Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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METHODS OF REDUCING PRODUCTION OF IgA, IgM AND/OR IgG USING
sBCMA VARIANTS AND FC FUSION PROTEINS THEREOF
T. FIELD OF THE INVENTION
[0001] This invention relates to methods of reducing
immunoglobulin production
(e.g. IgA, IgM, and/or IgG, etc.) in subjects diagnosed with autoimmune
disease(s) and/or
fibrosis comprising administering to the subjects compositions comprising
soluble B-cell
maturation antigen (sBCMA) variants and/or sBCMA variant ¨ Fc fusion proteins.
BACKGROUND OF THE INVENTION
[0002] B-cell maturation antigen (BCMA) is a member of the
tumor necrosis factor
receptor superfamily member. The amino acid sequence of the extracellular
domain of
BCMA is shown in Figure 30. For example, BCMA is a receptor for 3-cell
Activating
Factor of the TNF family (BAFF) and A Proliferation Inducing Ligand (APRIL).
Anti-
BCMA antibodies, including an antibody drug conjugate (ADC), have shown
initial success
in treating cancer in early testing, as have BCMA bispecific T cell engaging
antibodies and
CAR-T constructs using BCMA.
[0003] BAFF is previously described in WO/0012964 and US
9,650,430 B2, which
are incorporated by reference herein. The amino acid sequence of the
extracellular domain
of BAFF is shown in Figure 7. BAFF is a cell survival and maturation factor
for B cells, and
overproduction of BAFF is associated with systemic autoimmune disease. In
humans, high
levels of BAFF are detectable in the blood of a proportion of patients with
autoimmune
rheumatic diseases, particularly systemic lupus erythematosus and Sjogren's
syndrome
(Groom et al. I Clin. Invest., 2002,109:59; Zhang et al. I Immunol., 2001,
166:6; Cheema
et al. Arthritis Rheum. 2001, 44:1313, which are all incorporated by reference
herein).
BAFF is also an effective costimulator for T cells, and this costimulation
occurs entirely
through BAFF-R (Ng et al. I Immunol., 2004, 173:807, incorporated by reference
herein).
[0004] APRIL is previously described in WO 99 12965 and US
7,276,241 B2, which
are incorporated by reference herein. The amino acid sequence of the
extracellular domain
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of APRIL is shown in Figure 30. APRIL expression and functional studies
suggest that this
protein is utilized by tumor cells to induce rapid proliferation. In addition,
APRIL may act
in other disease settings, for example, in cell proliferative diseases, such
as those that occur
in connection with some autoimmune diseases (e.g., lupus) or in inflammatory
diseases
where cell populations expand rapidly (e.g. bacterial sepsis) (US 7,276,241B2,
which is
incorporated by reference herein).
[0005] Transmembrane activator and CAML interactor (TACT) also
known as tumor
necrosis factor receptor superfamily member 13B (TNFRSF13B) is a type III
transmembrane protein. Several proteins (BAFF/BLys, APRIL, Syndecan-2) have
been
identified as TACI ligands. The interaction of TACI with its ligands induces
activation of
the transcription factors NFAT, API_ and NF-ic B and plays a crucial role in
humoral
immunity by regulation of B cell proliferation and survival. TACI activation
of B cells leads
to their differentiation and maturation, including antibody isotype switch,
and T cell-
independent antibody production (Chinen et al. J Allergy Clin Innnunol. 2011,
127(6): 1579,
incorporated by reference herein).
[0006] APRIL and BAFF can bind to receptors, such as BCMA,
BAFF-receptor
(BAFFR) and TACI, and thus neutralizing APRIL and/or BAFF can be used for
treating the
diseases, e.g. cancers, autoimmune diseases and fibrosis arising from altered
signaling
pathways through BCMA, BAFFR and/or TACI.
[0007] Current treatments for autoimmune diseases and/or
fibrotic disorders in
patients especially those with elevated levels of immunoglobulin are
inadequate due to poor
efficacy, low impact on survivorship, toxicity that causes severe side
effects, or
combinations thereof Therefore, there is a need to develop additional methods
for treating
autoimmune disease(s) and/or fibrosis in patients with elevated levels of IgA,
IgM, and/or
IgG. The present invention satisfies at least this need.
[0008] It is an object of the present invention to provide
methods of reducing
immunoglobulin production (e.g. IgA, IgM, and/or IgG, etc.) in subjects
diagnosed with
autoimmune disease(s) and/or fibrotic disorder(s) comprising administering to
the subjects
compositions comprising soluble B-cell maturation antigen (sBCMA) variants
and/or
sBCMA variant ¨ Fc fusion proteins.
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BRIEF SUMMARY OF THE INVENTION
[0009] The present invention provides inter alia, a method of
reducing
immunoglobulin production in a subject diagnosed with an autoimmune disease
and/or
fibrosis, said method comprising administering to the subject a
therapeutically effective dose
of a soluble B-cell maturation antigen (sBCMA) variant protein and/or sBCMA
variant -Fc
fusion protein. In some embodiments, the present invention provides inter
alia, a method of
reducing immunoglobulin production in a subject diagnosed with an autoimmune
disease,
said method comprising administering to the subject a therapeutically
effective dose of an
sBCMA variant protein. In some embodiments, the present invention provides
inter alia, a
method of reducing immunoglobulin production in a subject diagnosed with an
autoimmune
disease, said method comprising administering to the subject a therapeutically
effective dose
of an sBCMA variant -Fc fusion protein. In some embodiments, the present
invention
provides inter alia, a method of reducing immunoglobulin production in a
subject diagnosed
with fibrosis, said method comprising administering to the subject a
therapeutically effective
dose of an sBCMA variant protein. In some embodiments, the present invention
provides
inter alia, a method of reducing immunoglobulin production in a subject
diagnosed with
fibrosis, said method comprising administering to the subject a
therapeutically effective dose
of an sBCMA variant-Fc fusion protein.
[0010] The present invention provides inter alia, a method of
reducing production of
IgA, IgM, and/or IgG in a subject diagnosed with an autoimmune disease and/or
fibrosis,
said method comprising administering to the subject a therapeutically
effective dose of a
soluble B-cell maturation antigen (sBCMA) variant protein and/or sBCMA variant
-Fc
fusion protein. In some embodiments, the present invention provides inter
alia, a method of
reducing production of IgA, IgM, and/or IgG in a subject diagnosed with an
autoimmune
disease, said method comprising administering to the subject a therapeutically
effective dose
of an sBCMA variant protein. In some embodiments, the present invention
provides inter
alia, a method of reducing production of IgA, IgM, and/or IgG in a subject
diagnosed with
an autoimmune disease, said method comprising administering to the subject a
therapeutically effective dose of an sBCMA variant -Fc fusion protein In some
embodiments, the present invention provides inter alia, a method of reducing
production of
IgA, IgM, and/or IgG in a subject diagnosed with fibrosis, said method
comprising
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administering to the subject a therapeutically effective dose of an sBCMA
variant protein.
In some embodiments, the present invention provides inter al/a, a method of
reducing
production of IgA, IgM, and/or IgG in a subject diagnosed with fibrosis, said
method
comprising administering to the subject a therapeutically effective dose of an
sBCMA
variant-Fc fusion protein. In some embodiments, the method as disclosed herein
reduces
production of IgA. In some embodiments, the method as disclosed herein reduces
production
of IgM. In some embodiments, the method as disclosed herein reduces production
of IgG. In
some embodiments, the method as disclosed herein reduces production of both
IgA and IgM.
