CD38
heavy chain binding domain and second CD38 light chain binding domain together
form a second Fab.
In various embodiments: the first and second polypeptides are identical in
sequence; the third
and fourth polypeptides are identical in sequence; the fifth and sixth
polypeptides are identical in
sequence; the seventh and eighth polypeptides are identical in sequence; the
first and second
polypeptides are identical in sequence, the third and fourth polypeptides are
identical in sequence, the
fifth and sixth polypeptides are identical in sequence, and the seventh and
eighth polypeptides are
identical in sequence; the CH3 domain of each of the Fc domain monomers
includes up to 8, 7, 6, 5, 4, 3,
2 or 1 single amino acid substitutions; the CH3 domain of each of the Fc
domain monomers includes up
to 8, 7, 6, 5, 4, 3, 2 or 1 single amino acid substitutions compared to the
amino acid sequence of human
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IgG1; the Fc domain monomers independently comprises the amino acid sequence
of any of SEQ ID
NOs:42, 43, 45, and 47 having up to 10, 8, 7, 6, 5, 4, 3, 2 or 1 single amino
acid substitutions; the single
amino acids substitutions are only in the CH3 domain; the second and fifth Fc
domain monomers
comprise up to 8, 7, 6, 5, 4, 3, 2 or 1 single amino acid substitutions that
promote homodimerization
between the second and fifth Fc domain monomers; the first and seventh Fc
domain monomers comprise
up to 8, 7, 6, 5, 4, 3, 2 or 1 single amino acid substitutions that promote
heterodimerization between the
first and seventh Fc domain monomers, the fourth and eighth Fc domain monomers
comprise up to 8, 7,
6, 5, 4, 3, 2 or 1 single amino acid substitutions that promote
heterodimerization between the fourth and
eighth Fc domain monomers, the third and ninth Fc domain monomers comprise up
to 8, 7, 6, 5, 4, 3, 2 or
1 single amino acid substitutions that promote heterodimerization between the
third and ninth Fc domain
monomers, and the sixth and tenth Fc domain monomers comprise up to 8, 7, 6,
5, 4, 3, 2 or 1 single
amino acid substitutions that promote heterodimerization between the sixth and
tenth Fc domain
monomers; the substitutions that promote homodimerization are selected from
substitutions in Table 4A
and 4B; the substitutions that promote heterodimerization are selected from
substitutions in Table 3.
Also described is an Fc-antigen binding domain construct, comprising:
a) a first polypeptide comprising:
i) a first Fc domain monomer,
ii) a second Fc domain monomer,
iii) a third Fc domain monomer,
iv) a first CD38 heavy chain binding domain,
v) a linker joining the first and he second Fc domain monomers, and
vi) a linker joining the second and third Fc domain monomers;
b) a second polypeptide comprising:
i) a fourth Fc domain monomer,
ii) a fifth Fc domain monomer,
iii) a sixth Fc domain monomer,
iv) a second CD38 heavy chain binding domain,
v) a linker joining the fourth and fifth Fc domain monomers, and
vi) a linker joining the fifth and sixth Fc domain monomers;
c) a third polypeptide comprising a seventh Fc domain monomer;
d) a fourth polypeptide comprising an eighth Fc domain monomer;
e) a fifth polypeptide comprising ninth Fc domain monomer and a first CD38
light chain binding
domain; and
0 a sixth polypeptide comprising a tenth Fc domain monomer and ; a second CD38
light chain
.. binding domain
wherein the first and seventh Fc domain monomers together form a first Fc
domain, the fourth
and eighth Fc domain monomers together form an second Fc domain, the second
and fifth Fc monomer
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together form a third Fc domain, the third and ninth Fc domain monomers
together form a fourth Fc
domain, the sixth and tenth Fc monomers together form a fifth Fc domain, the
first CD38 heavy chain
binding domain and first CD38 light chain binding domain together form a first
Fab; and the second CD38
heavy chain binding domain and second CD38 light chain binding domain together
form a second Fab.
Also described is an Fc-antigen binding domain construct, comprising:
a) a first polypeptide comprising:
i) a first Fc domain monomer,
ii) a second Fc domain monomer,
iii) a third Fc domain monomer,
iv) a first CD38 heavy chain binding domain,
v) a linker joining the first and second Fc domain monomers, and
vi) a linker joining the second and te third Fc domain monomers;
b) a second polypeptide comprising:
i) a fourth Fc domain monomer,
ii) a fifth Fc domain monomer,
iii) a sixth Fc domain monomer,
iv) a second CD38 heavy chain binding domain,
v) a linker joining the fourth and fifth Fc domain monomers, and
vi) a linker joining the fifth and sixth Fc domain monomers;
c) a third polypeptide comprising a seventh Fc domain monomer;
d) a fourth polypeptide comprising an eighth Fc domain monomer;
e) a fifth polypeptide comprising ninth Fc domain monomer;
0 a sixth polypeptide comprising a tenth Fc domain monomer;
g) a seventh polypeptide comprising a first CD38 light chain binding domain;
and
h) an eighth polypeptide comprising a second CD38 light chain binding domain;
wherein the first and fourth Fc domain monomers together form a first Fc
domain, the second and
seventh Fc domain monomers together form an second Fc domain, the fifth and
eighth Fc monomers
together form a third Fc domain, the third and ninth Fc domain monomers
together form a fourth Fc
domain, the sixth and tenth Fc monomers together form a fifth Fc domain, the
first CD38 heavy chain
binding domain and first CD38 light chain binding domain together form a first
Fab; and the second CD38
heavy chain binding domain and second CD38 light chain binding domain together
form a second Fab.
In various embodiments: the first and second polypeptides are identical in
sequence; the third
and fourth polypeptides are identical in sequence; the fifth and sixth
polypeptides are identical in
sequence; the seventh and eighth polypeptides are identical in sequence; the
first and second
polypeptides are identical in sequence, the third and fourth polypeptides are
identical in sequence, the
fifth and sixth polypeptides are identical in sequence, and the seventh and
eighth polypeptides are
identical in sequence; the CH3 domain of each of the Fc domain monomers
includes up to 8, 7, 6, 5, 4, 3,
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2 or 1 single amino acid substitutions; the CH3 domain of each of the Fc
domain monomers includes up
to 8, 7, 6, 5, 4, 3, 2 or 1 single amino acid substitutions compared to the
amino acid sequence of human
IgG1; each of the Fc domain monomers independently comprises the amino acid
sequence of any of
SEQ ID NOs:42, 43, 45, and 47 having up to 10, 8, 7, 6, 5, 4, 3, 2 or 1 single
amino acid substitutions; the
single amino acids substitutions are only in the CH3 domain; the first and
fourth Fc domain monomers
comprise up to 8, 7, 6, 5, 4, 3, 2 or 1 single amino acid substitutions that
promote homodimerization
between the first and fourth Fc domain monomers; the second and seventh Fc
domain monomers
comprise up to 8, 7, 6, 5, 4, 3, 2 or 1 single amino acid substitutions that
promote heterodimerization
between the second and seventh Fc domain monomers, the fifth and eighth Fc
domain monomers
comprise up to 8, 7, 6, 5, 4, 3, 2 or 1 single amino acid substitutions that
promote heterodimerization
between the fifth and eighth Fc domain monomers, the third and ninth Fc domain
monomers comprise up
to 8, 7, 6, 5, 4, 3, 2 or 1 single amino acid substitutions that promote
heterodimerization between the third
and ninth Fc domain monomers, and the sixth and tenth Fc domain monomers
comprise up to 8, 7, 6, 5,
4, 3, 2 or 1 single amino acid substitutions that promote heterodimerization
between the sixth and tenth
Fc domain monomers; the substitutions that promote homodimerization are
selected from substitutions in
Table 4A and 4B; and the substitutions that promote heterodimerization are
selected from substitutions in
Table 3.
Also described is an Fc-antigen binding domain construct, comprising:
a) a first polypeptide comprising:
i) a first Fc domain monomer,
ii) a second Fc domain monomer,
iii) a third Fc domain monomer,
iv) a first CD38 heavy chain binding domain,
v) a linker joining the first and second Fc domain monomers, and
vi) a linker joining the second and te third Fc domain monomers;
b) a second polypeptide comprising:
i) a fourth Fc domain monomer,
ii) a fifth Fc domain monomer,
iii) a sixth Fc domain monomer,
iv) a second CD38 heavy chain binding domain,
v) a linker joining the fourth and fifth Fc domain monomers, and
vi) a linker joining the fifth and sixth Fc domain monomers;
c) a third polypeptide comprising a seventh Fc domain monomer;
d) a fourth polypeptide comprising an eighth Fc domain monomer;
e) a fifth polypeptide comprising ninth Fc domain monomer and a first CD38
light chain binding
domain;
0 a sixth polypeptide comprising a tenth Fc domain monomer and a second CD38
light chain
binding domain;
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wherein the first and fourth Fc domain monomers together form a first Fc
domain, the second and
seventh Fc domain monomers together form an second Fc domain, the fifth and
eighth Fc monomers
together form a third Fc domain, the third and ninth Fc domain monomers
together form a fourth Fc
domain, the sixth and tenth Fc monomers together form a fifth Fc domain, the
first CD38 heavy chain
binding domain and first CD38 light chain binding domain together form a first
Fab; and the second CD38
heavy chain binding domain and second CD38 light chain binding domain together
form a second Fab.
Also described is an Fc-antigen binding domain construct, comprising:
a) a first polypeptide comprising:
i) a first Fc domain monomer,
ii) a second Fc domain monomer,
iii) a linker joining the first and second Fc domain monomers, and
b) a second polypeptide comprising:
i) a third Fc domain monomer,
ii) a fourth Fc domain monomer
iii) a linker joining the third and fourth Fc domain monomers;
c) a third polypeptide comprising a fifth Fc domain monomer and a first CD38
heavy chain binding
domain and;
d) a fourth polypeptide comprising a sixth Fc domain monomer a second CD38
heavy chain
binding domain;
e) a fifth polypeptide comprising a first CD38 light chain binding domain; and
0 a sixth polypeptide comprising a second CD38 light chain binding domain;
wherein the first and fifth Fc domain monomers together form a first Fc
domain, the third and sixth
Fc domain monomers together form an second Fc domain, the second and fourth Fc
domain monomers
together form a third Fc domain, the first CD38 heavy chain binding domain and
first CD38 light chain
.. binding domain together form a first Fab; and the second CD38 heavy chain
binding domain and second
CD38 light chain binding domain together form a second Fab.
In various embodiments: the first and second polypeptides are identical in
sequence; the third
and fourth polypeptides are identical in sequence; the fifth and sixth
polypeptides are identical in
sequence; the first and second polypeptides are identical in sequence, the
third and fourth polypeptides
are identical in sequence, and the fifth and sixth polypeptides are identical
in sequence; the CH3 domain
of each of the Fc domain monomers includes up to 8, 7, 6, 5, 4, 3, 2 or 1
single amino acid substitutions;
the CH3 domain of each of the Fc domain monomers includes up to 8, 7, 6, 5, 4,
3, 2 or 1 single amino
acid substitutions compared to the amino acid sequence of human IgG1; each of
the Fc domain
monomers independently comprises the amino acid sequence of any of SEQ ID
NOs:42, 43, 45, and 47
.. having up to 10, 8, 7, 6, 5, 4, 3, 2 or 1 single amino acid substitutions;
the single amino acids substitutions
are only in the CH3 domain; the second and fourth Fc domain monomers comprise
up to 8, 7, 6, 5, 4, 3, 2
or 1 single amino acid substitutions that promote homodimerization between the
second and fourth Fc
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domain monomers; the first and fifth Fc domain monomers comprise up to 8, 7,
6, 5, 4, 3, 2 or 1 single
amino acid substitutions that promote heterodimerization between the first and
fifth Fc domain monomers
and the third and sixth Fc domain monomers comprise up to 8, 7, 6, 5, 4, 3, 2
or 1 single amino acid
substitutions that promote heterodimerization between the third and sixth Fc
domain monomers; the
substitutions that promote homodimerization are selected from substitutions in
Table 4A and 4B; the
substitutions that promote heterodimerization are selected from substitutions
in Table 3.
Also described is an Fc-antigen binding domain construct, comprising:
a) a first polypeptide comprising:
i) a first Fc domain monomer,
ii) a second Fc domain monomer,
iii) a first CD38 heavy chain binding domain, and
iv) a linker joining the first and second Fc domain monomers,
b) a second polypeptide comprising:
i) a third Fc domain monomer,
ii) a fourth Fc domain monomer,
iii) a second CD38 heavy chain binding domain, and
iv) a linker joining the third and fourth Fc domain monomers,
c) a third polypeptide comprising a fifth Fc domain monomer and a third CD38
heavy chain
binding domain;
d) a fourth polypeptide comprising a sixth Fc domain monomer and a fourth CD38
light chain
binding domain;
e) a fifth polypeptide comprising a first CD38 light chain binding domain;
0 a sixth polypeptide comprising a second CD38 light chain binding domain;
g) a seventh polypeptide comprising a third CD38 light chain binding domain;
and
h) an eighth polypeptide comprising a fourth CD38 light chain binding domain;
wherein the first and fifth Fc domain monomers together form a first Fc
domain, the third and sixth
Fc domain monomers together form an second Fc domain, the second and fourth Fc
monomers together
form a third Fc domain, the first CD38 light chain binding domain and third
CD38 heavy chain binding
domain together form a first Fab, the second CD38 light chain binding domain
and fourth CD38 heavy
chain binding domain together form a second Fab, the third CD38 light chain
binding domain and first
CD38 heavy chain binding domain together form a third Fab; and the fourth CD38
light chain binding
domain and second CD38 heavy chain binding domain together form a second Fab
In various embodiments: the first and second polypeptides are identical in
sequence; the third
and fourth polypeptides are identical in sequence; the fifth, sixth, seventh
and eighth polypeptides are
identical in sequence; the first and second polypeptides are identical in
sequence, the third and fourth
polypeptides are identical in sequence, and the fifth, sixth, seventh and
eighth polypeptides are identical
in sequence; the CH3 domain of each of the Fc domain monomers includes up to
8, 7, 6, 5, 4, 3, 2 or 1
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single amino acid substitutions; the CH3 domain of each of the Fc domain
monomers includes up to 8, 7,
6, 5, 4, 3, 2 or 1 single amino acid substitutions compared to the amino acid
sequence of human IgG1;
each of the Fc domain monomers independently comprises the amino acid sequence
of any of SEQ ID
NOs:42, 43, 45, and 47 having up to 10, 8, 7, 6, 5, 4, 3, 2 or 1 single amino
acid substitutions; the single
amino acids substitutions are only in the CH3 domain; the second and fourth Fc
domain monomers
comprise up to 8, 7, 6, 5, 4, 3, 2 or 1 single amino acid substitutions that
promote homodimerization
between the second and fourth Fc domain monomers; wherein the first and fifth
Fc domain monomers
comprise up to 8, 7, 6, 5, 4, 3, 2 or 1 single amino acid substitutions that
promote heterodimerization
between the first and fifth Fc domain monomers and the third and sixth Fc
domain monomers comprise
up to 8, 7, 6, 5, 4, 3, 2 or 1 single amino acid substitutions that promote
heterodimerization between the
third and sixth Fc domain monomers; the substitutions that promote
homodimerization are selected from
substitutions in Table 4A and 4B; and the substitutions that promote
heterodimerization are selected from
substitutions in Table 3.
In various embodiments: each linker comprise3 or consist of an amino acid
sequence selected
from the group consisting of:GGGGGGGGGGGGGGGGGGGG, GGGGS, GGSG, SGGG, GSGS,
GSGSGS, GSGSGSGS, GSGSGSGSGS, GSGSGSGSGSGS, GGSGGS, GGSGGSGGS,
GGSGGSGGSGGS, GGSG, GGSG, GGSGGGSG,
GGSGGGSGGGSGGGGGSGGGGSGGGGSGGGGS, GENLYFQSGG, SACYCELS, RSIAT,
RPACKIPNDLKQKVMNH, GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG,
AAANSSIDLISVPVDSR, GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS,
GGGSGGGSGGGS, SGGGSGGGSGGGSGGGSGGG, GGSGGGSGGGSGGGSGGS, GGGG,
GGGGGGGG, GGGGGGGGGGGG and GGGGGGGGGGGGGGGG; at least one of the Fc domain
monomers comprises a substitution at EU position 1253; each amino acid
substitution at EU position 1253
is independently selected from the group consisting of 1253A, 1253C, 1253D,
1253E, 1253F, 1253G, 1253H,
12531, 1253K, 1253L, 1253M, 1253N, 1253P, 1253Q, 1253R, 1253S, 1253T, 1253V,
1253W, and 1253Y; at least
one of the Fc domain monomers comprises a substitution at EU position R292;
each amino acid
substitution at EU position R292 is independently selected from the group
consisting of R292D, R292E,
R292L, R292P, R292Q, R292R, R292T, and R292Y; at least one of the Fc domain
monomers comprises
a substitution selected from the group consisting of: T366Y, T366W, T394W,
T394Y, F405W, F405A,
Y407A, 5354C, Y349T, T394F, K409D, K409E, K392D, K392E, K370D, K370E, D399K,
D399R, E357K,
E357R, D356K, and D356R; and the hinge of each Fc domain monomer independently
comprises or
consists of an amino acid sequence selected from the group consisting of
EPKSCDKTHTCPPCPAPELL
and DKTHTCPPCPAPELL.
In all aspects of the disclosure, some or all of the Fc domain monomers (e.g.,
an Fc domain
monomer comprising the amino acid sequence of any of SEQ ID Nos; 42, 43, 45
and 47 having no more
than 10, 8, 6 0r4 single amino acid substitutions (e.g., in the CH3 domain
only) can have one or both of a
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E345K and E430G amino acid substitution in addition to other amino acid
substitutions or modifications.
The E345K and E430G amino acid substitutions can increase Fc domain
multimerization.
Also described is an Fc-antigen binding domain construct comprising:
a) a first polypeptide comprising:
i) a first Fc domain monomer,
ii) a second Fc domain monomer
iii) a first CD38 heavy chain binding domain (e.g., comprising an VH domain
and a CH1
domain), and
iv) a linker joining the first and second Fc domain monomers;
b) a second polypeptide comprising:
i) a third Fc domain monomer,
ii) a fourth Fc domain monomer
iii) a second CD38 heavy chain binding domain (e.g., comprising an VH domain
and a
CH1 domain) and
iv) a linker joining the third and fourth Fc domain monomers;
c) a third polypeptide comprising a fifth Fc domain monomer;
d) a fourth polypeptide comprising a sixth Fc domain monomer;
e) a fifth polypeptide comprising a first CD38 light chain binding domain
(e.g., comprising an VL
domain and a CL domain); and
0 a sixth polypeptide comprising a second CD38 light chain binding domain
(e.g., comprising an
VL domain and a CL domain);
wherein the first and third Fc domain monomers together form a first Fc
domain, the second and
fifth Fc domain monomers together form a second Fc domain, the fourth and
sixth Fc monomers together
form a third Fc domain, the first CD38 heavy chain binding domain and first
CD38 light chain binding
.. domain together form a first Fab; and the second CD38 heavy chain binding
domain and second CD38
light chain binding domain together form a second Fab.
In various embodiments: the first and second polypeptides are identical in
sequence; the third
and fourth polypeptides are identical in sequence; the fifth and sixth
polypeptides are identical in
sequence; the first and second polypeptides are at least 95% identical to SEQ
ID NO: B, the third and
fourth polypeptides are at least 95% identical to SEQ ID NO: C, and the fifth
and sixth polypeptides are at
least 95% identical to SEQ ID NO: A; the first and second polypeptides are at
least 98% identical to SEQ
ID NO: B, the third and fourth polypeptides are at least 98% identical to SEQ
ID NO: C, and the fifth and
sixth polypeptides are at least 98% identical to SEQ ID NO: A; the first and
second polypeptides comprise
or consist of SEQ ID NO: B, the third and fourth polypeptides comprise of
consist of SEQ ID NO: C, and
.. the fifth and sixth polypeptides comprise or consist of SEQ ID NO: A; and
the first and second
polypeptides are identical in sequence, the third and fourth polypeptides are
identical in sequence, and
the fifth and sixth polypeptides are identical in sequence.
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Also disclosed is composition comprising:
a) a first polypeptide comprising:
i) a first Fc domain monomer,
ii) a second Fc domain monomer
iii) a first CD38 heavy chain binding domain, and
iv) a linker joining the first and second Fc domain monomers;
b) a second polypeptide comprising a third Fc domain monomer; and
c) a third polypeptide comprising a CD38 light chain binding domain.
In various embodiments: the first polypeptide is at least 95% identical to SEQ
ID NO: B, the second
polypeptide is at least 95% identical to SEQ ID NO: C, and the third
polypeptide is at least 95% identical
to SEQ ID NO: A; the first polypeptide is at least 98% identical to SEQ ID NO:
B, the second polypeptide
is at least 98% identical to SEQ ID NO: C, and the third polypeptide is at
least 98% identical to SEQ ID
NO: A; and the first polypeptide comprises of consists of SEQ ID NO: B, the
second polypeptide
comprises or consists of SEQ ID NO: C, and the third polypeptide comprise or
consists of SEQ ID NO: A.
Definitions:
As used herein, the term "Fe domain monomer" refers to a polypeptide chain
that includes at
least a hinge domain and second and third antibody constant domains (CH2 and
CH3) or functional
fragments thereof (e.g., at least a hinge domain or functional fragment
thereof, a CH2 domain or
functional fragment thereof, and a CH3 domain or functional fragment thereof)
(e.g., fragments that that
capable of (i) dimerizing with another Fc domain monomer to form an Fc domain,
and (ii) binding to an Fc
receptor). A preferred Fc domain monomer comprises, from amino to carboxy
terminus, at least a portion
of IgG1 hinge, an IgG1 CH2 domain and an IgG1 CH3 domain. Thus, an Fc domain
monomer, e.g., aa
human IgG1 Fc domain monomer can extend from E316 to G446 or K447, from P317
to G446 or K447,
from K318 to G446 or K447, from K318 to G446 or K447, from S319 to G446 or
K447, from C320 to
G446 or K447, from D321 to G446 or K447, from K322 to G446 or K447, from T323
to G446 or K447,
from K323 to G446 or K447, from H324 to G446 or K447, from T325 to G446 or
K447, or from C326 to
G446 or K447. The Fc domain monomer can be any immunoglobulin antibody
isotype, including IgG, IgE,
IgM, IgA, or IgD (e.g., IgG). Additionally, the Fc domain monomer can be an
IgG subtype (e.g., IgG1,
IgG2a, IgG2b, IgG3, or IgG4) (e.g., human IgG1). The human IgG1 Fc domain
monomer is used in the
examples described herein. The full hinge domain of human IgG1 extends from EU
Numbering E316 to
P230 or L235, the CH2 domain extends from A231 or G236 to K340 and the CH3
domain extends from
G341 to K447. There are differing views of the position of the last amino acid
of the hinge domain. It is
either P230 or L235. In many examples herein the CH3 domain does not include
K347. Thus, a CH3
domain can be from G341 to G446. In many examples herein a hinge domain can
include E216 to L235.
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This is true, for example, when the hinge is carboxy terminal to a CH1 domain
or a CD38 binding domain.
In some case, for example when the hinge is at the amino terminus of a
polypeptide, the Asp at EU
Numbering 221 is mutated to Gln. An Fc domain monomer does not include any
portion of an
immunoglobulin that is capable of acting as an antigen-recognition region,
e.g., a variable domain or a
complementarity determining region (CDR). Fc domain monomers can contain as
many as ten changes
from a wild-type (e.g., human) Fc domain monomer sequence (e.g., 1-10, 1-8, 1-
6, 1-4 amino acid
substitutions, additions, or deletions) that alter the interaction between an
Fc domain and an Fc receptor.
Fc domain monomers can contain as many as ten changes (e.g., single amino acid
changes) from a wild-
type Fc domain monomer sequence (e.g., 1-10, 1-8, 1-6, 1-4 amino acid
substitutions, additions, or
deletions) that alter the interaction between Fc domain monomers. In certain
embodiments, there are up
to 10, 8, 6 or 5 single amino acid substitution on the CH3 domain compared to
the human IgG1 CH3
domain sequence:
GQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQ
GNVFSCSV
MHEALHNHYTQKSLSLSPG. Examples of suitable changes are known in the art.
As used herein, the term "Fe domain" refers to a dimer of two Fc domain
monomers that is
capable of binding an Fc receptor. In the wild-type Fc domain, the two Fc
domain monomers dimerize by
the interaction between the two CH3 antibody constant domains, as well as one
or more disulfide bonds
that form between the hinge domains of the two dimerizing Fc domain monomers.
In the present disclosure, the term "Fc-antigen binding domain construct"
refers to associated
polypeptide chains forming at least two Fc domains as described herein and
including at least one
"antigen binding domain." Fe-antigen binding domain constructs described
herein can include Fc domain
monomers that have the same or different sequences. For example, an Fe-antigen
binding domain
construct can have three Fc domains, two of which includes IgG1 or IgG1-
derived Fc domain monomers,
and a third which includes IgG2 or IgG2-derived Fc domain monomers. In another
example, an Fe-
antigen binding domain construct can have three Fc domains, two of which
include a "protuberance-into-
cavity pair" and a third which does not include a "protuberance-into-cavity
pair." An Fc domain forms the
minimum structure that binds to an Fc receptor, e.g., FeyRI, FeyRIla, FeyRIlb,
FeyRIlla, FeyR111b, or
FeyRIV.
As used herein, the term "antigen binding domain" refers to a peptide, a
polypeptide, or a set of
associated polypeptides that is capable of specifically binding a target
molecule. In some embodiments,
the "antigen binding domain" is the minimal sequence of an antibody that binds
with specificity to the
antigen bound by the antibody. Surface plasmon resonance (SPR) or various
immunoassays known in
the art, e.g., Western Blots or ELISAs, can be used to assess antibody
specificity for an antigen. In some
embodiments, the "antigen binding domain" includes a variable domain or a
complementarity determining
region (CDR) of an antibody, e.g., one or more CDRs of an antibody set forth
in Table 1, one or more
CDRs of an antibody set forth in Table 2, or the VH and/or VL domains of an
antibody set forth in Table 2.
In some embodiments, the CD38 binding domain can include a VH domain and a CH1
domain, optionally
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with a VL domain. In other embodiments, the antigen (e.g., CD38) binding
domain is a Fab fragment of an
antibody or a scFv. Thus, a CD38 binding domain can include a "CD38 heavy
chain binding domain" that
comprises or consists of a VH domain and a CH1 domain and a" CD38 light chain
binding domain" that
comprises or consists of a VL domain and a CL domain. A CD38 binding domain
may also be a
synthetically engineered peptide that binds a target specifically such as a
fibronectin-based binding
protein (e.g., a fibronectin type III domain (FN3) monobody).
As used herein, the term "Complementarity Determining Regions" (CDRs) refers
to the amino
acid residues of an antibody variable domain the presence of which are
necessary for CD38 binding.
Each variable domain typically has three CDR regions identified as CDR-L1, CDR-
L2 and CDR-L3, and
CDR-H1, CDR-H2, and CDR-H3). Each complementarity determining region may
include amino acid
residues from a "complementarity determining region" as defined by Kabat
(i.e., about residues 24-34
(CDR-L1), 50-56 (CDR-L2), and 89-97 (CDR-L3) in the light chain variable
domain and 31-35 (CDR-H1),
50-65 (CDR-H2), and 95-102 (CDR-H3) in the heavy chain variable domain; Kabat
et al., Sequences of
Proteins of Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda,
Md. (1991)) and/or those residues from a "hypervariable loop" (i.e., about
residues 26-32 (CDR-L1), 50-
52 (CDR-L2), and 91-96 (CDR-L3) in the light chain variable domain and 26-32
(CDR-H1), 53-55 (CDR-
H2), and 96-101 (CDR-H3) in the heavy chain variable domain; Chothia and Lesk
J. Mol. Biol. 196:901-
917 (1987)). In some instances, a complementarity determining region can
include amino acids from
both a CDR region defined according to Kabat and a hypervariable loop.
"Framework regions" (hereinafter FR) are those variable domain residues other
than the CDR
residues. Each variable domain typically has four FRs identified as FR1, FR2,
FR3 and FR4. If the CDRs
are defined according to Kabat, the light chain FR residues are positioned at
about residues 1-23
(LCFR1), 35-49 (LCFR2), 57-88 (LCFR3), and 98-107 (LCFR4) and the heavy chain
FR residues are
positioned about at residues 1-30 (HCFR1), 36-49 (HCFR2), 66-94 (HCFR3), and
103-113 (HCFR4) in
the heavy chain residues. If the CDRs include amino acid residues from
hypervariable loops, the light
chain FR residues are positioned about at residues 1-25 (LCFR1), 33-49
(LCFR2), 53-90 (LCFR3), and
97-107 (LCFR4) in the light chain and the heavy chain FR residues are
positioned about at residues 1-25
(HCFR1), 33-52 (HCFR2), 56-95 (HCFR3), and 102-113 (HCFR4) in the heavy chain
residues. In some
instances, when the CDR includes amino acids from both a CDR as defined by
Kabat and those of a
hypervariable loop, the FR residues will be adjusted accordingly.
An "Fv" fragment is an antibody fragment which contains a complete antigen
recognition and
binding site. This region consists of a dimer of one heavy and one light chain
variable domain in tight
association, which can be covalent in nature, for example, in a scFv. It is in
this configuration that the
three CDRs of each variable domain interact to define a CD38 binding site on
the surface of the VH-VL
dimer.
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The "Fab" fragment contains a variable and constant domain of the light chain
and a variable
domain and the first constant domain (CH1) of the heavy chain. F(ab')2
antibody fragments include a pair
of Fab fragments which are generally covalently linked near their carboxy
termini by hinge cysteines.
"Single-chain Fv" or "scFv" antibody fragments include the VH and VL domains
of antibody in a
single polypeptide chain. Generally, the scFv polypeptide further includes a
polypeptide linker between
the VH and VL domains, which enables the scFv to form the desired structure
for CD38 binding.
As used herein, the term "antibody constant domain" refers to a polypeptide
that corresponds to a
constant region domain of an antibody (e.g., a CL antibody constant domain, a
CH1 antibody constant
domain, a CH2 antibody constant domain, or a CH3 antibody constant domain).
As used herein, the term "promote" means to encourage and to favor, e.g., to
favor the formation
of an Fc domain from two Fc domain monomers which have higher binding affinity
for each other than for
other, distinct Fc domain monomers. As is described herein, two Fc domain
monomers that combine to
form an Fc domain can have compatible amino acid modifications (e.g.,
engineered protuberances and
engineered cavities, and/or electrostatic steering mutations) at the interface
of their respective CH3
antibody constant domains. The compatible amino acid modifications promote or
favor the selective
interaction of such Fc domain monomers with each other relative to with other
Fc domain monomers
which lack such amino acid modifications or with incompatible amino acid
modifications. This occurs
because, due to the amino acid modifications at the interface of the two
interacting CH3 antibody constant
domains, the Fc domain monomers to have a higher affinity toward each other
than to other Fc domain
monomers lacking amino acid modifications.
As used herein, the term "dimerization selectivity module" refers to a
sequence of the Fc domain
monomer that facilitates the favored pairing between two Fc domain monomers.
"Complementary"
dimerization selectivity modules are dimerization selectivity modules that
promote or favor the selective
interaction of two Fc domain monomers with each other. Complementary
dimerization selectivity modules
can have the same or different sequences. Exemplary complementary dimerization
selectivity modules
are described herein.
As used herein, the term "engineered cavity" refers to the substitution of at
least one of the
original amino acid residues in the CH3 antibody constant domain with a
different amino acid residue
having a smaller side chain volume than the original amino acid residue, thus
creating a three
dimensional cavity in the CH3 antibody constant domain. The term "original
amino acid residue" refers to
a naturally occurring amino acid residue encoded by the genetic code of a wild-
type CH3 antibody
constant domain.
As used herein, the term "engineered protuberance" refers to the substitution
of at least one of
the original amino acid residues in the CH3 antibody constant domain with a
different amino acid residue
having a larger side chain volume than the original amino acid residue, thus
creating a three dimensional
protuberance in the CH3 antibody constant domain. The term "original amino
acid residues" refers to
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naturally occurring amino acid residues encoded by the genetic code of a wild-
type CH3 antibody constant
domain.
As used herein, the term "protuberance-into-cavity pair" describes an Fc
domain including two Fc
domain monomers, wherein the first Fc domain monomer includes an engineered
cavity in its CH3
antibody constant domain, while the second Fc domain monomer includes an
engineered protuberance in
its CH3 antibody constant domain. In a protuberance-into-cavity pair, the
engineered protuberance in the
CH3 antibody constant domain of the first Fc domain monomer is positioned such
that it interacts with the
engineered cavity of the CH3 antibody constant domain of the second Fc domain
monomer without
significantly perturbing the normal association of the dimer at the inter-CH3
antibody constant domain
interface.
As used herein, the term "heterodimer Fc domain" refers to an Fc domain that
is formed by the
heterodimerization of two Fc domain monomers, wherein the two Fc domain
monomers contain different
reverse charge mutations (see, e.g., mutations in Tables 4A and 4B) that
promote the favorable formation
of these two Fc domain monomers. In an Fc construct having three Fc domains -
one carboxyl terminal
"stem" Fc domain and two amino terminal "branch" Fc domains ¨ each of the
amino terminal "branch" Fc
domains may be a heterodimeric Fc domain (also called a "branch heterodimeric
Fc domain").
As used herein, the term "structurally identical," in reference to a
population of Fc-antigen binding
domain constructs, refers to constructs that are assemblies of the same
polypeptide sequences in the
same ratio and configuration and does not refer to any post-translational
modification, such as
.. glycosylation.
As used herein, the term "homodimeric Fc domain" refers to an Fc domain that
is formed by the
homodimerization of two Fc domain monomers, wherein the two Fc domain monomers
contain the same
reverse charge mutations (see, e.g., mutations in Tables 5 and 6). In an Fc
construct having three Fc
domains - one carboxyl terminal "stem" Fc domain and two amino terminal
"branch" Fc domains ¨ the
carboxy terminal "stem" Fc domain may be a homodimeric Fc domain (also called
a "stem homodimeric
Fc domain").
As used herein, the term "heterodimerizing selectivity module" refers to
engineered
protuberances, engineered cavities, and certain reverse charge amino acid
substitutions that can be
made in the CH3 antibody constant domains of Fc domain monomers in order to
promote favorable
heterodimerization of two Fc domain monomers that have compatible
heterodimerizing selectivity
modules. Fc domain monomers containing heterodimerizing selectivity modules
may combine to form a
heterodimeric Fc domain. Examples of heterodimerizing selectivity modules are
shown in Tables 3 and 4.
As used herein, the term "homodimerizing selectivity module" refers to reverse
charge mutations
in an Fc domain monomer in at least two positions within the ring of charged
residues at the interface
.. between CH3 domains that promote homodimerization of the Fc domain monomer
to form a homodimeric
Fc domain. Examples of homodimerizing selectivity modules are shown in Tables
4 and 5.
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As used herein, the term "joined" is used to describe the combination or
attachment of two or
more elements, components, or protein domains, e.g., polypeptides, by means
including chemical
conjugation, recombinant means, and chemical bonds, e.g., peptide bonds,
disulfide bonds and amide
bonds. For example, two single polypeptides can be joined to form one
contiguous protein structure
through chemical conjugation, a chemical bond, a peptide linker, or any other
means of covalent linkage.
In some embodiments, a CD38 binding domain is joined to a Fc domain monomer by
being expressed
from a contiguous nucleic acid sequence encoding both the CD38 binding domain
and the Fc domain
monomer. In other embodiments, a CD38 binding domain is joined to a Fc domain
monomer by way of a
peptide linker, wherein the N-terminus of the peptide linker is joined to the
C-terminus of the CD38
binding domain through a chemical bond, e.g., a peptide bond, and the C-
terminus of the peptide linker is
joined to the N-terminus of the Fc domain monomer through a chemical bond,
e.g., a peptide bond.
As used herein, the term "associated" is used to describe the interaction,
e.g., hydrogen bonding,
hydrophobic interaction, or ionic interaction, between polypeptides (or
sequences within one single
polypeptide) such that the polypeptides (or sequences within one single
polypeptide) are positioned to
form an Fc-antigen binding domain construct described herein (e.g., an Fc-
antigen binding domain
construct having three Fc domains). For example, in some embodiments, four
polypeptides, e.g., two
polypeptides each including two Fc domain monomers and two polypeptides each
including one Fc
domain monomer, associate to form an Fc construct that has three Fc domains
(e.g., as depicted in FIGS.
50 and 51). The four polypeptides can associate through their respective Fc
domain monomers. The
association between polypeptides does not include covalent interactions.
As used herein, the term "linker" refers to a linkage between two elements,
e.g., protein domains.
A linker can be a covalent bond or a spacer. The term "bond" refers to a
chemical bond, e.g., an amide
bond or a disulfide bond, or any kind of bond created from a chemical
reaction, e.g., chemical
conjugation. The term "spacer" refers to a moiety (e.g., a polyethylene glycol
(PEG) polymer) or an amino
acid sequence (e.g., a 3-200 amino acid, 3-150 amino acid, or 3-100 amino acid
sequence) occurring
between two polypeptides or polypeptide domains to provide space and/or
flexibility between the two
polypeptides or polypeptide domains. An amino acid spacer is part of the
primary sequence of a
polypeptide (e.g., joined to the spaced polypeptides or polypeptide domains
via the polypeptide
backbone). The formation of disulfide bonds, e.g., between two hinge regions
or two Fc domain
monomers that form an Fc domain, is not considered a linker.
As used herein, the term "glycine spacer" refers to a linker containing only
glycines that joins two
Fc domain monomers in tandem series. A glycine spacer may contain at least 4,
8, or 12 glycines (e.g.,
4-30, 8-30, or 12-30 glycines; e.g., 12-30, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
14, 15, 16, 17, 18, 19, 20, 21, 22,
23, 24, 25, 26, 27, 28, 29, or 30 glycines). In some embodiments, a glycine
spacer has the sequence of
GGGGGGGGGGGGGGGGGGGG (SEQ ID NO: 27).
As used herein, the term "albumin-binding peptide" refers to an amino acid
sequence of 12 to 16
amino acids that has affinity for and functions to bind serum albumin. An
albumin-binding peptide can be
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of different origins, e.g., human, mouse, or rat. In some embodiments of the
present disclosure, an
albumin-binding peptide is fused to the C-terminus of an Fc domain monomer to
increase the serum half-
life of the Fc-antigen binding domain construct. An albumin-binding peptide
can be fused, either directly
or through a linker, to the N- or C-terminus of an Fc domain monomer.
As used herein, the term "purification peptide" refers to a peptide of any
length that can be used
for purification, isolation, or identification of a polypeptide. A
purification peptide may be joined to a
polypeptide to aid in purifying the polypeptide and/or isolating the
polypeptide from, e.g., a cell lysate
mixture. In some embodiments, the purification peptide binds to another moiety
that has a specific affinity
for the purification peptide. In some embodiments, such moieties which
specifically bind to the
purification peptide are attached to a solid support, such as a matrix, a
resin, or agarose beads.
Examples of purification peptides that may be joined to an Fc-antigen binding
domain construct are
described in detail further herein.
As used herein, the term "multimer" refers to a molecule including at least
two associated Fc
constructs or Fc-antigen binding domain constructs described herein.
As used herein, the term "polynucleotide" refers to an oligonucleotide, or
nucleotide, and
fragments or portions thereof, and to DNA or RNA of genomic or synthetic
origin, which may be single- or
double-stranded, and represent the sense or anti-sense strand. A single
polynucleotide is translated into
a single polypeptide.
As used herein, the term "polypeptide" describes a single polymer in which the
monomers are
amino acid residues which are joined together through amide bonds. A
polypeptide is intended to
encompass any amino acid sequence, either naturally occurring, recombinant, or
synthetically produced.
As used herein, the term "amino acid positions" refers to the position numbers
of amino acids in a
protein or protein domain. The amino acid positions are numbered using the
Kabat numbering system
(Kabat et al., Sequences of Proteins of Immunological Interest, National
Institutes of Health, Bethesda,
Md., ed 5, 1991) where indicated (eg.g., for CDR and FR regions), otherwise
the EU numbering is used.