In some embodiments, the method as disclosed herein reduces production of both
IgA and
IgG. In some embodiments, the method as disclosed herein reduces production of
both IgM
and IgG. In some embodiments, the method as disclosed herein reduces
production of IgA,
IgM and IgG.
[0011] In one aspect, the invention provides a method of
reducing production of IgA,
IgM, and/or IgG in a subject diagnosed with an autoimmune disease or fibrosis,
said method
comprising administering to the subject a therapeutically effective dose of a
soluble B-cell
maturation antigen (sBCMA) variant-Fc fusion protein, wherein the sBCMA
variant-Fc
fusion protein comprises:
a) a variant sBCMA domain comprising at least one amino acid substitution as
compared to SEQ ID NO:1, wherein said amino acid substitution is at a position
number selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 9, 10, 11,
12, 14, 16,
19_ 20, 22, 23, 25, 26, 29, 31, 32, 35, 36, 38, 39, 42, 43, 44, 45, 46, 47,
48, 49, 50, 51,
52, 53, and 54, wherein the numbering is according to the EU index;
b) an optional linker; and
c) an Fc domain.
[0012] In an additional aspect, the invention provides the
method as disclosed herein,
wherein normal B cell viability is not altered.
[0013] In a further aspect, the invention provides the method
as disclosed herein,
wherein the method reduces production of IgA.
[0014] In an additional aspect, the invention provides the
method as disclosed herein,
wherein the method reduces production of IgM.
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[0015] In a further aspect, the invention provides the method
as disclosed herein,
wherein the method reduces production of IgG.
100161 In a further aspect, the invention provides the method
as disclosed herein,
wherein the method reduces production of both IgA and IgM.
[0017] In a further aspect, the invention provides the method
as disclosed herein,
wherein the method reduces production of both IgA and IgG.
[0018] In a further aspect, the invention provides the method
as disclosed herein,
wherein the method reduces production of both IgM and IgG.
[0019] In a further aspect, the invention provides the method
as disclosed herein,
wherein the method reduces production of IgA, IgM and IgG.
[0020] In a further aspect, the invention provides the method
as disclosed herein,
wherein the subject is diagnosed with the autoimmune disease.
[0021] In an additional aspect, the invention provides the
method as disclosed herein,
wherein the autoimmune disease is selected from the group consisting of IgA
Nephropathy,
Systemic Lupus Erythematosus, Churg-Strauss Syndrome, Myasthenia Gravis,
Multiple
Sclerosis, and rheumatoid arthritis. In an additional aspect, the invention
provides the
method as disclosed herein, wherein the autoimmune disease is Lupus.
[0022] In a further aspect, the invention provides the method
as disclosed herein,
wherein the subject is diagnosed with the fibrosis.
[0023] In an additional aspect, the invention provides the
method as disclosed herein,
wherein the fibrosis is selected from the group consisting of idiopathic
pulmonary fibrosis,
non-alcoholic steatohepatitis, scleroderma, and kidney fibrosis.
[0024] In a further aspect, the invention provides the method
as disclosed herein,
wherein said fusion protein comprises, from N- to C-terminal:
a) said variant sBCMA domain;
b) said optional linker; and
c) said Fc domain.
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[0025] In a further aspect, the invention provides the method
as disclosed herein,
wherein said fusion protein comprises, from N- to C-terminal:
a) said Fc domain;
b) said optional linker; and
c) said variant sBCMA domain.
[0026] In an additional aspect, the invention provides the
method as disclosed herein,
wherein said variant sBCMA domain has at least 80%, at least 85%, at least
90%, or at least
95% sequence identity to SEQ ID NO: 1 .
[0027] In a further aspect, the invention provides the method
as disclosed herein,
wherein said amino acid substitution(s) occur at one of said positions, two of
said positions,
three of said positions, four of said positions, five of said positions, six
of said positions,
seven of said positions, eight of said positions, or nine of said positions.
[0028] In an additional aspect, the invention provides the
method as disclosed herein,
wherein said amino acid substitution(s) is selected from the group consisting
of M1A, M1C,
M1I, M1R, M1T, M1V, L2C, L2S, Q3P, Q3R, M4E, M4I, M4T, M4V, A5T, G6E, Q7R,
S9A, S9F, S9P, Q10H, Q10P, Q10R,N11D, N11S, E12K, F14L, S16G, S16N, S16R,
H19L,
H19Y, A20V, A20T, I22M, I22V, P23S, Q25R, L26F, S29A, N31D, N31S, T32A, T32I,
T32P, L35S, L35P, T36A, T36I, T36P, Q38R, R39H, N42D, N42R, N42S, A43T, A43V,
S44D, S44G, S44N, S44R, V45A, V45M, 146A, T46I, N47D, N47K, N47R, N47S, S48L,
S48P, S48T, V49A, V49M, K50E, K50G, K5OR, K50T, G51E, T52A, T52M, N53D, N53K,
N53S, A54V, and A54T.
[0029] In a further aspect, the invention provides the method
as disclosed herein,
wherein said amino acid substitution(s) is selected from the group consisting
of M1V, L2S,
Q3P, M4T, S9P, N11D, S16G, H19Y, N31S, N31D, T32I, T36A, R39H, N47S, K50E, and
N53E.
[0030] In an additional aspect, the invention provides the
method as disclosed herein,
wherein said amino acid substitution(s) is selected from the group consisting
of S16G, H19Y
and T36A.
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100311 In a further aspect, the invention provides the method
as disclosed herein,
wherein said amino acid substitutions are selected from the group consisting
of
L2 S/S 9P/E12K/N31D/T36A/N42S/N53 S, M1V/T32P/T36A/T461/N53D/A54V,
Q3R/S16N/T36A/A43T, Fl4L/S16G/T36A/V45A/N47D,
M1T/M4V/S9F/S16G/T32A/Q38R, M1A/S9A/Q38R, G6E/Q25R/Q38R,
M1V/M4I/G6E/S9P/N11DN49M/T52M/A54V, N11D/S16G/N31 is,
Nil D/H19Y/122M/T32P/N47S/N53S, G6E/Q7R/H19Y/L35S, H19Y/N42D/S48P/T52A,
M1VN31D/T321/T3 GA, M1V/A5T/H19L/T36A,
M 1 T/N31D/132A/136A/Q38R/S44DN49A/K5 OE, MIV/T36A/Q38R/A43V,
M1V/L2S/S9P/Q10H/T36A/Q38R/K50G, T36A/Q38R/N53S,
M1T/L2S/L35P/T36A/Q38R/T46A/K5OR, A5T/A20V/T36A/Q38R,
M1T/S16G/122V/T36 A/S44G/T46 AN49 A, Sl6G/T36A,
MIEN 11D/S16G/122M/S29A/T36A/S 44G/K5 OR, M1C/L2C/Q3R/M4E/N11D/S16G/T36P,
M1I/N11D/S16G/122M/S29A/T36A/S44G/K5OR,
N11D/N31D/T321/T36A/S44N/N47D/N53D, M1R/L2C/Q3R, H19Y/T36A/S44G,
H19Y/T321/T36AN49A, H19YN31 S/T36AN45 A, H19Y/N31S/T36A, H19Y/T36P/T52A,
H19Y/N31D/T52M, M1V/H19YN45M, Sl6G/H19Y/N47D, S 1 6G/H19Y/K50T,
S 1 6G/H19Y/S44N/K5OR. N11D/H19Y/S48T,
S9P/N11D/S16R/T32A/Q38R/S44G/T461/T52A/N53D/A54T, N11D/S16G/S44R,
H19L/T32A/S44G/G51E/T52A, Sl6N/H19Y/T36A/K5OR, M1V/H19Y/T36A/R39H/T46A,
M1V/H19Y/T36A, H19Y/T36A/N42D/N47S/S48P, M1 V/H19Y/T36A/S44G/N47D,
M1V/H19Y/T36A/N42R/N53S, H19Y/L35P/T36A/N42D/T461N49A,
Q3P/S 9P/H19Y/N31S/T36A/R39H/N47R/K5 OE, M1V/H19Y/T36A/N42R/N53S,
M1T/H19Y/T36A, M1V/S16N/H19Y/122M/T36A,
M1T/N11D/H19Y/T36A/N42SN45A/N53S, N11D/S16G/H19Y/T36A/N47S/N53D,
M1V/S9P/Q10P/S16G/H19Y/L26F/T36A/A43V/N53D, Sl6G/H19Y/T36A/V49A/N53D,
Sl6G/T36A/A43T/S44GN45M, M4V/S9P/S16G/T36A/Q38R,
S9P/N11S/S16G/T36A/Q38R, N11D/E12K/S16R/T36A/T52M,
M4V/T321/T36A/Q38R/A43TN45 A/SLIM), 59P/N11D/S16G/Q25R,
M1T/A5T/S 9P/S16G/Q25R/N31D/V 49M,
L2 S/S 9P/S16G/A20T/T321/Q38R/N42D/T46A/S48L, Sl6G/Q25R/T46A,
G6E/S9A/S16G/Q25R/N31D/N47S/T52M, H19Y/Q38R/T52M,
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N11D/H19Y/122M/T32P/N47S/N53S, Sl6G/H19Y/T36A, Sl6G/H19Y/T36A/N53D,
S9P/N11D/S16G/H19Y/T36A/N47S/N53D,
Q3P/S9P/H19Y/N31S/T36A/R39H/N47R/K50E, M1V/L2S/M4T/N11D/H19Y/T36A,
M1V/L2S/M4T/N11D/T36A, M1V/L2S/M4T/H19Y/T361/V45A/V49M,
M1V/L2S/M4T/N11D/H19Y/T36A, M1V/L2S/M4T/S 9P/Q 10R/H19Y/T36A/T46A/N47S,
M1V/L2S/M4T/S16G/N31D/T321/T36A, M1V/M4T/T36A/Q38R/N53K,
M1T/N11D/H19Y/T36A/N42SN45A/N53S,
M1T/N31D/T32A/T36A/A38R/S44DN49A/K50E,
M1T/S9P/P23S/Q38R/N42S/S48PN49A/A54V, H19Y/T36A/S44G, H19Y/T36A, and
M4T/T36A/Q38R/N42S/S44G/T46A/N47K/S48P/T52A.
[0032] In an additional aspect, the invention provides the
method as disclosed herein,
wherein said variant sBCMA domain comprises the amino acid substitutions
Sl6G/H19Y/T36A, and at least one further amino acid substitution selected from
the group
consisting of M1A, M1C, M1I, MIR, M1T, M1V, L2C, L2S, Q3P, Q3R, M4E, M4I, M4T,
M4V, A5T, G6E, Q7R, S9A, S9F, S9P, QI0H, QI0P, QIOR, NI ID, NI IS, El2K, F14L,
Sl6N, Sl6R, H19L, A20V, A20T, I22M, I22V, P23S, Q25R, L26F, S29A, N31D, N31S,
T32A, T32I, T32P, L35S, L35P, T36I, T36P, Q38R, R39H, N42D, N42R, N42S, A43T,
A43V, S44D, S44G, S44N, S44R, V45A, V45M, T46A, T46I, N47D, N47K, N47R, N47S,
S48L, S48P, S48T, V49A, V49M, K50E, K50G, K5OR, K50T, G51E, T52A, 152M, N53D,
N53K, N53S, A54V, and A54T.
[0033] In a further aspect, the invention provides the method
as disclosed herein,
wherein said variant sBCMA domain comprises the amino acid substitutions
Sl6G/H19Y/T36A/N53D, and at least one further amino acid substitution selected
from the
group consisting of M1A, M1C, M1I, M1R, M1T, M1V, L2C, L2S, Q3P, Q3R, M4E,
M4I,
M4V, A5'1', Ci6E, Q7R, S9A, S9F, S9P, QI0H, QI0P, Q1OR, NI Ill, N I IS, El2K,
Fl4L, Sl6N, Sl6R, H19L, A20V, A20T, I22M, I22V, P23S, Q25R, L26F, S29A, N31D,
N31S, T32A, T32I, T32P, L35S, L35P, T36I, T36P, Q38R, R39H, N42D, N42R, N42S,
A43T, A43V, S44D, S44G, S44N, S44R, V45A, V45M, T46A, T461, N47D, N47K, N47R,
N47S, S48L, S48P, S48T, V49A, V49M, K50E, K50G, K5OR, K50T, G51E, T52A, T52M,
N53K, N53S, A54V, and A54T.
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[0034] In an additional aspect, the invention provides the
method as disclosed herein,
wherein said variant sBCMA domain comprises the amino acid substitutions
S9P/N11D/S16G/H19Y/T36A/N47S/N53D, and at least one further amino acid
substitution
selected from the group consisting of M1A, M1C, Mu, M1R, MIT, M1V, L2C, L2S,
Q3P,
Q3R, M4E, M4I, M4T, M4V_ A5T, G6E, Q7R, S9A, S9F, Q10H, Q 10P, Q10R, NlIS,
E12K, F14L, S16N, S16R, H19L, A20V, A20T, I22M, I22V, P23S, Q25R, L26F, S29A,
N31D, N31S, T32A, T321, T32P, L35S, L35P, T36I, T36P, Q38R, R39H, N42D, N42R,
N42S, A43T, A43V, S44D, S44G, S44N, S44R, V45A, V45M, T46A, T46I, N47D, N47K,
N47R, S48L, S48P, S481, V49A, V49M, K50E, K50G, K5OR, K50T, G51E, 152A, 152M,
N53K, N53S, A54V, and A54T.
100351 In a further aspect, the invention provides the method
as disclosed herein,
wherein said variant sBCMA domain comprises the amino acid substitutions
Q3P/S9P/H19Y/N31S/T36A/R39H/N47R/K50E, and at least one further amino acid
substitution selected from the group consisting of M1A, M1C, M1I, M1R, M1T,
M1V, L2C,
L2S, Q3R, M4E, M4I, M4T, M4V, A5T, G6E, Q7R, S9A, S9F, QI0H, QI0P, Q 10R, NI
ID,
NI IS, E12K, F14L, S16G, S16N, S16R, H19L, A20V, A20T, I22M, I22V, P23S, Q25R,
L26F, S29A, N31D, T32A, T32I, T32P, L35S, L35P, T36I, T36P, Q38R, N42D, N42R,
N42S, A43T, A43V, S44D, S44G, S44N, S44R, V45A, V45M, T46A, T46I, N47D, N47K,
N47S, S48L, S48P, S48T, V49A, V49M, K50G, K5OR, K50T, G51E, T52A, T52M, N53D,
N53K, N53S, A54V, and A54T.