FIGs. 24A-24D depict human IgG1 Fc domains numbered using the EU numbering
system.
As used herein, the term "amino acid modification" or refers to an alteration
of an Fc domain
polypeptide sequence that, compared with a reference sequence (e.g., a wild-
type, unmutated, or
unmodified Fc sequence) may have an effect on the pharmacokinetics (PK) and/or
pharmacodynamics
(PD) properties, serum half-life, effector functions (e.g., cell lysis (e.g.,
antibody-dependent cell-mediated
toxicity(ADCC) and/or complement dependent cytotoxicity activity (CDC)),
phagocytosis (e.g., antibody
dependent cellular phagocytosis (ADCP) and/or complement-dependent cellular
cytotoxicity (CDCC)),
immune activation, and T-cell activation), affinity for Fc receptors (e.g., Fc-
gamma receptors (FcyR) (e.g.,
FcyRI (CD64), FcyRIla (CD32), FcyRIlb (CD32), FcyRIlla (CD16a), and/or
FcyRIllb (CD16b)), Fc-alpha
receptors (FcaR), Fc-epsilon receptors (FcER), and/or to the neonatal Fc
receptor (FcRn)), affinity for
proteins involved in the compliment cascade (e.g., C1q), post-translational
modifications (e.g.,
glycosylation, sialylation), aggregation properties (e.g., the ability to form
dimers (e.g., homo- and/or
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heterodimers) and/or multimers), and the biophysical properties (e.g., alters
the interaction between CH1
and CL, alters stability, and/or alters sensitivity to temperature and/or pH)
of an Fc construct, and may
promote improved efficacy of treatment of immunological and inflammatory
diseases. An amino acid
modification includes amino acid substitutions, deletions, and/or insertions.
In some embodiments, an
amino acid modification is the modification of a single amino acid. In other
embodiment, the amino acid
modification is the modification of multiple (e.g., more than one) amino
acids. The amino acid
modification may include a combination of amino acid substitutions, deletions,
and/or insertions. Included
in the description of amino acid modifications, are genetic (i.e., DNA and
RNA) alterations such as point
mutations (e.g., the exchange of a single nucleotide for another), insertions
and deletions (e.g., the
.. addition and/or removal of one or more nucleotides) of the nucleotide
sequence that codes for an Fc
polypeptide.
In certain embodiments, at least one (e.g., one, two, or three) Fc domain
monomers within an Fc
construct or Fc-antigen binding domain construct include an amino acid
modification (e.g., substitution).
In some instances, the at least one Fc domain monomers includes one or more
(e.g., no more than two,
three, four, five, six, seven, eight, nine, ten, or twenty) amino acid
modifications (e.g., substitutions).
As used herein, the term "percent (%) identity" refers to the percentage of
amino acid (or nucleic
acid) residues of a candidate sequence, e.g., the sequence of an Fc domain
monomer in an Fc-antigen
binding domain construct described herein, that are identical to the amino
acid (or nucleic acid) residues
of a reference sequence, e.g., the sequence of a wild-type Fc domain monomer,
after aligning the
sequences and introducing gaps, if necessary, to achieve the maximum percent
identity (i.e., gaps can be
introduced in one or both of the candidate and reference sequences for optimal
alignment and non-
homologous sequences can be disregarded for comparison purposes). Alignment
for purposes of
determining percent identity can be achieved in various ways that are within
the skill in the art, for
instance, using publicly available computer software such as BLAST, ALIGN, or
Megalign (DNASTAR)
.. software. Those skilled in the art can determine appropriate parameters for
measuring alignment,
including any algorithms needed to achieve maximal alignment over the full
length of the sequences
being compared. In some embodiments, the percent amino acid (or nucleic acid)
sequence identity of a
given candidate sequence to, with, or against a given reference sequence
(which can alternatively be
phrased as a given candidate sequence that has or includes a certain percent
amino acid (or nucleic
acid) sequence identity to, with, or against a given reference sequence) is
calculated as follows:
100 x (fraction of A/B)
where A is the number of amino acid (or nucleic acid) residues scored as
identical in the alignment of the
candidate sequence and the reference sequence, and where B is the total number
of amino acid (or
nucleic acid) residues in the reference sequence. In some embodiments where
the length of the
candidate sequence does not equal to the length of the reference sequence, the
percent amino acid (or
nucleic acid) sequence identity of the candidate sequence to the reference
sequence would not equal to
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the percent amino acid (or nucleic acid) sequence identity of the reference
sequence to the candidate
sequence.
In particular embodiments, a reference sequence aligned for comparison with a
candidate
sequence may show that the candidate sequence exhibits from 50% to 100%
identity (e.g., 50% to 100%,
60% to 100%, 70% to 100%, 80% to 100%, 90% to 100%, 92% to 100%, 95% to 100%,
97% to 100%,
99% to 100%, or 99.5% to 100% identity), across the full length of the
candidate sequence or a selected
portion of contiguous amino acid (or nucleic acid) residues of the candidate
sequence. The length of the
candidate sequence aligned for comparison purpose is at least 30%, e.g., at
least 40%, e.g., at least
50%, 60%, 70%, 80%, 90%, or 100% of the length of the reference sequence. When
a position in the
candidate sequence is occupied by the same amino acid (or nucleic acid)
residue as the corresponding
position in the reference sequence, then the molecules are identical at that
position.
In some embodiments, an Fc domain monomer in an Fc construct described herein
(e.g., an Fc-
antigen binding domain construct having three Fc domains) may have a sequence
that is at least 95%
identical (at least 97%, 99%, or 99.5% identical) to the sequence of a wild-
type Fc domain monomer (e.g.,
SEQ ID NO: 42). In some embodiments, an Fc domain monomer in an Fc construct
described herein
(e.g., an Fc-antigen binding domain construct having three Fc domains) may
have a sequence that is at
least 95% identical (at least 97%, 99%, or 99.5% identical) to the sequence of
any one of SEQ ID NOs:
44, 46, 48, and 50-53. In certain embodiments, an Fc domain monomer in the Fc
construct may have a
sequence that is at least 95% identical (at least 97%, 99%, or 99.5%
identical) to the sequence of SEQ ID
NO: 48, 52, and 53.
In some embodiments, a spacer between two Fc domain monomers may have a
sequence that is
at least 75% identical (at least 75%, 77%, 79%, 81%, 83%, 85%, 87%, 89%, 91%,
93%, 95%, 97%, 99%,
99.5%, or 100% identical) to the sequence of any one of SEQ ID NOs: 1-36
(e.g., SEQ ID NOs: 17, 18,
26, and 27) described further herein.
As used herein, the term "host cell" refers to a vehicle that includes the
necessary cellular
components, e.g., organelles, needed to express proteins from their
corresponding nucleic acids. The
nucleic acids are typically included in nucleic acid vectors that can be
introduced into the host cell by
conventional techniques known in the art (transformation, transfection,
electroporation, calcium
phosphate precipitation, direct microinjection, etc.). A host cell may be a
prokaryotic cell, e.g., a bacterial
cell, or a eukaryotic cell, e.g., a mammalian cell (e.g., a CHO cell). As
described herein, a host cell is
used to express one or more polypeptides encoding desired domains which can
then combine to form a
desired Fc-antigen binding domain construct.
As used herein, the term "pharmaceutical composition" refers to a medicinal or
pharmaceutical
formulation that contains an active ingredient as well as one or more
excipients and diluents to enable the
active ingredient to be suitable for the method of administration. The
pharmaceutical composition of the
present disclosure includes pharmaceutically acceptable components that are
compatible with the Fc-
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antigen binding domain construct. The pharmaceutical composition is typically
in aqueous form for
intravenous or subcutaneous administration.
As used herein, a "substantially homogenous population" of polypeptides or of
an Fc construct is
one in which at least 50% of the polypeptides or Fc constructs in a
composition (e.g., a cell culture
medium or a pharmaceutical composition) have the same number of Fc domains, as
determined by non-
reducing SDS gel electrophoresis or size exclusion chromatography. A
substantially homogenous
population of polypeptides or of an Fc construct may be obtained prior to
purification, or after Protein A or
Protein G purification, or after any Fab or Fc-specific affinity
chromatography only. In various
embodiments, at least 55%, 60%, 65%, 70%, 75%, 80%, or 85% of the polypeptides
or Fc constructs in
the composition have the same number of Fc domains. In other embodiments, up
to 85%, 90%, 92%, or
95% of the polypeptides or Fc constructs in the composition have the same
number of Fc domains.
As used herein, the term "pharmaceutically acceptable carrier" refers to an
excipient or diluent in
a pharmaceutical composition. The pharmaceutically acceptable carrier must be
compatible with the
other ingredients of the formulation and not deleterious to the recipient. In
the present disclosure, the
pharmaceutically acceptable carrier must provide adequate pharmaceutical
stability to the Fc-antigen
binding domain construct. The nature of the carrier differs with the mode of
administration. For example,
for oral administration, a solid carrier is preferred; for intravenous
administration, an aqueous solution
carrier (e.g., WFI, and/or a buffered solution) is generally used.
As used herein, "therapeutically effective amount" refers to an amount, e.g.,
pharmaceutical dose,
effective in inducing a desired biological effect in a subject or patient or
in treating a patient having a
condition or disorder described herein. It is also to be understood herein
that a "therapeutically effective
amount" may be interpreted as an amount giving a desired therapeutic effect,
either taken in one dose or
in any dosage or route, taken alone or in combination with other therapeutic
agents.
As used herein, the term fragment and the term portion can be used
interchangeably.
Brief Description of the Drawings
FIG. 1 is an illustration of an Fc-antigen binding domain construct (construct
1) containing two Fc
domains and a CD38 binding domain. Each Fc domain is a dimer of two Fc domain
monomers. Two of
the Fc domain monomers (106 and 108) contain a protuberance in its CH3
antibody constant domain,
while the other two Fc domain monomers (112 and 114) contain a cavity in the
juxtaposed position in its
CH3 antibody constant domain. The construct is formed from three Fc domain
monomer containing
polypeptides. The first polypeptide (102) contains two protuberance-containing
Fc domain monomers
(106 and 108) linked by a spacer in a tandem series to a CD38 binding domain
containing a VH domain
(110) on the N-terminus. A VL containing domain (104) is joined to the VH
domain. Each of the second
and third polypeptides (112 and 114) contains a cavity-containing Fc domain
monomer.
FIG. 2 is an illustration of an Fc-antigen binding domain construct (construct
2) containing three
Fc domains and a CD38 binding domain. The construct is formed from four Fc
domain monomer
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containing polypeptides. The first polypeptide (202) contains three
protuberance-containing Fc domains
(206, 208, and 210) linked by spacers in a tandem series to a CD38 binding
domain containing a VH
domain (212) on the N-terminus. A VL containing domain (204) is joined to the
VH domain. Each of the
second, third, and fourth polypeptides (214, 216, and 218) contains a cavity-
containing Fc domain
monomer.
FIG. 3 is an illustration of an Fc-antigen binding domain construct (construct
3) containing two Fc
domains and twoCD38 binding domains. The construct is formed from three Fc
domain monomer
containing polypeptides. The first polypeptide (302) contains two protuberance-
containing Fc domain
monomers (304 and 306) linked by a spacer in a tandem series. Each of the
second and third
polypeptides (320 and 322) contains a cavity-containing Fc domain monomer (310
and 314) joined in
tandem to a CD38 binding domain containing a VH domain (316 and 318) on the N-
terminus. A VL
containing domain (308 and 312) is joined to each VH domain.
FIG. 4 is an illustration of an Fc-antigen binding domain construct (construct
4) containing three
Fc domains and threeCD38 binding domains. The construct is formed from four Fc
domain monomer
containing polypeptides. The first polypeptide (402) contains three
protuberance-containing Fc domain
monomers (404, 406, and 408) linked by spacers in a tandem series. Each of the
second, third, and
fourth polypeptides (428, 430, and 432) contains a cavity-containing Fc domain
monomer (426, 420, and
414) joined in tandem to a CD38 binding domain containing a VH domain (422,
416, and 410) on the N-
terminus. A VL containing domain (424, 418, and 412) is joined to each VH
domain.
FIG. 5 is an illustration of an Fc-antigen binding domain construct (construct
5) containing two Fc
domains and threeCD38 binding domains. The construct is formed from three Fc
domain monomer
containing polypeptides. The first polypeptide (502) contains two protuberance-
containing Fc domain
monomers (508 and 506) linked by a spacer in a tandem series with a CD38
binding domain containing a
VH domain (510) at the N-terminus. Each of the second and third polypeptides
(524 and 526) contains a
cavity-containing Fc domain monomer (516 and 522) joined in tandem to a CD38
binding domain
containing a VH domain (512 and 518) on the N-terminus. A VL containing domain
(504, 514, and 520) is
joined to each VH domain.
FIG. 6 is an illustration of an Fc-antigen binding domain construct (construct
6) containing three
Fc domains and four CD38 binding domains. The construct is formed from four Fc
monomer containing
polypeptides. The first polypeptide (602) contains three protuberance-
containing Fc domain monomers
(606, 608, and 610) linked by spacers in a tandem series with a CD38 binding
domain containing a VH
domain (612) at the N-terminus. Each of the second, third, and fourth
polypeptides (632, 634, and 636)
contains a cavity-containing Fc domain monomer (618, 624, and 630) joined in
tandem to a CD38 binding
domain containing a VH domain (616, 622, and 628) on the N-terminus. A VL
containing domain (604,
616, 622, and 628) is joined to each VH domain.
FIG. 7 is an illustration of an Fc-antigen binding domain construct (construct
7) containing three
Fc domains and twoCD38 binding domains. This Fc-antigen binding domain
construct contains a dimer
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of two Fc domain monomers (706 and 718), wherein both Fc domain monomers
contain different charged
amino acids at their CH3-CH3 interface than the VVT sequence to promote
favorable electrostatic
interactions between the two Fc domain monomers. The construct is formed from
four Fc domain
monomer containing polypeptides. Two polypeptides (702 and 724) each contain a
protuberance-
containing Fc domain monomer (710 and 720) linked by a spacer in a tandem
series to an Fc domain
monomer containing different charged amino acids at the CH3-CH3 interface than
the \MT sequence (706
and 718) and a CD38 binding domain containing a VH domain (712 and 714) on the
N-terminus. The third
and fourth polypeptides (708 and 722) each contain a cavity-containing Fc
domain monomer. A VL
containing domain (704 and 716) is joined to each VH domain.
FIG. 8 is an illustration of an Fc-antigen binding domain construct (construct
8) containing three
Fc domains and twoCD38 binding domains. The construct is formed of four Fc
domain monomer
containing polypeptides. Two polypeptides (802 and 828) each contain a
protuberance-containing Fc
domain monomer (814 and 820) linked by a spacer in a tandem series to an Fc
domain monomer
containing different charged amino acids at the CH3-CH3 interface than the \MT
sequence (810 and 816).
The third and fourth polypeptides (804 and 826) each contain a cavity-
containing Fc domain monomer
(808 and 824) joined in tandem to a CD38 binding domain containing a VH domain
(812 and 818) at the
N-terminus. A VL containing domain (806 and 822) is joined to each VH domain.
FIG. 9 is an illustration of an Fc-antigen binding domain construct (construct
9) containing three
Fc domains and fourCD38 binding domains. The construct is formed of four Fc
domain monomer
containing polypeptides. Two polypeptides (902 and 936) each contain a
protuberance-containing Fc
domain monomer (918 and 928) linked by a spacer in a tandem series to an Fc
domain monomer
containing different charged amino acids at the CH3-CH3 interface than the \MT
sequence (910 and 924)
and a CD38 binding domain containing a VH domain (908 and 920) at the N-
terminus. The third and
fourth polypeptides (904 and 934) contain a cavity-containing Fc domain
monomer (916 and 932) joined
in a tandem series to a CD38 binding domain containing a VH domain (912 and
926) at the N-terminus. A
VL containing domain (906, 914, 922, and 930) is joined to each VH domain.
FIG. 10 is an illustration of an Fc-antigen binding domain construct
(construct 10) containing five
Fc domains and twoCD38 binding domains. The construct is formed of six Fc
domain monomer
containing polypeptides. Two polypeptides (1002 and 1032) each contain a
protuberance-containing Fc
domain monomer (1016 and 1030) linked by spacers in a tandem series to another
protuberance-
containing Fc domain monomer (1014 and 1028), an Fc domain monomer containing
different charged
amino acids at the CH3-CH3 interface than the VVT sequence (1008 and 1022) and
a CD38 binding
domain containing a VH domain (1006 and 1018) at the N-terminus. The third,
fourth, fifth, and sixth
polypeptides (1012, 1010, 1026, and 1024) each contain a cavity-containing Fc
domain monomer. A VL
containing domain (1004 and 1020) is joined to each VH domain.
FIG. 11 is an illustration of an Fc-antigen binding domain construct
(construct 11) containing five
Fc domains and fourCD38 binding domains. The construct is formed of six Fc
domain monomer
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containing polypeptides. Two polypeptides (1102 and 1148) contain a
protuberance-containing Fc
domain monomer (1118 and 1132) linked by spacers in a tandem series to another
protuberance-
containing Fc domain monomer (1120 and 1130) and an Fc domain monomer
containing different
charged amino acids at the CH3-CH3 interface than the VVT sequence (1124 and
1126). The third, fourth,
fifth, and sixth polypeptides (1106, 1104, 1144, and 1146) each contain a
cavity-containing Fc domain
monomer (1116, 1110, 1134, and 1140) joined in a tandem series to a CD38
binding domain containing a
VH domain (1112, 1122, 1138, and 1128) at the N-terminus. A VL containing
domain (1108, 1114, 1135,
and 1142) is joined to each VH domain.
FIG. 12 is an illustration of an Fc-antigen binding domain construct
(construct 12) containing five
Fc domains and sixCD38 binding domains. The construct is formed of six Fc
domain monomer
containing polypeptides. Two polypeptides (1202 and 1256) contain a
protuberance-containing Fc
domain monomer (1224 and 1230) linked by spacers in a tandem series to another
protuberance-
containing Fc domain monomer (1226 and 1228), an Fc domain monomer containing
different charged
amino acids at the CH3-CH3 interface than the VVT sequence (1210 and 1244),
and a CD38 binding
domain containing a VH domain (1250 and 1248) at the N-terminus. The third,
fourth, fifth, and sixth
polypeptides (1206, 1204, 1254, and 1252) each contain a cavity-containing Fc
domain monomer (1222,
1216, 1232, and 1238) joined in a tandem series to a CD38 binding domain
containing a VH domain
(1218, 1212,1236, and 1242) at the N-terminus. A VL containing domain (1208,
1214,1220, 1234, 1240,
and 1246) is joined to each VH domain.
FIG. 13 is an illustration of an Fc-antigen binding domain construct
(construct 13) containing three
Fc domains and twoCD38 binding domains. The construct is formed of four Fc
domain monomer
containing polypeptides. Two polypeptides (1302 and 1324) contain an Fc domain
monomer containing
different charged amino acids at the CH3-CH3 interface than the \MT sequence
(1308 and 1318) linked by
a spacer in a tandem series to a protuberance-containing Fc domain monomer
(1312 and 1316) and a
CD38 binding domain containing a VH domain (1310 and 1314) at the N-terminus.
The third and fourth
polypeptides (1306 and 1320) contain a cavity-containing Fc domain monomer. A
VL containing domain
(1304 and 1322) is joined to each VH domain.
FIG. 14 is an illustration of an Fc-antigen binding domain construct
(construct 14) containing three
Fc domains and two CD38 binding domains. The construct is formed of four Fc
domain monomer
containing polypeptides. Two polypeptides (1404 and 1426) contain an Fc domain
monomer containing
different charged amino acids at the CH3-CH3 interface than the \MT sequence
(1308 and 1318) linked by
a spacer in a tandem series to a protuberance-containing Fc domain monomer
(1414 and 1418). The
third and fourth polypeptides (1402 and 1428) each contain a cavity-containing
Fc domain monomer
(1410 and 1422) joined in a tandem series to a CD38 binding domain containing
a VH domain (1408 and
1416) at the N-terminus. A VL containing domain (1406 and 1424) is joined to
each VH domain.
FIG. 15 is an illustration of an Fc-antigen binding domain construct
(construct 15) containing three
Fc domains and fourCD38 binding domains. The construct is formed of four Fc
domain monomer
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containing polypeptides. Two polypeptides (1502 and 1536) contain an Fc domain
monomer containing
different charged amino acids at the CH3-CH3 interface than the \MT sequence
(1512 and 1524) linked by
a spacer in a tandem series to a protuberance-containing Fc domain monomer
(1518 and 1522) and a
CD38 binding domain containing a VH domain (1514 and 1532) at the N-terminus.
The third and fourth
polypeptides (1504 and 1534) contain a cavity-containing Fc domain monomer
(1510 and 1526) joined in
a tandem series toCD38 binding domain containing a VH domain (1508 and 1530)
at the N-terminus. A
VL containing domain (1506, 1516, 1520, and 1528) is joined to each VH domain.
FIG. 16 is an illustration of an Fc-antigen binding domain construct
(construct 16) containing five
Fc domains and twoCD38 binding domains. The construct is formed of six Fc
domain monomer
containing polypeptides. Two polypeptides (1602 and 1632) contain an Fc domain
monomer containing
different charged amino acids at the CH3-CH3 interface than the \MT sequence
(1610 and 1624) linked by
spacers in a tandem series to a protuberance-containing Fc domain monomer
(1612 and 1622), a second
protuberance-containing Fc domain monomer (1614 and 1620) and a CD38 binding
domain containing a
VH domain (1616 and 1618) at the N-terminus. The third, fourth, fifth, and
sixth polypeptides (1608, 1606,
1626, and 1628) each contain a cavity-containing Fc domain. A VL containing
domain (1604 and 1630) is
joined to each VH domain.
FIG. 17 is an illustration of an Fc-antigen binding domain construct
(construct 17) containing five
Fc domains and fourCD38 binding domains. The construct is formed of six Fc
monomer containing
polypeptides. Two polypeptides (1702 and 1748) contain an Fc domain monomer
containing different
charged amino acids at the CH3-CH3 interface than the \MT sequence (1718 and
1732) linked by spacers
in a tandem series to a protuberance-containing Fc domain monomer (1720 and
1730) and a second
protuberance-containing Fc domain monomer (1722 and 1728) at the N-terminus.
The third, fourth, fifth,
and sixth polypeptides (1706, 1704, 1746, and 1744) contain a cavity-
containing Fc domain monomer
(1716, 1710, 1734, and 1740) joined in a tandem series to a CD38 binding
domain containing a VH
domain (1712, 1724, 1738, and 1726) at the N-terminus. A VL containing domain
(1708, 1714, 1736, and
1742) is joined to each VH domain.
FIG. 18 is an illustration of an Fc-antigen binding domain construct
(construct 18) containing five
Fc domains and sixCD38 binding domains. The construct is formed of six Fc
domain monomer
containing polypeptides. Two polypeptides (1802 and 1856) contain an Fc domain
monomer containing
different charged amino acids at the CH3-CH3 interface than the \MT sequence
(1818 and 1838) linked by
spacers in a tandem series to a protuberance-containing Fc domain monomer
(1820 and 1836), a second
protuberance-containing Fc domain monomer (1822 and 1834) and a CD38 binding
domain containing a
VH domain (1826 and 1830) at the N-terminus. The third, fourth, fifth, and
sixth polypeptides (1806, 1804,
1854, and 1852) each contain a cavity-containing Fc domain monomer (1816,
1810, 1840, and 1846)
joined in a tandem series to a CD38 binding domain containing a VH domain
(1812, 1828, 1844, and
1850) at the N-terminus. A VL containing domain (1808, 1814, 1824, 1832, 1842,
and 1848) is joined to
each VH domain.
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FIG. 19 is an illustration of an Fc-antigen binding domain construct
(construct 19) containing five
Fc domains and twoCD38 binding domains. The construct is formed of six Fc
domain monomer
containing polypeptides. Two polypeptides (1902 and 1932) contain a
protuberance-containing Fc
domain monomer (1912 and 1930) linked by spacers in a tandem series to an Fc
domain monomer
containing different charged amino acids at the CH3-CH3 interface than the VVT
sequence (1908 and
1926), a protuberance-containing Fc domain monomer (1916 and 1918), and a CD38
binding domain
containing a VH domain (1914 and 1920) at the N-terminus. The third and fourth
polypeptides (1910 and
1928) contain cavity-containing Fc domain monomers and the fifth and sixth
polypeptides (1906 and
1924) contain cavity-containing Fc domain monomers. A VL containing domain
(1904 and 1922) is joined
to each VH domain.
FIG. 20 is an illustration of an Fc-antigen binding domain construct
(construct 20) containing five
Fc domains and fourCD38 binding domains. The construct is formed of six Fc
domain monomer
containing polypeptides. Two polypeptides (2002 and 2048) contain a
protuberance-containing Fc
domain monomer (2020 and 2022) linked by spacers in a tandem series to an Fc
domain monomer
containing different charged amino acids at the CH3-CH3 interface than the VVT
sequence (2012 and
2030), and a protuberance-containing Fc domain monomer (2040 and 2038) at the
N-terminus. The third,
fourth, fifth, and sixth polypeptides (2006, 2004, 2046, and 2044) each
contain a cavity-containing Fc
domain monomer (2018. 2010, 2024, and 2032) joined in a tandem series to a
CD38 binding domain
containing a VH domain (2014, 2042, 2028, and 2036) at the N-terminus. A VL
containing domain (2008,
2016, 2026, and 2034) is joined to each VH domain.
FIG. 21 is an illustration of an Fc-antigen binding domain construct
(construct 21) containing five
Fc domains and six CD38 binding domains. The construct is formed of six Fc
domain monomer
containing polypeptides. Two polypeptides (2102 and 2156) contain a
protuberance-containing Fc
domain monomer (2120 and 2122) linked by spacers in a tandem series to an Fc
domain monomer
containing different charged amino acids at the CH3-CH3 interface than the VVT
sequence (2112 and
2130), another protuberance-containing Fc domain monomer (2144 and 2142), and
a CD38 binding
domain containing a VH domain (2148 and 2138) at the N-terminus. The third,
fourth, fifth, and sixth
polypeptides (2106, 2104, 2154, and 2152) each contain a cavity-containing Fc
domain monomer (2118,
2110, 2124, and 2132) joined in a tandem series to a CD38 binding domain
containing a VH domain
(2114, 2150, 2128, and 2136) at the N-terminus. A VL containing domain (2108,
2116, 2126, 2134, 2140,
and 2146) is joined to each VH domain.
FIG. 22 is three graphs showing the results of CDC, ADCP, and ADCC assays with
various anti-
CD20 constructs targeting B cells. The first graph shows that the S3Y Fc-
antigen binding domain
construct can mediate CDC. The middle graph shows that both the SAI and S3Y Fc-
antigen binding
domain constructs exhibit >100-fold enhanced potency in an ADCP FcyRIla
reporter assay. The third
graph shows that the SAI and S3Y Fc-antigen binding domain constructs exhibit
enhanced ADCC activity
relative to the fucosylated mAb and similar activity to the afucosylated mAb.
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FIGS. 23A-C are a schematic representation of three exemplary ways the CD38
binding domain
can be joined to the Fc domain of an Fc construct. Panel A shows a heavy chain
component of a CD38
binding domain can be expressed as a fusion protein of an Fc chain and a light
chain component can be
expressed as a separate polypeptide. Panel B shows an scFv expressed as a
fusion protein of the long
Fc chain. Panel C shows heavy chain and light chain components expressed
separately and
exogenously added and joined to the Fc-antigen binding domain construct with a
chemical bond.
FIG. 24A depicts the amino acid sequence of a human IgG1 (SEQ ID NO: 43) with
EU
numbering. The hinge region is indicated by a double underline, the CH2 domain
is not underlined and
the CH3 region is underlined.
FIG. 24B depicts the amino acid sequence of a human IgG1 (SEQ ID NO: 45) with
EU
numbering. The hinge region, which lacks E216-C220, inclusive, is indicated by
a double underline, the
CH2 domain is not underlined and the CH3 region is underlined and lacks K447.
FIG. 24C depicts the amino acid sequence of a human IgG1 (SEQ ID NO: 47) with
EU
numbering. The hinge region is indicated by a double underline, the CH2 domain
is not underlined and
the CH3 region is underlined and lacks 447K.
FIG. 24D depicts the amino acid sequence of a human IgG1 (SEQ ID NO: 42) with
EU
numbering. The hinge region, which lacks E216-C220, inclusive, is indicated by
a double underline, the
CH2 domain is not underlined and the CH3 region is underlined.
FIG. 25. Depicts the results of an analysis of dose dependent binding of an
anti-CD38 antibody
showing relatively high, moderate, and low cell surface CD38 expression among
multiple hematological
tumor cell lines. VivoTag645-labeled anti-CD38 antibody binding to live cell
surface CD38. Cell surface
binding was assessed by FACS analysis.
FIGS. 26A-B. Depict the results of an analysis showing that anti-CD38
constructs have a similar
cell binding profile as IgG1 anti-CD38 antibodies that cross-react with the
human and cyno CD38. (A)
Human CD38 expressing Raji tumor cells were incubated with VivoTag645-labeled-
antibodies, SIA-AA-
Cyno (anti-Cyno CD38 mAb), 53Y-AA-Cyno ¨CD38 (Construct 13 with Cyno CD38
Fab), anti-CD38 mAb,
53Y-AA-CD38 (Construct 13 with anti-CD38 Fab), IgG isotype control and SIF1
Control (Fc trimer without
Fab regions) at 4 C for 1 hour. Extent of cell surface binding was assessed by
flow cytometry. (B) CHO
cells stably expressing cyno CD38 were harvested and cell suspensions were
incubated with
VivoTag645-labeled-antibodies, SIA-AA-Cyno (anti-Cyno CD38 mAb), 53Y-AA-Cyno
CD38 (Construct 13
with anti-Cyno CD38 Fab), anti-CD38 mAb, 53Y-AA-CD38 (Construct 13 with anti-
CD38 Fab), IgG
isotype control and SIF1 Control (Fc trimer without Fab regions) at 4 C for 1
hour. Extent of cell surface
binding was assessed by flow cytometry. Note: anti-Cyno CD38 mAb cross
reactive antibody (S1A-AA-
Cyno) and 53Y-AA-Cyno recognize both human and cyno CD38. In addition, 53Y-AA-
Cyno CD38 binds
cell surface CD38 better than S1A-AA-Cyno (anti-Cyno CD38 mAb).
FIGS. 27A-D. Depict the results of an assessment of CDC activity by anti-CD38
constructs in
Daudi cells and Raji cells.
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FIGS. 28A-B. Depict the results of an assessment of tumor cell killing by anti-
CD38 constructs in
whole human blood. Anti-CD38 Construct 13 (53Y-AA-CD38) demonstrates highly
potent tumor cell
killing capacity in human whole blood. (A) Effects of anti-CD38 mAb and 53Y-AA-
CD38 in killing of Daudi-
luciferase tumor cells in whole human blood. (B) Effects of anti-CD38 mAb and
53Y-AA-CD38 in killing of
tumor cells in human blood. In both (A) & (B), live Daudi-luciferase cells
were quantified by adding
luciferin substrate and measuring light emission on a luminometer. `)/0 Cell
killing is calculated by
normalizing the luminescence values of test samples with Spontaneous Lysis
Control (0% Cell Lysis) (No
Antibody Addition) and Total Lysis Control (100% Cell Lysis). Table show tumor
cell killing EC50 value
comparisons from whole blood from 3 separate human donors. Values represent
mean SD.
FIGS. 29A-C. Depict the results of an assessment of endogenous B cell
depletion from
cynomolgus monkey blood. (A) Dose-dependent binding of SIA-AA-Cyno (anti-Cyno
CD38 mAb), 53Y-
AA-Cyno-011 (Construct 13 with Cyno CD38 Fab), IgG isotype control and SIF1
Control (Fc trimer
without Fab regions) to cyno B cells. (B) Dose-dependent increase in frequency
of cyno B cell binding to
SIA-AA-Cyno, 53Y-AA-Cyno-011 (Construct 13 with Cyno CD38 Fab), IgG isotype
control and SIF1
Control (Fc trimer without Fab regions). (C) Dose-dependent increase in B cell
depletion with SIA-AA-
Cyno, 53Y-AA-Cyno-001 (Construct 13 with Cyno CD38 Fab), IgG isotype control
and SIF1 Control (Fc
trimer without Fab regions). Anti-CD38 construct (53Y-AA-CD38) treatment
resulted in much greater cell
depletion than anti-CD38 mAb. Values are normalized to B cell frequency in
untreated control group. (A,
B, C) Values plotted in these figures were generated from same monkey blood
donor.
FIG. 30. Depicts the results of an assessment of the impact of an anti-CD38
construct in a
lymphoma subcutaneous tumor model. SCID mice were subcutaneously inoculated
with human
lymphoma (Raji) tumor cells. Six days after tumor cell implantation mice were
randomized into treatment
groups (n = 10 in each) & treated intraperitoneally with 0.5 mL normal human
serum complement (HSC).
Next day (on day 7) mice were again injected intraperitoneally with HSC
followed by anti-CD38 mAb
(single iv dose of 5.94 mg/kg), or 53Y-AA-CD38 (single iv dose of 10 mg/kg),
or PBS (single IV injection).
Mice were given 31d ip injection of HSC on day 8th. Tumor growth was routinely
monitored by tumor
volume measurement. Points labeled with ** in 53Y-AA-CD38 group had p values
of <0.0022 relative to
corresponding treatment groups.
FIG. 31A. Depicts the results of a comparison of 53Y-AA-CD38 (inverted
triangles) and an anti-
CD38 mAb (circles) with respect to ADCC, ADCP and CDC activity in Daudi cells.
FIG. 31B Depicts the results of a comparison of 53Y-AA-CD38 (inverted
triangles) and an anti-
CD38 mAb (circles) with respect to ADCC, ADCP (measured using a reporter as a
surrogate for
phagocytosis by macrophages) and CDC activities against Raji tumor cells,
which are resistant to anti-
CD38 mAb mediated CDC.
FIG 32. Depicts the results of a study of tumor cell depletion from whole
human blood by 53Y-
AA-CD38 (inverted triangles) and an anti-CD38 mAb (circles).
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FIG. 33. Depicts the results of a study of the complement mediated
cytotoxicity of S3Y-AA-CD38
(inverted triangles) and an anti-CD38 mAb (circles) in Daudi cells (left
panel, relatively high CD38
expression and relatively low CD55 and CD59 expression) and in Raji cells
(right panel, relatively low
CD38 expression and relatively high CD55 and CD59 expression).
FIG. 34A. Depicts the results of a study of the ADCC activity (left panel) and
CDC activity (right
panel) of S3Y-AA-Cyno CD38 (inverted triangles) and an anti-cyno CD38 mAb
(circles).
FIG. 34B. Depicts the results of a study of the ADCC activity (left panel),
ADCP activity (center
panel), and CDC activity (right panel) of S3Y-AA-Cyno CD38 (inverted
triangles) and an anti-cyno CD38
mAb (circles). CDC activity was measured using Raji cells, which are resistant
to anti-CD38 mAb
mediated CDC.
FIG. 35. Depicts the results of a study of tumor cell depletion by S3Y-AA-Cyno
CD38 (inverted
triangles) and an anti-Cyno CD38 mAb (circles).
FIG. 36. Depicts the results of a study comparing B cell depletion by S3Y-AA-
Cyno CD38
(second bar in each pair) and an anti-cyno CD38 mAb (first bar in each pair)
in vitro (left panel) and in
vivo (right panel).
FIG. 37. Depicts the results of a study comparing plasma cell depletion by S3Y-
AA-CD38
(inverted triangles) and an anti-CD38 mAb (circles) in vitro. Percent plasma
cell depletion by either anti-
CD38 mAb or S3Y-AA-CD38 within total bone marrow mononuclear cells (BM-MNCs)
from multiple
myeloma patient MM536. Depletion was calculated as the total number of viable
CD138+ cells at each
concentration, relative to a baseline value from untreated BM-MNCs.
FIG. 38A. Depicts the results of a study showing that S3Y-AA-CD38 binding to
FcgRIla, FcgRIlla
and complement is at least 100-fold greater than an anti-CD38 mAb.
FIG. 38B. Depicts the results of a study showing that S3Y-AA-CD38 binding to
FcyRIIA, FcyRIIIA
is enhanced by >500X and S3Y-AA-CD38-opsonized tumor cells to human complement
protein C1q is
enhanced by 12X than an anti-CD38 mAb.
FIG. 39. Depicts the results of an analysis of various constructs on B cell
counts in
Cynomologous monkeys.
FIGS. 40A-B. A schematic depiction of an Fc-antigen binding domain construct
containing three
Fc domains and two CD38 binding domains. (A) The construct is formed of four
Fc domain monomer
containing polypeptides. Two polypeptides contain an Fc domain monomer
containing different charged
amino acids at the CH3-CH3 interface than the \MT sequence linked by a spacer
in a tandem series to a
protuberance-containing Fc domain monomer and a CD38 binding domain containing
a VH domain at the
N-terminus. The third and fourth polypeptides contain a cavity-containing Fc
domain monomer. A VL
containing domain and is joined to each VH domain. R292P mutations, signified
by diamonds, have been
introduced into all six CH2 domains. The six CH2 domains are part of the three
Fc domains, formed by
assembly of the four constituent polypeptides.53Y-CD38 (CC R292P) is an
example of this construct. (B)
A schematic depiction of an Fc-antigen binding domain construct (construct 14)
containing three Fc
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domains and twoCD38 binding domains. The construct is formed of four Fc domain
monomer containing
polypeptides. Two polypeptides contain an Fc domain monomer containing
different charged amino
acids at the CH3-CH3 interface than the VVT sequence linked by a spacer in a
tandem series to a
protuberance-containing Fc domain monomer. The third and fourth polypeptides
each contain a cavity-
containing Fc domain monomer joined in a tandem series to a CD38 binding
domain containing a VH
domain at the N-terminus. A VL containing domain is joined to each VH domain.
R292P mutations,
signified by diamonds, have been introduced into all six CH2 domains. The six
CH2 domains are part of
the three Fc domains formed by assembly of the four constituent polypeptides.
FIG. 41. Depicts the results of an analysis of human CD38 binding to anti-CD38
antibodies and
SIF-bodies measured by SPR. Binding sensorgrams for anti-CD38 molecules and
1:1 binding model fit
are shown on the left Y-axis is percent response and X-axis is time (500 and
1000 s). Kinetic and
equilibrium constants are shown on the center and upper right. Stoichiometry
of human CD38 binding to
anti-CD38 molecules is shown on the lower right.
FIG. 42. Depicts the results of an analysis of human CD38 binding to cyno-CD38
antibodies and
SIF-bodies measured by SPR. Binding sensorgrams for cyno-CD38 molecules and
1:1 binding model fit
are shown on the left. Kinetic and equilibrium constants are shown on the
center and upper right.
Stoichiometry of human CD38 binding to cyno-CD38 molecules is shown on the
lower right.
FIG. 43. Depicts the results of an analysis of cynomolgus CD38 binding to cyno-
CD38 antibodies,
cyno-CD38 SIF-bodies and CD38 mAb measured by SPR. Binding sensorgrams for
cyno-CD38
molecules and 1:1 binding model fit are shown on the left and lower center
panels. Binding sensorgrams
for CD38 mAb and 1:1 binding model fit are shown on the upper center panel.
Equilibrium constants are
shown on the right panel.
FIG. 44. Depicts the results of an analysis of S3Y-AA-CD38 and S3Y-CC-CD38 to
human FcgRs
relative to CD38 mAb using an Fc-gamma receptor homogeneous time-resolved
fluorescence (HTRF)
assay.
FIG. 45. Depicts the results of an analysis of CD38 expression on various cell
lines.
FIG. 46. Depicts the results of an analysis of anti-CD38 mAb and S3Y-CC-CD38
molecules to (A)
Daudi and (B) Raji cells.
FIGS. 47A-B. Depict the results of an analysis of 53Y-CC-CD38 on target cell
depletion in in
anti-CD38 mAb-Resistant Cells. Daudi cells (A) or Raji cells (B) were
incubated with human serum
complement and 53Y-CC-CD38 or 53Y-CC-CD38 or anti-CD38 mAb at 37 C for 2
hours in a 96 well
plate to facilitate complement mediated lysis. Alamar Blue (a cell viability
reagent) was then added to
each well and incubated at 37C for 18 hours and the viable cell fluorescence
was measured using a
fluorometer. The `)/0 cell lysis= (RFU test ¨ RFU background) x 100 (RFU at
total cell lysis ¨ RFU
background). Values represent mean SD (n = 3).