[0036] In an additional aspect, the invention provides the
method as disclosed herein,
wherein said variant sBCMA domain comprises the amino acid substitutions
M1V/L2S/M4T/S16G/N31D/T321/T36A, and at least one further amino acid
substitution
selected from the group consisting of M1A, M1C, M1I, M1R, M1T, L2C, Q3P, Q3R,
M4E,
M41, M4V, AST, G6E, Q7R, S9A, S9F, S9P, QI0H, QI0P, Q1OR, N I Ill, N I IS,
E12K,
F14L, S16N, S16R, H19L, H19Y, A20V, A20T, I22M, I22V, P23S, Q25R, L26F, S29A,
N31S, T32A, T32P, L35S, L35P, T36I, T36P, Q38R, R39H, N42D, N42R, N42S, A43T,
A43V, S44D, S44G, S44N, S44R, V45A, V45M, T46A, T46I, N47D, N47K, N47R, N47S,
S48L, S48P, S48T, V49A, V49M, K50E, K50G, K5OR, K50T, G51E, T52A, 152M, N53D,
N53K, N53S, A54V, and A54T.
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[0037] In a further aspect, the invention provides the method
as disclosed herein,
wherein said variant sBCMA domain has at least 90% sequence identity to SEQ ID
NO: 67.
100381 In an additional aspect, the invention provides the
method as disclosed herein,
wherein said variant sBCMA domain has at least 90% sequence identity to SEQ ID
NO: 68.
[0039] In a further aspect, the invention provides the method
as disclosed herein,
wherein said variant sBCMA domain has at least 90% sequence identity to SEQ ID
NO: 69.
[0040] In an additional aspect, the invention provides the
method as disclosed herein,
wherein said variant sBCMA domain has at least 90% sequence identity to SEQ ID
NO: 49.
[0041] In a further aspect, the invention provides the method
as disclosed herein,
wherein said variant sBCMA domain has at least 90% sequence identity to SEQ ID
NO: 74.
[0042] In an additional aspect, the invention provides the
method as disclosed herein,
wherein said variant sBCMA domain has the amino acid sequence of SEQ ID NO:
67.
[0043] In a further aspect, the invention provides the method
as disclosed herein,
wherein said variant sBCMA domain has the amino acid sequence of SEQ ID NO:
68.
[0044] In an additional aspect, the invention provides the
method as disclosed herein,
wherein said variant sBCMA domain has the amino acid sequence of SEQ ID NO:
69.
[0045] In a further aspect, the invention provides the method
as disclosed herein,
wherein said variant sBCMA domain has the amino acid sequence of SEQ ID NO:
49.
[0046] In an additional aspect, the invention provides the
method as disclosed herein,
wherein said variant sBCMA domain has the amino acid sequence of SEQ ID NO:
74.
[0047] In a further aspect, the invention provides the method
as disclosed herein,
wherein said Fc domain is a human IgG Fc domain or a variant human IgG Fc
domain.
[0048] In an additional aspect, the invention provides the
method as disclosed herein,
wherein said human IgG Fc domain comprises the hinge-CH2-CH3 of human IgGl.
[0049] In a further aspect, the invention provides the method
as disclosed herein,
wherein said Fc domain is a variant human IgG Fc domain.
100501 In an additional aspect, the invention provides the
method as disclosed herein,
wherein said Fc domain is a human IgG1 Fc domain.
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[0051] In a further aspect, the invention provides the method
as disclosed herein,
wherein said linker is SEQ ID NO:87.
100521 In an additional aspect, the invention provides the
method as disclosed herein,
wherein said linker is selected from the group consisting of (GS)n, (GSGGS)n,
(GGGGS)n,
and (GGGS)n, wherein n is selected from the group consisting of 1, 2, 3, 4 and
5.
[0053] In a further aspect, the invention provides the method
as disclosed herein,
wherein said linker is SEQ ID NO:88.
[0054] In an additional aspect, the invention provides the
method as disclosed herein,
wherein the sBCMA variant ¨ Fc fusion protein comprises the amino acid
sequence of SEQ
ID NO:80.
[0055] In a further aspect, the invention provides the method
as disclosed herein,
wherein the sBCMA variant ¨ Fc fusion protein comprises the amino acid
sequence of SEQ
ID NO:81.
100561 In an additional aspect, the invention provides the
method as disclosed herein,
wherein the sBCMA variant ¨ Fc fusion protein comprises the amino acid
sequence of SEQ
ID NO:82.
[0057] In a further aspect, the invention provides the method
as disclosed herein,
wherein the sBCMA variant ¨ Fc fusion protein comprises the amino acid
sequence of SEQ
ID NO:83.
[0058] In an additional aspect, the invention provides the
method as disclosed herein,
wherein the sBCMA variant ¨ Fc fusion protein comprises the amino acid
sequence of SEQ
ID NO:84.
IV. BRIEF DESCRIPTION OF THE DRAWINGS
[0059] Figure 1 shows nonhuman single dose toxicity study
design.
[0060] Figure 2 shows immune cell counts in male cynomolgus
monkeys in single
dose toxicity studies treated with vehicle control and variant sBCMA Fc at
0.1mg/kg,
lmg/kg, 10mg/kg and 100mg/kg. Figure 2A, CD3+CD4+ T Lymphocyte. Figure 2B,
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CD3+CD8+ Cytotoxic T Cells. Figure 2C, CD3-CD16+ NK Cells. Figure 2D, CD3-
CD19+
Pan B Cells. Figure 2E, CD3-CD20+ Mature B Cells.
100611 Figure 3 shows immune cell counts in female cynomolgus
monkeys in single
dose toxicity studies treated with vehicle control and variant sBCMA Fc at
0.1mg/kg,
lmg/kg, 10mg/kg and 100mg/kg. Figure 3A, CD3+CD4+ T Lymphocyte. Figure 3B,
CD3+CD8+ Cytotoxic T Cells. Figure 3C, CD3-CD16+ NK Cells. Figure 3D, CD3-
CD19+
Pan B Cells. Figure 3E, CD3-CD20+ Mature B Cells.
[0062] Figure 4 shows body weight over time in male 4A and
female 4B cynomolgus
monkeys in single dose toxicity studies treated with vehicle control and
variant sBCMA Fc
at 0.1mg/kg, lmg/kg, 10mg/kg and 100mg/kg.
[0063] Figure 5 shows total lymphocyte counts over time in
male 5A and female 5B
cynomolgus monkeys in single dose toxicity studies treated with vehicle
control and variant
sBCMA Fc at 0.1mg/kg, lmg/kg, 10mg/kg and 100mg/kg.
[0064] Figure 6 shows changes of immunoglobulin levels over
time in female
cynomolgus monkeys in single dose toxicity studies treated with vehicle
control and variant
sBCMA Fc at 0.1mg/kg, lmg/kg, 10mg/kg and 100mg/kg.
[0065] Figure 7 shows changes of immunoglobulin over time in
male cynomolgus
monkeys in single dose toxicity studies treated with vehicle control and
variant sBCMA Fc
at 0.1mg/kg, lmg/kg, 10mg/kg and 100mg/kg.