FIGS. 48A-B. Depict the results of an analysis of the cytolytic activity of
53Y-CC-CD38A. (A)
Primary human NK cells were added to Daudi tumor cells in a ratio of 5:1 in a
96 well plate. Cell mixtures
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were then incubated with either one of the drug molecules for 5 hours at 37:C
followed by detection of
dead cells by CytoTox Glo reagent. (B) Monocytes purified from human PBMCs,
were cultured in M-CSF
then with IL-10 at 37C to generate M2c macrophages. PHrodo Red labeled Raji
cells were then added to
the 96 well plate containing macrophage monolayers, then incubated with drug
molecules & IL-10 a 37cC
in the IncuCyte live cell imaging system for overnight. Phagocytosis of pHrodo
red labeled tumor cells by
macrophages leads to increase in pHrodo fluorescence captured by real time
imaging. Values in A, B
represent mean SD (n = 3).
FIG. 49. Depicts the results of an analysis of the whole blood tumor cell
depletion 53Y-CC-
CD38A. Daudi (A) and Raji (iB) lymphoma cells were labeled with CFSE were
added to whole blood and
incubated at 37 C in the presence of anti-CD38A mAb or 53Y-CC-CD38 or 53Y-AA-
CD38 for 18 hours.
RBCs were lysed and cells were then stained for surface markers, fixed, and
acquired on a flow
cytometer. Reduction in the frequency of CFSE+ (Daudi or Raji) cells in the
presence of drug molecules
when compared to without drug treatment was the indicator of selective cell
depletion. Values represent
mean SD.
FIG. 50. Depicts the result of an analysis of the impact of 53Y-CC-CD38 & 53Y-
AA-CD38 on
plasma cells from patient biopsies. Bone marrow (BM) biopsies were collected
from patients clinically
diagnosed with Multiple Myeloma (MM) by IMWG criteria. Fresh bone marrow
aspirates collected from
patients were treated with drug molecules (53Y & Darzalex) in the presence of
autologous plasma from
patients to maintain native microenvironment at 37C for 3 hours in a CO2
incubator. Samples were then
stained and analyzed by flow cytometry for depletion of CD138+ cells (used as
a surrogate marker for
CD38 expressing plasma/myeloma cells). Cell depletion was determined using the
viable CD138+ cell
frequency out of total single cells, with all relative cell frequencies
normalized to basal parameter of a
previous fit done using cell counts as dependent variable. Top asymptote of
each drug is used as
baseline (set to 100% live plasma cells).
FIG Si. Depicts the results of an analysis of 53Y-CC-Cyno-CD38, 53Y-AA-Cyno-
CD38, and anti-
Cyno-CD38 mAb binding to cell surface CD38 on human lymphoma cell lines. Daudi
or Raji Cells were
incubated with VivoTag 645-labeled 53Y or CD38 mAb at 4 C for 40 min. Cells
were then washed, fixed,
and single cell events were acquired on Cytek Aurora flow cytometer. Data was
unmixed using
SpectroFlo and analyzed on FlowJo. Values represent mean SD.
FIG 52. Depicts the results of an analysis of the effect of 53Y-CC-Cyno-CD38
and anti-Cyno-
CD38 on Fc effector function (A) Daudi cells were incubated with human serum
complement and 53Y or
CD38 mAb at 37C for 2 hours in a 96 well plate to facilitate complement
mediated lysis. Alamar Blue (a
cell viability reagent) was then added to each well and incubated at 37C for
18 hours and the viable cell
fluorescence was measured using a fluorometer. The `)/0 cell lysis= (RFU test
¨ RFU background) x 100
(RFU at total cell lysis ¨ RFU background). Values represent mean SD (n =
3). (B) Primary human NK
cells were added to Daudi tumor cells in a ratio of 5:1 in a 96 well plate.
Cell mixtures were then
incubated with either one of the drug molecule for 5 hours at 37cC for 5 hours
at 37cC followed by detection
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of dead cells by CytoTox Glo reagent. (C) Monocytes purified from human PBMCs,
were cultured in M-
CSF then with IL-10 at 37C to generate M2c macrophages. PHrodo Red labeled
Raji cells were then
added to the 96 well plate containing macrophage monolayers, then incubated
with drug molecules & IL-
a 37:C in the IncuCyte live cell imaging system for overnight. Phagocytosis of
pHrodo red labeled
5 tumor cells by macrophages leads to increase in pHrodo fluorescence
captured by real time imaging.
Values in A, B, C represent mean SD (n = 3).
FIG 53. Depicts the results of a single dose PK study. The graph shows serum
concentrations of
indicated drug molecules after single IV administration in Cyno monkeys
(n=4/group). Values represent
mean SEM.
10 FIG 54. Depicts the results of a single dose study. The graph shows B
cell changes in blood after
one time treatment with a one dose of either 53Y-AA-Cyno-CD38 (1.7 mg/kg), 3
dose groups of 53Y-CC-
Cyno-CD38 (0.51, 1.7, 5.1 mg/Kg) or one molar equivalent dose of anti-Cyno-
CD38-mAb (1.0 mg/Kg), or
saline (control). Percentage of change from pre-dose baseline in absolute cell
counts of CD3-CD20+ B
cells in peripheral blood of each monkey in a group after intravenous infusion
were plotted. Values
represent mean SEM.
FIGS. 55A-B. Depict the results of a multiple repeat dose study. (A) The graph
shows B cell
changes in blood after treatment with the single dose of 53Y-CC-Cyno-CD38 each
week. Percentage of
change from pre-dose baseline in absolute cell counts of CD3-CD20+ B cells in
peripheral blood of each
monkey in a group after intravenous infusion were plotted. Values represent
mean SEM. (B) Tissues
were harvested before terminal necropsy after 4 once weekly dosing of 53Y-CC-
Cyno-CD38. Single cell
suspension was generated from bone marrow and spleen and CD138+ plasma cells
counts were
determined by flow cytometry. Values represent mean SD.
FIGS. 56A-B. Depict an analysis of subcutaneous injection of 53Y-CC-Cyno-CD38
in
Cynomolgus monkeys. To evaluate the bioavailability, 53Y-CC-Cyno-CD38 was
administered
intravenously or by subcutaneous injection as a single dose to cynomolgus
monkeys. (A) The graph
shows serum concentrations of 53Y-CC-Cyno-CD38 after single IV or SC
administration in Cyno
monkeys (n=4/group). Values represent mean SEM. (B) The graph shows B cell
changes in blood after
one-time treatment with 53Y-CC-Cyno-CD38. Percentage of change from pre-dose
baseline in absolute
cell counts of CD3-CD20+ B cells in peripheral blood of each monkey in a group
after intravenous
infusion were plotted. Values represent mean SEM.
Detailed Description
Many therapeutic antibodies function by recruiting elements of the innate
immune system through
the effector function of the Fc domains, such as antibody-dependent
cytotoxicity (ADCC), antibody-
dependent cellular phagocytosis (ADCP), and complement-dependent cytotoxicity
(CDC). In some
instances, the present disclosure contemplates combining a CD38 binding domain
of a known single Fc-
domain containing therapeutic, e.g., a known therapeutic antibody, with at
least two Fc domains to
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generate a novel therapeutic with unique biological activity. In some
instances, a novel therapeutic
disclosed herein has a biological activity greater than that of the known Fc-
domain containing therapeutic,
e.g., a known therapeutic antibody. The presence of at least two Fc domains
can enhance effector
functions and to activate multiple effector functions, such as ADCC in
combination with ADCP and/or
CDC, thereby increasing the efficacy of the therapeutic molecules. In order to
generate a product with
consistent biological function, control of the number of Fc domains is
critical. The disclosure features a
set of Fc engineering tools to control homodimerization and heterodimerization
of the peptides encoding
the Fc domain, to assemble molecules of discrete size from a limited number of
polypeptide chains.
International Publication Nos. WO/2015/168643, W02017/151971, WO 2017/205436,
and WO
2017/205434 disclose Fc engineering tools and methods for assembling molecules
with two or more Fc
domains, and are herein incorporated by reference in their entirety. The
engineering tools include
structural features (for example, glycine linkers) that significantly improve
manufacturing outcome. The
properties of these constructs allow for the efficient generation of
substantially homogenous
pharmaceutical compositions. Such homogeneity in a pharmaceutical composition
is desirable in order to
ensure the safety, efficacy, uniformity, and reliability of the pharmaceutical
composition. Having a high
degree of homogeneity in a pharmaceutical composition also minimizes potential
aggregation or
degradation of the pharmaceutical product caused by unwanted materials (e.g.,
degradation products,
and/or aggregated products or multimers), as well as limiting off-target and
adverse side effects caused
by the unwanted materials.
As described in detail herein, we improved homogeneity of the composition by
engineering the Fc
domain components of the Fc-antigen binding domain constructs using approaches
including the use of
spacers including only glycine residues to join two Fc domain monomers in
tandem series, the use of
polypeptide sequences having the terminal lysine residue removed, and the use
of two sets of
heterodimerizing selectivity modules: (i) heterodimerizing selectivity modules
having different reverse
charge mutations and (ii) heterodimerizing selectivity modules having
engineered cavities and
protuberances.
We designed a series of Fc-antigen binding domain constructs in which Fc
domains were
connected in tandem, using one long peptide chain containing multiple Fc
sequences separated by
linkers, and multiple copies of a short chain containing a single Fc sequence
(Fc-antigen binding domain
constructs 1-6; FIG. 1-FIG. 6). Heterodimerizing mutations were introduced
into each Fc sequence to
ensure assembly into the desired tandem configuration with minimal formation
of smaller or larger
complexes. Any number of Fc domains can be connected in tandem in this
fashion, allowing the creation
of constructs with 2, 3, 4, 5, 6, 7, 8, 9, 10, or more Fc domains. For a
peptide with N Fc domains, such
constructs can be prepared with 1 to N+1 CD38 binding domains, depending
whether the CD38 binding
domains are introduced into the long peptide chain, the short peptide chain,
or both, respectively.
In Fc-antigen binding domain constructs 1-6 (FIG. 1-FIG. 6), Fc domains were
connected with a
single branch point between the Fc domains. These constructs include two
copies of a long peptide
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chain containing multiple Fc sequences separated by linkers, in which the
branching Fc sequence
contains homodimerizing mutations and the non-branching Fc domains contain
heterodimerizing
mutations. Multiple copies of short chains including a single Fc sequence with
mutations complementary
to the heterodimerizing mutations in the long chains are used to complete the
multimeric Fc scaffold.
Heterodimerizing Fc domains can be linked to the C-terminal end (e.g., Fc-
antigen binding domain
constructs 7-12; FIG. 7-FIG. 12), the N-terminal end (e.g., Fc-antigen binding
domain constructs 13-18;
FIG. 13-FIG. 18), or both ends of the branching Fc domain (e.g., Fc-antigen
binding domain constructs
19-21; FIG. 19-FIG. 21). Multiple Fc domains in tandem may be linked to either
end of the branching Fc
domain. CD38 binding domains may be introduced into the long peptide chains,
resulting in twoCD38
binding domains per assembled protein molecule. Alternatively,CD38 binding
domains may be
introduced into the short peptide chains, resulting in N-1CD38 binding domains
per assembled protein
molecule, where N is the number of Fc domains in the assembled protein
molecule. IfCD38 binding
domains are introduced into both the short and the long peptide chains, the
resulting assembled protein
molecule contains N+1CD38 binding domains.
Past engineering efforts for monoclonal antibodies (mAbs) and Fc domains
included making
mutations in the Fc domain to strengthen binding to FcyRIlla and thus
enhancing the antibody-dependent
cell-mediated cytotoxicity (ADCC) response, and afucosylation of the Fc domain
to strengthen binding to
FcyRIlla and thus enhances the ADCC response.
In comparison to antibodies with mutations in the Fc domain to strengthen
binding to FcyRIlla or
afucosylation of the Fc domain, the Fc-antigen binding domain constructs
disclosed in this disclosure
unexpectedly feature stronger binding to multiple classes of Fcy receptors and
enhanced activity of
multiple cytotoxicity pathways. The Fc-antigen binding domain constructs of
this disclosure can enhance
binding to both FcyRIla and FcyRIlla compared to their corresponding
fucosylated and afucosylated
parent monoclonal antibodies (see, Example 46). Further, the Fc-antigen
binding domain constructs of
this disclosure unexpectedly feature an ability to mediate the complement-
dependent cytotoxicity (CDC)
pathway and/or the antibody-dependent cellular phagocytosis (ADCP) pathway in
addition to enhancing
the ADCC pathway response (see, Example 47).
I. Fc domain monomers
An Fc domain monomer includes at least a portion of a hinge domain, a CH2
antibody constant
domain, and a CH3 antibody constant domain (e.g., a human IgG1 hinge, a CH2
antibody constant
domain, and a CH3 antibody constant domain with optional amino acid
substituions). The Fc domain
monomer can be of immunoglobulin antibody isotype IgG, IgE, IgM, IgA, or IgD.
The Fc domain monomer
may also be of any immunoglobulin antibody isotype (e.g., IgG1, IgG2a, IgG2b,
IgG3, or IgG4). The Fc
domain monomers may also be hybrids, e.g., with the hinge and CH2 from IgG1
and the CH3 from IgA, or
with the hinge and CH2 from IgG1 but the CH3 from IgG3. A dimer of Fc domain
monomers is an Fc
domain (further defined herein) that can bind to an Fc receptor, e.g.,
FcyRIlla, which is a receptor located
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on the surface of leukocytes. In the present disclosure, the CH3 antibody
constant domain of an Fc
domain monomer may contain amino acid substitutions at the interface of the
CH3-CH3 antibody constant
domains to promote their association with each other. In other embodiments, an
Fc domain monomer
includes an additional moiety, e.g., an albumin-binding peptide or a
purification peptide, attached to the
N- or C-terminus. In the present disclosure, an Fc domain monomer does not
contain any type of
antibody variable region, e.g., VH, VL, a complementarity determining region
(CDR), or a hypervariable
region (HVR).
In some embodiments, an Fc domain monomer in an Fc-antigen binding domain
construct
described herein (e.g., an Fc-antigen binding domain construct having three Fc
domains) may have a
sequence that is at least 95% identical (at least 97%, 99%, or 99.5%
identical) to the sequence of SEQ ID
NO:42. In some embodiments, an Fc domain monomer in an Fc-antigen binding
domain construct
described herein (e.g., an Fc-antigen binding domain construct having three Fc
domains) may have a
sequence that is at least 95% identical (at least 97%, 99%, or 99.5%
identical) to the sequence of any
one of SEQ ID NOs: 44, 46, 48, and 50-53. In certain embodiments, an Fc domain
monomer in the Fc-
antigen binding domain construct may have a sequence that is at least 95%
identical (at least 97%, 99%,
or 99.5% identical) to the sequence of any one of SEQ ID NOs: 48, 52, and 53.
SEQ ID NO: 42
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV
DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK
AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO: 44
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV
HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
CTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTV
DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO: 46
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEV
HNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQV
CTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTV
DKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
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SEQ ID NO: 48
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV
DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK
AKGQPREPQVCTLPPSRDELTKNQVSLSCAVDGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
SEQ ID NO: 50
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV
DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK
AKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO: 51
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV
DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK
AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLKSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO: 52
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV
DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK
AKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLKSDGSFFLYSDLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG
SEQ ID NO: 53
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYV
DGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISK
AKGQPREPQVYTLPPCRDKLTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
II. Fc domains
As defined herein, an Fc domain includes two Fc domain monomers that are
dimerized by the
interaction between the CH3 antibody constant domains. An Fc domain forms the
minimum structure that
binds to an Fc receptor, e.g., Fc-gamma receptors (i.e., Fcy receptors
(FcyR)), Fc-alpha receptors (i.e.,
Fca receptors (FcaR)), Fc-epsilon receptors (i.e., FCE receptors (FcER)),
and/or the neonatal Fc receptor
(FcRn). In some embodiments, an Fc domain of the present disclosure binds to
an Fcy receptor (e.g.,
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FcyRI (CD64), FcyRIla (CD32), FcyRIlb (CD32), FcyRIlla (CD16a), FcyRIllb
(CD16b)), and/or FcyRIV
and/or the neonatal Fc receptor (FcRn).
III. CD38 binding domains
Antigen binding domains include one or more peptides or polypeptides that
specifically bind a
target molecule. CD38 binding domains may include the CD38 binding domain of
an antibody. In some
embodiments, the CD38 binding domain may be a fragment of an antibody or an
antibody-construct, e.g.,
the minimal portion of the antibody that binds to the target antigen. A CD38
binding domain may also be
a synthetically engineered peptide that binds a target specifically such as a
fibronectin-based binding
protein (e.g., a FN3 monobody).
A fragment antigen-binding (Fab) fragment is a region on an antibody that
binds to a target
antigen. It is composed of one constant and one variable domain of each of the
heavy and the light
chain. A Fab fragment includes a VH, VL, CH1 and CL domains. The variable
domains VH and VL each
contain a set of 3 complementarity-determining regions (CDRs) at the amino
terminal end of the
monomer. The Fab fragment can be of immunoglobulin antibody isotype IgG, IgE,
IgM, IgA, or IgD. The
Fab fragment monomer may also be of any immunoglobulin antibody isotype (e.g.,
IgG1, IgG2a, IgG2b,
IgG3, or IgG4). In some embodiments, a Fab fragment may be covalently attached
to a second identical
Fab fragment following protease treatment (e.g., pepsin) of an immunoglobulin,
forming an F(a13')2
fragment. In some embodiments, the Fab may be expressed as a single
polypeptide, which includes both
the variable and constant domains fused, e.g. with a linker between the
domains.
In some embodiments, only a portion of a Fab fragment may be used as a CD38
binding domain.
In some embodiments, only the light chain component (VL + CL) of a Fab may be
used, or only the heavy
chain component (VH + CH) of a Fab may be used. In some embodiments, a single-
chain variable
fragment (scFv), which is a fusion protein of the the VH and VL chains of the
Fab variable region, may be
used. In other embodiments, a linear antibody, which includes a pair of tandem
Fd segments (VH-CH1-
VH-CH1), which, together with complementary light chain polypeptides form a
pair ofCD38 binding regions,
may be used.
In some embodiments, a CD38 binding domain of the present disclosure includes
for a target or
antigen listed in Table 1, one, two, three, four, five, or all six of the CDR
sequences listed in Table 1 for
the listed target or antigen, as provided in further detail below Table 1.
Table 1: CDR Sequences
anti-CD38 GFTFNSF ISGSGGG AKDKILWF QSVSSY DAS QQRSNW
A (SEQ ID T (SEQ ID GEPVFDY (SEQ ID PPT
NO: 85) NO: 115) (SEQ ID NO: 180) (SEQ ID
NO: 148) NO:
211)
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Isatuximab GYTFTDY IYPGDGD ARGDYYG QDVSTV SAS QQHYSPP
W (SEQ ID T (SEQ ID SNSLDY (SEQ ID YT
NO: 86) NO: 109) (SEQ ID NO: 181) (SEQ ID
NO: 149) NO:
212)
M0R202 SYYMN GISGDPS DLPLVYT SGDNLRH GDSKRPS QTYTGGA
(Kabat NTYYADS GFAY YYVY
Numbering) VKG
Table 2: VH and VL Sequences
ltibodyinalikiningininiginigininiMMFIMENEMENNEMiginiginieMAILMENENNina
Isatuximab (VH + CH1)
QVQLVQSGAEVAKPGTSVKLSCK DIVMTQSHLSMSTSLGDPVSITCK
ASGYTFTDYWMQVVVKQRPGQGL ASQDVSTVVAWYQQKPGQSPRRL
EWIGTIYPGDGDTGYAQKFQGKAT IYSASYRYIGVPDRFTGSGAGTDF
LTADKSSKTVYMHLSSLASEDSAV TFTISSVQAEDLAVYYCQQHYSPP
YYCARGDYYGSNSLDYWGQGTS YTFGGGTKLEIKRTVAAPSVFIFPP
VTVSSASTKGPSVFPLAPSSKSTS SDEQLKSGTASVVCLLNNFYPREA
GGTAALGCLVKDYFPEPVTVSWN KVQWKVDNALQSGNSQESVTEQ
SGALTSGVHTFPAVLQSSGLYSLS DSKDSTYSLSSTLTLSKADYEKHK
SVVTVPSSSLGTQTYICNVNHKPS VYACEVTHQGLSSPVTKSFNRGE
NTKVDKKVEPKSCDKTHTCPPCP C
APELLGGPSVFLFPPKPKDTLMISR
TPEVTCVVVDVSHEDPEVKFNWY
VDGVEVHNAKTKPREEQYN 300
STYRVVSVLTVLHQDWLNGKEYK
CKVSNKALPAPIEKTISKAKGQPRE
PQVYTLPPSRDELTKNQVSLTCLV
KGFYPSDIAVEWESNGQPENNYK
TTPPVLDSDGSFFLYSKLTVDKSR
WQQGNVFSCSVMHEALHNHYTQ
KSLSLSPGK
M0R202 QVQLVESGGGLVQPGGSLRLSCAASGF
DIELTQPPSVSVAPGQTARISCSGDNLR
TFSSYYMNWVRQAPGKGLEWVSGISG HYYVYWYQQKPGQAPVLVIYGDSKRP
DPSNTYYADSVKGRFTISRDNSKNTLYL SGIPERFSGSNSGNTATLTISGTQAEDE
QMNSLRAEDTAVYYCARDLPLVYTGFA ADYYCQTYTGGASLVFGGGTKLTVLGQ
YWGQGTLVTV
The CD38 binding domain of Fc-antigen binding domain construct 1 (110/104 in
FIG. 1) can
include the three heavy chain and the three light chain CDR sequences of any
one of the antibodies listed
in Table 1.
The CD38 binding domain of Fc-antigen binding domain construct 2 (212/204 in
FIG. 2) can
include the three heavy chain and the three light chain CDR sequences of any
one of the antibodies listed
in Table 1.
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The CD38 binding domains of Fc-antigen binding domain construct 3 (308/316 and
312/318 in
FIG. 3) each can include the three heavy chain and the three light chain CDR
sequences of any one of
the antibodies listed in Table 1.
The CD38 binding domains of Fc-antigen binding domain construct 4 (410/412,
416/418 and
422/424 in FIG. 4) each can include the three heavy chain and the three light
chain CDR sequences of
any one of the antibodies listed in Table 1.
The CD38 binding domains of Fc-antigen binding domain construct 5 (510/504,
512/514 and
518/520 in FIG. 5) each can include the three heavy chain and the three light
chain CDR sequences of
any one of the antibodies listed in Table 1.
The CD38 binding domains of Fc-antigen binding domain construct 6 (612/604,
614/616,
620/622, and 626/628 in FIG. 6) each can include the three heavy chain and the
three light chain CDR
sequences of any one of the antibodies listed in Table 1.
The CD38 binding domains of Fc-antigen binding domain construct 7 (712/714 and
714/716 in
FIG. 7) each can include the three heavy chain and the three light chain CDR
sequences of any one of
the antibodies listed in Table 1.
The CD38 binding domains of Fc-antigen binding domain construct 8 (812/806 and
818/822 in
FIG. 8) each can include the three heavy chain and the three light chain CDR
sequences of any one of
the antibodies listed in Table 1.
The CD38 binding domains of Fc-antigen binding domain construct 9 (908/906,
920/922,
912/914, and 926/930 in FIG. 9) each can include the three heavy chain and the
three light chain CDR
sequences of any one of the antibodies listed in Table 1.
The CD38 binding domains of Fc-antigen binding domain construct 10 (1006/1004
and
1018/1020 in FIG. 10) each can include the three heavy chain and the three
light chain CDR sequences
of any one of the antibodies listed in Table 1.
The CD38 binding domains of Fc-antigen binding domain construct 11 (1112/1114,
1122/1108,
1128/1142, and 1138/1136 in FIG. 11) each can include the three heavy chain
and the three light chain
CDR sequences of any one of the antibodies listed in Table 1.
The CD38 binding domains of Fc-antigen binding domain construct 12(1218/1220,
1212/1214,
1250/1208, 1248/1246, 1242/1240, and 1236/1234 in FIG. 12) each can include
the three heavy chain
and the three light chain CDR sequences of any one of the antibodies listed in
Table 1.
The CD38 binding domains of Fc-antigen binding domain construct 13 (1310/1304
and
1314/1322 in FIG. 13) each can include the three heavy chain and the three
light chain CDR sequences
of any one of the antibodies listed in Table 1.
The CD38 binding domains of Fc-antigen binding domain construct 14 (1408/1406
and
1416/1424 in FIG. 14) each can include the three heavy chain and the three
light chain CDR sequences
of any one of the antibodies listed in Table 1.
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The CD38 binding domains of Fc-antigen binding domain construct 15(1508/1506,
1514/1516,
1532/1520, and 1530/1528 in FIG. 15) each can include the three heavy chain
and the three light chain
CDR sequences of any one of the antibodies listed in Table 1.
The CD38 binding domains of Fc-antigen binding domain construct 16 (1616/1604
and
1618/1630 in FIG. 16) each can include the three heavy chain and the three
light chain CDR sequences
of any one of the antibodies listed in Table 1.
The CD38 binding domains of Fc-antigen binding domain construct 17(1712/1714,
1724/1708,
1726/1742, and 1738/1736 in FIG. 17) each can include the three heavy chain
and the three light chain
CDR sequences of any one of the antibodies listed in Table 1.
The CD38 binding domains of Fc-antigen binding domain construct 18(1812/1814,
1828/1808,
1826/1824, 1830/1832, 1850/1848, and 1844/1842 in FIG. 18) each can include
the three heavy chain
and the three light chain CDR sequences of any one of the antibodies listed in
Table 1.
The CD38 binding domains of Fc-antigen binding domain construct 19 (1914/1904
and
1920/1922 in FIG. 19) each can include the three heavy chain and the three
light chain CDR sequences
.. of any one of the antibodies listed in Table 1.
The CD38 binding domains of Fc-antigen binding domain construct 20 (2014/2016,
2042/2008,
2036/2034, and 2028/2026 in FIG. 20) each can include the three heavy chain
and the three light chain
CDR sequences of any one of the antibodies listed in Table 1.
The CD38 binding domains of Fc-antigen binding domain construct 21 (2114/2116,
2150/2108,
.. 2148/2146, 2138/2140, 2136/2134, and 2128/2126 in FIG. 21) each can include
the three heavy chain
and the three light chain CDR sequences of any one of the antibodies listed in
Table 1.
IV. Dimerization selectivity modules
In the present disclosure, a dimerization selectivity module includes
components or select amino
acids within the Fc domain monomer that facilitate the preferred pairing of
two Fc domain monomers to
form an Fc domain. Specifically, a dimerization selectivity module is that
part of the CH3 antibody
constant domain of an Fc domain monomer which includes amino acid
substitutions positioned at the
interface between interacting CH3 antibody constant domains of two Fc domain
monomers. In a
dimerization selectivity module, the amino acid substitutions make favorable
the dimerization of the two
CH3 antibody constant domains as a result of the compatibility of amino acids
chosen for those
substitutions. The ultimate formation of the favored Fc domain is selective
over other Fc domains which
form from Fc domain monomers lacking dimerization selectivity modules or with
incompatible amino acid
substitutions in the dimerization selectivity modules. This type of amino acid
substitution can be made
using conventional molecular cloning techniques well-known in the art, such as
QuikChange
mutagenesis.
In some embodiments, a dimerization selectivity module includes an engineered
cavity (of "hole"
described further herein) in the CH3 antibody constant domain. In other
embodiments, a dimerization
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selectivity module includes an engineered protuberance (or "knob" described
further herein) in the CH3
antibody constant domain. To selectively form an Fc domain, two Fc domain
monomers with compatible
dimerization selectivity modules, e.g., one CH3 antibody constant domain
containing an engineered cavity
and the other CH3 antibody constant domain containing an engineered
protuberance, combine to form a
protuberance-into-cavity (or "knob and hole") pair of Fc domain monomers.
Engineered protuberances
and engineered cavities are examples of heterodimerizing selectivity modules,
which can be made in the
CH3 antibody constant domains of Fc domain monomers in order to promote
favorable heterodimerization
of two Fc domain monomers that have compatible heterodimerizing selectivity
modules. Table 3 lists
suitable mutation.
In other embodiments, heterodimerization is achieved by use of an Fc domain
monomer with a
dimerization selectivity module containing positively-charged amino acid
substitutions and an Fc domain
monomer with a dimerization selectivity module containing negatively-charged
amino acid substitutions
may selectively combine to form an Fc domain through the favorable
electrostatic steering (described
further herein) of the charged amino acids. In some embodiments, an Fc domain
monomer may include
one of the following positively-charged and negatively-charged amino acid
substitutions: K392D, K392E,
D399K, K409D, K409E, K439D, and K439E. In one example, an Fc domain monomer
containing a
positively-charged amino acid substitution, e.g., D356K or E357K, and an Fc
domain monomer containing
a negatively-charged amino acid substitution, e.g., K370D or K370E, may
selectively combine to form an
Fc domain through favorable electrostatic steering of the charged amino acids.
In another example, an
Fc domain monomer containing E357K and an Fc domain monomer containing K370D
may selectively
combine to form an Fc domain through favorable electrostatic steering of the
charged amino acids. In
some embodiments, reverse charge amino acid substitutions may be used as
heterodimerizing selectivity
modules, wherein two Fc domain monomers containing different, but compatible,
reverse charge amino
acid substitutions combine to form a heterodimeric Fc domain. Table 3 lists
various reverse charged
dimerization selectivity modules for promoting heterodimerization.
There are additional types of mutations, beyond knob and hole mutations and
electrostatic
steering mutations, than can be employed to promoting heterodimerization.
These mutations are also
listed in Table 3.
In other embodiments, two Fc domain monomers include homodimerizing
selectivity modules
containing identical reverse charge mutations in at least two positions within
the ring of charged residues
at the interface between CH3 domains. Homodimerizing selectivity modules are
reverse charge amino
acid substitutions that promote the homodimerization of Fc domain monomers to
form a homodimeric Fc
domain. By reversing the charge of both members of two or more complementary
pairs of residues in the
two Fc domain monomers, mutated Fc domain monomers remain complementary to Fc
domain
monomers of the same mutated sequence, but have a lower complementarity to Fc
domain monomers
without those mutations. In one embodiment, an Fc domain includes Fc domain
monomers including the
double mutants K409D/D399K, K392D/D399K, E357K/K370E, D356K/K439D,
K409E/D399K,
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K392E/D399K, E357K/K370D, or D356K/K439E. In another embodiment, an Fc domain
includes Fc
domain monomers including quadruple mutants combining any pair of the double
mutants, e.g.,
K409D/D399K/E357K/K370E. Tables 4A and 4B lists various selectivity that
promote homodimerization.
In further embodiments, an Fc domain monomer containing (i) at least one
reverse charge
mutation and (ii) at least one engineered cavity or at least one engineered
protuberance may selectively
combine with another Fc domain monomer containing (i) at least one reverse
charge mutation and (ii) at
least one engineered protuberance or at least one engineered cavity to form an
Fc domain. For example,
an Fc domain monomer containing reversed charge mutation K370D and engineered
cavities Y349C,
T366S, L368A, and Y407V and another Fc domain monomer containing reversed
charge mutation E357K
and engineered protuberances S354C and T366W may selectively combine to form
an Fc domain.
The formation of such Fc domains is promoted by the compatible amino acid
substitutions in the
CH3 antibody constant domains. Two dimerization selectivity modules containing
incompatible amino acid
substitutions, e.g., both containing engineered cavities, both containing
engineered protuberances, or
both containing the same charged amino acids at the CH3-CH3 interface, will
not promote the formation of
.. a heterodimeric Fc domain.
Furthermore, other methods used to promote the formation of Fc domains with
defined Fc domain
monomers include, without limitation, the LUZ-Y approach (U.S. Patent
Application Publication No.
W02011034605) which includes C-terminal fusion of a monomer a¨helices of a
leucine zipper to each of
the Fc domain monomers to allow heterodimer formation, as well as strand-
exchange engineered domain
(SEED) body approach (Davis et al., Protein Eng Des Se!. 23:195-202, 2010)
that generates Fc domain
with heterodimeric Fc domain monomers each including alternating segments of
IgA and IgG CH3
sequences.
V. Engineered cavities and engineered protuberances
The use of engineered cavities and engineered protuberances (or the "knob-into-
hole" strategy) is
described by Carter and co-workers (Ridgway et al., Protein Eng. 9:617-612,
1996; Atwell et al., J Mol
Biol. 270:26-35, 1997; Merchant et al., Nat Biotechnol. 16:677-681, 1998). The
knob and hole interaction
favors heterodimer formation, whereas the knob-knob and the hole-hole
interaction hinder homodimer
formation due to steric clash and deletion of favorable interactions. The
"knob-into-hole" technique is also
disclosed in U.S. Patent No. 5,731,168.
In the present disclosure, engineered cavities and engineered protuberances
are used in the
preparation of the Fc-antigen binding domain constructs described herein. An
engineered cavity is a void
that is created when an original amino acid in a protein is replaced with a
different amino acid having a
smaller side-chain volume. An engineered protuberance is a bump that is
created when an original
amino acid in a protein is replaced with a different amino acid having a
larger side-chain volume.
Specifically, the amino acid being replaced is in the CH3 antibody constant
domain of an Fc domain
monomer and is involved in the dimerization of two Fc domain monomers. In some
embodiments, an
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engineered cavity in one CH3 antibody constant domain is created to
accommodate an engineered
protuberance in another CH3 antibody constant domain, such that both CH3
antibody constant domains
act as dimerization selectivity modules (e.g., heterodimerizing selectivity
modules) (described above) that
promote or favor the dimerization of the two Fc domain monomers. In other
embodiments, an engineered
cavity in one CH3 antibody constant domain is created to better accommodate an
original amino acid in
another CH3 antibody constant domain. In yet other embodiments, an engineered
protuberance in one
CH3 antibody constant domain is created to form additional interactions with
original amino acids in
another CH3 antibody constant domain.
An engineered cavity can be constructed by replacing amino acids containing
larger side chains
such as tyrosine or tryptophan with amino acids containing smaller side chains
such as alanine, valine, or
threonine. Specifically, some dimerization selectivity modules (e.g.,
heterodimerizing selectivity modules)
(described further above) contain engineered cavities such as Y407V mutation
in the CH3 antibody
constant domain. Similarly, an engineered protuberance can be constructed by
replacing amino acids
containing smaller side chains with amino acids containing larger side chains.
Specifically, some
dimerization selectivity modules (e.g., heterodimerizing selectivity modules)
(described further above)
contain engineered protuberances such as T366W mutation in the CH3 antibody
constant domain. In the
present disclosure, engineered cavities and engineered protuberances are also
combined with inter-CH3
domain disulfide bond engineering to enhance heterodimer formation. In one
example, an Fc domain
monomer containing engineered cavities Y349C, T3665, L368A, and Y407V may
selectively combine
with another Fc domain monomer containing engineered protuberances 5354C and
T366W to form an Fc
domain. In another example, an Fc domain monomer containing an engineered
cavity with the addition of
Y349C and an Fc domain monomer containing an engineered protuberance with the
addition of 5354C
may selectively combine to form an Fc domain. Other engineered cavities and
engineered
protuberances, in combination with either disulfide bond engineering or
structural calculations (mixed HA-
TF) are included, without limitation, in Table 3.
Replacing an original amino acid residue in the CH3 antibody constant domain
with a different
amino acid residue can be achieved by altering the nucleic acid encoding the
original amino acid residue.
The upper limit for the number of original amino acid residues that can be
replaced is the total number of
residues in the interface of the CH3 antibody constant domains, given that
sufficient interaction at the
interface is still maintained.
Combining engineered cavities and engineered protuberances with electrostatic
steering
Electrostatic steering can be combined with knob-in-hole technology to favor
heterominerization,
for example, between Fc domain monomers in two different polypeptides.
Electrostatic steering,
described in greater detail below, is the utilization of favorable
electrostatic interactions between
oppositely charged amino acids in peptides, protein domains, and proteins to
control the formation of
higher ordered protein molecules. Electrostatic steering can be used to
promote either homodimerization
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or heterodimerization, the latter of which can be usefully combined with knob-
in-hole technology. In the
case of heterodimerization, different, but compatible, mutations are
introduced in each of the Fc domain
monomers which are to heterodimerize. Thus, an Fc domain monomer can be
modified to include one of
the following positively-charged and negatively-charged amino acid
substitutions: D356K, D356R, E357K,
E357R, K370D, K370E, K392D, K392E, D399K, K409D, K409E, K439D, and K439E. For
example, one
Fc domain monomer, for example, an Fc domain monomer having a cavity (Y349C,
T366S, L368A and
Y407V), can also include K370D mutation and the other Fc domain monomer, for
example, an Fc domain
monomer having a protuberance (S354C and T366VV) can include E357K.
More generally, any of the cavity mutations (or mutation combinations): Y407T,
Y407A, F405A,
Y407T, T394S, T394W:Y407A, T366W:T394S, T366S1368A:Y407V:Y349C, and
S3364H:F405 can be
combined with an electrostatic steering mutation in Table 3 and any of the
protuberance mutations (or
mutation combinations): T366Y, T366W, T394W, F405W, T366Y:F405A, T366W:Y407A,
T366W:S354C,
and Y349T:T394F can be combined with an electrostatic steering mutation in
Table 3.
VI. Electrostatic steering
Electrostatic steering is the utilization of favorable electrostatic
interactions between oppositely
charged amino acids in peptides, protein domains, and proteins to control the
formation of higher ordered
protein molecules. A method of using electrostatic steering effects to alter
the interaction of antibody
domains to reduce for formation of homodimer in favor of heterodimer formation
in the generation of
bi-specific antibodies is disclosed in U.S. Patent Application Publication No.
2014-0024111.
In the present disclosure, electrostatic steering is used to control the
dimerization of Fc domain
monomers and the formation of Fc-antigen binding domain constructs. In
particular, to control the
dimerization of Fc domain monomers using electrostatic steering, one or more
amino acid residues that
make up the CH3-CH3 interface are replaced with positively- or negatively-
charged amino acid residues
such that the interaction becomes electrostatically favorable or unfavorable
depending on the specific
charged amino acids introduced. In some embodiments, a positively-charged
amino acid in the interface,
such as lysine, arginine, or histidine, is replaced with a negatively-charged
amino acid such as aspartic
acid or glutamic acid. In other embodiments, a negatively-charged amino acid
in the interface is replaced
with a positively-charged amino acid. The charged amino acids may be
introduced to one of the
interacting CH3 antibody constant domains, or both. By introducing charged
amino acids to the
interacting CH3 antibody constant domains, dimerization selectivity modules
(described further above) are
created that can selectively form dimers of Fc domain monomers as controlled
by the electrostatic
steering effects resulting from the interaction between charged amino acids.
In some embodiments, to create a dimerization selectivity module including
reversed charges that
can selectively form dimers of Fc domain monomers as controlled by the
electrostatic steering effects, the
two Fc domain monomers may be selectively formed through heterodimerization or
homodimerization.
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Heterodimerization of Fc domain monomers
Heterodimerization of Fc domain monomers can be promoted by introducing
different, but
compatible, mutations in the two Fc domain monomers, such as the charge
residue pairs included,
without limitation, in Table 3. In some embodiments, an Fc domain monomer may
include one of the
following positively-charged and negatively-charged amino acid substitutions:
D356K, D356R, E357K,
E357R, K370D, K370E, K392D, K392E, D399K, K409D, K409E, K439D, and K439E. In
one example, an
Fc domain monomer containing a positively-charged amino acid substitution,
e.g., D356K or E357K, and
an Fc domain monomer containing a negatively-charged amino acid substitution,
e.g., K370D or K370E,
may selectively combine to form an Fc domain through favorable electrostatic
steering of the charged
amino acids. In another example, an Fc domain monomer containing E357K and an
Fc domain monomer
containing K370D may selectively combine to form an Fc domain through
favorable electrostatic steering
of the charged amino acids.