[0066] Figure 8 shows hematology panel I in male cynomolgus
monkeys in single
dose toxicity studies treated with vehicle control and variant sBCMA Fc at
0.1mg/kg,
lmg/kg, 10mg/kg and 100mg/kg. Abbreviations: RBC (Red Blood Cells), HGB
(Hemoglobin), HCT (Hematocrit), MCV (Mean Corpuscular Volume), MCH (Mean
Corpuscular Hemoglobin), MCHC (Mean Corpuscular Hemoglobin Concentration), RDW
(Red Cell Distribution Width), RET (Reticulocytes (Absolute)).
[0067] Figure 9 shows hematology panel II in male cynomolgus
monkeys in single
dose toxicity studies treated with vehicle control and variant sBCMA Fc at
0.1mg/kg,
lmg/kg, 10mg/kg and 100mg/kg. Abbreviations: PLT (Platelets), WBC (White Blood
Cells), NEUT (Neutrophils (Absolute)), LYNIP (Lymphocytes (Absolute)), MONO
(Monocytes (Absolute)), EOS (Eosinophils (Absolute)), BASO (Basophils
(Absolute)).
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[0068] Figure 10 shows hematology panel Tin female cynomolgus
monkeys in single
dose toxicity studies treated with vehicle control and variant sBCMA Fc at
0.1mg/kg,
lmg/kg, 10mg/kg and 100mg/kg. Abbreviations: RBC (Red Blood Cells), HGB
(Hemoglobin), HCT (Hematocrit), MCV (Mean Corpuscular Volume), MCH (Mean
Corpuscular Hemoglobin), MCHC (Mean Corpuscular Hemoglobin Concentration), RDW
(Red Cell Distribution Width), RET (Reticulocytes (Absolute)).
[0069] Figure 11 shows hematology panel II in female
cynomolgus monkeys in
single dose toxicity studies treated with vehicle control and variant sBCMA Fc
at 0.1mg/kg,
lmg/kg, 10mg/kg and 100mg/kg. Abbreviations: PLT (Platelets), WBC (White Blood
Cells), NEUT (Neutrophils (Absolute)), LYMP (Lymphocytes (Absolute)), MONO
(Monocytes (Absolute)), EOS (Eosinophils (Absolute)), BASO (Basophils
(Absolute)).
[0070] Figure 12 shows coagulation panel in male cynomolgus
monkeys in single
dose toxicity studies treated with vehicle control and variant sBCMA Fc at
0.1mg/kg,
lmg/kg, 10mg/kg and 100mg/kg. Abbreviations: PT (Prothrombin Time), APTT
(Activated
Partial Thromboplastin Time), FIB (Fibrinogen).
[0071] Figure 13 shows coagulation panel in female cynomolgus
monkeys in single
dose toxicity studies treated with vehicle control and variant sBCMA Fc at
0.1mg/kg,
lmg/kg, 10mg/kg and 100mg/kg. Abbreviations: PT (Prothrombin Time), APTT
(Activated
Partial Thromboplastin Time), FIB (Fibrinogen).
[0072] Figure 14 shows chemistry panel Tin male cynomolgus
monkeys in single
dose toxicity studies treated with vehicle control and variant sBCMA Fc at
0.1mg/kg,
lmg/kg, 10mg/kg and 100mg/kg. Abbreviations: ALT (Alanine Aminotransferase),
AST
(Aspartate Aminotransferase), ALP (Alkaline Phosphatase), GGT (Gamma Glutamyl
Transferase), CK (Creatine Kinase), TB1L (Total Bilirubin), GLU (Glucose).
[0073] Figure 15 shows chemistry panel II in male cynomolgus
monkeys in single
dose toxicity studies treated with vehicle control and variant sBCMA Fc at
0.1mg/kg,
lmg/kg, 10mg/kg and 100mg/kg. Abbreviations: UREA (Urea), CREA (Creatinine),
TG
(Triglycerides), CHOL (Total Cholesterol), TP (Total Protein), ALB (Albumin),
GLOB
(Globulin).
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[0074] Figure 16 shows chemistry panel III in male cynomolgus
monkeys in single
dose toxicity studies treated with vehicle control and variant sBCMA Fc at
0.1mg/kg,
lmg/kg, 10mg/kg and 100mg/kg. Abbreviations: A/G (Albumin/Globulin Ratio), Na
(Sodium), K (Potassium Chloride), Cl (Chloride), IgA (Immunoglobulin A), IgG
(Immunoglobulin G), IgM (Immunoglobulin M).
[0075] Figure 17 shows chemistry panel I in female cynomolgus
monkeys in single
dose toxicity studies treated with vehicle control and variant sBCMA Fc at
0.1mg/kg,
lmg/kg, 10mg/kg and 100mg/kg. Abbreviations: ALT (Alanine Aminotransferase),
AST
(Aspartate Aminotransferase), ALP (Alkaline Phosphatase), GGT (Gamma Glutamyl
Transferase), CK (Creatine Kinase), TBIL (Total Bilirubin), GLU (Glucose).
[0076] Figure 18 shows chemistry panel II in female cynomolgus
monkeys in single
dose toxicity studies treated with vehicle control and variant sBCMA Fc at
0.1mg/kg,
lmg/kg, 10mg/kg and 100mg/kg. Abbreviations: UREA (Urea), CREA (Creatinine),
TG
(Triglycerides), CHOL (Total Cholesterol), TP (Total Protein), ALB (Albumin),
GLOB
(Globulin).
[0077] Figure 19 shows chemistry panel III in female
cynomolgus monkeys in single
dose toxicity studies treated with vehicle control and variant sBCMA Fc at
0.1mg/kg,
lmg/kg, 10mg/kg and 100mg/kg. Abbreviations: A/G (Albumin/Globulin Ratio), Na
(Sodium), K (Potassium Chloride), Cl (Chloride), IgA (Immunoglobulin A), IgG
(Immunoglobulin G), IgM (Immunoglobulin M).
[0078] Figure 20 shows cytokine panel Tin male cynomolgus
monkeys in single dose
toxicity studies treated with vehicle control and variant sBCMA Fc at
0.1mg/kg, lmg/kg,
10mg/kg and 100mg/kg. Abbreviations: PrDOM (Pre Dose 0 min), 1HPD (1 Hr Post
Dose),
3HPD (3 Hr Post Dose), 8HPD (8 Hr Post Dose).
[0079] Figure 21 shows cytokine panel II in male cynomolgus
monkeys in single
dose toxicity studies treated with vehicle control and variant sBCMA Fc at
0.1mg/kg,
lmg/kg, 10mg/kg and 100mg/kg. Abbreviations: PrDOM (Pre Dose 0 min), 1HPD (1
Hr Post
Dose), 3HPD (3 Hr Post Dose), 8HPD (8 Hr Post Dose).
[0080] Figure 22 shows cytokine panel I in female cynomolgus
monkeys in single
dose toxicity studies treated with vehicle control and variant sBCMA Fc at
0.1mg/kg,
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lmg/kg, 10mg/kg and 100mg/kg. Abbreviations: PrDOM (Pre Dose 0 min), 1HPD (1
Hr Post
Dose), 3HPD (3 Hr Post Dose), 8HPD (8 Hr Post Dose).