For example, in an Fc-antigen binding domain construct having three Fc
domains, two of the
three Fc domains may be formed by the heterodimerization of two Fc domain
monomers, as promoted by
the electrostatic steering effects. A "heterodimeric Fc domain" refers to an
Fc domain that is formed by
the heterodimerization of two Fc domain monomers, wherein the two Fc domain
monomers contain
different reverse charge mutations (heterodimerizing selectivity modules)
(see, e.g., mutations in Tables
4A and 4B) that promote the favorable formation of these two Fc domain
monomers. In an Fc-antigen
binding domain construct having three Fc domains - one carboxyl terminal
"stem" Fc domain and two
amino terminal "branch" Fc domains ¨ each of the amino terminal "branch" Fc
domains may be a
heterodimeric Fc domain (also called a "branch heterodimeric Fc domain")
(e.g., a heterodimeric Fc
domain formed by Fc domain monomers 106 and 114 or Fc domain monomers 112 and
116 in FIG. 1; a
heterodimeric Fc domain formed by Fc domain monomers 206 and 214 or Fc domain
monomers 212 and
216 in FIG. 2). A branch heterodimeric Fc domain may be formed by an Fc domain
monomer containing
E357K and another Fc domain monomer containing K370D.
Table 3. Fc heterodimerization methods
EmMOtriddiniiimeimiEMOOtiOW(gboinAmminiminfikitaitideitiNboakeyomminigiRoforeog
emmigi
Knobs-into- Y407T T336Y US Pat. #
Holes (Y-T) 8,216,805
Knobs-into- Y407A T336W US Pat. #
Holes 8,216,805
Knobs-into- F405A T394W US Pat. #
Holes 8,216,805
Knobs-into- Y407T T366Y US Pat. #
Holes 8,216,805
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Table 3. Fc heterodimerization methods
Method Mutations (Chain A) Mutations {Chair B}
Reference
Knobs-into- T394S F405W US Pat. #
Holes 8,216,805
Knobs-into- T394W, Y407T T366Y, F406A US Pat. #
Holes 8,216,805
Knobs-into- T394S, Y407A T366W, F405W US Pat. #
Holes 8,216,805
Knobs-into- T366W, T3945 F405W, T407A US Pat. #
Holes 8,216,805
Knobs-into- 5354C, T366W Y349C, T3665, L368A,
Holes Y407V
Knobs-into- Y349C, T3665, L368A, Y407V 5354C, T366W
Zeidler et al, J
Holes (CW- lmmunol.
163:
CSAV) 1246-52, 1999
HA-TF 5364H, F405A Y349T, T394F W02011028952
Electrostatic K409D D399K US 2014/0024111
Steering
Electrostatic K409D D399R US 2014/0024111
Steering
Electrostatic K409E D399K U52014/0024111
Steering
Electrostatic K409E D399R U52014/0024111
Steering
Electrostatic K392D D399K US 2014/0024111
Steering
Electrostatic K392D D399R US 2014/0024111
Steering
Electrostatic K392E D399K U52014/0024111
Steering
Electrostatic K392E D399R U52014/0024111
Steering
Electrostatic K392D, K409D E356K, D399K Gunasekaran et
Steering (DD- al., J Biol Chem.
KK) 285: 19637-46,
2010
Electrostatic K370E, K409D, K439E E356K, E357K, D399K WO 2006/106905
Steering
Knobs-into- 5354C, E357K, T366W Y349C, T3665, L368A, WO 2015/168643
Holes plus K370D, Y407V
Electrostatic
Steering
VYAV-VLLW T350V, L351Y, F405A, Y407V T350V, T366L,
K392L, Von Kreudenstein
T394W et al, MAbs,
5:646-
54, 2013
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Table 3. Fc heterodimerization methods
iiMiAkUthtitiMiinMniMMittAtiOnS1ChafilAyniginiEiniMMiiitatiOirMitptigitiTiyinin
iiniMRefeteneeNigig
EEE-RRR D221E, P228E, L368E D221R, P228R, K409R Strop et al, J
Mol
Biol, 420:204-19,
2012
EW-RVT K360E, K409W Q347R, D399V, F405T Choi et al, Mol
Cancer Ther,
12:2748-59, 2013
EW-RVTs_s K360E, K409W, Y349C Q347R, D399V, F405T, Choi et al,
Mol
5354C Immunol, 65:377-
83, 2015
Charge L351D T366K De Nardis, J
Biol
Introduction (DK) Chem, 292:14706-
17, 2017
Charge L351D, L368E L351K, T366K De Nardis, J
Biol
Introduction Chem, 292:14706-
(DEKK) 17, 2017
L-R F405L K409R Labrijn et al,
Proc
Natl Acad Sci
USA, 110:5145-
50, 2013
IgG/A chimera IgG/A chimera Davis et al,
Protein
Eng Des Sel,
23:195-202, 2010
S364K, T366V, K370T, K392Y, Q347E, Y349A, L351F, Skegro et al, J
Biol
F4055, Y407V, K409W, T411N 5364T, T366V, K370T, Chem, 292:9745-
T394D, V397L, D399E, 59, 2017
F405A, Y4075, K409R,
T411R
S364K, T366V, K370T, K392Y, F405A, Y4075 Skegro et al, J
Biol
K409W, T411N Chem, 292:9745-
59, 2017
Q347A, S364K, T366V, K370T, Q347E, Y349A, L351F, Skegro et al, J
Biol
K392Y, F4055, Y407V, 5364T, T366V, K370T, Chem, 292:9745-
K409W, T411N T394D, V397L, D399E, 59, 2017
F405A, Y4075, K409R,
T411R
BEAT (A/B - T) S364K, T366V, K370T, K392Y, Q347E, Y349A, L351F, Skegro
et al, J Biol
F4055, Y407V, K409W, T411N 5364T, T366V, K370T, Chem, 292:9745-
T394D, V397L, D399E, 59, 2017
F405A, Y4075, K409R
DMA-RRVV K360D, D399M, Y407A E345R, Q347R, T366V, Leaver-Fay et
al,
K409V Structure,
24:641-
51, 2016
SYMV-GDQA Y3495, K370Y, T366M, K409V E356G, E357D, 5364Q, Leaver-Fay
et al,
Y407A Structure,
24:641-
51, 2016
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Table 3. Fc heterodimerization methods
EMMOthtitiniMMnaMtitAtideit1ChafitArinigiNHAVItitatittiiittiptialitByginiNinnFt
eiditiltdain
Electrostatic
K370D E357K
Steering
Electrostatic
K370D E357R
Steering
Electrostatic
K370E E357K
Steering
Electrostatic
K370E E357R
Steering
Electrostatic
K370D D356K
Steering
Electrostatic
K370D D356R
Steering
Electrostatic
K370E D356K
Steering
Electrostatic
K370E D356R
Steering
Note: All residues numbered per the EU numbering scheme (Edelman et al., Proc
Nail Acad Sci USA,
63:78-85, 1969)
Homodimerization of Fc domain monomers
Homodimerization of Fc domain monomers can be promoted by introducing the same
electrostatic steering mutations (homodimerizing selectivity modules) in both
Fc domain monomers in a
symmetric fashion. In some embodiments, two Fc domain monomers include
homodimerizing selectivity
modules containing identical reverse charge mutations in at least two
positions within the ring of charged
residues at the interface between CH3 domains. By reversing the charge of both
members of two or more
complementary pairs of residues in the two Fc domain monomers, mutated Fc
domain monomers remain
complementary to Fc domain monomers of the same mutated sequence, but have a
lower
complementarity to Fc domain monomers without those mutations. Electrostatic
steering mutations that
may be introduced into an Fc domain monomer to promote its homodimerization
are shown, without
limitation, in Tables 4A and 4B In one embodiment, an Fc domain includes two
Fc domain monomers
each including the double reverse charge mutants (Tables 4A and 4B), e.g.,
K409D/D399K. In another
embodiment, an Fc domain includes two Fc domain monomers each including
quadruple reverse mutants
(Tables 4A and 4B), e.g., K409D/D399K/K370D/E357K.
For example, in an Fc-antigen binding domain construct having three Fc
domains, one of the
three Fc domains may be formed by the homodimerization of two Fc domain
monomers, as promoted by
the electrostatic steering effects. A "homodimeric Fc domain" refers to an Fc
domain that is formed by the
homodimerization of two Fc domain monomers, wherein the two Fc domain monomers
contain the same
reverse charge mutations (see, e.g., mutations in Tables 5 and 6). In an Fc-
antigen binding domain
construct having three Fc domains - one carboxyl terminal "stem" Fc domain and
two amino terminal
"branch" Fc domains ¨ the carbon/ terminal "stem" Fc domain may be a
homodimeric Fc domain (also
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called a "stem homodimeric Fc domain"). A stem homodimeric Fc domain may be
formed by two Fc
domain monomers each containing the double mutants K409D/D399K.
Table 4A. Fc homodimerization methods ¨ two mutations in each chain
Wild Type None US Pat. #8,216,805
Electrostatic Steering (KD) D399K/K409D Gunasekaran et al., J
Biol
Chem. 285: 19637-46, 2010,
WO 2015/168643
Electrostatic Steering D399K/K409E Gunasekaran et al., J
Biol
Chem. 285: 19637-46, 2010,
WO 2015/168643
Electrostatic Steering E357KK370D Gunasekaran et al., J
Biol
Chem. 285: 19637-46, 2010,
WO 2015/168643
Electrostatic Steering E357K/K370E Gunasekaran et al., J
Biol
Chem. 285: 19637-46, 2010,
WO 2015/168643
Electrostatic Steering D356K/K439D Gunasekaran et al., J
Biol
Chem. 285: 19637-46, 2010,
WO 2015/168643
Electrostatic Steering D356K/K439E Gunasekaran et al., J
Biol
Chem. 285: 19637-46, 2010,
WO 2015/168643
Electrostatic Steering K392D/D399K Gunasekaran et al., J
Biol Chem.
285: 19637-46, 2010, WO
2015/168643
Electrostatic Steering K392E/D399K Gunasekaran et al., J
Biol
Chem. 285: 19637-46, 2010,
WO 2015/168643
Electrostatic Steering K409D/D399R
Electrostatic Steering K409E/D399R
Electrostatic Steering K392D/D399R
Table 4B. Fc homodimerization methods ¨ four mutations in each chain
mggyorgpiiigvrgpiiifniptgtiippigkfpip.fA4otigipdyim:
iixonstatittibiiidittitifitAdWattidWitifedothAiiiV
K409D/D399K/K370D/E357K K392D/D399K/K370D/E357K
K409D/D399K/K370D/E357R K392D/D399K/K370D/E357R
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lig $00.01i#0#0.ffii.0104.00.tpicootpoyd.
iiiiifogootigpoivoilowoogokwoogpoolli
ilIllrgoolo.00tigtookjoiof ooi.5No-fr lpollfopotooloIII
Illpoogtotoroto4iIormggtptrootwo÷Idroo#iqi
K409D/D399K/K370E/E357K K392D/D399K/K370E/E357K
K409D/D399K/K370E/E357R K392D/D399K/K370E/E357R
K409D/D399K/K370D/D356K K392D/D399K/K370D/D356K
K409D/D399K/K370D/D356R K392D/D399K/K370D/D356R
K409D/D399K/K370E/D356K K392D/D399K/K370E/D356K
K409D/D399K/K370E/D356R K392D/D399K/K370E/D356R
K409D/D399R/K370D/E357K K392D/D399R/K370D/E357K
K409D/D399R/K370D/E357R K392D/D399R/K370D/E357R
K409D/D399R/K370E/E357K K392D/D399R/K370E/E357K
K409D/D399R/K370E/E357R K392D/D399R/K370E/E357R
K409D/D399R/K370D/D356K K392D/D399R/K370D/D356K
K409D/D399R/K370D/D356R K392D/D399R/K370D/D356R
K409D/D399R/K370E/D356K K392D/D399R/K370E/D356K
K409D/D399R/K370E/D356R K392D/D399R/K370E/D356R
K409E/D399K/K370D/E357K K392E/D399K/K370D/E357K
K409E/D399K/K370D/E357R K392E/D399K/K370D/E357R
K409E/D399K/K370E/E357K K392E/D399K/K370E/E357K
K409E/D399K/K370E/E357R K392E/D399K/K370E/E357R
K409E/D399K/K370D/D356K K392E/D399K/K370D/D356K
K409E/D399K/K370D/D356R K392E/D399K/K370D/D356R
K409E/D399K/K370E/D356K K392E/D399K/K370E/D356K
K409E/D399K/K370E/D356R K392E/D399K/K370E/D356R
K409E/D399R/K370D/E357K K392E/D399R/K370D/E357K
K409E/D399R/K370D/E357R K392E/D399R/K370D/E357R
K409E/D399R/K370E/E357K K392E/D399R/K370E/E357K
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Rererse charge mutaturns) in C3 antibody
Reverse ctiarge mutation(s) in C antbdy
zonittaiititiomaitcbteadltitififetionight
iiiconstatitdbitialititiftgdfiltittlietikieifedortigit
K409E/D399R/K370E/E357R K392E/D399R/K370E/E357R
K409E/D399R/K370D/D356K K392E/D399R/K370D/D356K
K409E/D399R/K370D/D356R K392E/D399R/K370D/D356R
K409E/D399R/K370E/D356K K392E/D399R/K370E/D356K
K409E/D399R/K370E/D356R K392E/D399R/K370E/D356R
VII. Linkers
In the present disclosure, a linker is used to describe a linkage or
connection between
polypeptides or protein domains and/or associated non-protein moieties. In
some embodiments, a linker
is a linkage or connection between at least two Fc domain monomers, for which
the linker connects the
C-terminus of the CH3 antibody constant domain of a first Fc domain monomer to
the N-terminus of the
hinge domain of a second Fc domain monomer, such that the two Fc domain
monomers are joined to
each other in tandem series. In other embodiments, a linker is a linkage
between an Fc domain
monomer and any other protein domains that are attached to it. For example, a
linker can attach the C-
terminus of the CH3 antibody constant domain of an Fc domain monomer to the N-
terminus of an
albumin-binding peptide.
A linker can be a simple covalent bond, e.g., a peptide bond, a synthetic
polymer, e.g., a
polyethylene glycol (PEG) polymer, or any kind of bond created from a chemical
reaction, e.g., chemical
conjugation. In the case that a linker is a peptide bond, the carboxylic acid
group at the C-terminus of
one protein domain can react with the amino group at the N-terminus of another
protein domain in a
condensation reaction to form a peptide bond. Specifically, the peptide bond
can be formed from
synthetic means through a conventional organic chemistry reaction well-known
in the art, or by natural
production from a host cell, wherein a polynucleotide sequence encoding the
DNA sequences of both
proteins, e.g., two Fc domain monomer, in tandem series can be directly
transcribed and translated into a
contiguous polypeptide encoding both proteins by the necessary molecular
machineries, e.g., DNA
polymerase and ribosome, in the host cell.
In the case that a linker is a synthetic polymer, e.g., a PEG polymer, the
polymer can be
functionalized with reactive chemical functional groups at each end to react
with the terminal amino acids
at the connecting ends of two proteins.
In the case that a linker (except peptide bond mentioned above) is made from a
chemical
reaction, chemical functional groups, e.g., amine, carboxylic acid, ester,
azide, or other functional groups
commonly used in the art, can be attached synthetically to the C-terminus of
one protein and the N-
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terminus of another protein, respectively. The two functional groups can then
react to through synthetic
chemistry means to form a chemical bond, thus connecting the two proteins
together. Such chemical
conjugation procedures are routine for those skilled in the art.
Spacer
In the present disclosure, a linker between two Fc domain monomers can be an
amino acid
spacer including 3-200 amino acids (e.g., 3-200, 3-180, 3-160, 3-140, 3-120, 3-
100, 3-90, 3-80, 3-70, 3-
60, 3-50, 3-45, 3-40, 3-35, 3-30, 3-25, 3-20, 3-15, 3-10, 3-9, 3-8, 3-7, 3-6,
3-5, 3-4, 4-200, 5-200, 6-200,
7-200, 8-200, 9-200, 10-200, 15-200, 20-200, 25-200, 30-200, 35-200, 40-200,
45-200, 50-200, 60-200,
.. 70-200, 80-200, 90-200, 100-200, 120-200, 140-200, 160-200, or 180-200
amino acids). In some
embodiments, a linker between two Fc domain monomers is an amino acid spacer
containing at least 12
amino acids, such as 12-200 amino acids (e.g., 12-200, 12-180, 12-160, 12-140,
12-120, 12-100, 12-90,
12-80, 12-70, 12-60, 12-50, 12-40, 12-30, 12-20, 12-19, 12-18, 12-17, 12-16,
12-15, 12-14, or 12-13
amino acids) (e.g., 14-200, 16-200, 18-200, 20-200, 30-200, 40-200, 50-200, 60-
200, 70-200, 80-200, 90-
200, 100-200, 120-200, 140-200, 160-200, 180-200, or 190-200 amino acids). In
some embodiments, a
linker between two Fc domain monomers is an amino acid spacer containing 12-30
amino acids (e.g., 12,
13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 0r30 amino
acids). Suitable peptide
spacers are known in the art, and include, for example, peptide linkers
containing flexible amino acid
residues such as glycine and serine. In certain embodiments, a spacer can
contain motifs, e.g., multiple
or repeating motifs, of GS, GGS, GGGGS (SEQ ID NO: 1), GGSG (SEQ ID NO: 2), or
SGGG (SEQ ID
NO: 3). In certain embodiments, a spacer can contain 2t0 12 amino acids
including motifs of GS, e.g.,
GS, GSGS (SEQ ID NO: 4), GSGSGS (SEQ ID NO: 5), GSGSGSGS (SEQ ID NO: 6),
GSGSGSGSGS
(SEQ ID NO: 7), or GSGSGSGSGSGS (SEQ ID NO: 8). In certain other embodiments,
a spacer can
contain 3 to 12 amino acids including motifs of GGS, e.g., GGS, GGSGGS (SEQ ID
NO: 9),
GGSGGSGGS (SEQ ID NO: 10), and GGSGGSGGSGGS (SEQ ID NO: 11). In yet other
embodiments,
a spacer can contain 4 to 20 amino acids including motifs of GGSG (SEQ ID NO:
2), e.g., GGSGGGSG
(SEQ ID NO: 12), GGSGGGSGGGSG (SEQ ID NO: 13), GGSGGGSGGGSGGGSG (SEQ ID NO:
14), or
GGSGGGSGGGSGGGSGGGSG (SEQ ID NO: 15). In other embodiments, a spacer can
contain motifs
of GGGGS (SEQ ID NO: 1), e.g., GGGGSGGGGS (SEQ ID NO: 16) or GGGGSGGGGSGGGGS
(SEQ
ID NO: 17). In certain embodiments, a spacer is SGGGSGGGSGGGSGGGSGGG (SEQ ID
NO: 18).
In some embodiments, a spacer between two Fc domain monomers contains only
glycine
residues, e.g., at least 4 glycine residues (e.g., 4-200, 4-180, 4-160, 4-140,
4-40, 4-100, 4-90, 4-80, 4-70,
4-60, 4-50, 4-40, 4-30, 4-20, 4-19, 4-18, 4-17, 4-16, 4-15, 4-14, 4-13, 4-12,
4-11, 4-10, 4-9, 4-8, 4-7, 4-6
or 4-5 glycine residues) (e.g., 4-200, 6-200, 8-200, 10-200, 12-200, 14-200,
16-200, 18-200, 20-200, 30-
200, 40-200, 50-200, 60-200, 70-200, 80-200, 90-200, 100-200, 120-200, 140-
200, 160-200, 180-200, or
190-200 glycine residues). In certain embodiments, a spacer has 4-30 glycine
residues (e.g., 4, 5, 6, 7,
8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, 0r30 glycine residues).
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In some embodiments, a spacer containing only glycine residues may not be
glycosylated (e.g., 0-linked
glycosylation, also referred to as 0-glycosylation) or may have a decreased
level of glycosylation (e.g., a
decreased level of 0-glycosylation) (e.g., a decreased level of 0-
glycosylation with glycans such as
xylose, mannose, sialic acids, fucose (Fuc), and/or galactose (Gal) (e.g.,
xylose)) as compared to, e.g., a
spacer containing one or more serine residues (e.g., SGGGSGGGSGGGSGGGSGGG (SEQ
ID NO: 18)).
In some embodiments, a spacer containing only glycine residues may not be 0-
glycosylated
(e.g., 0-xylosylation) or may have a decreased level of 0-glycosylation (e.g.,
a decreased level of 0-
xylosylation) as compared to, e.g., a spacer containing one or more serine
residues (e.g.,
SGGGSGGGSGGGSGGGSGGG (SEQ ID NO: 18)).
In some embodiments, a spacer containing only glycine residues may not undergo
proteolysis or
may have a decreased rate of proteolysis as compared to, e.g., a spacer
containing one or more serine
residues (e.g., SGGGSGGGSGGGSGGGSGGG (SEQ ID NO: 18)).
In certain embodiments, a spacer can contain motifs of GGGG (SEQ ID NO: 19),
e.g.,
GGGGGGGG (SEQ ID NO: 20), GGGGGGGGGGGG (SEQ ID NO: 21), GGGGGGGGGGGGGGGG
(SEQ ID NO: 22), or GGGGGGGGGGGGGGGGGGGG (SEQ ID NO: 23). In certain
embodiments, a
spacer can contain motifs of GGGGG (SEQ ID NO: 24), e.g., GGGGGGGGGG (SEQ ID
NO: 25), or
GGGGGGGGGGGGGGG (SEQ ID NO: 26). In certain embodiments, a spacer is
GGGGGGGGGGGGGGGGGGGG (SEQ ID NO: 27).
In other embodiments, a spacer can also contain amino acids other than glycine
and serine, e.g.,
GENLYFQSGG (SEQ ID NO: 28), SACYCELS (SEQ ID NO: 29), RSIAT (SEQ ID NO: 30),
RPACKIPNDLKQKVMNH (SEQ ID NO: 31), GGSAGGSGSGSSGGSSGASGTGTAGGTGSGSGTGSG
(SEQ ID NO: 32), AAANSSIDLISVPVDSR (SEQ ID NO: 33), or
GGSGGGSEGGGSEGGGSEGGGSEGGGSEGGGSGGGS (SEQ ID NO: 34).
In certain embodiments in the present disclosure, a 12- or 20-amino acid
peptide spacer is used
to connect two Fc domain monomers in tandem series, the 12- and 20-amino acid
peptide spacers
consisting of sequences GGGSGGGSGGGS (SEQ ID NO: 35) and SGGGSGGGSGGGSGGGSGGG
(SEQ ID NO: 18), respectively. In other embodiments, an 18-amino acid peptide
spacer consisting of
sequence GGSGGGSGGGSGGGSGGS (SEQ ID NO: 36) may be used.
In some embodiments, a spacer between two Fc domain monomers may have a
sequence that is
at least 75% identical (e.g., at least 77%, 79%, 81%, 83%, 85%, 87%, 89%, 91%,
93%, 95%, 97%, 99%,
or 99.5% identical) to the sequence of any one of SEQ ID NOs: 1-36 described
above. In certain
embodiments, a spacer between two Fc domain monomers may have a sequence that
is at least 80%
identical (e.g., at least 82%, 85%, 87%, 90%, 92%, 95%, 97%, 99%, or 99.5%
identical) to the sequence
of any one of SEQ ID NOs: 17, 18, 26, and 27. In certain embodiments, a spacer
between two Fc
domain monomers may have a sequence that is at least 80% identical (e.g., at
least 82%, 85%, 87%,
90%, 92%, 95%, 97%, 99%, or 99.5%) to the sequence of SEQ ID NO: 18 or 27.
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In certain embodiments, the linker between the amino terminus of the hinge of
an Fc domain
monomer and the carboxy terminus of a Fc monomer that is in the same
polypeptide (i.e., the linker
connects the C-terminus of the CH3 antibody constant domain of a first Fc
domain monomer to the N-
terminus of the hinge domain of a second Fc domain monomer, such that the two
Fc domain monomers
are joined to each other in tandem series) is a spacer having 3 or more amino
acids rather than a
covalent bond (e.g., 3-200 amino acids (e.g., 3-200, 3-180, 3-160, 3-140, 3-
120, 3-100, 3-90, 3-80, 3-70,
3-60, 3-50, 3-45, 3-40, 3-35, 3-30, 3-25, 3-20, 3-15, 3-10, 3-9, 3-8, 3-7, 3-
6, 3-5, 3-4, 4-200, 5-200, 6-200,
7-200, 8-200, 9-200, 10-200, 15-200, 20-200, 25-200, 30-200, 35-200, 40-200,
45-200, 50-200, 60-200,
70-200, 80-200, 90-200, 100-200, 120-200, 140-200, 160-200, or 180-200 amino
acids) or an amino acid
spacer containing at least 12 amino acids, such as 12-200 amino acids (e.g.,
12-200, 12-180, 12-160, 12-
140, 12-120, 12-100, 12-90, 12-80, 12-70, 12-60, 12-50, 12-40, 12-30, 12-20,
12-19, 12-18, 12-17, 12-16,
12-15, 12-14, or 12-13 amino acids) (e.g., 14-200, 16-200, 18-200, 20-200, 30-
200, 40-200, 50-200, 60-
200, 70-200, 80-200, 90-200, 100-200, 120-200, 140-200, 160-200, 180-200, or
190-200 amino acids)).
A spacer can also be present between the N-terminus of the hinge domain of a
Fc domain
monomer and the carboxy terminus of a CD38 binding domain (e.g., a CH1 domain
of a CD38 heavy
chain binding domain or the CL domain of a CD38 light chain binding domain)
such that the domains are
joined by a spacer of 3 or more amino acids (e.g., 3-200 amino acids (e.g., 3-
200, 3-180, 3-160, 3-140, 3-
120, 3-100, 3-90, 3-80, 3-70, 3-60, 3-50, 3-45, 3-40, 3-35, 3-30, 3-25, 3-20,
3-15, 3-10, 3-9, 3-8, 3-7, 3-6,
3-5, 3-4, 4-200, 5-200, 6-200, 7-200, 8-200, 9-200, 10-200, 15-200, 20-200, 25-
200, 30-200, 35-200, 40-
200, 45-200, 50-200, 60-200, 70-200, 80-200, 90-200, 100-200, 120-200, 140-
200, 160-200, or 180-200
amino acids) or an amino acid spacer containing at least 12 amino acids, such
as 12-200 amino acids
(e.g., 12-200, 12-180, 12-160, 12-140, 12-120, 12-100, 12-90, 12-80, 12-70, 12-
60, 12-50, 12-40, 12-30,
12-20, 12-19, 12-18, 12-17, 12-16, 12-15, 12-14, or 12-13 amino acids) (e.g.,
14-200, 16-200, 18-200, 20-
200, 30-200, 40-200, 50-200, 60-200, 70-200, 80-200, 90-200, 100-200, 120-200,
140-200, 160-200,
180-200, or 190-200 amino acids)).
VIII. Serum protein-binding peptides
Binding to serum protein peptides can improve the pharmacokinetics of protein
pharmaceuticals,
and in particular the Fc-antigen binding domain constructs described here may
be fused with serum
protein-binding peptides
As one example, albumin-binding peptides that can be used in the methods and
compositions
described here are generally known in the art. In one embodiment, the albumin
binding peptide includes
the sequence DICLPRWGCLW (SEQ ID NO: 37). In some embodiments, the albumin
binding peptide
has a sequence that is at least 80% identical (e.g., 80%, 90%, or 100%
identical) to the sequence of SEQ
ID NO: 37.
In the present disclosure, albumin-binding peptides may be attached to the N-
or C-terminus of
certain polypeptides in the Fc-antigen binding domain construct. In one
embodiment, an albumin-binding
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peptide may be attached to the C-terminus of one or more polypeptides in Fc
constructs containing a
CD38 binding domain. In another embodiment, an albumin-binding peptide can be
fused to the C-
terminus of the polypeptide encoding two Fc domain monomers linked in tandem
series in Fc constructs
containing a CD38 binding domain. In yet another embodiment, an albumin-
binding peptide can be
attached to the C-terminus of Fc domain monomer (e.g., Fc domain monomers 114
and 116 in FIG. 1; Fc
domain monomers 214 and 216 in FIG. 2) which is joined to the second Fc domain
monomer in the
polypeptide encoding the two Fc domain monomers linked in tandem series.
Albumin-binding peptides
can be fused genetically to Fc-antigen binding domain constructs or attached
to Fc-antigen binding
domain constructs through chemical means, e.g., chemical conjugation. If
desired, a spacer can be
inserted between the Fc-antigen binding domain construct and the albumin-
binding peptide. Without
being bound to a theory, it is expected that inclusion of an albumin-binding
peptide in an Fc-antigen
binding domain construct of the disclosure may lead to prolonged retention of
the therapeutic protein
through its binding to serum albumin.
I. Fc-antigen binding domain constructs
In general, the disclosure features Fc-antigen binding domain constructs
having 2-10 Fc domains
and one or more CD38 binding domains attached. These may have greater binding
affinity and/or avidity
than a single wild-type Fc domain for an Fc receptor, e.g., FcyRIlla. The
disclosure discloses methods of
engineering amino acids at the interface of two interacting CH3 antibody
constant domains such that the
two Fc domain monomers of an Fc domain selectively form a dimer with each
other, thus preventing the
formation of unwanted multimers or aggregates. An Fc-antigen binding domain
construct includes an
even number of Fc domain monomers, with each pair of Fc domain monomers
forming an Fc domain. An
Fc-antigen binding domain construct includes, at a minimum, two functional Fc
domains formed from
dimer of four Fc domain monomers and oneCD38 binding domain. The CD38 binding
domain may be
joined to an Fc domain e.g., with a linker, a spacer, a peptide bond, a
chemical bond or chemical moiety.
The Fc-antigen binding domain constructs can be assembled in many ways. The Fc-
antigen
binding domain constructs can be assembled from asymmetrical tandem Fc domains
(FIG. 1 - FIG. 6).
The Fc-antigen binding domain constructs can be assembled from singly branched
Fc domains, where
the branch point is at the N-terminal Fc domain (FIG. 7 - FIG.12). The Fc-
antigen binding domain
constructs can be assembled from singly branched Fc domains, where the branch
point is at the C-
terminal Fc domain (FIG. 13 - FIG. 18). The Fc-antigen binding domain
constructs can be assembled
from singly branched Fc domains, where the branch point is neither at the N-
or C-terminal Fc domain
(FIG. 19- FIG. 21).
The CD38 binding domain can be joined to the Fc-antigen binding domain
construct in many
ways. The CD38 binding domain can be expressed as a fusion protein of an Fc
chain. The heavy chain
component of a CD38 binding Fab can be expressed as a fusion protein of an Fc
chain and the light
chain component can be expressed as a separate polypeptide (FIG. 50, panel A).
In some embodiments,
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a scFv is used as a CD38 binding domain. The scFv can be expressed as a fusion
protein of the long Fc
chain (FIG. 50, panel B). In some embodiments, the heavy chain and light chain
components are
expressed separately and exogenously added to the Fc-antigen binding domain
construct. In some
embodiments, the CD38 binding domain is expressed separately and later joined
to the Fc-antigen
binding domain construct with a chemical bond (FIG. 50, panel C).
In some embodiments, one or more Fc polypeptides in an Fc-antigen binding
domain construct
lack a C-terminal lysine residue. In some embodiments, all of the Fc
polypeptides in an Fc-antigen
binding domain construct lack a C-terminal lysine residue. In some
embodiments, the absence of a C-
terminal lysine in one or more Fc polypeptides in an Fc-antigen binding domain
construct may improve
the homogeneity of a population of an Fc-antigen binding domain construct
(e.g., an Fc-antigen binding
domain construct having three Fc domains), e.g., a population of an Fc-antigen
binding domain construct
having three Fc domains that is at least 85%, 90%, 95%, 98%, or 99%
homogeneous.
In some embodiments, the N-terminal Asp in one or more of the first, second,
third, fourth, fifth, or
sixth polypeptides in an Fc-antigen binding domain construct described herein
(e.g., polypeptides 102,
112, and 114 in FIG. 1, 202, 214, 216 and 218 in FIG. 2, 302, 320, and 322 in
FIG. 3, 402, 428, 430, and
432 in FIG. 4, 502, 524, and 526 in FIG. 5, 602, 632, 634, and 636 in FIG 6,
702, 708, 722, and 724 in
FIG. 7, 802, 804, 826, and 828 in FIG. 8, 902, 904, 934, and 936 in FIG. 9,
1002, 1010, 1012, 1024,
1026, and 1032 in FIG. 10, 1102, 1104, 1106, 1144,1146, and 1148 in FIG. 11,
1202, 1204, 1206, 1252,
1254, and 1256 in FIG. 12, 1302, 1306 1320, and 1324 in FIG. 13, 1402, 1404,
1426, and 1428 in FIG.
14, 1502, 1504, 1534, and 1536 in FIG. 15, 1602, 1606, 1608, 1626, 1628, and
1632 in FIG. 16, 1702,
1704, 1706, 1744, 1746, and 1748 in FIG. 17, 1802, 1804, 1806, 1852, 1854, and
1856 in FIG. 18, 1902,
1906, 1910, 1924, 1928, and 1932 in FIG. 19, 2002, 2004, 2006, 2044, 2046, and
2048 in FIG. 20, 2102,
2104, 2106, 2152, 2154, and 2156 in FIG. 21 may be mutated to Gln.
For the exemplary Fc-antigen binding domain constructs described in the
Examples herein, Fc-
antigen binding domain constructs 1-21may contain the E357K and K370D charge
pairs in the Knobs and
Holes subunits, respectively.
Any one of the exemplary Fc-antigen binding domain constructs described herein
(e.g. Fc-antigen
binding domain constructs 1-21) can have enhanced effector function in an
antibody-dependent
cytotoxicity (ADCC) assay, an antibody-dependent cellular phagocytosis (ADCP)
and/or complement-
dependent cytotoxicity (CDC) assay relative to a construct having a single Fc
domain and the CD38
binding domain, or can include a biological activity that is not exhibited by
a construct having a single Fc
domain and the CD38 binding domain.
X. Host cells and protein production
In the present disclosure, a host cell refers to a vehicle that includes the
necessary cellular
components, e.g., organelles, needed to express the polypeptides and
constructs described herein from
their corresponding nucleic acids. The nucleic acids may be included in
nucleic acid vectors that can be
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introduced into the host cell by conventional techniques known in the art
(transformation, transfection,
electroporation, calcium phosphate precipitation, direct microinjection,
etc.). Host cells can be of
mammalian, bacterial, fungal or insect origin. Mammalian host cells include,
but are not limited to, CHO
(or CHO-derived cell strains, e.g., CHO-K1, CHO-DX611 CHO-DG44), murine host
cells (e.g., NSO,
Sp2/0), VERY, HEK (e.g., HEK293), BHK, HeLa, COS, MDCK, 293, 3T3, W138, BT483,
Hs578T, HTB2,
BT20 and T47D, CRL7030 and HsS78Bst cells. Host cells can also be chosen that
modulate the
expression of the protein constructs, or modify and process the protein
product in the specific fashion
desired. Different host cells have characteristic and specific mechanisms for
the post-translational
processing and modification of protein products. Appropriate cell lines or
host systems can be chosen to
ensure the correct modification and processing of the protein expressed.
For expression and secretion of protein products from their corresponding DNA
plasmid
constructs, host cells may be transfected or transformed with DNA controlled
by appropriate expression
control elements known in the art, including promoter, enhancer, sequences,
transcription terminators,
polyadenylation sites, and selectable markers. Methods for expression of
therapeutic proteins are known
in the art. See, for example, Paulina Balbas, Argelia Lorence (eds.)
Recombinant Gene Expression:
Reviews and Protocols (Methods in Molecular Biology), Humana Press; 2nd ed.
2004 edition (July 20,
2004); Vladimir Voynov and Justin A. Caravella (eds.) Therapeutic Proteins:
Methods and Protocols
(Methods in Molecular Biology) Humana Press; 2nd ed. 2012 edition (June 28,
2012).
XI. Afucosylation
Each Fc monomer includes an N-glycosylation site at Asn 297. The glycan can be
present in a
number of different forms on a given Fc monomer. In a composition containing
antibodies or the antigen-
binding Fc constructs described herein, the glycans can be quite heterogeneous
and the nature of the
glycan present can depend on, among other things, the type of cells used to
produce the antibodies or
antigen-binding Fc constructs, the growth conditions for the cells (including
the growth media) and post-
production purification. In various instances, compositions containing a
construct or polypeptide complex
or polypeptide described herein are afucosylated to at least some extent. For
example, at least 5%, 10%,
15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90% or 95% of the
glycans (e.g., the Fc
glycans) present in the composition lack a fucose residue. Thus, 5%-60%, 5%-
50%, 5%-40%, 10%-50%,
10%-50%, 10%-40%, 20%-50%, 0r20%-40% of the glycans lack a fucose residue.
Compositions that
are afucosylated to at least some extent can be produced by culturing cells
producing the antibody in the
presence of 1,3,4-Tri-O-acetyl-2-deoxy-2-fluoro-L-fucose inhibitor. Relatively
afucosylated forms of the
constructs and polypeptides described herein can be produced using a variety
of other methods,
including: expressing in cells with reduced or no expression of FUT8 (e.gõ by
knocking out FUT8 or
reducing expression with RNAi (siRNA, miRNA or shRNA) and expressing in cells
that overexpress beta-
1,4-mannosyl-glycoprotein 4-beta-N-acetylglucosaminyltransferase (GnT-III).
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XII. Purification
An Fc-antigen binding domain construct can be purified by any method known in
the art of protein
purification, for example, by chromatography (e.g., ion exchange, affinity
(e.g., Protein A affinity), and
size-exclusion column chromatography), centrifugation, differential
solubility, or by any other standard
technique for the purification of proteins. For example, an Fc-antigen binding
domain construct can be
isolated and purified by appropriately selecting and combining affinity
columns such as Protein A column
with chromatography columns, filtration, ultrafiltration, salting-out and
dialysis procedures (see, e.g.,
Process Scale Purification of Antibodies, Uwe Gottschalk (ed.) John Wiley &
Sons, Inc., 2009; and
Subramanian (ed.) Antibodies-Volume I-Production and Purification, Kluwer
Academic/Plenum
Publishers, New York (2004)).
In some instances, an Fc-antigen binding domain construct can be conjugated to
one or more
purification peptides to facilitate purification and isolation of the Fc-
antigen binding domain construct from,
e.g., a whole cell lysate mixture. In some embodiments, the purification
peptide binds to another moiety
that has a specific affinity for the purification peptide. In some
embodiments, such moieties which
specifically bind to the purification peptide are attached to a solid support,
such as a matrix, a resin, or
agarose beads. Examples of purification peptides that may be joined to an Fc-
antigen binding domain
construct include, but are not limited to, a hexa-histidine peptide, a FLAG
peptide, a myc peptide, and a
hemagglutinin (HA) peptide. A hexa-histidine peptide (HHHHHH (SEQ ID NO: 38))
binds to nickel-
functionalized agarose affinity column with micromolar affinity. In some
embodiments, a FLAG peptide
includes the sequence DYKDDDDK (SEQ ID NO: 39). In some embodiments, a FLAG
peptide includes
integer multiples of the sequence DYKDDDDK in tandem series, e.g., 3xDYKDDDDK.
In some
embodiments, a myc peptide includes the sequence EQKLISEEDL (SEQ ID NO: 40).
In some
embodiments, a myc peptide includes integer multiples of the sequence
EQKLISEEDL in tandem series,
e.g., 3xEQKLISEEDL. In some embodiments, an HA peptide includes the sequence
YPYDVPDYA (SEQ
ID NO: 41). In some embodiments, an HA peptide includes integer multiples of
the sequence
YPYDVPDYA in tandem series, e.g., 3xYPYDVPDYA. Antibodies that specifically
recognize and bind to
the FLAG, myc, or HA purification peptide are well-known in the art and often
commercially available. A
solid support (e.g., a matrix, a resin, or agarose beads) functionalized with
these antibodies may be used
to purify an Fc-antigen binding domain construct that includes a FLAG, myc, or
HA peptide.
For the Fc-antigen binding domain constructs, Protein A column chromatography
may be
employed as a purification process. Protein A ligands interact with Fc-antigen
binding domain constructs
through the Fc region, making Protein A chromatography a highly selective
capture process that is able to
remove most of the host cell proteins. In the present disclosure, Fc-antigen
binding domain constructs
may be purified using Protein A column chromatography as described in Example
2.