100811 Figure 23 shows cytokine panel II in female cynomolgus
monkeys in single
dose toxicity studies treated with vehicle control and variant sBCMA Fc at
0.1mg/kg,
lmg/kg, 10mg/kg and 100mg/kg. Abbreviations: PrDOM (Pre Dose 0 min), 1HPD (1
Hr Post
Dose), 3HPD (3 Hr Post Dose), 8HPD (8 Hr Post Dose).
[0082] Figure 24A shows viable cell density of sBCMA variant
clone pools at 11
days during Feb batch culture. Each line represents cell growth of pooled
clones grown in
HyCell CHO or BalanCD CHO. Figure 24B shows viability of sBCMA variant clone
pools
at 11 days during Feb batch culture. Each line represents cell growth of
pooled clones grown
in HyCell CHO or BalanCD CHO.
[0083] Figure 25 shows ProA-purified materials and
glycosylation study of sBCMA
variant clones in HyCell CHO or BalanCD CHO in the presence and the absence of
PNGaseF using SDS-PAGE in non-Reduced (top) or Reduced (bottom) form.
[0084] Figure 26 shows titer, IVCD and viability of sBCMA
variant single cell
clones at 12 day Fed batch experiment. IVCD (The Integral of Viable Cell
Density).
[0085] Figure 27 shows N-glycan profiles of sBCMA variant top
10 clones at 12 day
Fed batch experiment.
[0086] Figures 28A-280 show the sequences of sBCMA variant
clones as compared
to the sequence of the extracellular domain of wild-type human BCMA as set
forth in SEQ
ID NO: 1. Figure 28A shows the sequences of S3 clones# 1-12. Figure 28B shows
the
sequences of S4 clones# 13-41. Figure 28C shows the sequences of S5 clones# 42-
74.
Figure 2811 shows the sequences of S6 clones# 75-118.
[0087] Figure 29 shows the amino acid sequences of sBCMA
variant ¨ Fc fusion
proteins as set forth in SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID
NO:83,
and SEQ ID NO:84. The variant sBCMA domain is underlined, the linker domain is
bolded
and the human IgG1 Fc domain is italic.
[0088] Figure 30 shows the amino acid sequences of the
extracellular domain of wild
type human BCMA (SEQ ID NO:1), the extracellular domain of APRIL (SEQ ID
NO:85),
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the extracellular domain of BAFF (SEQ ID NO:86), a linker domain (SEQ ID
NO:87) and
another linker domain (SEQ ID NO:88).
100891 Figures 31 and 32 show results from evaluation of sBCMA
Variants on lupus
model in NZBWF1/J mice + Pristane
V. DETAILED DESCRIPTION OF THE INVENTION
[0090] In order to more clearly and concisely point out the
subject matter of the
claimed invention, the following definitions are provided for specific terms
used in the
following written description and appended claims.
A. Introduction
[0091] The present invention is directed to the use of soluble
forms of human BCMA
that contain amino acid modifications, e.g. variant sBCMA proteins. These
variant sBCMA
proteins bind to either one or both of the BCMA ligands, human BAFF and/or
human
APRIL, with tighter affinity than wild type human BCMA. APRIL and BAFF can
bind to
receptors, such as BCMA, BAFFR and TACI, and thus neutralizing APRIL and/or
BAFF
can be used for treating diseases arising from altered signaling pathways
through BCMA,
BAFFR and/or TACI. These diseases include autoimmune diseases and fibrosis.
Neutralizing APRIL alone can be effective in treating autoimmune diseases and
fibrosis
expressing high levels of BCMA and TACT or other receptors that are activated
through
binding to APRIL. Neutralizing BAFF alone can be effective in treating
fibrosis and
autoimmune diseases expressing BCMA, BAFFR and TACI or other receptors that
are
activated through binding to BAFF. Therefore, the variant sBCMA as described
herein can
be used to treat immunomodulatory disorders and/or fibrotic diseases
expressing BCMA,
BAFFR, TACI and/or any other receptors that are activated through binding to
APRIL
and/or BAFF by binding more tightly, and thus preferentially, to the ligand(s)
e.g. APRIL
and/or BAFF and thus altering the normal receptor signaling that would
otherwise occur
between BCMA, BAFFR and/or TACI on the surface of a cell with APRIL or BAFF.
[0092] In some embodiments, the present invention provides
methods of reducing
immunoglobulin production in subjects diagnosed with autoimmune disease(s)
and/or
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fibrosis comprising administering to the subjects a therapeutically effective
dose of sBCMA
variant protein(s). In some embodiments, the present invention provides
methods of
reducing production of IgA, IgM and/or IgG in subjects diagnosed with
autoimmune
disease(s) and/or fibrosis comprising administering to the subjects a
therapeutically effective
dose of sBCMA variant protein(s).
[0093] In some embodiments, the present invention provides
methods of reducing
production of IgA, IgM or both in subjects diagnosed with autoimmune
disease(s) and/or
fibrosis comprising administering to the subjects a therapeutically effective
dose of sBCMA
variant protein(s). In some embodiments, the present invention provides
methods of
reducing production of IgA, IgG or both in subjects diagnosed with autoimmune
disease(s)
and/or fibrosis comprising administering to the subjects a therapeutically
effective dose of
sBCMA variant protein(s). In some embodiments, the present invention provides
methods of
reducing production of IgG. IgM or both in subjects diagnosed with autoimmune
disease(s)
and/or fibrosis comprising administering to the subjects a therapeutically
effective dose of
sBCMA variant protein(s).
[0094] In some embodiments, the present invention provides
methods of reducing
production of IgA in subjects diagnosed with autoimmune disease(s) and/or
fibrosis
comprising administering to the subjects a therapeutically effective dose of
sBCMA variant
protein(s). In some embodiments, the present invention provides methods of
reducing
production of IgM in subjects diagnosed with autoimmune disease(s) and/or
fibrosis
comprising administering to the subjects a therapeutically effective dose of
sBCMA variant
protein(s). In some embodiments, the present invention provides methods of
reducing
production of IgG in subjects diagnosed with autoimmune disease(s) and/or
fibrosis
comprising administering to the subjects a therapeutically effective dose of
sBCMA variant
protein(s).
[0095] In some embodiments, the present invention provides
methods of reducing
production of IgA and IgM in subjects diagnosed with autoimmune disease(s)
and/or fibrosis
comprising administering to the subjects a therapeutically effective dose of
sBCMA variant
protein(s). In some embodiments, the present invention provides methods of
reducing
production of IgA and IgG in subjects diagnosed with autoimmune disease(s)
and/or fibrosis
comprising administering to the subjects a therapeutically effective dose of
sBCMA variant
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protein(s). In some embodiments, the present invention provides methods of
reducing
production of IgG and IgM in subjects diagnosed with autoimmune disease(s)
and/or fibrosis
comprising administering to the subjects a therapeutically effective dose of
sBCMA variant
protein(s). In some embodiments, the present invention provides methods of
reducing
production of IgA, IgM and IgG in subjects diagnosed with autoimmune
disease(s) and/or
fibrosis comprising administering to the subjects a therapeutically effective
dose of sBCMA
variant protein(s).