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XIII. Pharmaceutical compositions/preparations
The disclosure features pharmaceutical compositions that include one or more
Fc-antigen binding
domain constructs described herein. In one embodiment, a pharmaceutical
composition includes a
substantially homogenous population of Fc-antigen binding domain constructs
that are identical or
substantially identical in structure. In various examples, the pharmaceutical
composition includes a
substantially homogenous population of any one of Fc-antigen binding domain
constructs 1-42.
A therapeutic protein construct, e.g., an Fc-antigen binding domain construct
described herein
(e.g., an Fc-antigen binding domain construct having three Fc domains), of the
present disclosure can be
incorporated into a pharmaceutical composition. Pharmaceutical compositions
including therapeutic
proteins can be formulated by methods know to those skilled in the art. The
pharmaceutical composition
can be administered parenterally in the form of an injectable formulation
including a sterile solution or
suspension in water or another pharmaceutically acceptable liquid. For
example, the pharmaceutical
composition can be formulated by suitably combining the Fc-antigen binding
domain construct with
pharmaceutically acceptable vehicles or media, such as sterile water for
injection (NFI), physiological
saline, emulsifier, suspension agent, surfactant, stabilizer, diluent, binder,
excipient, followed by mixing in
a unit dose form required for generally accepted pharmaceutical practices. The
amount of active
ingredient included in the pharmaceutical preparations is such that a suitable
dose within the designated
range is provided.
The sterile composition for injection can be formulated in accordance with
conventional
pharmaceutical practices using distilled water for injection as a vehicle. For
example, physiological saline
or an isotonic solution containing glucose and other supplements such as D-
sorbitol, D-mannose, D-
mannitol, and sodium chloride may be used as an aqueous solution for
injection, optionally in combination
with a suitable solubilizing agent, for example, alcohol such as ethanol and
polyalcohol such as propylene
glycol or polyethylene glycol, and a nonionic surfactant such as polysorbate
80TM HCO-50, and the like
commonly known in the art. Formulation methods for therapeutic protein
products are known in the art,
see e.g., Banga (ed.) Therapeutic Peptides and Proteins: Formulation,
Processing and Delivery Systems
(2d ed.) Taylor & Francis Group, CRC Press (2006).
XIV. Methods of Treatment and Dosage
The Fc antigen binding domain constructs described here in can be used to
treat a variety of
cancers (e.g., hematologic malignancies and solid tumors) and autoimmune
diseases.
The cancer can be one that is resistant to daratumumab or any other
therapeutic anti-CD38
monoclonal antibody treatment. The cancer can be selected from: gastric
cancer, breast cancer, colon
cancer, lung cancer, mantle cell lymphoma, acute lymphoblastic leukemia, acute
myeloid leukemia, NK
cell leukemia, NK/T-cell lymphoma, chronic lymphocytic leukemia, plasma cell
leukemia, and multiple
myeloma. The constructs can also be used to treat: Amyloid light chain
Amyloidosis, Castleman's
disease, Monoclonal gammopathy of undetermined significance (MGUS), Biclonal
gammopathy of
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undetermined significance, Heavy chain diseases, Solitary plasmacytome,
Extramedullary plasmacytoma.
In some cases, the constructs can be used to augment immunoregulatory
functions against cancer cells
by immune complex mediated induction of preventative and/or therapeutic
vaccinal effects.
The constructs can also be used to treat: plasma cell dyscrasias or monoclonal
gammopathies
such as: Light chain deposition disease, Membranoproliferative
Glomerulonephritis (MGRS), Autoimmune
hemolytic anemia, Tempi Syndrome (Telangiectasia-Erythrocytosis-Monoclonal
Gammopathy
Perinephric-Fluid Collections-Intrapulmonary Shunting), Rheumatoid Arthritis,
Lupus Erythematosus
POEMS Syndrome (Polyneuropathy-Organomegaly-Endocrinopathy-Monoclonal
plasmaproliferative
disorder-Skin) and Waldenstrom Macroglobulinemia
The constructs can be used to treat autoantibody-mediated diseases such as:
Myasthenia Gravis
(MG), MuSK-MG, Myocarditis, Lambert Eaton, Myasthenic Syndrome, Neuromyotonia,
Neuromyelitis
optica, Narcolepsy, Acute motor axonal neuropathy, Guillain-Barre syndrome,
Fisher Syndrome, Acute
Sensory Ataxic Neuropathy, Paraneoplastic Stiff Person Syndrome, Chronic
Neuropathy, Peripheral
Neuropathy, Acute disseminated encephalomyelitis, Multiple sclerosis,
Goodpasture Syndrome,
Membranous Nephropathy, Glomerulonephritis, Pulmonary Alveolar Proteinosis,
CIPD, Autoimmune
hemolytic anemia, Autoimmune Thrombocytopenic purpura, Pemphigus vulgaris,
Pemphigus foliaceus,
Bullous pemphigoid, pemphigoid gestationis, Epidermolysis bullosa aquisita,
Neonatal lupus
erythematosus, Dermatitis herpetiformis, Graves Disease, Addison's Disease,
Ovarian insufficiency,
Autoimune Orchitis, Sjogren's Disease, Autoimmune gastritis, Rheumatoid
Arthritis, SLE, Dry eye
disease, Vasulitis (Acute), Carditis, and Antibody-mediated rejection.
The pharmaceutical compositions are administered in a manner compatible with
the dosage
formulation and in such amount as is therapeutically effective to result in an
improvement or remediation
of the symptoms. The pharmaceutical compositions are administered in a variety
of dosage forms, e.g.,
intravenous dosage forms, subcutaneous dosage forms, oral dosage forms such as
ingestible solutions,
drug release capsules, and the like. The appropriate dosage for the individual
subject depends on the
therapeutic objectives, the route of administration, and the condition of the
patient. Generally,
recombinant proteins are dosed at 1-200 mg/kg, e.g., 1-100 mg/kg, e.g., 20-100
mg/kg. Accordingly, it
will be necessary for a healthcare provider to tailor and titer the dosage and
modify the route of
administration as required to obtain the optimal therapeutic effect.
In addition to treating humans, the constructs can be used to treat companion
animals such as
dogs and cats as well as other veterinary subjects.
XV. Complement-dependent cytotoxicity (CDC)
Fc-antigen binding domain constructs described in this disclosure are able to
activate various Fc
receptor mediated effector functions. One component of the immune system is
the complement-
dependent cytotoxicity (CDC) system, a part of the innate immune system that
enhances the ability of
antibodies and phagocytic cells to clear foreign pathogens. Three biochemical
pathways activate the
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complement system: the classical complement pathway, the alternative
complement pathway, and the
lectin pathway, all of which entail a set of complex activation and signaling
cascades.
In the classical complement pathway, IgG or IgM trigger complement activation.
The C1q protein
binds to these antibodies after they have bound an antigen, forming the Cl
complex. This complex
generates Cis esterase, which cleaves and activates the C4 and C2 proteins
into C4a and C4b, and C2a
and C2b. The C2a and C4b fragments then form a protein complex called C3
convertase, which cleaves
C3 into C3a and C3b, leading to a signal amplification and formation of the
membrane attack complex.
The Fc-antigen binding domain constructs of this disclosure are able to
enhance CDC activity by
the immune system.
CDC may be evaluated by using a colorimetric assay in which Raji cells (ATCC)
are coated with a
serially diluted antibody, Fc-antigen binding domain construct, or IVIg. Human
serum complement
(Quidel) can be added to all wells at 25% v/v and incubated for 2 h at 37 C.
Cells can be incubated for
12 h at 37 C after addition of WST-1 cell proliferation reagent (Roche
Applied Science). Plates can then
be placed on a shaker for 2 min and absorbance at 450 nm can be measured.
XVI. Antibody-dependent cell-mediated cytotoxicity (ADCC)
The Fc-antigen binding domain constructs of this disclosure are also able to
enhance antibody-
dependent cell-mediated cytotoxicity (ADCC) activity by the immune system.
ADCC is a part of the
adaptive immune system where antibodies bind surface antigens of foreign
pathogens and target them
for death. ADCC involves activation of natural killer (NK) cells by
antibodies. NK cells express Fc
receptors, which bind to Fc portions of antibodies such as IgG and IgM. When
the antibodies are bound
to the surface of a pathogen-infected target cell, they then subsequently bind
the NK cells and activate
them. The NK cells release cytokines such as IFN-y, and proteins such as
perforin and granzymes.
Perforin is a pore forming cytolysin that oligomerizes in the presence of
calcium. Granzymes are serine
proteases that induce programmed cell death in target cells. In addition to NK
cells, macrophages,
neutrophils and eosinophils can also mediate ADCC.
ADCC may be evaluated using a luminescence assay. Human primary NK effector
cells
(Hemacare) are thawed and rested overnight at 37 C in lymphocyte growth medium-
3 (Lonza) at
5x105/mL. The next day, the human lymphoblastoid cell line Raji target cells
(ATCC CCL-86) are
harvested, resuspended in assay media (phenol red free RPMI, 10% FBSA,
GlutaMA)(Tm), and plated in
the presence of various concentrations of each probe of interest for 30
minutes at 37 C. The rested NK
cells are then harvested, resuspended in assay media, and added to the plates
containing the anti-CD20
coated Raji cells. The plates are incubated at 37 C for 6 hours with the final
ratio of effector-to-target
cells at 5:1 (5x104 NK cells: 1x104 Raji).
The CytoTox-Glo TM Cytotoxicity Assay kit (Promega) is used to determined ADCC
activity. The
CytoTox-Glo TM assay uses a luminogenic peptide substrate to measure dead cell
protease activity which
is released by cells that have lost membrane integrity e.g. lysed Raji cells.
After the 6 hour incubation
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period, the prepared reagent (substrate) is added to each well of the plate
and placed on an orbital plate
shaker for 15 minutes at room temperature. Luminescence is measured using the
PHERAstar F5 plate
reader (BMG Labtech). The data is analyzed after the readings from the control
conditions (NK cells +
Raji only) are subtracted from the test conditions to eliminate background.
XVII. Antibody-dependent cellular phagocytosis (ADCP)
The Fc-antigen binding domain constructs of this disclosure are also able to
enhance antibody-
dependent cellular phagocytosis (ADCP) activity by the immune system. ADCP,
also known as antibody
opsonization, is the process by which a pathogen is marked for ingestion and
elimination by a phagocyte.
Phagocytes are cells that protect the body by ingesting harmful foreign
pathogens and dead or dying
cells. The process is activated by pathogen-associated molecular patterns
(PAMPS), which leads to NF-
KB activation. Opsonins such as C3b and antibodies can then attach to target
pathogens. When a target
is coated in opsonin, the Fc domains attract phagocytes via their Fc
receptors. The phagocytes then
engulf the cells, and the phagosome of ingested material is fused with the
lysosome. The subsequent
phagolysosome then proteolytically digests the cellular material.
ADCP may be evaluated using a bioluminescence assay. Antibody-dependent cell-
mediated
phagocytosis (ADCP) is an important mechanism of action of therapeutic
antibodies. ADCP can be
mediated by monocytes, macrophages, neutrophils and dendritic cells via
FcyRIla (CD32a), FcyRI
(CD64), and FcyRIlla (CD16a). All three receptors can participate in antibody
recognition, immune
receptor clustering, and signaling events that result in ADCP; however,
blocking studies suggest that
FcyRIla is the predominant Fcy receptor involved in this process.
The FcyRIla-H ADCP Reporter Bioassay is a bioluminescent cell-based assay that
can be used
to measure the potency and stability of antibodies and other biologics with Fc
domains that specifically
bind and activate FcyRIla. The assay consists of a genetically engineered
Jurkat T cell line that
expresses the high-affinity human FcyRIla-H variant that contains a Histidine
(H) at amino acid 131 and a
luciferase reporter driven by an NFAT-response element (NFAT-RE).
When co-cultured with a target cell and relevant antibody, the FcyRIla-H
effector cells bind the Fc
domain of the antibody, resulting in FcyRIla signaling and NFAT-RE-mediated
luciferase activity. The
bioluminescent signal is detected and quantified with a Luciferase assay and a
standard luminometer.
Examples
The following examples are put forth so as to provide those of ordinary skill
in the art with a
complete disclosure and description of how the methods and compounds claimed
herein are performed,
made, and evaluated, and are intended to be purely exemplary of the disclosure
and are not intended to
limit the scope of what the inventors regard as their disclosure.
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Example 1. Design and purification of Fc-antigen binding domain construct 7
with a CD38 binding
domain
Protein Expression
Fc-antigen binding domain constructs are designed to increase folding
efficiencies, to minimize
.. uncontrolled association of subunits, which may create unwanted high
molecular weight oligomers and
multimers, and to generate compositions for pharmaceutical use that are
substantially homogenous (e.g.,
at least 85%, 90%, 95%, 98%, or 99% homogeneous). With these goals in mind, a
construct formed from
a singly branched Fc domain where the branch point is at the N-terminal Fc
domain is made as
described below. Fc-antigen binding domain construct 7 (CD38) each include two
distinct Fc domain
monomer containing polypeptides (two copies of an anti-CD38 long Fc chain (SEQ
ID NO:ZZ1), and two
copies of a short Fc chain (SEQ ID NO: ZZ2)), and two copies of an anti-CD38
light chain polypeptide
(SEQ ID NO: ZZ3). The long Fc chain contains an Fc domain monomer with an
E357K charge mutation
and 5354C and T366W protuberance-forming mutations (to promote
heterodimerization) in a tandem
series with a charge-mutated (K409D/D399K mutations) Fc domain monomer (to
promote
homodimerization), and anti-CD38 VH and CH1 domains (EU positions 1-220) at
the N-terminus
(construct 7 (CD38)). The short Fc chain contains an Fc domain monomer with a
K370D charge mutation
and Y349C, T3665, L368A, and Y407V cavity-forming mutations (to promote
heterodimerization). The
anti-CD38 light chain can also be expressed fused to the N-terminus of the
long Fc chain as part of an
scFv. DNA sequences are optimized for expression in mammalian cells and cloned
into the pcDNA3.4
mammalian expression vector. The DNA plasmid constructs are transfected via
liposomes into human
embryonic kidney (HEK) 293 cells. The amino acid sequences in Table 7 are
encoded by three separate
plasmids (one plasmid encoding the light chain (anti-CD38), one plasmid
encoding the long Fc chain
(anti-CD38) and one plasmid encoding the short Fc chain).
Table 5. Construct 7 (CD38) sequences
Construct Light chain Long Fc chain Short Fc chain
(with anti-CD38 VH and
CH1)
Construct 7 SEQ ID NO: SEQ ID NO: SEQ ID NO:
(CD38)
EIVLTQSPATLSLSPGERATLS QLLESGGGLVQPGGSLRL DKTHTCPPCPAPELLGGPSVF
CRASQSVSSYLAWYQQKPG SCAASGFTFDDYGMSWV LFPPKPKDTLMISRTPEVTCV
QAPRLLIYDASNRATGIPARF RQAPGKGLEWVSDISWN VVDVSHEDPEVKFNWYVDG
SGSGSGTDFTLTISSLEPEDFA GGKTHYVDSVKGQFTISR VEVHNAKTKPREEQYNSTYR
VYYCQQRSNWPPTFGQGTK DNSKNTLYLQM NSLRAED VVSVLTVLHQDWLNGKEYK
VEIKRTVAAPSVFIFPPSDEQL
CKVSNKALPAPIEKTISKAKG
KSGTASVVCLLNNFYPREAK TAVYYCARGSLFHDSSGFYQPREPQVCTLPPSRDELTKN
FGHWGQGTLVTVSSASTK
VQWKVDNALQSGNSQESVT QVSLSCAVDGFYPSDIAVEW
EQDSKDSTYSLSSTLTLSKAD GPSVFPLAPSSKSTSGGTA ESNGQPENNYKTTPPVLDSD
YEKHKVYACEVTHQGLSSPV ALGCLVKDYFPEPVTVSW GSFFLVSKLTVDKSRWQQG
TKSFNRGEC NSGALTSGVHTFPAVLQS NVFSCSVMHEALHNHYTQK
SGLYSLSSVVTVPSSSLGT SLSLSPG
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QTYICNVNHKPSNTKVDK
RVEPKSCDKTHTCPPCPA
PELLGGPSVFLFPPKPKDT
LMISRTPEVTCVVVDVSH
EDPEVKFNWYVDGVEVH
NAKTKPREEQYNSTYRVV
SVLTVLHQDWLNGKEYKC
KVSNKALPAPIEKTISKAKG
QPREPQVYTLPPSRDELTK
NQVSLTCLVKGFYPSDIAV
EWESNGQPENNYKTTPP
VLKSDGSFFLYSDLTVDKS
RWQQGNVFSCSVMHEA
LHNHYTQKSLSLSPGKGG
GGGGGGGGGGGGGGG
GGGDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMIS
RTPEVTCVVVDVSHEDPE
VKFNWYVDGVEVHNAKT
KPREEQYNSTYRVVSVLTV
LHQDWLNGKEYKCKVSN
KALPAPIEKTISKAKGQPRE
PQVYTLPPCRDKLTKNQV
SLWCLVKGFYPSDIAVEW
ESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRW
QQGNVFSCSVMHEALHN
HYTQKSLSLSPG
The expressed proteins are purified from the cell culture supernatant by
Protein A-based affinity
column chromatography, using a Poros MabCapture A (LifeTechnologies) column
and then further
fractionated by ion exchange chromatography. Purified sample are concentrated
to approximately 30
mg/mL and sterile filtered through a 0.2 pm filter.
Example 2. Design and purification of Fc-antigen binding domain construct 13
with a
CD38 binding domain
Protein Expression
A construct formed from a singly branched Fc domain where the branch point is
at the C-terminal
Fc domain is made as described below. Fc-antigen binding domain construct 13
(CD38) each include
two distinct Fc domain monomer containing polypeptides (two copies of an anti-
CD38 long Fc chain (any
one of SEQ ID NOs: ZZ, and two copies of a short Fc chain (SEQ ID NO: ZZ)) and
two copies of an anti-
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CD38 light chain polypeptide (SEQ ID NO: ZZ). The long Fc chain contains a
charge-mutated
(K409D/D399K mutations) Fc domain monomer (to promote homodimerization) in a
tandem series with
an Fc domain monomer with an E357K charge mutation and 5354C and T366W
protuberance-forming
mutations (to promote heterodimerization), and anti-CD38 VH and CH1 domains
(EU positions 1-220) at
the N-terminus (construct 13 (CD38)). The short Fc chain contains an Fc domain
monomer with a K370D
charge mutation and Y349C, T3665, L368A, and Y407V cavity-forming mutations
(to promote
heterodimerization). The anti-CD38 light chain and the anti-CD38 VH and CH1
are taken from an ant-
CD38 monoclonal antibody. Constructs with this light chain and anti-CD38 VH
and CH1 are indicated by
the abbreviation CD38. A related construct can be produced using the anti-CD38
light chain and the anti-
CD38 VH and CH1 taken from a fully human monoclonal antibody that cross-reacts
with CD38 expressed
by cynomolgus monkeys. These constructs are indicated by the abbreviation
Cyno. The CD38 light chain
can also be expressed fused to the N-terminus of the long Fc chain as part of
an scFv. Other versions of
construct 13 can be made with the anti-CD38 heavy chain, wherein each version
carries a different sized
glycine spacer (G4, G10, G15 or G20 linkers) between the Fc domain monomers in
the long Fc chain
polypeptide. DNA sequences are optimized for expression in mammalian cells and
cloned into the
pcDNA3.4 mammalian expression vector. The DNA plasmid constructs are
transfected via liposomes into
human embryonic kidney (HEK) 293 cells. The amino acid sequences for each of
the following
constructs are encoded by three separate plasmids (one plasmid encoding the
light chain (anti-CD38),
one plasmid encoding the long Fc chain (anti-CD38) and one plasmid encoding
the short Fc chain):
Table 6. Construct 13 (CD38) sequences
Construct Light chain Long Fc chain Short Fc chain
(anti-CD38 VH and CH1)
Construct 13 SEQ ID NO: W SEQ ID NO: X SEQ ID NO: Y
(CD38), G20
linker EIVLTQSPATLSLSPGERATLS EVQLLESGGGLVQPGGSLRL
DKTHTCPPCPAPELLGGPSVF
53Y-CD38 CRASQSVSSYLAWYQQKPG SCAVSGFTFNSFAMSWVRQ LFPPKPKDTLMISRTPEVTCV
QAPRLLIYDASNRATGIPARF APGKGLEWVSAISGSGGGTY VVDVSHEDPEVKFNWYVDG
SGSGSGTDFTLTISSLEPEDFA YADSVKGRFTISRDNSKNTLY VEVHNAKTKPREEQYNSTYR
VYYCQQRSNWPPTFGQGTK LQMNSLRAEDTAVYFCAKDK VVSVLTVLHQDWLNGKEYK
VEIKRTVAAPSVFIFPPSDEQL ILWFGEPVFDYWGQGTLVT CKVSNKALPAPIEKTISKAKG
KSGTASVVCLLNNFYPREAK VSSASTKGPSVFPLAPSSKSTS QPREPQVCTLPPSRDELTKN
VQWKVDNALQSGNSQESVT GGTAALGCLVKDYFPEPVTV QVSLSCAVDGFYPSDIAVEW
EQDSKDSTYSLSSTLTLSKAD SWNSGALTSGVHTFPAVLQS ESNGQPENNYKTTPPVLDSD
YEKHKVYACEVTHQGLSSPV SGLYSLSSVVTVPSSSLGTQTY GSFFLVSKLTVDKSRWQQG
TKSFNRGEC ICNVNHKPSNTKVDKRVEPK NVFSCSVMHEALHNHYTQK
SCDKTHTCPPCPAPELLGGPS SLSLSPG
VFLFPPKPKDTLMISRTPEVT
CVVVDVSHEDPEVKFNWYV
DGVEVHNAKTKPREEQYNST
YRVVSVLTVLHQDWLNGKE
YKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPCRDKLTK
NQVSLWCLVKGFYPSDIAVE
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WESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQ
GNVFSCSVMHEALHNHYTQ
KSLSLSPGKGGGGGGGGGG
GGGGGGGGGGDKTHTCPP
CPAPELLGGPSVFLFPPKPKD
TLM ISRTPEVTCVVVDVSHE
DPEVKFNWYVDGVEVHNAK
TKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKAL
PAPIEKTISKAKGQPREPQVY
TLPPSRDELTKNQVSLTCLVK
GFYPSDIAVEWESNGQPEN
NYKTTPPVLKSDGSFFLYSDL
TVDKSRWQQGNVFSCSVM
HEALHNHYTQKSLSLSPG
Construct 13 SEQ ID NO: SEQ ID NO: SEQ ID NO:
(CD38), G20 QSVLTQPPSASGTPGQRV QLLESGGGLVQPGGSLRL DKTHTCP PCPAP ELLGG PS
linker TISCSGSSSN IGDNYVSWY SCAASGFTFDDYGMSWV VFLFPPKPKDTLM ISRTPE
53Y-Cyno-001 QQLPGTAPKLLIYRDSQRP RQAPG KG LEWVSD ISWN VTCVVVDVSH EDP EVKF N
SGVPDRFSGSKSGTSASLA GGKTHYVDSVKGQFTISR WYVDGVEVH NAKTKPRE
ISGLRSEDEADYYCQSYDS DNSKNTLYLQM NSLRAED EQYNSTYRVVSVLTVLHQ
SLSGSVFGGGTKLTVLGQ TAVYYCARGSLFHDSSGFY DWLNGKEYKCKVSN KALP
PKAN PTVTLFPPSSEELQA FGHWGQGTLVTVSSASTK API E KTIS KAKGQPREPQV
N KATLVCLISDFYPGAVTV GPSVFPLAPSSKSTSGGTA CTLPPSRDELTKNQVSLSC
AWKADGSPVKAGVETTK ALGCLVKDYFPEPVTVSW AVDGFYPSDIAVEWESNG
PSKQSN N KYAASSYLSLTP NSGALTSGVHTFPAVLQS QPENNYKTTPPVLDSDGS
EQWKSH RSYSCQVTH EG SGLYSLSSVVTVPSSSLGT FFLVSKLTVDKSRWQQGN
STV E KTVA PT ECS QTYICNVN H KPSNTKVDK VFSCSVM H EALH NHYTQ
RVEPKSCDKTHTCPPCPA KSLSLSPG
PE LLGG PSVF LF P PKPKDT
LM ISRTPEVTCVVVDVSH
EDP EVKF NWYVDGVEVH
NAKTKPREEQYNSTYRVV
SVLTVLHQDWLNGKEYKC
KVSN KALPAPI EKTISKAKG
UP RE PQVYTLPPCR D KLTK
NQVSLWCLVKGFYPSDIA
VEWESNGQP EN NYKTTP
PVLDS DGS F F LYS KLTVD K
SRWQQGNVFSCSVM H E
ALH N HYTQKSLSLSPG KG
GGGGGGGGGGGGGGG
GGGG DKTHTCP PCPAP EL
LGGPSVFLFPPKPKDTLM I
S RT PEVTCVVVDVSH ED P
EVKFNWYVDGVEVHNAK
TKPREEQYNSTYRVVSVLT
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VLHQDWLNGKEYKCKVS
NKALPAPIEKTISKAKGQP
REPQVYTLPPSRDELTKN
QVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPV
LKSDGSFFLYSDLTVDKSR
WQQGNVFSCSVMHEAL
HNHYTQKSLSLSPG
The expressed proteins were purified from the cell culture supernatant by
Protein A-based affinity
column chromatography, using a Poros MabCapture A (LifeTechnologies) column
and then Purified
sample were concentrated to approximately 30 mg/mL and sterile filtered
through a 0.2 pm filter.
Example 3. Design and purification of Fc-antigen binding domain construct 1
An unbranched construct formed from asymmetrical tandem Fc domains is made as
described
below. Fc-antigen binding domain construct 1 (FIG. 1) includes two distinct Fc
domain monomer
.. containing polypeptides (a long Fc chain and two copies of a short Fc
chain) and a light chain
polypeptide. The long Fc chain contains two Fc domain monomers in a tandem
series, wherein each Fc
domain monomer has an engineered protuberance that is made by introducing at
least one protuberance-
forming mutation selected from Table 3 (e.g., the S354C and T366W mutations)
and, optionally, one or
more reverse charge mutation selected from Table 4A or 4B (e.g., E357K) (to
promote
heterodimerization), and a CD38 binding domain at the N-terminus. The CD38
binding domain may be
expressed as part of the same amino acid sequence as the long Fc chain (e.g.,
to form a scFv). The
short Fc chain contains an Fc domain monomer with an engineered cavity that is
made by introducing at
least one cavity-forming mutation selected from Table 3 (e.g., the Y349C,
T366S, L368A, and Y407V
mutations), and, optionally, a reverse charge mutation selected from Table 4A
or 4B (e.g., K370D) (to
promote heterodimerization). DNA sequences are optimized for expression in
mammalian cells and
cloned into the pcDNA3.4 mammalian expression vector. The DNA plasmid
constructs are transfected
via liposomes into human embryonic kidney (HEK) 293 cells. The amino acid
sequences for the short
and the long Fc chains are encoded by two separate plasmids. In this Example,
and in each of the
following Examples for Fc-antigen binding domain constructs 2-42, the cell may
contain a third plasmid
expressing an antibody variable light chain.
The expressed proteins are purified from the cell culture supernatant by
Protein A-based affinity
column chromatography, using a Poros MabCapture A (LifeTechnologies) column.
Captured Fc-antigen
binding domain constructs are washed with phosphate buffered saline (low-salt
wash) and eluted with
100mM glycine, pH 3. The eluate is quickly neutralized by the addition of 1 M
TRIS pH 7.4 and sterile
.. filtered through a 0.2 pm filter. The proteins are further fractionated by
ion exchange chromatography
using Poros XS resin (Applied Biosciences). The column is pre-equilibrated
with 50 mM MES, pH 6
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(buffer A), and the sample is eluted with a step gradient using 50 mM MES, 400
mM sodium chloride, pH
6 (buffer B) as the elution buffer. After ion exchange, the target fraction is
buffer exchanged into PBS
buffer using a 10 kDa cut-off polyether sulfone (PES) membrane cartridge on a
tangential flow filtration
system. The samples are concentrated to approximately 30 mg/mL and sterile
filtered through a 0.2 pm
filter.
Samples are denatured in Laemmli sample buffer (4% SDS, Bio-Rad) at 95 C for
10 min.
Samples are run on a Criterion TGX stain-free gel (4-15% polyacrylamide, Bio-
Rad). Protein bands are
visualized by UV illumination or Coommassie blue staining. Gels are imaged by
ChemiDoc MP Imaging
System (Bio-Rad). Quantification of bands is performed using Imagelab 4Ø1
software (Bio-Rad).
Example 4. Design and purification of Fc-antigen binding domain construct 2
An unbranched construct formed from asymmetrical tandem Fc domains is made as
described
below. Fc-antigen binding domain construct 2 (FIG. 2) includes two distinct Fc
monomer containing
polypeptides (a long Fc chain and three copies of a short Fc chain) and a
light chain polypeptide. The
long Fc chain contains three Fc domain monomers in a tandem series with a CD38
binding domain at N-
terminus, wherein each Fc domain monomer has an engineered protuberance that
is made by introducing
at least one protuberance-forming mutation selected from Table 3 (e.g., the
5354C and T366W
mutations) and, optionally, one or more reverse charge mutation selected from
Table 4A or 4B (e.g.,
E357K). The short Fc chain contains an Fc domain monomer with an engineered
cavity that is made by
introducing at least one cavity-forming mutation selected from Table 3 (e.g.,
the Y349C, T3665, L368A,
and Y407V mutations), and, optionally, one or more reverse charge mutation
selected from Table 4A or
4B (e.g., K370D). DNA sequences are optimized for expression in mammalian
cells and cloned into the
pcDNA3.4 mammalian expression vector. The DNA plasmid constructs are
transfected via liposomes into
human embryonic kidney (HEK) 293 cells. The amino acid sequences for the short
and long Fc chains
are encoded by two separate plasmids. The expressed proteins are purified as
in Example 3.
Example 5. Design and purification of Fc-antigen binding domain construct 3
A construct formed from asymmetrical tandem Fc domains is made as described
below. Fc-
antigen binding domain construct 3 (FIG. 3) includes two distinct Fc monomer
containing polypeptides (a
long Fc chain and two copies of a short Fc chain) and a light chain
polypeptide. The long Fc chain
contains two Fc domain monomers in a tandem series, wherein each Fc domain
monomer has an
engineered protuberance that is made by introducing at least one protuberance-
forming mutation
selected from Table 3 (e.g., the 5354C and T366W mutations) and, optionally,
one or more reverse
charge mutation selected from Table 4A or 4B (e.g., E357K). The short Fc chain
contains an Fc domain
monomer with an engineered cavity that is made by introducing at least one
cavity-forming mutation
selected from Table 3 (e.g., the Y349C, T3665, L368A, and Y407V mutations),
and, optionally, one or
more reverse charge mutation selected from Table 4A or 4B (e.g., K370D), and a
CD38 binding domain
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at N-terminus. DNA sequences are optimized for expression in mammalian cells
and cloned into the
pcDNA3.4 mammalian expression vector. The DNA plasmid constructs are
transfected via liposomes into
human embryonic kidney (HEK) 293 cells. The amino acid sequences for the short
and long Fc chains
are encoded by two separate plasmids. The expressed proteins are purified as
in Example 3.
Example 6. Design and purification of Fc-antigen binding domain construct 4
A construct formed from asymmetrical tandem Fc domains is made as described
below. Fc-
antigen binding domain construct 4 (FIG. 4) includes two distinct Fc monomer
containing polypeptides (a
long Fc chain and three copies of a short Fc chain) and a light chain
polypeptide. The long Fc chain
contains three Fc domain monomers in a tandem series, wherein each Fc domain
monomer has an
engineered protuberance that is made by introducing at least one protuberance-
forming mutation
selected from Table 3 (e.g., the S354C and T366W mutations) and, optionally,
one or more reverse
charge mutations selected from Table 4A or 4B (e.g., E357K). The short Fc
chain contains an Fc domain
monomer with an engineered cavity that is made by introducing at least one
cavity-forming mutation
selected from Table 3 (e.g., the Y349C, T366S, L368A, and Y407V mutations),
and, optionally, a reverse
charge mutation selected from Table 4A or 4B (e.g., K370D), and a CD38 binding
domain at the N-
terminus. DNA sequences are optimized for expression in mammalian cells and
cloned into the
pcDNA3.4 mammalian expression vector. The DNA plasmid constructs are
transfected via liposomes into
human embryonic kidney (HEK) 293 cells. The amino acid sequences for the short
and long Fc chains
are encoded by two separate plasmids. The expressed proteins are purified as
in Example 3.
Example 7. Design and purification of Fc-antigen binding domain construct 5
A construct formed from asymmetrical tandem Fc domains is made as described
below. Fc-
antigen binding domain construct 5 (FIG. 5) includes two distinct Fc monomer
containing polypeptides (a
long Fc chain and two copies of a short Fc chain) and a light chain
polypeptide. The long Fc chain
contains two Fc domain monomers in a tandem series with a CD38 binding domain
at the N-terminus,
wherein each Fc domain monomer has an engineered protuberance that is made by
introducing at least
one protuberance-forming mutation selected from Table 3 (e.g., the S354C and
T366W mutations) and,
optionally, one or more reverse charge mutations selected from Table 4A or 4B
(e.g., E357K). The short
Fc chain contains an Fc domain monomer with an engineered cavity that is made
by introducing at least
one cavity-forming mutation selected from Table 3 (e.g., the Y349C, T366S,
L368A, and Y407V
mutations), and, optionally, a reverse charge mutation selected from Table 4A
or 4B (e.g., K370D), and a
CD38 binding domain at N-terminus. DNA sequences are optimized for expression
in mammalian cells
and cloned into the pcDNA3.4 mammalian expression vector. The DNA plasmid
constructs are
transfected via liposomes into human embryonic kidney (HEK) 293 cells. The
amino acid sequences for
the short and long Fc chains are encoded by two separate plasmids. The
expressed proteins are purified
as in Example 3.
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Example 8. Design and purification of Fc-antigen binding domain construct 6
A construct formed from asymmetrical tandem Fc domains is made as described
below. Fc-
antigen binding domain construct 6 (FIG. 6) includes two distinct Fc monomer
containing polypeptides (a
long Fc chain and three copies of a short Fc chain) and a light chain
polypeptide. The long Fc chain
contains three Fc domain monomers in a tandem series with a CD38 binding
domain at the N-terminus,
wherein each Fc domain monomer has an engineered protuberance that is made by
introducing at least
one protuberance-forming mutation selected from Table 3 (e.g., the S354C and
T366W mutations) and,
optionally, one or more reverse charge mutations selected from Table 4A or 4B
(e.g., E357K). The short
Fc chain contains an Fc domain monomer with an engineered cavity that is made
by introducing at least
one cavity-forming mutation selected from Table 3 (e.g., the Y349C, T366S,
L368A, and Y407V
mutations), and, optionally, a reverse charge mutation selected from Table 4A
or 4B (e.g., K370D), and a
CD38 binding domain at N-terminus. DNA sequences are optimized for expression
in mammalian cells
and cloned into the pcDNA3.4 mammalian expression vector. The DNA plasmid
constructs are
transfected via liposomes into human embryonic kidney (HEK) 293 cells. The
amino acid sequences for
the short and long Fc chains are encoded by two separate plasmids. The
expressed proteins are purified
as in Example 3.
Example 9. Design and purification of Fc-antigen binding domain construct 7
A construct formed from a singly branched Fc domain where the branch point is
at the N-terminal
Fc domain is made as described below. Fc-antigen binding domain construct 7
(FIG. 7) includes two
distinct Fc monomer containing polypeptides (two copies of a long Fc chain and
two copies of a short Fc
chain) and a light chain polypeptide. The long Fc chain contains an Fc domain
monomer with an
engineered protuberance that is made by introducing at least one protuberance-
forming mutation
selected from Table 3 (e.g., the S354C and T366W mutations) and, optionally,
one or more reverse
charge mutation selected from Table 4A or 4B (e.g., E357K), in a tandem series
with an Fc domain
monomer with reverse charge mutations selected from Table 4A or 4B (e.g., the
K409D/D399K
mutations), and a CD38 binding domain at the N-terminus. The short Fc chain
contains an Fc domain
monomer with an engineered cavity that is made by introducing at least one
cavity-forming mutation
selected from Table 3 (e.g., the Y349C, T366S, L368A, and Y407V mutations),
and, optionally, one or
more reverse charge mutation selected from Table 4A or 4B (e.g., K370D). DNA
sequences are
optimized for expression in mammalian cells and cloned into the pcDNA3.4
mammalian expression
vector. The DNA plasmid constructs are transfected via liposomes into human
embryonic kidney (HEK)
293 cells. The amino acid sequences for the short and long Fc chains are
encoded by two separate
plasmids. The expressed proteins are purified as in Example 3.
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Example 10. Design and purification of Fc-antigen binding domain construct 8
A construct formed from a singly branched Fc domain where the branch point is
at the N-terminal
Fc domain is made as described below. Fc-antigen binding domain construct 8
(FIG. 8) includes two
distinct Fc monomer containing polypeptides (two copies of a long Fc chain and
two copies of a short Fc
chain) and a light chain polypeptide. The long Fc chain contains an Fc domain
monomer with an
engineered protuberance that is made by introducing at least one protuberance-
forming mutation
selected from Table 3 (e.g., the S354C and T366W mutations) and, optionally,
one or more reverse
charge mutation selected from Table 4A or 4B (e.g., E357K), in a tandem series
with an Fc domain
monomer with reverse charge mutations selected from Table 4A or 4B (e.g., the
K409D/D399K
mutations). The short Fc chain contains an Fc domain monomer with an
engineered cavity that is made
by introducing at least one cavity-forming mutation selected from Table 3
(e.g., the Y349C, T366S,
L368A, and Y407V mutations), and, optionally, one or more reverse charge
mutation selected from Table
4A or 4B (e.g., K370D), and a CD38 binding domain at the N-terminus. DNA
sequences are optimized
for expression in mammalian cells and cloned into the pcDNA3.4 mammalian
expression vector. The
DNA plasmid constructs are transfected via liposomes into human embryonic
kidney (HEK) 293 cells.
The amino acid sequences for the short and long Fc chains are encoded by two
separate plasmids. The
expressed proteins are purified as in Example 3.
Example 11. Design and purification of Fc-antigen binding domain construct 9
A construct formed from a singly branched Fc domain where the branch point is
at the N-terminal
Fc domain is made as described below. Fc-antigen binding domain construct 9
(FIG. 9) includes two
distinct Fc monomer containing polypeptides (two copies of a long Fc chain and
two copies of a short Fc
chain) and a light chain polypeptide. The long Fc chain contains an Fc domain
monomer with an
engineered protuberance that is made by introducing at least one protuberance-
forming mutation
selected from Table 3 (e.g., the S354C and T366W mutations) and, optionally,
one or more reverse
charge mutation selected from Table 4A or 4B (e.g., E357K), in a tandem series
with an Fc domain
monomer with reverse charge mutations selected from Table 4A or 4B (e.g., the
K409D/D399K
mutations), and a CD38 binding domain at the N-terminus. The short Fc chain
contains an Fc domain
monomer with an engineered cavity that is made by introducing at least one
cavity-forming mutation
selected from Table 3 (e.g., the Y349C, T366S, L368A, and Y407V mutations),
and, optionally, one or
more reverse charge mutation selected from Table 4A or 4B (e.g., K370D), and a
CD38 binding domain
at the N-terminus. DNA sequences are optimized for expression in mammalian
cells and cloned into the
pcDNA3.4 mammalian expression vector. The DNA plasmid constructs are
transfected via liposomes into
human embryonic kidney (HEK) 293 cells. The amino acid sequences for the short
and long Fc chains
are encoded by two separate plasmids. The expressed proteins are purified as
in Example 3.