[0096] Additionally, in some embodiments, since the sBCMA
variants are small
proteins that generally are cleared rapidly from the bloodstream, the
invention provides
fusion proteins that link the sBCMA variant to a human or variant Fc domain as
discussed
herein. Since Fc domains, through binding to the FcRn receptor, confer
extended half-life in
serum, the creation of an sBCMA variant-Fc domain fusion proteins results in
improved
therapies. Thus, the invention provides sBCMA domain-Fe domain fusion
proteins, referred
sometimes herein as "fusion proteins". In some embodiments, the sBCMA variant
or the
variant sBCMA domain of the fusion protein as described herein exhibits
enhanced binding
affinity for APRIL as compared to SEQ ID NO: 1. In some embodiments, the sBCMA
variant or the variant sBCMA domain of the fusion protein as described herein
exhibits
enhanced binding affinity for BAFF as compared to SEQ ID NO: 1. In some
embodiments,
the sBCMA variant or the variant sBCMA domain of the fusion protein as
described herein
exhibits enhanced binding affinity for APRIL and BAFF as compared to SEQ ID
NO: 1.
[0097] In some embodiments, the present invention provides
methods of reducing
immunoglobulin production in subjects diagnosed with autoimmune disease(s)
and/or
fibrosis comprising administering to the subjects a therapeutically effective
dose of sBCMA
variant ¨ Fc fusion protein(s). In some embodiments, the present invention
provides methods
of reducing production of IgA, 1gM and/or IgG in subjects diagnosed with
autoimmune
disease(s) and/or fibrosis comprising administering to the subjects a
therapeutically effective
dose of sBCMA variant ¨ Fc fusion protein(s).
[0098] In some embodiments, the present invention provides
methods of reducing
production of IgA, IgM or both in subjects diagnosed with autoimmune
disease(s) and/or
fibrosis comprising administering to the subjects a therapeutically effective
dose of sBCMA
variant ¨ Fc fusion protein(s). In some embodiments, the present invention
provides methods
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of reducing production of IgA, IgG or both in subjects diagnosed with
autoimmune
disease(s) and/or fibrosis comprising administering to the subjects a
therapeutically effective
dose of sBCMA variant ¨ Fc fusion protein(s). In some embodiments, the present
invention
provides methods of reducing production of IgG. IgM or both in subjects
diagnosed with
autoimmune disease(s) and/or fibrosis comprising administering to the subjects
a
therapeutically effective dose of sBCMA variant ¨ Fc fusion protein(s).
[0099] In some embodiments, the present invention provides
methods of reducing
production of IgA in subjects diagnosed with autoimmune disease(s) and/or
fibrosis
comprising administering to the subjects a therapeutically effective dose of
sBCMA variant
¨ Fc fusion protein(s). In some embodiments, the present invention provides
methods of
reducing production of IgM in subjects diagnosed with autoimmune disease(s)
and/or
fibrosis comprising administering to the subjects a therapeutically effective
dose of sBCMA
variant ¨ Fc fusion protein(s). In some embodiments, the present invention
provides methods
of reducing production of IgG in subjects diagnosed with autoimmune disease(s)
and/or
fibrosis comprising administering to the subjects a therapeutically effective
dose of sBCMA
variant ¨ Fc fusion protein(s).
[00100] In some embodiments, the present invention provides
methods of reducing
production of IgA and IgM in subjects diagnosed with autoimmune disease(s)
and/or fibrosis
comprising administering to the subjects a therapeutically effective dose of
sBCMA variant
¨ Fc fusion protein(s). In some embodiments, the present invention provides
methods of
reducing production of IgA and IgG in subjects diagnosed with autoimmune
disease(s)
and/or fibrosis comprising administering to the subjects a therapeutically
effective dose of
sBCMA variant ¨ Fc fusion protein(s). In some embodiments, the present
invention provides
methods of reducing production of IgG and IgM in subjects diagnosed with
autoimmune
disease(s) and/or fibrosis comprising administering to the subjects a
therapeutically effective
dose of sBCMA variant ¨ Fc fusion protein(s). In some embodiments, the present
invention
provides methods of reducing production of IgA, IgM and IgG in subjects
diagnosed with
autoimmune disease(s) and/or fibrosis comprising administering to the subjects
a
therapeutically effective dose of sBCMA variant ¨ Fc fusion protein(s).
B. Definitions
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[00101] As used herein, the following terms have the meanings
ascribed to them unless
specified otherwise.
1001021 The terms "a", "an", or "the" as used herein not only
include aspects with one
member, but also include aspects with more than one member. For instance, the
singular
forms "a", "an", and "the" include plural referents unless the context clearly
dictates
otherwise. Thus, for example, reference to "a cell" includes a plurality of
such cells and
reference to "the agent" includes reference to one or more agents known to
those skilled in
the art, and so forth.
1001031 As used herein, "protein" herein is meant at least two
covalently attached
amino acids, which includes proteins, polypeptides, oligopeptides and
peptides.
[00104] The term "isolated" refers to a molecule that is
substantially free of its natural
environment and devoid of other proteins. For instance, an isolated protein is
substantially
free of cellular material or other proteins from the cell or tissue source
from which it is
derived. The term "isolated" also refers to preparations where the isolated
protein is
sufficiently pure to be administered as a pharmaceutical composition, or at
least about 70-
80%, 80-90%, or 90-95% (w/w) pure, or at least about 95%, 96%, 97%, 98%, 99%,
or 100%
(w/w) pure. In particular, it is preferred that the polypeptides are in
"essentially pure form",
i.e., that the polypeptide preparation is essentially free of other
polypeptide material with
which it is natively associated. This can be accomplished, for example, by
preparing the
polypeptide by means of well-known recombinant methods or by classical
purification
methods.
[00105] The term B-cell maturation antigen "BCMA" refers to the
protein for B cell
maturation as described in Gras et al. International Immunology, 1995, 7:1093;
Y. Laabi et
al. EMBO J., 1992, 11:3897. BCMA is a member of the TNF-receptor superfamily.
For
example, BCMA is a receptor for APRIL and BAFF. The amino acid sequence of the
extracellular domain of the wild type human BCMA (SEQ ID NO:1) is shown in
Table 3
and Figure 30.
[00106] The term "ligand" refers to a biomolecule that is able
to bind to and form a
complex with a second biomolecule such as a receptor present on the surface of
target cells
to serve a biological purpose. A ligand is generally an effector molecule that
binds to a site
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on a target protein, e.g., by intermolecular forces such as ionic bonds,
hydrogen bonds,
hydrophobic interactions, dipole-dipole bonds, or Van der Waals forces. In the
present
invention, APRIL and BAFF are ligand proteins.
[00107] The term "receptor" refers to a biomolecule present on
the surface of a target
cell that is able to bind to and form a complex with a second biomolecule such
as a ligand. A
receptor generally activates a specific signal transduction pathway. For
example, BCMA is a
receptor for APRIL and BAFF, members of the TNF family.
[00108] By "position" as used herein is meant a location in the
sequence of a protein.
In some embodiments of the present invention, positions are numbered
sequentially starting
with the first amino acid of the mature protein, for example for the human
BCMA protein
shown in Figure 28. In some cases, for example for the Fc domain portion of
the fusion
proteins described herein, the Fc domain positions may be numbered
sequentially, or
according to an established format, for example the EU index. The EU index or
EU index as
in Kabat or EU numbering scheme refers to the EU numbering (see SEQUENCES OF
IMMUNOLOGICAL INTEREST, 5th edition, NIH publication, No. 91-3242, E.A. Kabat
et
al., entirely incorporated by reference; and see also Edelman et al., 1969,
Proc Natl Acad Sci
USA 63:78-85, hereby entirely incorporated by reference).