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Example 12. Design and purification of Fc-antigen binding domain construct 10
A construct formed from a singly branched Fc domain where the branch point is
at the N-terminal
Fc domain is made as described below. Fc-antigen binding domain construct 10
(FIG. 10) includes two
distinct Fc monomer containing polypeptides (two copies of a long Fc chain and
four copies of a short Fc
chain) and a light chain polypeptide. The long Fc chain contains two Fc domain
monomers in a tandem
series, wherein each Fc domain monomer has an engineered protuberance that is
made by introducing at
least one protuberance-forming mutation selected from Table 3 (e.g., the S354C
and T366W mutations)
and, optionally, one or more reverse charge mutation selected from Table 4A or
4B (e.g., E357K), in a
tandem series with an Fc domain monomer with reverse charge mutations selected
from Table 4A or 4B
(e.g., the K409D/D399K mutations), and a CD38 binding domain at the N-
terminus. The short Fc chain
contains an Fc domain monomer with an engineered cavity that is made by
introducing at least one
cavity-forming mutation selected from Table 3 (e.g., the Y349C, T366S, L368A,
and Y407V mutations),
and, optionally, one or more reverse charge mutation selected from Table 4A or
4B (e.g., K370D). DNA
sequences are optimized for expression in mammalian cells and cloned into the
pcDNA3.4 mammalian
expression vector. The DNA plasmid constructs are transfected via liposomes
into human embryonic
kidney (HEK) 293 cells. The amino acid sequences for the short and long Fc
chains are encoded by two
separate plasmids. The expressed proteins are purified as in Example 3.
Example 13. Design and purification of Fc-antigen binding domain construct 11
A construct formed from a singly branched Fc domain where the branch point is
at the N-terminal
Fc domain is made as described below. Fc-antigen binding domain construct 11
(FIG. 11) includes two
distinct Fc monomer containing polypeptides (two copies of a long Fc chain and
four copies of a short Fc
chain) and a light chain polypeptide. The long Fc chain contains two Fc domain
monomers in a tandem
series, wherein each Fc domain monomer has an engineered protuberance that is
made by introducing at
least one protuberance-forming mutation selected from Table 3 (e.g., the S354C
and T366W mutations)
and, optionally, one or more reverse charge mutation selected from Table 4A or
4B (e.g., E357K), in a
tandem series with an Fc domain monomer with reverse charge mutations selected
from Table 4A or 4B
(e.g., the K409D/D399K mutations) at the N-terminus. The short Fc chain
contains an Fc domain
monomer with an engineered cavity that is made by introducing at least one
cavity-forming mutation
selected from Table 3 (e.g., the Y349C, T366S, L368A, and Y407V mutations),
and, optionally, one or
more reverse charge mutation selected from Table 4A or 4B (e.g., K370D), and
an antigen-binding
domain at the N-terminus. DNA sequences are optimized for expression in
mammalian cells and cloned
into the pcDNA3.4 mammalian expression vector. The DNA plasmid constructs are
transfected via
liposomes into human embryonic kidney (HEK) 293 cells. The amino acid
sequences for the short and
long Fc chains are encoded by two separate plasmids. The expressed proteins
are purified as in
Example 3.
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Example 14. Design and purification of Fc-antigen binding domain construct 12
A construct formed from a singly branched Fc domain where the branch point is
at the N-terminal
Fc domain is made as described below. Fc-antigen binding domain construct 12
(FIG. 12) includes two
distinct Fc monomer containing polypeptides (two copies of a long Fc chain and
four copies of a short Fc
chain) and a light chain polypeptide. The long Fc chain contains two Fc domain
monomers in a tandem
series, wherein each Fc domain monomer has an engineered protuberance that is
made by introducing at
least one protuberance-forming mutation selected from Table 3 (e.g., the S354C
and T366W mutations)
and, optionally, one or more reverse charge mutation selected from Table 4A or
4B (e.g., E357K), in a
tandem series with an Fc domain monomer with reverse charge mutations selected
from Table 4A or 4B
(e.g., the K409D/D399K mutations), and a CD38 binding domain at the N-
terminus. The short Fc chain
contains an Fc domain monomer with an engineered cavity that is made by
introducing at least one
cavity-forming mutation selected from Table 3 (e.g., the Y349C, T366S, L368A,
and Y407V mutations),
and, optionally, one or more reverse charge mutation selected from Table 4A or
4B (e.g., K370D), and an
antigen-binding domain at the N-terminus. DNA sequences are optimized for
expression in mammalian
cells and cloned into the pcDNA3.4 mammalian expression vector. The DNA
plasmid constructs are
transfected via liposomes into human embryonic kidney (HEK) 293 cells. The
amino acid sequences for
the short and long Fc chains are encoded by two separate plasmids. The
expressed proteins are purified
as in Example 3.
Example 15. Design and purification of Fc-antigen binding domain construct 13
A construct formed from a singly branched Fc domain where the branch point is
at the C-terminal
Fc domain is made as described below. Fc-antigen binding domain construct 13
(FIG. 13) includes two
distinct Fc monomer containing polypeptides (two copies of a long Fc chain and
two copies of a short Fc
chain) and a light chain polypeptide. The long Fc chain contains an Fc domain
monomer with reverse
charge mutations selected from Table 4A or 4B (e.g., the K409D/D399K
mutations), in a tandem series
with an Fc domain monomer with an engineered protuberance that is made by
introducing at least one
protuberance-forming mutation selected from Table 3 (e.g., the S354C and T366W
mutations) and,
optionally, one or more reverse charge mutation selected from Table 4A or 4B
(e.g., E357K), and a CD38
binding domain at the N-terminus. The short Fc chain contains an Fc domain
monomer with an
engineered cavity that is made by introducing at least one cavity-forming
mutation selected from Table 3
(e.g., the Y349C, T366S, L368A, and Y407V mutations), and, optionally, one or
more reverse charge
mutation selected from Table 4A or 4B (e.g., K370D). DNA sequences are
optimized for expression in
mammalian cells and cloned into the pcDNA3.4 mammalian expression vector. The
DNA plasmid
constructs are transfected via liposomes into human embryonic kidney (HEK) 293
cells. The amino acid
sequences for the short and long Fc chains are encoded by two separate
plasmids. The expressed
proteins are purified as in Example 3.
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Example 16. Design and purification of Fc-antigen binding domain construct 14
A construct formed from a singly branched Fc domain where the branch point is
at the C-terminal
Fc domain is made as described below. Fc-antigen binding domain construct 14
(FIG. 14) includes two
distinct Fc monomer containing polypeptides (two copies of a long Fc chain and
two copies of a short Fc
chain) and a light chain polypeptide. The long Fc chain contains an Fc domain
monomer with reverse
charge mutations selected from Table 4A or 4B (e.g., the K409D/D399K
mutations), in a tandem series
with an Fc domain monomer with an engineered protuberance that is made by
introducing at least one
protuberance-forming mutation selected from Table 3 (e.g., the S354C and T366W
mutations) and,
optionally, one or more reverse charge mutation selected from Table 4A or 4B
(e.g., E357K) at the N-
terminus. The short Fc chain contains an Fc domain monomer with an engineered
cavity that is made by
introducing at least one cavity-forming mutation selected from Table 3 (e.g.,
the Y349C, T366S, L368A,
and Y407V mutations), and, optionally, one or more reverse charge mutation
selected from Table 4A or
4B (e.g., K370D), and a CD38 binding domain at the N-terminus. DNA sequences
are optimized for
expression in mammalian cells and cloned into the pcDNA3.4 mammalian
expression vector. The DNA
plasmid constructs are transfected via liposomes into human embryonic kidney
(HEK) 293 cells. The
amino acid sequences for the short and long Fc chains are encoded by two
separate plasmids. The
expressed proteins are purified as in Example 3.
Example 17. Design and purification of Fc-antigen binding domain construct 15
A construct formed from a singly branched Fc domain where the branch point is
at the C-terminal
Fc domain is made as described below. Fc-antigen binding domain construct 15
(FIG. 15) includes two
distinct Fc monomer containing polypeptides (two copies of a long Fc chain and
two copies of a short Fc
chain) and a light chain polypeptide. The long Fc chain contains an Fc domain
monomer with reverse
charge mutations selected from Table 4A or 4B (e.g., the K409D/D399K
mutations), in a tandem series
with an Fc domain monomer with an engineered protuberance that is made by
introducing at least one
protuberance-forming mutation selected from Table 3 (e.g., the S354C and T366W
mutations) and,
optionally, one or more reverse charge mutation selected from Table 4A or 4B
(e.g., E357K), and a CD38
binding domain at the N-terminus. The short Fc chain contains an Fc domain
monomer with an
engineered cavity that is made by introducing at least one cavity-forming
mutation selected from Table 3
(e.g., the Y349C, T366S, L368A, and Y407V mutations), and, optionally, one or
more reverse charge
mutation selected from Table 4A or 4B (e.g., K370D), and a CD38 binding domain
at the N-terminus.
DNA sequences are optimized for expression in mammalian cells and cloned into
the pcDNA3.4
mammalian expression vector. The DNA plasmid constructs are transfected via
liposomes into human
embryonic kidney (HEK) 293 cells. The amino acid sequences for the short and
long Fc chains are
encoded by two separate plasmids. The expressed proteins are purified as in
Example 3.
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Example 18. Design and purification of Fc-antigen binding domain construct 16
A construct formed from a singly branched Fc domain where the branch point is
at the C-terminal
Fc domain is made as described below. Fc-antigen binding domain construct 16
(FIG. 16) includes two
distinct Fc monomer containing polypeptides (two copies of a long Fc chain and
four copies of a short Fc
chain) and a light chain polypeptide. The long Fc chain contains an Fc domain
monomer with reverse
charge mutations selected from Table 4A or 4B (e.g., the K409D/D399K
mutations), in a tandem series
with two Fc domain monomers, each with an engineered protuberance that is made
by introducing at
least one protuberance-forming mutation selected from Table 3 (e.g., the S354C
and T366W mutations)
and, optionally, one or more reverse charge mutation selected from Table 4A or
4B (e.g., E357K), and a
CD38 binding domain at the N-terminus. The short Fc chain contains an Fc
domain monomer with an
engineered cavity that is made by introducing at least one cavity-forming
mutation selected from Table 3
(e.g., the Y349C, T366S, L368A, and Y407V mutations), and, optionally, one or
more reverse charge
mutation selected from Table 4A or 4B (e.g., K370D). DNA sequences are
optimized for expression in
mammalian cells and cloned into the pcDNA3.4 mammalian expression vector. The
DNA plasmid
constructs are transfected via liposomes into human embryonic kidney (HEK) 293
cells. The amino acid
sequences for the short and long Fc chains are encoded by two separate
plasmids. The expressed
proteins are purified as in Example 3.
Example 19. Design and purification of Fc-antigen binding domain construct 17
A construct formed from a singly branched Fc domain where the branch point is
at the C-terminal
Fc domain is made as described below. Fc-antigen binding domain construct 17
(FIG. 17) includes two
distinct Fc monomer containing polypeptides (two copies of a long Fc chain and
four copies of a short Fc
chain) and a light chain polypeptide. The long Fc chain contains an Fc domain
monomer with reverse
charge mutations selected from Table 4A or 4B (e.g., the K409D/D399K
mutations), in a tandem series
with two Fc domain monomers, each with an engineered protuberance that is made
by introducing at
least one protuberance-forming mutation selected from Table 3 (e.g., the S354C
and T366W mutations)
and, optionally, one or more reverse charge mutation selected from Table 4A or
4B (e.g., E357K), at the
N-terminus. The short Fc chain contains an Fc domain monomer with an
engineered cavity that is made
by introducing at least one cavity-forming mutation selected from Table 3
(e.g., the Y349C, T366S,
L368A, and Y407V mutations), and, optionally, one or more reverse charge
mutation selected from Table
4A or 4B (e.g., K370D), andCD38 binding domain at the N-terminus. DNA
sequences are optimized for
expression in mammalian cells and cloned into the pcDNA3.4 mammalian
expression vector. The DNA
plasmid constructs are transfected via liposomes into human embryonic kidney
(HEK) 293 cells. The
amino acid sequences for the short and long Fc chains are encoded by two
separate plasmids. The
expressed proteins are purified as in Example 3.
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Example 20. Design and purification of Fc-antigen binding domain construct 18
A construct formed from a singly branched Fc domain where the branch point is
at the C-terminal Fc
domain is made as described below. Fc-antigen binding domain construct 18
(FIG. 18) includes two
distinct Fc monomer containing polypeptides (two copies of a long Fc chain and
four copies of a short Fc
chain) and a light chain polypeptide. The long Fc chain contains an Fc domain
monomer with reverse
charge mutations selected from Table 4A or 4B (e.g., the K409D/D399K
mutations), in a tandem series
with two Fc domain monomers, each with an engineered protuberance that is made
by introducing at
least one protuberance-forming mutation selected from Table 3 (e.g., the S354C
and T366W mutations)
and, optionally, one or more reverse charge mutation selected from Table 4A or
4B (e.g., E357K), and a
CD38 binding domain at the N-terminus. The short Fc chain contains an Fc
domain monomer with an
engineered cavity that is made by introducing at least one cavity-forming
mutation selected from Table 3
(e.g., the Y349C, T366S, L368A, and Y407V mutations), and, optionally, one or
more reverse charge
mutation selected from Table 4A or 4B (e.g., K370D), and a CD38 binding domain
at the N-terminus.
DNA sequences are optimized for expression in mammalian cells and cloned into
the pcDNA3.4
mammalian expression vector. The DNA plasmid constructs are transfected via
liposomes into human
embryonic kidney (HEK) 293 cells. The amino acid sequences for the short and
long Fc chains are
encoded by two separate plasmids. The expressed proteins are purified as in
Example 3.
Example 21. Design and purification of Fc-antigen binding domain construct 19
A construct formed from a singly branched Fc domain where the branch point is
neither at the N-
or C-terminal Fc domain is made as described below. Fc-antigen binding domain
construct 19 (FIG. 19)
includes two distinct Fc monomer containing polypeptides (two copies of a long
Fc chain and four copies
of a short Fc chain) and a light chain polypeptide. The long Fc chain contains
an Fc domain monomer
with an engineered protuberance that is made by introducing at least one
protuberance-forming mutation
selected from Table 3 (e.g., the S354C and T366W mutations) and, optionally,
one or more reverse
charge mutation selected from Table 4A or 4B (e.g., E357K), in a tandem series
with an Fc domain
monomer with reverse charge mutations selected from Table 4A or 4B (e.g., the
K409D/D399K
mutations), and another Fc domain monomer with an engineered protuberance that
is made by
introducing at least one protuberance-forming mutation selected from Table 3
(e.g., the S354C and
T366W mutations) and, optionally, one or more reverse charge mutation selected
from Table 4A or 4B
(e.g., E357K), and a CD38 binding domain at the N-terminus. The short Fc chain
contains an Fc domain
monomer with an engineered cavity that is made by introducing at least one
cavity-forming mutation
selected from Table 3 (e.g., the Y349C, T366S, L368A, and Y407V mutations),
and, optionally, one or
more reverse charge mutation selected from Table 4A or 4B (e.g., K370D). DNA
sequences are
optimized for expression in mammalian cells and cloned into the pcDNA3.4
mammalian expression
vector. The DNA plasmid constructs are transfected via liposomes into human
embryonic kidney (HEK)
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293 cells. The amino acid sequences for the short and long Fc chains are
encoded by two separate
plasmids. The expressed proteins are purified as in Example 3.
Example 22. Design and purification of Fc-antigen binding domain construct 20
A construct formed from a singly branched Fc domain where the branch point is
at the C-terminal
Fc domain is made as described below. Fc-antigen binding domain construct 20
(FIG. 20) includes two
distinct Fc monomer containing polypeptides (two copies of a long Fc chain and
four copies of a short Fc
chain) and a light chain polypeptide. The long Fc chain contains an Fc domain
monomer with an
engineered protuberance that is made by introducing at least one protuberance-
forming mutation
selected from Table 3 (e.g., the S354C and T366W mutations) and, optionally,
one or more reverse
charge mutation selected from Table 4A or 4B (e.g., E357K), in a tandem series
with an Fc domain
monomer with reverse charge mutations selected from Table 4A or 4B (e.g., the
K409D/D399K
mutations), and another Fc domain monomer with an engineered protuberance that
is made by
introducing at least one protuberance-forming mutation selected from Table 3
(e.g., the S354C and
T366W mutations) and, optionally, one or more reverse charge mutation selected
from Table 4A or 4B
(e.g., E357K), at the N-terminus. The short Fc chain contains an Fc domain
monomer with an
engineered cavity that is made by introducing at least one cavity-forming
mutation selected from Table 3
(e.g., the Y349C, T366S, L368A, and Y407V mutations), and, optionally, one or
more reverse charge
mutation selected from Table 4A or 4B (e.g., K370D), and a CD38 binding domain
at the N-terminus.
DNA sequences are optimized for expression in mammalian cells and cloned into
the pcDNA3.4
mammalian expression vector. The DNA plasmid constructs are transfected via
liposomes into human
embryonic kidney (HEK) 293 cells. The amino acid sequences for the short and
long Fc chains are
encoded by two separate plasmids. The expressed proteins are purified as in
Example 3.
Example 23. Design and purification of Fc-antigen binding domain construct 21
A construct formed from a singly branched Fc domain where the branch point is
at the C-terminal
Fc domain is made as described below. Fc-antigen binding domain construct 21
(FIG. 21) includes two
distinct Fc monomer containing polypeptides (two copies of a long Fc chain and
four copies of a short Fc
chain) and a light chain polypeptide. The long Fc chain contains an Fc domain
monomer with an
engineered protuberance that is made by introducing at least one protuberance-
forming mutation
selected from Table 3 (e.g., the S354C and T366W mutations) and, optionally,
one or more reverse
charge mutation selected from Table 4A or 4B (e.g., E357K), in a tandem series
with an Fc domain
monomer with reverse charge mutations selected from Table 4A or 4B (e.g., the
K409D/D399K
mutations), another Fc domain monomer with an engineered protuberance that is
made by introducing at
least one protuberance-forming mutation selected from Table 3 (e.g., the S354C
and T366W mutations)
and, optionally, one or more reverse charge mutation selected from Table 4A or
4B (e.g., E357K), and a
CD38 binding domain at the N-terminus. The short Fc chain contains an Fc
domain monomer with an
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engineered cavity that is made by introducing at least one cavity-forming
mutation selected from Table 3
(e.g., the Y349C, T366S, L368A, and Y407V mutations), and, optionally, one or
more reverse charge
mutation selected from Table 4A or 4B (e.g., K370D), and a CD38 binding domain
at the N-terminus.
DNA sequences are optimized for expression in mammalian cells and cloned into
the pcDNA3.4
mammalian expression vector. The DNA plasmid constructs are transfected via
liposomes into human
embryonic kidney (HEK) 293 cells. The amino acid sequences for the short and
long Fc chains are
encoded by two separate plasmids. The expressed proteins are purified as in
Example 3.
Example 24. CDC, ADCP, and ADCC activation by Fc-antigen binding domain
constructs
Three assays are used to test the activation of CDC, ADCP, and ADCC pathways
by parent
mAbs and various Fc-antigen binding domain constructs. Four constructs are
created containing the
CDRs from Gazyva (obinutuzumab), an anti-CD20 mAb. Both fucosylated and
afucosylated anti-CD20
mAbs were made as well as S3Y-AA-CD20 (structure of Construct 13, FIG. 13, as
described in Example
2) and SAI-AA-CD20 (structure of Construct 7, FIG. 7, as described in Example
1) Fc-antigen binding
domain constructs.
A CDC assay is performed as follows:
1. The target cells used in the anti-CD20 CDC assay are the Raji cells (ATCC
CCL-86). Raji
cells (CD20 expressing tumor cells) were resuspended in X-VIVO 15 media at 6 x
105 cells/ml. Cells were
then transferred to a 96 well flat-bottom assay plate in a volume of 100 pl
per well (6 x 104 cells/well).
2. Anti-CD20 mAbs and Fc-antigen binding domain constructs were diluted to
3.33 pM in X-VIVO
15 media. Serial 1:3 dilutions were then performed with each molecule in 1.5
ml polypropylene tubes
resulting in an 11 point dilution series.
3. Each dilution of the molecules were transferred at 50 p1/well to the
appropriate wells in the
assay plate. Immediately following the transfer to assay plate, 50 pl of
normal human serum complement
were added to each well.
4. The assay plate was incubated at 37 C and 5% CO2 for 2 h. Following the 2 h
incubation, 20
pl of WST-1 proliferation reagent was added to each well of the assay plate.
The plate was returned to
the 37 C, 5% CO2 incubator for 14 h.
5. Following the 14 h incubation, the plate was shaken for 1 min on a plate
shaker and the
absorbance of the wells was immediately determined at 450 nm with 600 nm
correction using a
spectrophotometer.
In a CDC assay in which the target cells were Raji (FIG. 22, left panel), the
53Y-AA-CD20 (construct 13
with anti-CD20 Fab) was able to mediate cytotoxicity, while the other
constructs were not.
An ADCP assay was performed as follows:
The FcyRIla-H ADCP Reporter Bioassay, Complete Kit (Promega Cat # G9901), is a
bioluminescent cell-
based assay that can be used to measure the potency and stability of
antibodies and other biologics with
Fc domains that specifically bind and activate FcyRIla. The assay consisted of
a genetically engineered
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Jurkat T cell line that expresses the high-affinity human FcyRIla-H variant
that contains a Histidine (H) at
amino acid 131 and a luciferase reporter driven by an NFAT-response element
(NFAT-RE). When co-
cultured with a target cell and relevant antibody, the FcyRIla-H effector
cells upon binding to Fc domain of
an antibody results in FcyRIla signaling and NFAT-RE-mediated luciferase
activity. The bioluminescent
signal was detected and quantified using Bio-Glo TM Luciferase Assay System
and a luminometer.
Increasing concentrations of anti-CD20 mAbs and construct 7 (with an anti-CD20
Fab) or construct 13
(with an anti-CD20 Fab) were incubated with Raji target cells and FcyRIla-H
effector cells (in 2:1 ratio).
After 6 hours of incubation at 37 C Bio-Glo TM reagent was added, and
luminescence was measured in a
PHERAstar FS instrument. Data was fitted to a 4PL curve using GraphPad Prism
software (FIG. 22,
middle panel). Both the 53I-AA-CD20 (construct 7 with anti-CD20 Fab) and 53Y-
AA-CD20 (construct 13
with anti-CD20 Fab) constructs showed enhanced potency (EC50) >100-fold
relative to the anti-CD20
mAbs.
An ADCC assay was performed as follows:
Human primary NK effector cells were thawed and rested overnight at 37 C in
lymphocyte growth
medium-3 (Lonza) at 5x105/mL. The next day, the Raji cells were harvested,
resuspended in assay
media (phenol red free RPMI, 10% FBS, GlutaMAXTm), and plated in the presence
of various
concentrations of each molecule of interest for 30 minutes at 37 C. The rested
NK cells were then
harvested, resuspended in assay media, and added to the plates containing the
anti-CD20 coated Raji
cells. The plates were incubated at 37 C for 6 hours with the final ratio of
effector-to-target cells at 5:1
(5x104 NK: 1x104 Raji cells).
The CytoTox-Glo TM Cytotoxicity Assay kit (Promega) was used to determined
ADCC activity. The
CytoTox-Glo TM assay uses a luminogenic peptide substrate to measure dead cell
protease activity which
is released by cells that have lost membrane integrity e.g. lysed Raji cells.
After the 6 hour incubation
period, the prepared reagent (substrate) was added to each well of the plate
and placed on an orbital
plate shaker for 15 minutes at room temperature. Luminescence was measured
using the PHERAstar F5
plate reader (BMG Labtech). The data was analyzed after the readings from the
control conditions (NK
cells + Raji only) were subtracted from the test conditions to eliminate
background. (FIG. 47, right panel).
Both the S3I (construct 7 with anti-CD20 Fab) and 53Y (construct 13 with anti-
CD20 Fab) constructs
showed enhanced cytotoxicity relative to the fucosylated mAb and similar
cytotoxicity relative to the
afucosylated mAb.
Example 25. Experimental assays used to characterize Fc-antigen binding domain
constructs
Peptide and Glycopeptide Liquid Chromatography-MS/MS
The proteins were diluted to 1 pg/pL in 6M guanidine (Sigma). Dithiothreitol
(DTT) was added to
a concentration of 10 mM, to reduce the disulfide bonds under denaturing
conditions at 65 C for 30 min.
After cooling on ice, the samples were incubated with 30 mM iodoacetamide
(IAM) for 1 h in the dark to
alkylate (carbamidomethylate) the free thiols. The protein was then dialyzed
across a 10-kDa membrane
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into 25 mM ammonium bicarbonate buffer (pH 7.8) to remove IAM, DTT and
guanidine. The protein was
digested with trypsin in a Barocycler (NEP 2320; Pressure Biosciences, Inc.).
The pressure was cycled
between 20,000 psi and ambient pressure at 37 C for a total of 30 cycles in 1
h. LC-MS/MS analysis of
the peptides was performed on an Ultimate 3000 (Dionex) Chromatography System
and an Q-Exactive
(Thermo Fisher Scientific) Mass Spectrometer. Peptides were separated on a BEH
PepMap (Waters)
Column using 0.1% FA in water and 0.1% FA in acetonitrile as the mobile
phases. The singly xylosylated
linker peptide was targeted based on the doubly charged ion (z=2) m/z 842.5
with a quadrupole isolation
width of 1.5 Da.
Intact Mass Spectrometry
The protein was diluted to a concentration of 2 pg/pL in the running buffer
consisting of 78.98%
water, 20% acetonitrile, 1% formic acid (FA), and 0.02% trifluoroacetic acid.
Size exclusion
chromatography separation was performed on two Zenix-C SEC-300 (Sepax
Technologies, Newark, DE)
2.1 x 350 mm in tandem for a total length column length of 700 mm. The
proteins were eluted from the
SEC column using the running buffer described above at a flow rate of 80
pL/min. Mass spectra were
acquired on an QSTAR Elite (Applied Biosystems) Q-ToF mass spectrometer
operated in positive mode.
The neutral masses under the individual size fractions were deconvoluted using
Bayesian peak
deconvolution by summing the spectra across the entire width of the
chromatographic peak.
Capillary electrophoresis-sodium dodecyl sulfate (CE-SDS) assay
Samples were diluted to 1 mg/mL and mixed with the HT Protein Express
denaturing buffer
(PerkinElmer). The mixture was incubated at 40 C for 20 min. Samples were
diluted with 70 pL of water
and transferred to a 96-well plate. Samples were analyzed by a Caliper GXII
instrument (PerkinElmer)
equipped with the HT Protein Express LabChip (PerkinElmer). Fluorescence
intensity was used to
calculate the relative abundance of each size variant.
Non-reducing SDS-PAGE
Samples were denatured in Laemmli sample buffer (4% SDS, Bio-Rad) at 95 C for
10 min.
Samples were run on a Criterion TGX stain-free gel (4-15% polyacrylamide, Bio-
Rad). Protein bands
were visualized by UV illumination or Coommassie blue staining. Gels were
imaged by ChemiDoc MP
Imaging System (Bio-Rad). Quantification of bands was performed using Imagelab
4Ø1 software (Bio-
Rad).
Complement Dependent Cytotoxicity (CDC)
CDC was evaluated as described before in Example 24.
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Example 26. Design and purification of Fc-antigen binding domain construct 4
with CD38 binding
domain
Protein Expression
A construct formed from asymmetrical tandem Fc domains was made as described
below. Fc-
antigen binding domain construct 4 (CD38) each includes two distinct Fc domain
monomer containing
polypeptides (a long Fc chain (SEQ ID NO: 66), and three copies an anti-CD38
Fc chain (SEQ ID NO:
68)) and three copies of an anti-CD38 light chain polypeptide (SEQ ID NO: 49).
The long Fc chain
contains three Fc domain monomers in a tandem series, wherein each Fc domain
monomer has an
E357K charge mutation and 5354C and T366W protuberance-forming mutations (to
promote
heterodimerization). The short Fc chain contains an Fc domain monomer with a
K370D charge mutation
and Y349C, T3665, L368A, and Y407V cavity-forming mutations (to promote
heterodimerization), and
anti-CD38 VH and CH1 domains (EU positions 1-220) at the N-terminus (construct
4 (CD38)). The CD38
light chain can also be expressed fused to the N-terminus of the short Fc
chain as part of an scFv. DNA
sequences are optimized for expression in mammalian cells and cloned into the
pcDNA3.4 mammalian
expression vector. The DNA plasmid constructs are transfected via liposomes
into human embryonic
kidney (HEK) 293 cells. The following amino acid sequences for each construct
in Table 7 are encoded
by three separate plasmids (one plasmid encoding the light chain (anti-CD38),
one plasmid encoding the
long Fc chain and one plasmid encoding the short Fc chain (anti-CD38)):
Table 7. Construct 4 (CD38) sequences
Construct Light chain Long Fe chain Short Fe chain
(with anti-CD38 VH and
CH1)
Construct 4 SEQ ID NO: SEQ ID NO: SEQ ID NO:
S3L
(CD38) EIVLTQSPATLSLSPGERATLS DKTHTCPPCPAPELLGGPSVF
EVQLLESGGGLVQPGGS
CRASQSVSSYLAWYQQKPG LFPPKPKDTLMISRTPEVTCV LRLSCAVSGFTFNSFAMS
WVRQAPGKGLEWVSAIS
QAPRLLIYDASNRATGIPARF VVDVSHEDPEVKFNWYVDG
GSGGGTYYADSVKGRFTI
SGSGSGTDFTLTISSLEPEDFA VEVHNAKTKPREEQYNSTYR SRDNSKNTLYLQMNSLRA
VYYCQQRSNWPPTFGQGTK VVSVLTVLHCIDwi_NGKEYK EDTAVYFCAKDKILWFGE
VEIKRTVAAPSVFIFPPSDEQL CKVSNKALPAPIEKTISKAKG PVFDYWGQGTLVTVSSA
KSGTASVVCLLNNFYPREAK QPREPQVYTLPPCRDKLTKN STKGPSVFPLAPSSKSTS
GGTAALGCLVKDYFPEPV
VQWKVDNALQSGNSQESVT QVSLWCLVKGFYPSDIAVEW
TVSWNSGALTSGVHTFPA
EQDSKDSTYSLSSTLTLSKAD ESNGQPENNYKTTPPVLDSD VLQSSGLYSLSSVVTVPS
YEKHKVYACEVTHQGLSSPV GSFFLYSKLTVDKSRWQQGN SSLGTQTYICNVNHKPSN
TKSFNRGEC VFSCSVMHEALHNHYTQKSL TKVDKRVEPKSCDKTHTC
SLSPGKGGGGGGGGGGGG
PPCPAPELLGGPSVFLFPPKP
GGGGGGGGDKTHTCPPCPA KDTLMISRTPEVTCVVVDVS
PELLGGPSVFLFPPKPKDTLM HEDPEVKFNWYVDGVEVHN
ISRTPEVTCVVVDVSHEDPEV AKTKPREEQYNSTYRVVSVLT
KFNWYVDGVEVHNAKTKPR VLHQDWLNGKEYKCKVSNK
EEQYNSTYRVVSVLTVLHQD ALPAPIEKTISKAKGQPREPQ
WLNGKEYKCKVSNKALPAPI VCTLPPSRDELTKNQVSLSCA
EKTISKAKGQPREPQVYTLPP VDGFYPSDIAVEWESNGQPE
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CRDKLTKNQVSLWCLVKGFY NNYKTTPPVLDSDGSFFLVSK
PSDIAVEWESNGQPENNYKT LTVDKSRWQQGNVFSCSVM
TPPVLDSDGSFFLYSKLTVDK HEALHNHYTQKSLSLSPG
SRWQQGNVFSCSVMHEAL
HNHYTQKSLSLSPGKGGGG
GGGGGGGGGGGGGGGGD
KTHTCPPCPAPELLGGPSVFL
FPPKPKDTLMISRTPEVTCVV
VDVSHEDPEVKFNWYVDGV
EVHNAKTKPREEQYNSTYRV
VSVLTVLHQDWLNGKEYKCK
VSNKALPAPIEKTISKAKGQP
REPQVYTLPPCRDKLTKNQV
SLWCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLDSDGS
FFLYSKLTVDKSRWQQGNVF
SCSVMHEALHNHYTQKSLSL
SPG
The expressed proteins are purified from the cell culture supernatant by
Protein A-based affinity
column chromatography, using a Poros MabCapture A (Life Technologies) column.
Captured Fc-antigen
binding domain constructs are washed with phosphate buffered saline (low-salt
wash) and eluted with
100mM glycine, pH 3. The eluate was quickly neutralized by the addition of 1 M
TRIS pH 7.4 and sterile
filtered through a 0.2 pm filter. The proteins are further fractionated by ion
exchange chromatography
using Poros XS resin (Applied Biosciences). The column was pre-equilibrated
with 50 mM MES, pH 6
(buffer A), and the sample was eluted with a step gradient using 50 mM MES,
400 mM sodium chloride,
pH 6 (buffer B) as the elution buffer. After ion exchange, the target fraction
was buffer exchanged into
PBS buffer using a 10 kDa cut-off polyether sulfone (PES) membrane cartridge
on a tangential flow
filtration system. The samples are concentrated to approximately 30 mg/mL and
sterile filtered through a
0.2 pm filter.
Example 27. Design and purification of Fc-antigen binding domain construct 8
with a CD38
binding domain
Protein Expression
A construct formed from a singly branched Fc domain where the branch point is
at the N-terminal
Fc domain was made as described below. Fc-antigen binding domain construct 8
(CD38) each include
two distinct Fc domain monomer containing polypeptides (two copies of a long
Fc chain (SEQ ID NO: 69),
and two copies of an anti-CD38 short Fc chain (SEQ ID NO: 68)) and copies of
an anti-CD38 light chain
polypeptide (SEQ ID NO: 49). The long Fc chain contains an Fc domain monomer
with an E357K charge
mutation and 5354C and T366W protuberance-forming mutations (to promote
heterodimerization) in a
tandem series with an Fc domain monomer with reverse charge mutations K409D
and D399K (to
promote homodimerization). The short Fc chain contains an Fc domain monomer
with a K370D charge
mutation and Y349C, T3665, L368A, and Y407V cavity-forming mutations (to
promote
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heterodimerization), and anti-CD38 VH and CH1 domains (EU positions 1-220) at
the N-terminus
(construct 8 (CD38)). The CD38 light chain can also be expressed fused to the
N-terminus of the short
Fc chain as part of an scFv. DNA sequences are optimized for expression in
mammalian cells and
cloned into the pcDNA3.4 mammalian expression vector. The DNA plasmid
constructs are transfected
via liposomes into human embryonic kidney (HEK) 293 cells. The following amino
acid sequences for
each construct in Table 8 are encoded by three separate plasmids (one plasmid
encoding the light chain
(anti-CD38), one plasmid encoding the long Fc chain and one plasmid encoding
the short Fc chain (anti-
CD38)):
Table 8. Construct 8 (CD38) sequences
Construct Light chain Long Fc chain Short Fc chain
(with anti-CD38 VH and
CH1)
Construct 8 SEQ ID NO: SEQ ID NO: SEQ ID NO:
(CD38)
EIVLTQSPATLSLSPG ERATLS DKTHTCPPCPAPELLGGPSVF EVQLLESGGGLVQPGGS
CRASQSVSSYLAWYQQKPG LFPPKPKDTLM ISRTPEVTCV LRLSCAVSGFTFNSFAMS
WVRQAPGKGLEWVSAIS
QAPRLLIYDASNRATG I PARF VVDVSH E DP EVKF NWYVDG GSGGGTYYADSVKGRFTI
SGSGSGTDFTLTISSLEPEDFA VEVHNAKTKPREEQYNSTYR SRDNSKNTLYLQMNSLRA
VYYCQQRSNWPPTFGQGTK VVSVLTVLHQDWLNGKEYK EDTAVYFCAKDKILWFGE
VEI KRTVAAPSVF I FPPSDEQL CKVSN KALPAP I EKTISKAKG PVFDYWGQGTLVTVSSA
KSGTASVVCLLNNFYPREAK QPREPQVYTLPPSRDELTKN STKGPSVFPLAPSSKSTS
GGTAALGCLVKDYFPEPV
VQWKVDNALQSGNSQESVT QVSLTCLVKGFYPSDIAVEW
TVSWNSGALTSGVHTFPA
EQDSKDSTYSLSSTLTLSKAD ESNGQP EN NYKTTPPVLKSD VLQSSGLYSLSSVVTVPS
YEKHKVYACEVTHQGLSSPV GSFFLYSDLTVDKSRWQQG SSLGTQTYICNVNHKPSN
TKSF NRG EC NVFSCSVM HEALHNHYTQK TKVDKRVEPKSCDKTHTC
SLSLSPG KGGGGGGGGGGG PPCPAPELLGGPSVF LFPPKP
GGGGGGGGG DKTHTCP PCP KDTLM ISRTPEVTCVVVDVS
APE LLGG PSVF LF P PKPKDTL HEDPEVKFNWYVDGVEVHN
M ISRTPEVTCVVVDVSH EDP AKTKPREEQYNSTYRVVSVLT
EVKFNWYVDGVEVHNAKTK VLHQDW LNG KEYKCKVSNK
PREEQYNSTYRVVSVLTVLH ALPAP I
EKTISKAKGQPREPQ
QDW LNG KEYKCKVSN KALP VCTLPPSRDELTKNQVSLSCA
API EKTISKAKGQPREPQVYT VDGFYPSDIAVEWESNGQPE
LP PCRDKLTKNQVSLWCLVK NNYKTTPPVLDSDGSFFLVSK
GFYPSDIAVEWESNGQPEN LTVDKSRWQQGNVFSCSVM
NYKTTPPVLDSDGSFFLYSKL HEALHNHYTQKSLSLSPG
TVDKSRWQQGNVFSCSVM
HEALHNHYTQKSLSLSPG
The expressed proteins are purified from the cell culture supernatant by
Protein A-based affinity
column chromatography, using a Poros MabCapture A (LifeTechnologies) column.
Captured Fc-antigen
binding domain constructs are washed with phosphate buffered saline (low-salt
wash) and eluted with
100mM glycine, pH 3. The eluate is quickly neutralized by the addition of 1 M
TRIS pH 7.4 and sterile
filtered through a 0.2 pm filter. The proteins are further fractionated by ion
exchange chromatography
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using Poros XS resin (Applied Biosciences). The column is pre-equilibrated
with 50 mM MES, pH 6
(buffer A), and the sample is eluted with a step gradient using 50 mM MES, 400
mM sodium chloride, pH
6 (buffer B) as the elution buffer. After ion exchange, the target fraction is
buffer exchanged into PBS
buffer using a 10 kDa cut-off polyether sulfone (PES) membrane cartridge on a
tangential flow filtration
system. The samples are concentrated to approximately 30 mg/mL and sterile
filtered through a 0.2 pm
filter.
Example 28. Design and purification of Fc-antigen binding domain construct 9
with CD38 binding
domain
Protein Expression
A construct formed from a singly branched Fc domain where the branch point is
at the N-terminal
Fc domain was made as described below. Fc-antigen binding domain construct 9
(CD38) include two
distinct Fc domain monomer containing polypeptides (two copies an anti-CD38
long Fc chain (SEQ ID
NO: 54), and two copies of an anti-CD38 short Fc chain (SEQ ID NO: 68)) and
copies of an anti-CD38
light chain polypeptide (SEQ ID NO: 49). The long Fc chain contains an Fc
domain monomer with an
E357K charge mutation and 5354C and T366W protuberance-forming mutations (to
promote
heterodimerization) in a tandem series with an Fc domain monomer with reverse
charge mutations
K409D and D399K (to promote homodimerization), and anti-CD38 VH and CH1
domains (EU positions 1-
220) at the N-terminus (construct 9 (CD38)). The short Fc chain contains an Fc
domain monomer with a
K370D charge mutation and Y349C, T3665, L368A, and Y407V cavity-forming
mutations (to promote
heterodimerization), and an anti-CD38 heavy chain at the N-terminus (construct
9 (CD38)). The CD38
light chain can also be expressed fused to the N-terminus of the long Fc chain
and/or short Fc chain as
part of an scFv. DNA sequences were optimized for expression in mammalian
cells and cloned into the
pcDNA3.4 mammalian expression vector. The DNA plasmid constructs were
transfected via liposomes
into human embryonic kidney (HEK) 293 cells. The following amino acid
sequences for each construct in
Table 9 were encoded by three separate plasmids (one plasmid encoding the
light chain (anti-CD38), one
plasmid encoding the long Fc chain (anti-CD38) and one plasmid encoding the
short Fc chain (anti-
CD38)):
Table 9. Construct 9 (CD38) sequences
Construct Light chain Long Fc chain Short Fc chain
(with anti-CD38 VH and (with anti-CD38 VH
and
CH1) CH1)
Construct 9 SEQ ID NO: SEQ ID NO: SEQ ID NO:
S3A
(CD38) EIVLTQSPATLSLSPGERATLS EVQLLESGGGLVQPGGSL
EVQLLESGGGLVQPGGSLRL
CRASQSVSSYLAWYQQKPG RLSCAVSGFTFNSFAMSW SCAVSGFTFNSFAMSWVRQ
QAPRLLIYDASNRATGIPARF VRQAPGKGLEWVSAISGS APGKGLEWVSAISGSGGGTY
SGSGSGTDFTLTISSLEPEDFA GGGTYYADSVKGRFTISR YADSVKGRFTISRDNSKNTLY
VYYCQQRSNWPPTFGQGTK
LQMNSLRAEDTAVYFCAKDK
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VEI KRTVAAPSVF I F P PSDEQL DNSKNTLYLQM NSLRAED I LWFG E PVF DYWGQGTLVT
KSGTASVVCLLNNFYPREAK TAVYFCAKDKILWFGEPVF VSSAST KG PSVF P LA PSS KSTS
VQWKVDNALQSGNSQESVT DYWGQGTLVTVSSASTKG GGTAALGCLVKDYF PE PVTV
EQDSKDSTYS LSSTLTLSKAD PSVFPLAPSSKSTSGGTAA SWNSGALTSGVHTFPAVLQS
YEKHKVYACEVTHQGLSSPV SG LYS LSSVVTV PSSS LG TQTY
LGCLVKDYFPEPVTVSWN
TKSF NRG EC ICNVN H KPSNTKVDKRVE
PK
SGALTSGVHTFPAVLQSS SCDKTHTCPPCPAPELLGG PS
G LYS LSSVVTV PSSS LGTQ VFLFPPKPKDTLMISRTPEVT
TYICNVNH KPSNTKVDKR CVVVDVSH E DP EVKF NWYV
VEPKSCDKTHTCPPCPAPE DGVEVH NAKTKP RE EQYNST
LLGGPSVFLFPPKPKDTLM YRVVSVLTVLHQDW LNG KE
ISRTPEVTCVVVDVSH EDP YKCKVSN KALPAP I E KTISKAK
EVKFNWYVDGVEVHNAK GQP RE PQVCTLPPSRDE LTK
TKPREEQYNSTYRVVSVLT NQVSLSCAVDGFYPSDIAVE
VLHQDWLNGKEYKCKVS WESNGQPENNYKTTPPVLD
SDGSFFLVSKLTVDKSRWQQ
N KAL PAP I E KTISKAKGQP
GNVFSCWMHEALHNHYTQ
RE PQVYTLP PSRDE LTKN KS LSLSPG
QVS LTCLVKG FYPS D !AVE
WESNGQP EN NYKTTPPV
LKS DGS F F LYS D LTVD KS R
WQQGNVFSCSVM H [AL
HN HYTQKSLSLSPGKGGG
GGGGGGGGGGGGGGG
GGDKTHTCPPCPAPELLG
GPSVFLFPPKPKDTLM IS R
TPEVTCVVVDVSH EDP EV
KFNWYVDGVEVH NAKTK
PRE EQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSN K
ALPAP I E KTIS KAKGQP REP
QVYTLP PC RDK LTKN QVS
LWCLVKGFYPSDIAVEWE
SNGQP EN NYKTTPPVLDS
DGSFFLYSKLTVDKSRWQ
QGNVFSCSVM HEALH NH
YTQKS LS LS PG
Example 29. Design and purification of Fc-antigen binding domain construct 10
with CD38
binding domain
Protein Expression
A construct formed from a singly branched Fc domain where the branch point is
at the N-terminal
Fc domain is made as described below. Fc-antigen binding domain construct 10
(CD38) each include
two distinct Fc domain monomer containing polypeptides (two copies of an anti-
CD38 long fc chain (SEQ
ID NO: 71), and four copies of a short Fc chain (SEQ ID NO: 63)) and copies of
an anti-CD38 light chain
polypeptide (SEQ ID NO: 49), respectively. The long Fc chain contains two Fc
domain monomers in a
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tandem series, wherein each Fc domain monomer has an E357K charge mutation and
S354C and
T366W protuberance-forming mutations (to promote heterodimerization), in
tandem series with an Fc
domain monomer with reverse charge mutations K409D and D399K (to promote
homodimerization), and
anti-CD38 VH and CH1 domains (EU positions 1-220) at the N-terminus (construct
10 (CD38)). The short
Fc chain contains an Fc domain monomer with a K370D charge mutation and Y349C,
T366S, L368A, and
Y407V cavity-forming mutations (to promote heterodimerization). The anti- CD38
light chain can also be
expressed fused to the N-terminus of the long Fc chain as part of an scFv. DNA
sequences were
optimized for expression in mammalian cells and cloned into the pcDNA3.4
mammalian expression
vector. The DNA plasmid constructs were transfected via liposomes into human
embryonic kidney (HEK)
293 cells. The following amino acid sequences for each construct in Table 10
were encoded by three
separate plasmids (one plasmid encoding the light chain (anti-CD38), one
plasmid encoding the long Fc
chain (anti-CD38) and one plasmid encoding the short Fc chain:
Table 10. Construct 10 (CD38) sequences
Construct Light chain Long Fc chain Short Fc chain
(anti-CD38 VH and CH1)
Construct 10 SEQ ID NO: SEQ ID NO: 71 SEQ ID NO: 63
(CD38)
EIVLTQSPATLSLSPG ERATLS EVQLLESGGGLVCIPGGSLRL DKTHTCPPCPAPELLGGPS
CRASQSVSSYLAWYQQKPG SCAVSGFTFNSFAMSWVRQ VFLFPPKPKDTLMISRTPE
QAPRLLIYDASNRATG I PARF APG KG LEWVSAISGSGGGTY VTCVVVDVSHEDPEVKFN
SGSGSGTDFTLTISSLEPEDFA YADSVKGRFTISRDNSKNTLY WYVDGVEVHNAKTKPRE
VYYCQQRSNWPPTFGQGTK LQM NSLRAEDTAVYFCAKDK
EQYNSTYRVVSVLTVLHQ
VEIKRTVAAPSVFIFPPSDEQL I LW FG EPVF DYWGQGTLVT
KSGTASVVCLLNNFYPREAK VSSASTKGPSVFPLAPSSKSTS DWLNGKEYKCKVSNKALP
VQWKVDNALQSGNSQESVT GGTAALGCLVKDYF PVTV APIEKTISKAKGQPREPQV
EQDSKDSTYSLSSTLTLSKAD SWNSGALTSGVHTFPAVLQS CTLPPSRDELTKNQVSLSC
YE KH KVYACEVTHQG LSSPV SG LYSLSSVVTVPSSS LGTQTY AVDGFYPSDIAVEWESNG
TKSF NRG EC ICNVNHKPSNTKVDKRVEPK QPENNYKTTPPVLDSDGS
SCDKTHTCPPCPAPELLGG FFLVSKLTVDKSRWQQGN
PSVFLFPPKPKDTLMISRT VFSCSVMHEALHNHYTQ
PEVTCVVVDVSHEDPEVK KSLSLSPG
FNWYVDGVEVHNAKTKP
REEQYNSTYRVVSVLTVLH
QDWLNGKEYKCKVSNKA
LPAPIEKTISKAKGQPREP
QVYTLPPSRDELTKNQVSL
TCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLKSD
GSFFLYSDLTVDKSRWQQ
GNVFSCSVMHEALHNHY
TQKSLSLSPGKGGGGGGG
GGGGGGGGGGGGGDKT
HTCPPCPAPELLGGPSVFL
FPPKPKDTLMISRTPEVTC
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VVVDVSHEDPEVKFNWY
VDGVEVHNAKTKPREEQY
NSTYRVVSVLTVLHQDWL
NGKEYKCKVSNKALPAPIE
KTISKAKGQPREPQVYTLP
PCRDKLTKNQVSLWCLVK
GFYPSDIAVEWESNGQPE
NNYKTTPPVLDSDGSFFLY
SKLTVDKSRWQQGNVFS
CSVMHEALHNHYTQKSLS
LSPGKGGGGGGGGGGG
GGGGGGGGGDKTHTCPP
CPAPELLGGPSVFLFPPKP
KDTLMISRTPEVTCVVVD
VSHEDPEVKFNWYVDGV
EVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTIS
KAKGQPREPQVYTLPPCR
DKLTKNQVSLWCLVKGFY
PSDIAVEWESNGQPENNY
KTTPPVLDSDGSFFLYSKLT
VDKSRWQQGNVFSCSV
MHEALHNHYTQKSLSLSP
The expressed proteins were purified from the cell culture supernatant by
Protein A-based affinity
column chromatography, using a Poros MabCapture A (LifeTechnologies) column.
Captured Fc-antigen
binding domain constructs were washed with phosphate buffered saline (low-salt
wash) and eluted with
100mM glycine, pH 3. The eluate is quickly neutralized by the addition of 1 M
TRIS pH 7.4 and sterile
filtered through a 0.2 pm filter. The proteins are further fractionated by ion
exchange chromatography
using Poros XS resin (Applied Biosciences). The column is pre-equilibrated
with 50 mM MES, pH 6
(buffer A), and the sample is eluted with a step gradient using 50 mM MES, 400
mM sodium chloride, pH
6 (buffer B) as the elution buffer. After ion exchange, the target fraction is
buffer exchanged into PBS
.. buffer using a 10 kDa cut-off polyether sulfone (PES) membrane cartridge on
a tangential flow filtration
system. The samples are concentrated to approximately 30 mg/mL and sterile
filtered through a 0.2 pm
filter.
Example 30. Design and purification of Fc-antigen binding domain construct 16
with a CD38
.. binding domain
Protein Expression
A construct formed from a singly branched Fc domain where the branch point is
at the C-terminal
Fc domain is made as described below. Fc-antigen binding domain construct 16
(CD38) each includes
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two distinct Fc domain monomer containing polypeptides (two copies of an anti-
CD38 long Fc chain (SEQ
ID NO: 73), and four copies of a short Fc chain (SEQ ID NO: 63)) and three
copies of an anti-CD38 light
chain polypeptide (SEQ ID NO: 49), respectively. The long Fc chain contains an
Fc domain monomer
with reverse charge mutations K409D and D399K (to promote homodimerization) in
a tandem series with
two Fc domain monomers, in tandem, that each have an E357K charge mutation and
5354C and T366W
protuberance-forming mutations (to promote heterodimerization), and anti-CD38
VH and CH1 domains
(EU positions 1-220) at the N-terminus (construct 10 (CD38)). The short Fc
chain contains an Fc domain
monomer with a K370D charge mutation and Y349C, T3665, L368A, and Y407V cavity-
forming mutations
(to promote heterodimerization). The anti- CD38 light chain can also be
expressed fused to the N-
terminus of the long Fc chain as part of an scFv. DNA sequences are optimized
for expression in
mammalian cells and cloned into the pcDNA3.4 mammalian expression vector. The
DNA plasmid
constructs are transfected via liposomes into human embryonic kidney (HEK) 293
cells. The following
amino acid sequences for each construct in Table 11 are encoded by three
separate plasmids (one
plasmid encoding the light chain (anti-CD38), one plasmid encoding the long Fc
chain (anti-CD38) and
one plasmid encoding the short Fc chain:
Table 11. Construct 16 (CD38) sequences
Construct Light chain Long Fc chain Short Fc chain
(with anti-CD38 VH and
CH1)
Construct 16 SEQ ID NO: SEQ ID NO: 73 SEQ ID NO: 63
S5Y
(CD38) EIVLTQSPATLSLSPG ERATLS EVQLLESGGGLVCIPGGSLRL
DKTHTCPPCPAPELLGGPS
CRASQSVSSYLAWYQQKPG SCAVSGFTFNSFAMSWVRQ VFLFPPKPKDTLMISRTPE
QAPRLLIYDASNRATG I PARF APG KG LEWVSAISGSGGGTY VTCVVVDVSHEDPEVKFN
SGSGSGTDFTLTISSLEPEDFA YADSVKGRFTISRDNSKNTLY WYVDGVEVHNAKTKPRE
VYYCQQRSNWPPTFGQGTK LQM NSLRAEDTAVYFCAKDK
EQYNSTYRVVSVLTVLHQ
VEIKRTVAAPSVFIFPPSDEQL I LW FG EPVF DYWGQGTLVT
KSGTASVVCLLNNFYPREAK VSSASTKGPSVFPLAPSSKSTS DWLNGKEYKCKVSNKALP
VQWKVDNALQSGNSQESVT GGTAALGCLVKDYF PVTV APIEKTISKAKGQPREPQV
EQDSKDSTYSLSSTLTLSKAD SWNSGALTSGVHTFPAVLQS CTLPPSRDELTKNQVSLSC
YE KH KVYACEVTHQG LSSPV SG LYSLSSVVTVPSSS LGTQTY AVDGFYPSDIAVEWESNG
TKSF NRG EC ICNVNHKPSNTKVDKRVEPK QPENNYKTTPPVLDSDGS
SCDKTHTCPPCPAPELLGG FFLVSKLTVDKSRWQQGN
PSVFLFPPKPKDTLMISRT VFSCSVMHEALHNHYTQ
PEVTCVVVDVSHEDPEVK KSLSLSPG
FNWYVDGVEVHNAKTKP
REEQYNSTYRVVSVLTVLH
QDWLNGKEYKCKVSNKA
LPAPIEKTISKAKGQPREP
QVYTLPPCRDKLTKNQVS
LWCLVKGFYPSDIAVEWE
SNGQPENNYKTTPPVLDS
DGSFFLYSKLTVDKSRWQ
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QGNVFSCSVM HEALH NH
YTQKSLSLSPGKGGGGGG
GGGGGGGGGGGGGGDK
THTCP PC PAP E LLGG PS VF
LFPPKPKDTLM ISRTPEVT
CVVVDVSHEDPEVKFNW
YVDGVEVHNAKTKPREEQ
YNSTYRVVSVLTVLHQDW
LNGKEYKCKVSNKALPAPI
EKTISKAKGQPREPQVYTL
PPCRDKLTKNQVSLWCLV
KG FYPSDIAVEWESNGQP
EN NYKTTPPVLDS DGSFFL
YSKLTVDKSRWQQGNVF
SCSVMHEALHNHYTQKSL
SLSPGKGGGGGGGGGGG
GGGGGGGGGDKTHTCPP
CPAPELLGGPSVFLFPPKP
KDTLMISRTPEVTCVVVD
VSHEDPEVKFNWYVDGV
EVHNAKTKPREEQYNSTY
RVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTIS
KAKGQPREPQVYTLPPSR
DE LTKN QVS LTC LVKG FYP
SDIAVEWES N GQP EN NYK
TTP PVLKS DGS FF LYS D LTV
DKSRWQQGNVFSCSVM
HEALHNHYTQKSLSLSPG
The expressed proteins are purified from the cell culture supernatant by
Protein A-based affinity
column chromatography, using a Poros MabCapture A (LifeTechnologies) column.
Captured Fc-antigen
binding domain constructs are washed with phosphate buffered saline (low-salt
wash) and eluted with
100mM glycine, pH 3. The eluate is quickly neutralized by the addition of 1 M
TRIS pH 7.4 and sterile
filtered through a 0.2 pm filter. The proteins are further fractionated by ion
exchange chromatography
using Poros XS resin (Applied Biosciences). The column is pre-equilibrated
with 50 mM MES, pH 6
(buffer A), and the sample is eluted with a step gradient using 50 mM MES, 400
mM sodium chloride, pH
6 (buffer B) as the elution buffer. After ion exchange, the target fraction is
buffer exchanged into PBS
buffer using a 10 kDa cut-off polyether sulfone (PES) membrane cartridge on a
tangential flow filtration
system. The samples are concentrated to approximately 30 mg/mL and sterile
filtered through a 0.2 pm
filter.
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Example 31. Design and purification of Fc-antigen binding domain construct 19
with a CD38
binding domain
Protein Expression
A construct formed from a singly branched Fc domain where the branch point is
at neither the N-
terminal or C-terminal Fc domain was made as described below. Fc-antigen
binding domain construct 19
(CD38) includes two distinct Fc domain monomer containing polypeptides (two
copies of an anti-CD38
long Fc chain (SEQ ID NO: 75), and four copies of a short Fc chain (SEQ ID NO:
63)) and copies of an
anti-CD38 light chain polypeptide (SEQ ID NO: 49), respectively. The long Fc
chain contains an Fc
domain monomer with an E357K charge mutation and 5354C and T366W protuberance-
forming
mutations (to promote heterodimerization), in a tandem series with an Fc
domain monomer with reverse
charge mutations K409D and D399K (to promote homodimerization), in a tandem
series with an Fc
domain monomer with an E357K charge mutation and 5354C and T366W protuberance-
forming
mutations (to promote heterodimerization), and anti-CD38 VH and CH1 domains
(EU positions 1-220) at
the N-terminus (construct 19 (CD38)) . The short Fc chain contains an Fc
domain monomer with a K370D
charge mutation and Y349C, T3665, L368A, and Y407V cavity-forming mutations
(to promote
heterodimerization). The anti-CD38 light chain can also be expressed fused to
the N-terminus of the long
Fc chain as part of an scFv. DNA sequences were optimized for expression in
mammalian cells and
cloned into the pcDNA3.4 mammalian expression vector. The DNA plasmid
constructs were transfected
via liposomes into human embryonic kidney (HEK) 293 cells. The following amino
acid sequences for
each construct in Table 12 were encoded by three separate plasmids (one
plasmid encoding the light
chain (anti-CD38), one plasmid encoding the long Fc chain (anti-CD38) and one
plasmid encoding the
short Fc chain:
Table 12. Construct 19 (CD38) sequences
Construct Light chain Long Fc chain Short Fc chain
(with anti-CD38 VH and
CH1)
Construct 19 SEQ ID NO: SEQ ID NO: SEQ ID NO:
S5X
(CD38) EIVLTQSPATLSLSPG ERATLS EVQLLESGGGLVQPGGSLRL
DKTHTCPPCPAPELLGGPSVF
CRASQSVSSYLAWYQQKPG SCAVSGFTFNSFAMSWVRQ LFPPKPKDTLMISRTPEVTCV
QAPRLLIYDASNRATG I PARF APG KG LEWVSAISGSGGGTY VVDVSHEDPEVKF NWYVDG
SGSGSGTDFTLTISSLEPEDFA YADSVKGRFTISRDNSKNTLY VEVHNAKTKPREEQYNSTYR
VYYCQQRSNWPPTFGQGTK LQM NSLRAEDTAVYFCAKDK VVSVLTVLHQDWLNGKEYK
VEI KRTVAAPSVF I FPPSDEQL I LW FG EPVF DYWGQGTLVT CKVSN KALPAP I E KTISKAKG
KSGTASVVCLLNNFYPREAK VSSASTKGPSVFPLAPSSKSTS QPREPQVCTLPPSRDELTKN
VQWKVDNALQSGNSQESVT GGTAALG CLVKDYF PE PVTV QVSLSCAVDGFYPSDIAVEW
EQDSKDSTYSLSSTLTLSKAD SWNSGALTSGVHTFPAVLQS ESNGQPENNYKTTPPVLDSD
YEKHKVYACEVTHQGLSSPV SG LYSLSSVVTVPSSS LGTQTY GSFF LVSKLTVDKSRWQQG
TKSF NRG EC ICNVNHKPSNTKVDKRVEPK NVFSCSVMHEALHNHYTQK
SCDKTHTCPPCPAPE LLGG PS SLSLSPG
VFLFPPKPKDTLM ISRTPEVT
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CVVVDVSHEDPEVKFNWYV
DGVEVHNAKTKPREEQYNST
YRVVSVLTVLHQDWLNGKE
YKCKVSNKALPAPIEKTISKAK
GQPREPQVYTLPPCRDKLTK
NQVSLWCLVKGFYPSDIAVE
WESNGQPENNYKTTPPVLD
SDGSFFLYSKLTVDKSRWQQ
GNVFSCSVMHEALHNHYTQ
KSLSLSPGKGGGGGGGGGG
GGGGGGGGGGDKTHTCPP
CPAPELLGGPSVFLFPPKPKD
TLMISRTPEVTCVVVDVSHE
DPEVKFNWYVDGVEVHNAK
TKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKAL
PAPIEKTISKAKGQPREPQVY
TLPPSRDELTKNQVSLTCLVK
GFYPSDIAVEWESNGQPEN
NYKTTPPVLKSDGSFFLYSDL
TVDKSRWQQGNVFSCSVM
HEALHNHYTQKSLSLSPGKG
GGGGGGGGGGGGGGGGG
GGDKTHTCPPCPAPELLGGP
SVFLFPPKPKDTLMISRTPEV
TCVVVDVSHEDPEVKFNWY
VDGVEVHNAKTKPREEQYN
STYRVVSVLTVLHQDWLNGK
EYKCKVSNKALPAPIEKTISKA
KGQPREPQVYTLPPCRDKLT
KNQVSLWCLVKGFYPSDIAV
EWESNGQPENNYKTTPPVL
DSDGSFFLYSKLTVDKSRWQ
QGNVFSCSVMHEALHNHYT
QKSLSLSPG
Example 32. Binding of anti-CD38 constructs to human tumor cell lines and
stable cell lines
expressing human and cynomolgus monkey CD38
Tumor cell suspension in media containing 10% FBS was incubated with
increasing
concentrations of VivoTag645-labeled anti-CD38 antibody at 4 C for 1 hour.
Cells were then washed in
cold buffer and suspended in FACS buffer. Labeled cell suspensions were then
read on APC channel on
BD FACS Verse flow cytometer. Live cell population were gated using unlabeled
cells. Geometric mean
fluorescence intensity (gMFI) values were calculated from the gated population
using FlowJo software.
The results of this analysis are presented in FIG. 25.
Raji cells were used to evaluate dose-dependent relative binding of parental
IgG1 anti-CD38
antibody and the corresponding anti-CD38 constructs. Since the anti-CD38 mAb
(that was the source of
the Fabs for the various anti-CD38 Fc constructs) does not cross react with
monkey CD38, we generated
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a surrogate anti-CD38 human monoclonal IgG1 antibody that reacts with the
cynomolgus monkey CD38
(S1A-AA-Cyno CD38) and a surrogate anti-CD38 construct 13 using the same Fab
sequences, that
reacts with cynomolgus monkey CD38 (S3Y-AA-Cyno CD38); this was used for
evaluating CDC activity in
the presence of cynomolgus monkey serum complement and pharmacodynamic
response of targeting
endogenous cynomolgus monkey CD38 in non-human primate whole blood. The
results of these binding
studies are presented in FIG. 26.
Example 33. CDC activity of anti-CD38 constructs
The ability of anti-CD38 antibodies and anti-CD38 Fc constructs to promote
cell killing of a CD38-
expressing tumor cell lines (Daudi and Raji), was assessed by an in vitro CDC
assay. Human serum
complement was used as the complement source. RPMI-1640 media containing 0.1%
BSA was used as
a buffer for preparing cell suspensions, antibody, and serum dilutions. CD38
positive tumor cells were first
washed in buffer and resuspended at a density of 106 cells/ml. In a typical
assay, 50 pl of antibody or anti-
CD38 Fc construct, 50 pl of diluted complement (5X dilution), and 50 pl of a
cell suspension (50,000
.. cells/well) were added to a flat-bottom tissue culture 96-well plate. The
mixture was then incubated for 2
hours at 37 C in a 5% CO2 incubator to facilitate complement-mediated cell
lysis. Then, 50 pl of Alamar
Blue was added to each well and incubated for 18 hours at 37 C. Fluorescence
was read using a 96-well
fluorometer with excitation at 530 nm and emission at 590 nm.
The assay was performed with Daudi cells and Raji cells in the presence of
human or cyno serum
.. complement to evaluate relative CDC mediated tumor cell lysis induced by
either anti-CD38 mAb or anti-
CD38 constructs. The result, presented in Table 13, are expressed in relative
fluorescence units (RFU)
that are proportional to the number of viable cells. The activity of the
various mutants was examined by
plotting the percent CDC activity against the log of Ab concentration (final
concentration before the
addition of Alamar Blue). The percent CDC activity was calculated as follows:
`)/0 CDC activity = (RFU test
¨ RFU background) x 100 (RFU at total cell lysis ¨ RFU background). Values
represent mean SD from
a representative experiment (from n = 3 separate experiments). This study
demonstrates that the anti-
CD38 constructs exhibit greater efficacy (maximum tumor cell killing) and
potency than anti-CD38 mAb in
in anti-CD38 mAb-CDC sensitive cells (Daudi) as well as in anti-CD38 mAb-CDC
resistant cells (Raji).
Anti-CD38 mAb-sensitive or -resistant term refers to sensitivity or resistance
towards anti-CD38 mAb
mediated target cell lysis in cell based CDC assays.
Table 13: CDC activity of CD38 constructs in Daudi cell and Raji Cells
Daudi cells Raji cells
Treatment Max EC50 EC50, fold- Max % Max % lysis, fold-
change
nM change vs. anti- lysis vs. anti-CD38
mAb
lysis CD38 mAb
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anti-CD38 mAb 100 0.7749 1.00 14.7 1.00
Construct 13 100 0.0725 10.685 92.1 6.28
S3Y-AA-CD38
Construct 7 100 0.1960 3.954 15.5 1.06
S31-AA-CD38
Construct 9 100 0.0372 20.831 39.9 2.72
S3A-AA-CD38
Construct 19 100 0.0993 7.801 83.3 5.67
S5X-AA-CD38
1A11 constructs included G20 linkers unless otherwise noted.
A Cynomolgus monkey CD38 cross-reactive anti-CD38 construct 13 (S3Y-AA-Cyno
CD38)
showed significantly high potency and efficacy in inducing CDC in both
sensitive and resistant tumor cells
than the corresponding mAb (S1A-AA-Cyno (anti-Cyno CD38 mAb). This assay was
performed in a
similar fashion as described above, but using Daudi tumor cells and monkey
serum complement (FIG. 27,
panel A), Raji tumor cells and monkey serum complement (FIG. 27, panel B),
Daudi tumor cells and
human serum complement (FIG. 27, panel C), Raji tumor cells and human serum
complement (FIG. 27,
panel D). The CDC activities of these constructs are presented in Table 14
show significant enhancement
in efficacy and potency of S3Y-AA-Cyno CD38 over S1A-AA-Cyno (anti-Cyno CD38
mAb) in inducing
CDC against Daudi and Raji cells.
Table 14: CDC activity of cynomolgus monkey CD38 reactive constructs in Daudi
cell and Raji
Cells
Daudi cells Raji cells
Treatment Max % EC50 EC50, fold- Max % Max % lysis,
fold-
(in the presence of lysis nM change vs. S1A- lysis change vs.
S1A-
cyno serum AA-Cyno AA-Cyno
complement)
S1A-AA-Cyno 78.62 0.9593 12.76
Construct 13 97.84 0.1486 6.46 92.78 7.27
S3Y-AA-Cyno
Treatment Max % EC50 EC50, fold- Max % Max % lysis,
fold-
(in the presence of lysis nM change vs. S1A- lysis change vs.
S1A-
human serum AA-Cyno AA-Cyno
complement)
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S1A-AA-Cyno 67.96 0.6301 12.7
Construct 13 102.4 0.1547 4.07 100.6 7.92
S3Y-AA-Cyno
Example 34. Antibody-Dependent Cellular Phagocytosis (ADCP) activation by anti-
CD38 Fc
constructs
Monocytes were isolated from human whole blood and allowed to differentiate
into macrophages
by treating them with human M-CSF and IL-10 in a 6-well plate. These adherent
macrophages were then
detached using chilled PBS + 2 mM EDTA for subsequent seeding into assay
wells. 2 x 105 macrophages
were seeded in a 96 well flat bottom plate in RPMI-1640 media containing 2%
ultra-low FBS. Plates were
briefly centrifuged and incubated for 1 hour at 37 C to adhere macrophages to
the bottom of the 96-well
plate. Raji tumor cells were stained with Calcein-AM followed by addition on
macrophage containing plate
at an effector (macrophages): target (tumor cells) ratio of 3:1 in the
presence of serial dilutions of anti-
CD38 mAb or various anti-CD38 constructs. Plates were then incubated for 2
hours at 37 C in a CO2
incubator. Supernatants were collected in a V-bottom 96 well plate. Adherent
cells were collected by
detachment with chilled PBS containing 2 mM EDTA. Cells from supernatants and
detached adherent
cells were pooled together. These cells were then stained with anti-CD11 b APC
and -CD19 BV421
antibodies by incubating with these antibodies for 1 hour at 4 C. The labeled
cell suspensions were read
on FACS Verse flow cytometer. Double positive macrophages (CD11b+/Calcein-AM+)
that are negative
for surface CD19 staining were considered as phagocytic events. The results
are in Table 15 show
superior potencies of anti-CD38 constructs in inducing phagocytosis of
opsonized Raji cells by primary
human macrophages.
Table 15. Potency of anti-CD38 Fc constructs in an ADCP assay
Construct EC50 (nM)
Numberl Range Mean SD
anti-CD38 mAb 2 0.14-0.08 0.11 0.045
S3Y-AA-CD38 2 0.1-0.03 0.069 0.05
Construct 13
S3A-AA-CD38 2 0.034-0.028 0.031 0.004
Construct 9
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Construct EC50 (nM)
Numberl Range Mean SD
S5X-AA-CD38 2 0.02-0.04 0.03 0.011
Construct 19
1A11 constructs included G20 linkers unless otherwise noted.
Example 35. Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) activation by
anti-CD38 Fc
constructs
Raji cells were suspended in RPM! media containing 10% ultralow IgG FBS at
concentration of
5000 cells/50 pL media/well in a 96 well plate. Samples were then incubated
for 15 minutes at 25 C in
with increasing concentrations of antibodies and constructs (10 uL/well).
Primary human NK cells (effector
cells) were added in effector to target ratio of 5:1. Effector and target
cells mix were then incubated for 5
hours at 37 C in a 5% CO2 incubator. CytoTox Glo reagent (50 pL) was added and
plates were incubated
for 15 minutes at 25 C to label dead cells. Samples were then read on
Pherastar Luminometer to
measure luminescence signal from the dead cells. Our results demonstrate 5 ¨
7X higher potency of 53Y
(construct 13) molecule over the anti-CD38 mAb in inducing ADCC. As shown in
Table 15, below, anti-
CD38 Construct 13 demonstrated superior potency than anti-CD38 mAb in inducing
primary human NK
cell-mediated ADCC against Raji tumor cells. Target and effector cells were
treated with drug molecules
for 5 hours at 37C followed by detection of dead cells by CytoTox Glo reagent.
Assay controls,
Spontaneous Release Control (Target Cells Only); No Antibody Control; NK cells
only + Antibody; IgGk
Isotype Control
Table 15. Potency of anti-CD38 Fc constructs in an ADCC assay
Construct EC50 (nM)
Numberl Range Mean SD
anti-CD38 mAb 2 0.013-0.003 0.01567 0.007541
S3Y-AA-CD38 2 0.0019- 0.0012 0.0009
0.0006
Construct 13
G20 linker
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Example 36: Tumor cell killing by anti-CD38 constructs in human whole blood
Daudi cells were suspended in 50 pl of media (RPMI-1640 + 10% ULow IgG FBS)
and seeded
into each well of 96 well plate. 50 pL of whole human blood or ACK-Iysed human
whole blood cells
(without serum and RBCs) were added to the tumor cell suspension. This was
followed by addition of 50
pL of antibody and anti-CD38 construct dilutions (in RPMI-1640 media+10`)/0
FBS). Samples were mixed
and then incubated for 4 hours at 37 C in a CO2 incubator. After the
incubation, remaining live Daudi cells
were assessed by adding 50 pL of freshly prepared luciferin solution (stock
concentration, 50 mg/mL).
Plate was then placed on a plate shaker for 5 minutes. Luminescence emitted
from live Daudi-luciferase
cells was read using Pherastar Luminometer.
Results presented in FIG. 28 suggest that anti-CD38 construct 13 (53Y-AA-CD38)
is 10X ¨ 36X
more potent than anti-CD38 mAb in target cell killing in whole human blood
collected from 3 separate
donors. However, with RBC lysed & washed whole blood no tumor cell depletion
was observed with anti-
CD38 mAb or anti-CD38 constructs. Replenishing RBC lysed & washed whole human
blood cells with
autologous serum prepared from the same donor restored tumor cell depletion,
suggesting a role for
serum proteins in facilitating anti-CD38 mAb and anti-CD38 construct-induced
tumor cell killing in the
whole blood.
Example 37: Depletion of endogenous CD38 expressing B cells from monkey whole
blood
Cyno whole blood was mixed with serial dilutions of each VivoTag645-labeled
molecules (SIF1,
IgG isotype control, S1A-AA-Cyno-001 (anti-cyno CD38 mAb), anti-cyno CD38
construct 13 53Y-AA-
Cyno-001) separately along with cell surface marker antibody cocktail. Blood
samples were then either
incubated at 4 C for 30 min to determine cell surface binding or separately
incubated at 37 C for 3 hours
in a CO2 incubator for determining effect of treatment on cell depletion.
After these treatments, RBCs
were lysed by mixing samples with cold ammonium chloride solution. Samples
were then washed and re-
suspended in buffer containing 1% paraformaldehyde and FACS analysis was
performed the following
day. CD38+ B cell population was assessed based on CD38-binding & binding-
frequency data.
Frequency of CD38+ B cell type was measured to determine depletion due to
treatment with construct
molecule for 3 hours. B cell depletion was observed for anti-CD38 construct 13
(53Y-AA-Cyno-001) at
doses 10 nM (1 Log nM) and above, in a dose-dependent manner. Depletion with
begins to appear at
100-1000 nM (2 ¨ 3 Log nM). Greater depletion was observed with anti-cynoCD38
construct 13 (53Y-AA-
Cyno-001) compared to anti-cyno CD38 mAb (S1A-AA-Cyno-001).
Example 38: In Vivo Lymphoma Model
Effects of agents on disease progression and therapeutic response was
evaluated in a
subcutaneous tumor model for human lymphoma by tumor volume measurements. CB17-
severe
combined immunodeficiency (SCID) mice (female, 6-7 weeks old, average weight
of 20 grams, strain 236
from Charles River Laboratories) were housed in Momenta animal care facility
for 48 hours prior to use
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according to IACUC protocol. Water and food were provided ad libitum. All
experiments were approved
by the institutional animal ethics committee. Mice were checked daily for
signs of discomfort and for
general appearance. For subcutaneous tumor xenograft model, 5 X 106 human
Burkitt's lymphoma Raji
cells suspended in high concentration Matrigel were injected subcutaneously
into the right flank of mice.
Tumor volume was measured twice weekly until tumors reach approximately 250
mm3 (approximately by
day 6-7) at which time mice were assigned into treatment groups (8
mice/group). Mice in all 3 groups
were injected intraperitoneally with 0.5 mL normal human serum complement a
day before treatment,
immediately prior to intravenous treatment injections (with PBS, anti-CD38
mAb, or 53Y-AA-CD38), and a
day after treatment. Body weight and tumor volume was recorded twice weekly.
Tumors were measured
daily when volume approached 2000 mm3. All animals were observed daily; morbid
animals were
euthanized according to the IACUC protocol. The results shown in FIG. 30
suggest that the anti-CD38
construct 13 (53Y-AA-CD38) is more efficacious than anti-CD38 mAb in this
human lymphoma mouse
model when the treatment was given in the presence of human serum complement.
Example 39: Fc Domains in Constructs Retain Similar Binding to Fc Gamma
Receptors to that of
Fc Domains in Antibodies
Anti-CD20 and anti-CD38 constructs were utilized to evaluate whether the
various combinations
of homodimerization mutations, heterodimerization mutations, polypeptide
linkers, and Fab domains
affected the binding to Fc gamma receptors. Surface Plasmon Resonance (SPR)
was utilized to assess
1:1 binding with CD64 (Fc gamma receptor I). The constructs were captured on
the chip surface, and
binding to the soluble receptor was measured to ensure 1:1 binding. In this
format, binding valency is the
most sensitive readout to alterations in Fc function; kinetic and equilibrium
constants are insensitive to
alterations in a subset of Fc domains.
Cell Culture
DNA sequences were optimized for expression in mammalian cells and cloned into
the pcDNA3.4
mammalian expression vector. The DNA plasmid constructs were transfected via
liposomes into human
embryonic kidney (HEK) 293 cells. Antibodies were expressed from two different
plasmids: one encoding
the heavy chain and a second one encoding the light chain. SIF-bodies were
expressed from three
separate plasmids: in most cases one plasmid encoded the antibody light chain,
one plasmid encoded
the long Fc chain containing the CH1-VH FAB portion attached to the amino-
terminal Fc and a third
plasmid encoded the short Fc chain. The exceptions were the 53A and S3W Sif-
Bodies. For S3W, one
plasmid encoded the antibody light chain, the second plasmid encoded the long
chain containing two Fc
domains and a third plasmid encoded a single Fc chain containing a CH1-VH FAB
portion. For 53A, one
plasmid encoded the antibody light chain, a second plasmid encoded the long Fc
chain containing the
CH1-VH FAB portion attached to the amino-terminal Fc and one plasmid encoded
the short Fc chain also
containing a CH1-VH FAB portion.
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Protein Purification
The expressed proteins were purified from the cell culture supernatant by
Protein A-based affinity
column chromatography, using a Poros MabCapture A column. Captured SIF-Body
constructs were
washed with phosphate buffered saline (PBS, pH 7.0) after loading and further
washed with intermediate
wash buffer 50mM citrate buffer (pH 5.5) to remove additional process related
impurities. The bound SIF-
Body material is eluted with 100 mM glycine, pH 3 and the eluate was quickly
neutralized by the addition
of 1 M TRIS pH 7.4 then centrifuged and sterile filtered through a 0.2 pm
filter.
The proteins were further fractionated by ion exchange chromatography using
Poros XS resin. The
column was pre-equilibrated with 50 mM MES, pH 6 (buffer A), and the sample
was diluted (1:3) in the
equilibration buffer for loading. The sample was eluted using a 12-15CV's
linear gradient from 50 mM
MES (100% A) to 400 mM sodium chloride, pH 6 (100%B) as the elution buffer.
All fractions collected
during elution were analyzed by analytical size exclusion chromatography (SEC)
and target fractions
were pooled to produce the purified SIF-Body material.
After ion-exchange, the pooled material was buffer exchanged into 1X-PBS
buffer using a 30 kDa
cutoff polyether sulfone (PES) membrane cartridge on a tangential flow
filtration system. The samples
were concentrated to approximately 10-15 mg/mL and sterile filtered through a
0.2 pm filter.
Physicochemical Analyses
Analytical size exclusion chromatography (SEC) was used for the purity
assessment on post
Protein A, pooled ion-exchange fractions, and the final purified material.
The purified material was diluted to 1mg/m1 using 1X-PBS and analyzed on
Agilent 1200 system
with UV & FLD detector using Zenix SEC-300 (4.6 x 300 mm, 3pm, 300A, Sepax,
Cat. #213300-4630) as
the analytical column.
The column was equilibrated with 100mM sodium phosphate, 200mM arginine, 300mM
sodium
chloride pH=6.7 with 0.05% w/v sodium azide buffer at 0.3m1/min for an hour
before the analysis.
Injection amount approx. 10-15u1, column temperature: 300C with UV detection
at 280nm and FLD with
Excitation at 280mm and Emission at 330nm with total run time of 15min.
The size purity results are shown in Table 16. All materials showed only low
levels of high order species
(HOS).
Table 16: Size purity of constructs used in Fc binding assays
Size Purity by SEC Size Purity
by SEC
Construct Antigen
(Target Species %) (HOS %)
mAb CD20 97.0% 1.7%
Construct 13 (53Y) CD20 89.6% 0.0%
Construct 7 (S31) CD20 89.0% 1.7%
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Size Purity by SEC Size Purity
by SEC
Construct Antigen
(Target Species %) (HOS %)
Construct 8 (S3VV) CD20 83.4% 0.0%
Construct 9 (S3A) CD20 92.4% 1.5%
Construct 10 (S5I) CD20 98.4% 1.6%
(Construct 19 (S5X) CD20 90.0% 0.4%
Construct 16 (S5Y) CD20 73.8% 1.6%
mAb CD20 97.0% 1.7%
Binding Analyses
Binding experiments were performed on a Biacore T200 instrument (GE
Healthcare) using a CM3
Series S sensor chip. For valency analyses of FcgR binding, native Protein A
was immobilized via direct
amine coupling. Ligands were diluted in running buffer and captured. A 6-point
dilution series of human
recombinant CD32a or CD64 (R&D Systems) was flowed over the captured ligands.
The valency of each
ligand was calculated as:
Ligand Valency = Rmax/[(MW analyte/MW ligand)*Ligand Capture Level].
The results from analyses of CD64 binding to anti-CD20 constructs are shown in
Table 17. In all cases,
the CD64 binding valency was equal to the number of Fc domains, indicating
that all Fc domains were
functional to bind CD64. A control compound identical in sequence to 53Y-AA-
OBI and 53Y-AA-AVE,
but lacking the Fab domains, bound CD64 comparably to those constructs,
demonstrating that the
inclusion of Fab domains did not alter the binding to Fc receptors.
Table 17: Valency of certain anti-CD20 constructs
Number of Fc CD64 Valency by
Construct Antigen
Domains SPR
mAb CD20 1 1.5
Construct 13 CD20 3 3.4
(S3Y)
Construct 7 CD20 3 3.0
(S3I)
Construct 8 CD20 3 2.9
(S3W)
Construct 9 CD20 3 3.1
(S3A)
Construct 10 CD20 5 5.5
(S5I)
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(Construct 19 CD20 5 4.9
(S5X)
Construct 16 CD20 5 5.5
(S5Y)
Control (S3Y) No antigen binding 3 3.
domains
Example 40: Constructs Bind More Avidly to Cell Surface Fc Gamma Receptors
Relative binding of constructs to cell surface CD32a was evaluated in a time-
resolved
.. fluorescence resonance energy transfer (TR-FRET) assay (CisBio) using anti-
CD20 constructs. Assay
reagents were prepared according to the manufacturer's instructions. A Freedom
EVOware 150
automated liquid handler (Tecan) was used to generate a 10-point, 3-fold
serial dilution series for each
sample which were added to the cells bearing the labeled receptor. The labeled
competitor antibody was
then added and the plates incubated at room temperature. A PHERAstar
fluorescent reader (BMG
Labtech GmbH) was used to read assay plates at 665 and 620 nm. Log-transformed
sample
concentrations were plotted against corresponding HTRF signal ratios
(665nm/620nm). A four-parameter
non-linear regression analysis (least squares fit) was performed on the XY-
plot to calculate EC50 of the
unlabeled sample, with EC50 being inversely proportional to the sample's
affinity for Fc gamma receptor.
Measurements of competitive binding to CD32a determined by TR-FRET are
summarized in Table 17.
.. Increasing the number of Fc domains greatly increased the ability of
constructs to compete with
immunoglobulin for CD32a, as reflected by the decreased IC50 values. A control
compound identical in
sequence to S3Y-AA-OBI and S3Y-AA-AVE, but lacking the Fab domains, competed
for cell surface
CD32a comparably to those constructs, demonstrating that the inclusion of Fab
domains did not alter the
binding to Fc receptors.
Table 17: Fc receptor binding of certain anti-CD20 constructs
FcyRIllaV158 FcyRIlaH131 FcyRIlb IC50
Construct Antigen
IC50 (nM) IC50 (nM) (nM)
mAb CD20 428 1273 3291
Construct 13 CD20 0.076 0.009 2.146
(S3Y)
Construct 7 CD20 0.230 0.014 29.220
(S3 I)
Construct 8 CD20 0.476 0.026 34.925
(S3W)
Construct 9 CD20 0.539 0.018 17.361
(S3A)
Construct 10 CD20 0.045 0.002 4.427
(S5 I)
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(Construct 15 CD20 0.055 0.012 0.086
(S5X)
Construct 1 CD20 0.017 0.014 1.231
(S5Y)
Control (S3Y) No antigen 0.097 0.025 3.297
binding
domains
Example 41: Antigen binding is preserved in anti-CD38 constructs
Antigen binding was evaluated using SPR. Recombinant, Histidine tagged, CD38
(9049-B7 R&D
Systems) protein was captured on the sensor using a previously immobilized
anti-6X His antibody.
Dilution series of the cognate antibodies and SIF-bodies were passed over the
sensors, which were
regenerated with a low pH glycine solution between analyte injections. Binding
was calculated using a
1:1 Langmuir interaction model.
The binding of anti-CD38 constructs is shown in Table 18. All of the tested
compounds were no
less than 93% pure by SEC. Constructs had comparable antigen binding to that
of the corresponding
monoclonal antibody in an assay that favored 1:1 binding.
Table 18. CD38 binding to anti-CD38 constructs by SPR
Construct KD (nM)
mAb 670
S3Y 703
S3A 757
Table 19 provides data on binding of anti-CD38 constructs in a separate study.
Table 19: Human CD38 binding by certain anti-CD38 constructs
Construct KD (nM) at 25C KD (nM) at 37C
anti-CD38 mAb 129 410
53Y-AA-CD38 142 661
53I-AA-CD38 132 442
55X-AA-CD38 166 553
53A-AA-CD38 126 410
Example 42: Anti-CD38 Fc construct exhibits increased cytolytic activity
against human
lymphoma cells
As shown in FIG 31A and FIG 31B, 53Y-AA-CD38 anti-CD38 Fc construct was more
potent than
an anti-CD38 mAb having the same Fabs in ADCC (primary human NK cell
mediated), ADCP (primary
human macrophage mediated) and CDC.
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Example 43: Anti-CD38 Fc construct enhances tumor cell depletion from whole
blood with better
potency and efficacy that an anti-CD38 antibody
In this assay, the results of which are shown in FIG 32, human whole blood was
spiked with
CFSE-labeled Daudi cells and then treated with S3Y-AA-CD38 or an anti-CD38 mAb
having the same
Fabs. The change in tumor cell population (CFSE+CD19+) in whole blood from
baseline was measured
by flow cytometry. The anti-CD38 Fc construct demonstrated 40 ¨ 100 X higher
potency than the anti-
anti-CD38 mAb (n = 5 donors).
Example 44: Anti-CD38 Fc construct mediates cytotoxicity in both high and low
CD38 complement
inhibitory protein expressing tumor cell lines
Response to the CD38-targeting antibody anti-CD38 mAb is correlated with CD38
expression
levels on tumor cells. In addition, increased expression of complement
inhibitory proteins (CD55, CD59)
significantly decreases anti-CD38 mAb induced tumor cell depletion resulting
in disease progression
(Nijhof et al. (2016) Blood 128:959). As shown in FIG 33, S3Y-AA-CD38 anti-
CD38 Fc construct (inverted
triangles) was more potent CDC activity than an anti-CD38 mAb having the same
Fabs (circles) in both
Daudi cells (relatively high CD38 expression and relatively low CD55 and CD59
expression) and,
importantly, in Raji cells (relatively low CD38 expression and relatively high
CD55 and CD59 expression).
Example 45: Anti-CD38 Fc construct mediates cytotoxicity in both high and low
CD38 complement
inhibitory protein expressing tumor cell lines
S3Y-AA-Cyno, the anti-cyno CD38 Fc construct described above in Table 6, which
binds to both
human and cynomolgus monkey CD38, and demonstrated improved ADCC, ADCP, and
CDC activities
against human lymphoma cells compared to a mAb with the same Fabs (anti-cyno
CD38 mAb), as shown
in FIG 34A and 34B.
Example 46: Anti-Cyno CD38 Fc construct enhances tumor cell depletion from
Cynomolgus
monkey whole blood with better potency and efficacy than an anti-Cyno CD38
antibody
In this assay, the results of which are shown in FIG 35, Cynomolgus monkey
whole blood was
spiked with CFSE-labeled Daudi cells and then treated with S3Y-AA-Cyno CD38 or
an anti-CD38 mAb
having the same Fabs. The change in tumor cell population (CFSE+CD19+) in
whole blood from baseline
was measured by flow cytometry. The anti-Cyno CD38 Fc construct demonstrated
significantly higher
potency than anti-cyno CD38 mAb (n = 3).
Example 47: Anti-Cyno CD38 Fc construct demonstrates superior CD38hi9h B cell
depletion than
anti-Cyno CD38 mAb in Cynomolgus Monkeys
In this assay, the results of which are shown in FIG 36, S3A-AA-Cyno was
superior to the anti-
Cyno CD38 mAb both in vitro, as measured by B cell depletion from peripheral
blood collected from
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Cynomolgus monkeys (left panel), and in vivo, as measured in a single dose PD
study in Cynomolgus
monkeys that examined B cell depletion after 4 hours (right panel)
Example 48: Anti-CD38 Fc construct demonstrates superior depletion of plasma
cells from a
multiple myeloma patient with a high bone marrow plasma cell load
In this assay, the results of which are shown in FIG. 37, S3Y-AA-CD38 was
superior to an anti-
CD38 mAb having the same Fab sequences. Frozen bone marrow mononuclear cells
(BM-MNCs) from
multiple myeloma patient MM536, a relapsed patient with a BM plasma cell load
of 82%, were obtained
from the vendor. BM-MNCs were thawed and incubated in RPM! 1640 + 20% human
serum complement
(to allow for CDC mediated cell killing), in the presence or absence of
varying concentrations of either
anti-CD38 mAb or S3Y-AA-CD38 for 18 hours. The following day, samples were
stained and analyzed by
FACS to assess depletion of CD138+ cells, used as a surrogate marker for CD38
expressing
plasma/myeloma cells based on co-expression of the two markers determined by
phenotyping of
untreated cells. Cell depletion was determined using the viable CD138+ cell
frequency out of total single
cells, with all relative cell frequencies normalized to a baseline frequency
observed in untreated controls
(set to 0% change).
Depletion of CD138+ cells from total BM-MNCs of patient MM536 was observed
following either
S3Y-AA-CD38 or anti-CD38 mAb treatment at 100 or 1000 nM, while no depletion
was observed for
either treatment at a concentration of 10 nM. Saturating depletion of >90% of
viable CD138+ cells at
53Y-AA-CD38 concentrations of 100 or 1000 nM was observed. Anti-CD38 mAb-
mediated depletion was
considerably lower than that observed by 53Y-AA-CD38, with maximum depletion
levels 24% at
concentrations of 100 and 1000 nM, which appear to be at or near saturating.
Considering the high BM
plasma cell frequency in patient MM536 (about 82%), these results may indicate
potential for greater
response using an anti-CD38 Fc construct in MM patients with high bone marrow
plasma cell loads,
which have been shown to have lower objective response rate to anti-CD38 mAb
treatment in clinical
applications.
Example 48: Anti-CD38 Fc construct demonstrates enhanced binding to cell
surface FcyRs and
human serum complement
FIG. 38A depicts the results of a study showing that 53Y-AA-CD38 binding to
FcgRIla, FcgRIlla and
complement is at least 100-fold greater than an anti-CD38 mAb.
FIG. 38B depicts the results of a study showing >500X enhanced binding of 53Y-
AA-CD38 to FcyRIla,
FcyRIlla on immune cell surface and 12X enhanced C1q complement protein
binding than an anti-CD38
mAb.
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Example 49: Anti-CD38 Fc construct with mutant Fc CH2 domains exhibits longer
lasting B cell
depletion
Similarly to Example 38, Cyno whole blood was mixed with serial dilutions of
each an anti-cyno CD38
mAb, anti-cyno CD38 construct 13 (53Y-AA-Cyno-001) and B cell depletion was
measured. In addition,
as a variant anti-cyno CD 13 construct was tested. Each CH2 domain had a R292P
mutation (see FIGs:
24A- 24B for Fc domain numbering). Thus, this construct had an Fc domain
mutation noted above. Table
20, below, provides the amino acid sequence of the polypeptides making up this
variant CD38 Construct
13 variant (mutation is bold, highlighted and underlined). 53Y-CD38 (CC R292P)
is the version that binds
to human CD38 and 53Y-Cyno-001 (CC R292P) binds to cynomolgus monkey CD38. As
can be seen be
seen in FIG. 39, 53Y-Cyno-001 (CC R292P) ("2nd Gen SIFBody") exhibited
superior durability of B cell
depletion compared to 53Y-Cyno-001 (no R292P mutation) ("1st Gen SIFbody").
Table 20. Construct 13 (CD38) sequences with R292P Fc CH2 domain mutation
Construct Light chain Long Fc chain Short Fc chain
(anti-CD38 VH and CH1)
Construct 13 SEQ ID NO: A SEQ ID NO: B SEQ ID NO: C
(CD38), G20
linker EIVLTQSPATLSLSPGERAT EVQLLESGGGLVQPGGSL DKTHTCPPCPAPELLGGPS
53Y-CD38 LSCRASQSVSSYLAWYQQ RLSCAVSGFTFNSFAMSW VFLFPPKPKDTLMISRTPE
KPGQAPRLLIYDASNRAT VRQAPGKGLEWVSAISGS VTCVVVDVSHEDPEVKFN
(S3Y-CC- GIPARFSGSGSGTDFTLTIS GGGTYYADSVKGRFTISR WYVDGVEVHNAKTKPPE
CD38) SLE PE DFAVYYCQQRSNW DNSKNTLYLQM NSLRAED EQYNSTYRVVSVLTVLHQ
PPTFGQGTKVEIKRTVAAP TAVYFCAKDKILWFGEPVF DWLNGKEYKCKVSNKALP
SVFIFPPSDEQLKSGTASV DYWGQGTLVTVSSASTKG APIEKTISKAKGQPREPQV
VCLLNNFYPREAKVQWKV PSVFPLAPSSKSTSGGTAA CTLPPSRDELTKNQVSLSC
DNALQSGNSQESVTEQDS LGCLVKDYFPEPVTVSWN AVDGFYPSDIAVEWESNG
KDSTYSLSSTLTLSKADYEK SGALTSGVHTFPAVLQSS QPENNYKTTPPVLDSDGS
HKVYACEVTHQGLSSPVT GLYSLSSVVTVPSSSLGTQ FFLVSKLTVDKSRWQQGN
KSFNRGEC TYICNVNHKPSNTKVDKR VFSCSVMHEALHNHYTQ
VEPKSCDKTHTCPPCPAPE KSLSLSPG
LLGGPSVFLFPPKPKDTLM
ISRTPEVTCVVVDVSH EDP
EVKFNWYVDGVEVHNAK
TKPPEEQYNSTYRVVSVLT
VLHQDWLNGKEYKCKVS
NKALPAPIEKTISKAKGQP
REPQVYTLPPCRDKLTKN
QVSLWCLVKGFYPSDIAV
EWESNGQPENNYKTTPP
VLDSDGSFFLYSKLTVDKS
RWQQGNVFSCSVMHEA
LHNHYTQKSLSLSPGKGG
GGGGGGGGGGGGGGG
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GGGDKTHTCPPCPAPELL
GGPSVFLFPPKPKDTLMIS
RTPEVTCVVVDVSHEDPE
VKFNWYVDGVEVHNAKT
KPPEEQYNSTYRVVSVLTV
LHQDWLNGKEYKCKVSN
KALPAPIEKTISKAKGQPRE
PQVYTLPPSRDELTKNQVS
LTCLVKGFYPSDIAVEWES
NGQPENNYKTTPPVLKSD
GSFFLYSDLTVDKSRWQQ
GNVFSCSVM HEALH N HY
TQKSLSLSPG
Construct 13 SEQ ID NO: SEQ ID NO: SEQ ID NO:
(CD38), G20 QSVLTQPPSASGTPGQRV QLLESGGGLVQPGGSLRL DKTHTCPPCPAPELLGGPS
linker TISCSGSSSNIGDNYVSWY SCAASGFTFDDYGMSWV VFLFPPKPKDTLMISRTPE
53Y-Cyno-001 QQLPGTAPKLLIYRDSQRP RQAPGKGLEWVSDISWN VTCVVVDVSHEDPEVKFN
SGVPDRFSGSKSGTSASLA GGKTHYVDSVKGQFTISR WYVDGVEVHNAKTKPPE
S3Y-CC-CD38- ISGLRSEDEADYYCQSYDS DNSKNTLYLQM NSLRAED EQYNSTYRVVSVLTVLHQ
Cyno SLSGSVFGGGTKLTVLGQ TAVYYCARGSLFHDSSGFY DWLNGKEYKCKVSNKALP
PKANPTVTLFPPSSEELQA FGHWGQGTLVTVSSASTK APIEKTISKAKGQPREPQV
NKATLVCLISDFYPGAVTV GPSVFPLAPSSKSTSGGTA CTLPPSRDELTKNQVSLSC
AWKADGSPVKAGVETTK ALGCLVKDYFPEPVTVSW AVDGFYPSDIAVEWESNG
PSKQSNNKYAASSYLSLTP NSGALTSGVHTFPAVLQS QPENNYKTTPPVLDSDGS
EQWKSHRSYSCQVTHEG SGLYSLSSVVTVPSSSLGT FFLVSKLTVDKSRWQQGN
STVEKTVAPTECS QTYICNVNHKPSNTKVDK VFSCSVMHEALHNHYTQ
RVEPKSCDKTHTCPPCPA KSLSLSPG
PELLGGPSVFLFPPKPKDT
LMISRTPEVTCVVVDVSH
EDPEVKFNWYVDGVEVH
NAKTKPPEEQYNSTYRVV
SVLTVLHQDWLNGKEYKC
KVSNKALPAPIEKTISKAKG
QPREPQVYTLPPCRDKLTK
NQVSLWCLVKGFYPSDIA
VEWESNGQPENNYKTTP
PVLDSDGSFFLYSKLTVDK
SRWQQGNVFSCSVMHE
ALHNHYTQKSLSLSPGKG
GGGGGGGGGGGGGGG
GGGGDKTHTCPPCPAPEL
LGGPSVFLFPPKPKDTLMI
SRTPEVTCVVVDVSHEDP
EVKFNWYVDGVEVHNAK
TKPPEEQYNSTYRVVSVLT
VLHQDWLNGKEYKCKVS
NKALPAPIEKTISKAKGQP
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REPQVYTLPPSRDELTKN
QVSLTCLVKGFYPSDIAVE
WESNGQPENNYKTTPPV
LKSDGSFFLYSDLTVDKSR
WQQGNVFSCSVMHEAL
HNHYTQKSLSLSPG
Example 50: Binding of CD38 mAb and SIFbodies to CD38
A Biacore 8K instrument (GE Healthcare) was used to measure CD38 binding to
mAbs and SIF-
bodies. Protein A immobilized on a CM5 sensor chip was used to capture test
molecules (SIF-bodies or
antibodies) from solution. A dilution series of CD38 was flowed over the
captured test molecule. The
assay was run as a single-cycle kinetics assay where increasing concentrations
of CD38 are injected
over the captured test molecule and dissociation monitored at the end.
Sensorgrams were double-
reference subtracted and fit to a 1:1 binding model. The kinetic association
(ka) and dissociation (kd)
constants were measured and the equilibrium dissociation constant (KD)
computed by dividing the kd by
the ka. Stoichiometry of binding for test molecules was also measured. The
capture level of each test
molecule, measured in Response Units (RU), was multiplied by a ratio of the
molecular weights of the
CD38 to the test molecule to obtain a theoretical maximum binding level. The
RMax value for the
experiment was divided by the theoretical maximum binding level to obtain the
stoichiometry.
Cynomolgus monkey CD38 binding was tested by measuring the binding of 400nM
and 200nM protein to
each test molecule.
FIG. 41 depicts the results of an analysis of human CD38 binding to cyno anti-
CD38 antibodies and cyno
SIF-bodies measured by SPR. Binding sensorgrams for anti-CD38 molecules and
1:1 binding model fit
are shown on the left. Kinetic and equilibrium constants are shown on the
center and upper right.
Stoichiometry of human CD38 binding to anti-CD38 molecules is shown on the
lower right. All constructs
bind human CD38 equivalently. S3Y-CC-CD38 is Construct 13 with the R292P
mutation in the Fc CH2
mutation and S3Y-AA-CD38 lacks the R292P mutation, but is otherwise identical.
FIG. 42 depicts the results of an analysis of human CD38 binding to Cyno-CD38
antibodies and Cyno-
SIF-bodies measured by SPR. Binding sensorgrams for cyno-CD38 molecules and
1:1 binding model fit
are shown on the left. Kinetic and equilibrium constants are shown on the
center and upper right.
Stoichiometry of human CD38 binding to cyno-CD38 molecules is shown on the
lower right. All constructs
bind human CD38 equivalently.
FIG. 43 depicts the results of an analysis of cynomolgus CD38 binding to cyno-
CD38 antibodies, cyno-
CD38 SIF-bodies and CD38 mAb measured by SPR. Binding sensorgrams for cyno-
CD38 molecules
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and 1:1 binding model fit are shown on the left and lower center panels.
Binding sensorgrams for CD38
mAb and 1:1 binding model fit are shown on the upper center panel. Equilibrium
constants are shown on
the right panel. All cyno-CD38 constructs bind cynomolgus CD38 equivalently.
CD38 mAb does not bind
cynomolgus CD38.
Example 51: Binding of S3Y-AA-CD38 and S3Y-CC-CD38 to human FcyRs
FIG. 44 depicts the results of an analysis of binding S3Y-AA-CD38 and S3Y-CC-
CD38 to human FcyRs
relative to CD38 mAb using a Fc-gamma receptor homogeneous time-resolved
fluorescence (HTRF)
assay. The competitive HTRF assay was used to determine binding of S3Y-AA-
CD38, S3Y-CC-CD38 and
CD38 mAb to human for FCGR1A, FCGR2A (H167), FCGR2A (R167), FCGR2B, FCGR3A
(F176) and
FCGR3A (V176). ICso inhibition constants were derived from the data. Binding
of S3Y-AA-CD38 and S3Y-
CC-CD38 is expressed as fold change in IC50 relative to the CD38 MAb IC50. S3Y
SIF-bodies show
greatly enhanced avid binding to cell surface receptors. The R292P mutations
in S3Y-CC-CD38 result in
a greater than 100 fold reduction in binding to FcgR2b compared to the
parental S3Y-AA-CD38 molecule.
The Fc-gamma receptor competitive HTRF assays were purchased from Cis-Bio and
run according to
manufacturer's instructions. Assays for FCGR1A, FCGR2A (H167), FCGR2A (R167),
FCGR2B,
FCGR3A (F176) and FCGR3A (V176) were performed. Fluorescence measurements were
taken at
620nm and 655nm with the BMG Pharastar fluorometer.The ratio of 655/620 *10000
was computed for
each well. Data were copied into GraphPad Prism for analysis. Concentration
values were log-
transformed and the data was fit to a four-parameter curve constraining the
top and bottom values to be
common among all tested samples. IC50 values were estimated and reported for
each independent
dilution.
Example 52: Binding of anti-CD38 mAb and S3Y-CC-CD38 molecules to endogenous
CD38
receptor on human tumor cells.
CD38 is a cell surface type ll glycoprotein receptor with a large
extracellular region and very short
cytosolic domain. This receptor is expressed in multiple hematologic
malignancies derived from both B
cell and plasma cell lineages, including multiple myeloma (MM). Cell lines
derived from lymphoma and
myeloma patients were selected for CD38 expression analysis. CD38 levels were
determined by binding
of fluorescently labeled anti-CD38 mAb by flow cytometry. Cells were incubated
with VivoTag 645-labeled
anti-CD38 mAb at 4 C for 30 min. Cells were then washed, fixed, and single
cell events were acquired on
Verse flow cytometer. Values represent mean SD. The hierarchy of cell
surface CD38 expression is
Daudi cells (lymphoma cell line) > Raji (lymphoma cell line) > SU-DHL
(lymphoma cell line) > MM.1S
(myeloma cell line) > RPMI8226 (myeloma cell line) (FIG 45).
Daudi and Raji cell lines with high and moderate levels of cell surface CD38
were selected for cell
based functional assays. There was dose-dependent increase in binding followed
by saturation with anti-
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CD38 mAb and S3Y molecules in both Daudi and Raji cell lines (FIG 46). Binding
profiles of anti-CD38
mAb, S3Y-CC-CD38, and S3Y-AA-CD38 were similar suggesting that mutations in Fc
domain of S3Y-CC-
CD38 had no effect on interactions of these molecules with endogenous cell
surface CD38. Cells were
incubated with VivoTag 645-labeled anti-CD38 mAb or S3Y-AA or S3Y-CC molecules
at 4 C for 30 min.
Cells were then washed, fixed, and single cell events were acquired on Cytek
Aurora flow cytometer.
Values represent mean SD. As shown in FIG 46, 53Y-CC-CD38 and anti-CD38 mAb
show similar
binding to lymphoma cell surface CD38.
Example 53: Impact of S3Y-CC-CD38 and anti-CD38 mAb Fc Effector Function Based
Cell
Depletion Assays
Therapeutic anti-CD38 antibody daratumumab (Darzalex) kills tumor cells via Fe-
dependent
immune effector mechanisms including complement-dependent cytotoxicity (CDC),
antibody-dependent
cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis
(ADCP). To assess relative
cytotoxicity of 53Y-CC-CD38 to its parent version 53Y-AA-CD38 and anti-CD38
mAb CDC, ADCC, and
ADCP assays were performed.
To assess CDC activity, Daudi or Raji cells were incubated with human serum
complement and
drug molecules at 37 C for 2 hours to facilitate complement mediated lysis.
Alamar Blue (a cell viability
reagent) was then added to each well and incubated at 37 C for 18 hours and
the viable cell fluorescence
was measured using a fluorometer to determine extent of cell lysis. Both 53Y
and anti-CD38 mAb
demonstrated dose-dependent increase in complement mediated Daudi cell lysis
(FIG. 47A, left panel).
Raji cells have lower CD38 expression than Daudi cells, however they also have
higher levels of
complement inhibitory proteins on their cell surface. Anti-CD38 mAb fail to
induce CDC against Raji cells
whereas 53Y molecules demonstrated clear dose-dependent cytotoxicity (FIG.
47B, right panel). Overall,
53Y-CC-CD38 demonstrated better potency and maximum target cell lysis in cells
where an anti-CD38
mAb fails to induce lysis by CDC.
Example 54: Enhanced Potency & Efficacy of S3Y-CC-CD38A in Inducing Cytolytic
Activity against
CD38+ Human Lymphoma Cells.
ADCC and ADCP are dependent on effective interactions between Fc region of an
antibody with the Fey
receptors (FeyRs) expressed on immune effector cells. During the induction of
ADCC lysis of antibody
coated tumor cells by effector cells occurs. NK cells play a critical role in
ADCC mediated by therapeutic
antibodies. Importantly, the CD38-targeting 53Y molecules are differentiated
from anti-CD38 mAb by their
ability to bind with much stronger affinity due to avid binding to FeyRs.
Relative ADCC activities of 53Y-
CC-CD38, 53Y-AA-CD38, and anti-CD38 mAb were assessed. For this assay, primary
human NK cells
were added to tumor cells. Cell mixtures were then incubated with either one
of the drug molecules for 5
hours at 37 C followed by detection of dead cells by CytoTox Glo reagent. 53Y
molecules clearly
demonstrates significantly enhanced potency than reference anti-CD38 mAb (FIG.
48A).
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In the process of ADCP, phagocytosis of antibody-opsonized tumor cells occurs
via binding of
FcyRs (such as FcyRIIA and FcyRIIIA), which are present on monocytes and
macrophages.
Phagocytosis contributes to the anti-tumor activity of CD38-targeting
antibodies. Interestingly, individual
macrophages have the ability to quickly and sequentially engulf multiple
daratumumab-coated tumor
cells, indicating that ADCP is an efficient killing mechanism of daratumumab
(Darzalex). We selected M2c
phenotype of macrophages as an effector cell because they are more prevalent
in bone marrow
microenvironment in hematologic malignancies. To determine relative ADCP
activities of S3Y molecules
and reference anti-CD38 mAb, monocytes purified from human PBMCs, were
cultured in M-CSF and then
with IL-10 to generate M2c macrophages. Phagocytosis of tumor cells by
macrophages was captured by
real time imaging. S3Y-CC-CD38 and S3Y-AA-CD38 clearly demonstrates
significantly enhanced potency
in inducing ADCP than reference anti-CD38 mAb (FIG 48B).
Example 55: S3Y-CC-CD38 Enhances Lymphoma Cell Depletion From Whole Blood With
Better
Potency And Cytotoxicity.
The relative cytotoxic activities of the S3Y molecules to an anti-CD38 mAb was
further compared
in whole-blood tumor cell (CD38-high Daudi cells, and moderate-CD38 expressing
Raji cells) depletion
assays, which integrate different antibody modes of action (CDC, ADCC, and
induction of cell death).
Heparinized blood samples from healthy volunteers were spiked with Daudi or
Raji lymphoma cells
labeled with CFSE dye and incubated at 37 C in the presence of anti-CD38 mAb
or S3Y-CC-CD38 or
S3Y-AA-CD38 for 18 hours. Blood cells were then stained for surface markers,
fixed and acquired on a
flow cytometer. Reduction in the frequency of CFSE+ (Daudi or Raji) cells in
the presence of anti-CD38
mAb or S3Y-molecules when compared to without drug treatment was the indicator
of selective cell
depletion. The superiority of S3Y-CC-CD38 compared with anti-CD38 mAb is shown
both by higher
potency values and by higher maximal CFSE+ Daudi or Raji cell depletion in
whole blood (Figure 49
.. A&B).
Example 56: S3Y-CC-CD38 & S3Y-AA-CD38 Mediates Enhanced Cytotoxicity Against
Plasma Cells
in Bone Marrow Biopsies from Multiple Myeloma Patients in an Ex-Vivo Native
Environment
Assay.
Therapeutic anti-CD38 mAb Darzalex was approved by FDA based on monotherapy
trial in
relapsed and refractory multiple myeloma (RRMM) patient population with >4
lines of prior therapy with
31% clinical response rate. Nowadays, Darzalex is commonly used in combination
with iMIDs and
proteasomal inhibitors in frontline and relapsed and refractory settings for
MM patients. In these combo
trials achieving higher minimal residual disease (MRD) status by flow
cytometry is considered as one of
the major predictor of better progression free survival rates in patients.
Patients are considered MRD-
negative if they have < 1 plasma cell (CD138+)/100,000 bone marrow cells when
tested by validated
clinical flow cytometry assay. Retrospective data from Darzalex combo trial
(POLLUX study) show there
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are significant number of patients who do not achieve MRD-negative status.
Therefore, a better plasma
cell depleting agent may achieve better disease-free duration of remission
than current SOC in RRMM
patients.
The plasma cell depletion activity of 53Y-CC-CD38 and its parent molecule 53Y-
AA-CD38 in
comparison with daratumumab (Darzalex) were investigated in an ex-vivo assay
using bone marrow
aspirates from multiple myeloma (MM) patients. Bone marrow aspirates were
collected from relapsed
and refractory MM patients (n=5) who had prior lines of therapy including
corticosteroids, stem cell
transplant, proteasomal inhibitors (Velcade), iMIDs (lenalidomide). All these
patients had varying levels of
plasma cell load in their bone marrow as determined by a panel of plasma cell
surface makers using flow
cytometry. An ex vivo plasma cell depletion assay was performed with fresh
bone marrow aspirates (FIG
50). Bone marrow samples were extracted into Vacutainer tubes containing
heparin as an anticoagulant
and further diluted with IMDM supplemented with 20% (v/v) autologous plasma to
maintain native
environment. The mixture was dispensed into 96-well plates containing various
concentrations of either
daratumumab (Darzalex) or 53Y-CC-CD38 or its parent molecule 53Y-AA-CD38. The
plates containing
samples were then incubated for 3 hours at 37 C in humidified air containing
5% CO2. To prepare the
samples for flow cytometric analysis, at the end of incubation RBCs were lysed
and then stained with
fluorescently-labeled monoclonal antibody panel against cell surface markers
(CD38, CD16, CD319,
CD45, CD56, CD3) and Annexin V to identify and quantitate remaining live
plasma cells in each well.
As expected, Darzalex depleted plasma cells from bone marrow in a dose-
dependent manner (FIG. 50).
Both 53Y molecules (53Y-CC-CD38 and 53Y-AA-CD38) demonstrated superior dose-
dependent
depletion of Multiple Myeloma tumor cells in all patients. 53Y-CC-CD38 and 53Y-
AA-CD38
demonstrated better potency than Darzalex (FIG. 50). In addition, in 2 out of
5 patient bone marrow
samples, 53Y molecules eliminated most of the plasma cells (>95%). Overall,
data from these 5 patients
suggests 53Y-CC-CD38 & 53Y-AA-CD38 molecules are superior to Darzalex in
depleting MM tumor cells
from bone marrow aspirates (FIG. 50).
Example 57: S3Y-CC-Cyno-CD38, S3Y-AA-Cyno-CD38, and anti-Cyno-CD38 mAb show
similar
binding to cell surface CD38 on human lymphoma cell lines.
Mutations (R292P) were introduced in Fc domains of 53Y-AA-Cyno-CD38 molecule
to generate
53Y-CC-Cyno-CD38. The Fc mutation reduces its binding to Fc gamma RIIB (see
FIG 45). Functional
activity was conform in cell-based assays. The PK optimized molecule was then
tested in Cynomolgus
monkeys to determine if Fc mutations enhances the depth and duration of PD
response and improves the
serum half-life of 53Y-CC-Cyno-CD38 molecule.
53Y-CC-Cyno-CD38, 53Y-AA-Cyno-CD38, and anti-Cyno-CD38 mAb binds both human
and
cynomolgus monkey CD38 (FIG. 51). To confirm that R292P Fc mutations in 53Y-CC-
Cyno-CD38
molecule has no effect on its ability to bind cell surface CD38, binding of
labeled 53Y-CC-Cyno-CD38 to
its parent molecule (53Y-AA-Cyno-CD38) and reference anti-Cyno-CD38 mAb was
compared in Daudi
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and Raji cells. S3Y-CC-Cyno-CD38 show similar binding and EC50 values to
parent S3Y-AA-Cyno-CD38
and reference anti-Cyno-CD38 mAb.
Example 58: S3Y-CC-Cyno-CD38 demonstrated enhancement in Fc effector function
mediated
target cell cytotoxicity than an anti-Cyno-CD38 mAb.
Both S3Y-CC-Cyno-CD38 and reference anti-Cyno-CD38 mAb bind human and Cyno
CD38. Fc
effector function was assessed based cell depletion activity with this S3Y
molecule with Fc mutations.
Many Fc mutations affects complement binding to antibody-opsonized cell
surface and also interactions
with Fc gamma receptor on NK cells and macrophages. Therefore, Daudi and Raji
cells were used as
target cell lines. The impact of Fc mutations in S3Y-CC-Cyno-CD38 on cell
killing activity in CDC, ADCC,
and ADCP assays was examined as previously described. S3Y-CC-Cyno-CD38
demonstrated
significantly superior potency in inducing cellular cytotoxicity in all Fc
effector function based assays using
human complement, primary human NK cells, and M2c macrophages (FIG. 52).
Example 59: Exploratory PK/PD & Tolerability Studies in Cynomolgus Monkeys
with 53Y-CC-
Cyno-CD38
A single dose study in Cynomolgus monkeys was conducted to assess
pharmacokinetics (PK) of
S3Y-CC-Cyno-CD38 (with R292P mutations in Fc domains), tolerability, and
durability of
pharmacodynamic effect (B cell depletion). Superiority and differentiation
from parent molecule 53Y-AA-
Cyno-CD38 and reference anti-Cyno-CD38 mAb was also examined. To this end, a
15-Day single-dose
pharmacokinetics, pharmacodynamics, and tolerability study of 53Y-CC-Cyno-
CD38, 53Y-AA-Cyno-
CD38, and anti-Cyno-CD38 mAb following intravenous infusion in cynomolgus
monkeys was conducted.
Exposure (the area under the serum concentration-time curve from dose
administration to 360 hours
post-dose) at steady state generally increased in a dose-proportional manner
(FIG. 53). 53Y-CC-Cyno-
CD38 demonstrated significantly improved PK profile with higher serum drug
concentrations from its
parent molecule 53Y-AA-Cyno-CD38 (FIG. 53).
Cell immunophenotyping was performed on peripheral blood of 53Y-CC-Cyno-CD38,
53Y-AA-
Cyno-CD38, and anti-Cyno-CD38 mAb-treated animals using antibodies against
CD138, CD159a, CD27,
CD20, CD19, and CD3. Assessment of the relative percentage of positive cells
was measured using flow
cytometry and used in the calculation of absolute cell counts. Absolute cell
counts at each time point is
normalized to pre-dose level for individual animal. Results discussed here
represent percent change in B
cell counts in peripheral blood normalized to pre-dose baseline (set at 100%).
Flow cytometry analysis
indicated that administration of drug molecules resulted in a mild to marked
decrease in absolute cell
counts of CD19+CD20+/- (total B cells) in all treated groups (FIG 54). 53Y-CC-
Cyno-CD38 (1.7 mg/Kg)
demonstrated much higher potency and efficacy in terms of its pharmacodynamic
effect on B cells than
its parent molecule 53Y-AA-Cyno-CD38 (1.7 mg/Kg) and anti-Cyno-CD38 mAb (1.0
mg/Kg) at equimolar
doses (Figure 54).
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Example 60: Multiple Repeat-Dose Study: All S3Y-CC-Cyno-CD38 doses were potent
and
efficacious in repeat dose setting; Plasma cells (CD138+) represent only a
small population of all
lymphoid cells. 53Y-CC-Cyno-CD38 can deplete plasma cells from tissues in a
dose-dependent
manner.
Pharmacodynamic effects of S3Y-CC-Cyno-CD38 were examined in a 4-weeks repeat-
dose
(once a week for 4 weeks) study in Cynomolgus monkeys. For this study 10X
higher doses for S3Y-CC-
Cyno-CD38 (1.7, 17, 51 mg/Kg) were used compared to earlier study (FIG. 53 and
FIG. 54) to assess
maximum tolerability, safety, as well as multiple PD readouts of efficacy,
cell depletion in blood, and
plasma cell depletion from lymphoid tissues. Blood samples were collected
during the study for
hematological assessments, flow cytometry, and tissues were harvested after
completion of dosing to
assess effects of treatment on plasma cells depletion. All treatment groups
were well tolerated. S3Y-CC-
CD38B treatment depleted circulating B cells in a dose-dependent manner with
improved durability of
effect with increasing dose (FIG 55A). Bone marrow and spleen represent the
major lymphoid
tissues. Plasma cells (CD138+) represent only a small population of all
lymphoid cells (Fig 55B). S3Y-
CC-Cyno-CD38 depleted plasma cells in a dose-dependent manner from bone marrow
and spleen (Fig
55B).
S3Y-CC-Cyno-CD38 are novel protein structures for which there is no evidence
of feasibility of
subcutaneous (SC) dosing. The increased molecular weight of this molecule
could be to an impediment
to absorption via SC routes. CD38 therapeutics are commercially available in
both IV and SC
presentations and it will be desirable for 53Y-CC-Cyno-CD38 to be available in
both presentations. A
non-GLP single IV and SC dose pharmacokinetics (PK), pharmacodynamics (PD) and
tolerability
assessment of 53Y-CC-Cyno-CD38 in cynomolgus monkeys was conducted. This study
demonstrated no
tolerability findings, PK and PD results demonstrated feasibility of
subcutaneous delivery of 53Y-CC-
Cyno-CD38 molecule in non-human primates. Data also suggests relatively better
bioavailability and PD
effect with high dose SC injection (FIG. 56A, FIG 56B).
All publications, patents, and patent applications mentioned in this
specification are incorporated
herein by reference to the same extent as if each independent publication or
patent application was
specifically and individually indicated to be incorporated by reference.
While the disclosure has been described in connection with specific
embodiments thereof, it will
be understood that it is capable of further modifications and this application
is intended to cover any
variations, uses, or adaptations of the disclosure following, in general, the
principles of the disclosure and
including such departures from the disclosure that come within known or
customary practice within the art
to which the disclosure pertains and may be applied to the essential features
hereinbefore set forth, and
follows in the scope of the claims.
Other embodiments are within the claims.
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