[00109] By "amino acid modification" or "amino acid sequence
modification" herein is
meant an amino acid substitution, insertion, and/or deletion in a polypeptide
sequence.
[00110] By "parent protein" as used herein is meant a starting
protein that is
subsequently modified to generate a variant. The parent protein may be a
naturally occurring
protein, or a variant or engineered version of a naturally occurring protein.
Parent protein
may refer to the protein itself, compositions that comprise the parent
protein, or the amino
acid sequence that encodes it. In this context, a -parent Fc domain" will be
relative to the
recited variant; thus, a "variant human IgG Fc domain" is compared to the
parent Fc domain
of human IgG, for example, a "variant human IgG1 Fc domain" is compared to the
parent Fc
domain of human IgG1 , etc.
[00111] By "wild type" or "WT" herein is meant an amino acid
sequence or a
nucleotide sequence that is found in nature, including allelic variations. A
WT protein has an
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amino acid sequence or a nucleotide sequence that has not been intentionally
modified into a
non-naturally occurring sequence.
1001121 By "variant protein" or "protein variant", or "variant"
as used herein is meant a
protein with an amino acid sequence which differs from that of a parent
protein by virtue of
at least one amino acid sequence modification. For example, -variant sBCMA" or
"sBCMA
variant" as used herein is meant a protein with an amino acid sequence which
differs from
that of a parent sBCMA protein by virtue of at least one amino acid sequence
modification
yet still retains the ability to bind to a cognate ligand, as outlined below.
In some
embodiments, the parent proteins are human wild type sequences. In some
embodiments,
the parent proteins are human sequences with variants. Protein variant may
refer to the
protein itself, a composition comprising the protein, or the amino sequence
that encodes it.
In some embodiments, the protein variant has amino acid substitution(s) at one
position, two
positions, three positions, four positions, five positions, six positions,
seven positions, eight
positions, nine positions or ten positions. The protein variant sequence
herein will possess at
least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, or 98% sequence
identity with a parent protein sequence, and preferably at least about 85%,
86%, 88%, 90%,
93% or 95% sequence identity. The relatedness between two amino acid sequences
or
between two nucleotide sequences is described by the parameter "sequence
identity" or
-identity". The degree of identity between an amino acid sequence of the
present invention
("invention sequence") and the parent amino acid sequence referred to in the
claims (e.g.
SEQ ID NO:1) is calculated as the number of exact matches in an alignment of
the two
sequences, divided by the length of the "invention sequence," or the length of
the parent
amino acid sequence, whichever is the shortest. The result is expressed in
percent identity as
calculated below.
1001131 For purposes of the present invention, the
extracellular domain of sBCMA as
set forth in SEQ ID NO:1 is used as a parent protein to determine the
corresponding amino
acid sequence modification in sBCMA variants. The amino acid sequence of an
sBCMA
variant protein is aligned with the amino acid sequence of SEQ ID NO:1, and
based on the
alignment, the amino acid position number corresponding to any amino acid
residue as
disclosed in SEQ ID NO:1 is determined using the Needleman-Wunsch algorithm
(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as implemented in the
Needle
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program of the EMBOSS package (EMBOSS: The European Molecular Biology Open
Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277), preferably
version 5Ø0 or
later. The parameters used are gap open penalty of 10, gap extension penalty
of 0.5, and the
EBLOSUM62 (EMBOSS version of BLOSUM62) substitution matrix. The output of
Needle labeled -longest identity" (obtained using the -nobrief option) is used
as the percent
identity and is calculated as follows:
[00114] (Identical Residues x 100)/(Length of Alignment - Total
Number of Gaps in
Alignment)
1001151 Identification of the corresponding amino acid residue
in another sBCMA
variant can be determined by an alignment of multiple polypeptide sequences
using several
computer programs including, but not limited to, MUSCLE (multiple sequence
comparison
by log-expectation; version 3.5 or later; Edgar, 2004, Nucleic Acids Research
32: 1792-
1797), MAFFT (version 6.857 or later; Katoh and Kuma, 2002, Nucleic Acids
Research 30:
3059-3066; Katoh et al. , 2005, Nucleic Acids Research 33: 511 -518; Katoh and
Toh, 2007,
Bioinformatics 23: 372-374; Katoh et al., 2009, Methods in Molecular Biology
537: 39-64;
Katoh and Toh, 2010, Bioinformatics 26: 1899-1900), EMBOSS EMMA employing
ClustalW (1.83 or later; Thompson et al., 1994, Nucleic Acids Research 22:
4673-4680), and
EMBL-EBI employing Clustal Omega (Sievers and Higgins, 2014, Methods Mol Biol.
2014,1079:105-16), using their respective default parameters.
[00116] When the other variant polypeptides have diverged from
the wild type
sBCMA such that traditional sequence-based comparison fails to detect their
relationship
(Lindahl and Elofsson, 2000, J. Mol. Biol. 295: 613-615), other pairwise
sequence
comparison algorithms can be used. Greater sensitivity in sequence-based
searching can be
attained using search programs that utilize probabilistic representations of
polypeptide
families (profiles) to search databases. For example, the PSI-BLAST program
generates
profiles through an iterative database search process and is capable of
detecting remote
homologs (Atschul et al., 1997, Nucleic Acids Res. 25: 3389-3402). Even
greater sensitivity
can be achieved if the family or superfamily for the polypeptide has one or
more
representatives in the protein structure databases. Programs such as
GenTHREADER (Jones,
1999, J. Mol. Biol. 287: 797-815; McGuffin and Jones, 2003, Bioinformatics 19:
874-881)
utilize information from a variety of sources (PSI-BLAST, secondary structure
prediction,
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structural alignment profiles, and solvation potentials) as input to a neural
network that
predicts the structural fold for a query sequence. Similarly, the method of
Gough et al.,
2000, J. Mol. Biol. 313: 903-919, can be used to align a sequence of unknown
structure with
the superfamily models present in the SCOP database. These alignments can in
turn be used
to generate homology models for the polypeptide, and such models can be
assessed for
accuracy using a variety of tools developed for that purpose.
[00117] For proteins of known structure, several tools and
resources are available for
retrieving and generating structural alignments. For example, the SCOP
superfamilies of
proteins have been structurally aligned, and those alignments are accessible
and
downloadable. Two or more protein structures can be aligned using a variety of
algorithms
such as the distance alignment matrix (Holm and Sander, 1998, Proteins 33: 88-
96) or
combinatorial extension (Shindyalov and Bourne, 1998, Protein Engineering 11:
739-747),
and implementation of these algorithms can additionally be utilized to query
structure
databases with a structure of interest in order to discover possible
structural homologs (e.g.,
Holm and Park, 2000, Bioinformatics 16: 566-567).
[00118] In describing the variants of the present invention,
the nomenclature described
below is adapted for ease of reference. The standardly accepted IUPAC single
letter or three
letter amino acid abbreviation is employed.
1001191 For an amino acid substitution, the following
nomenclature is used herein:
Original amino acid, position, substituted amino acid. Accordingly, the
substitution of
alanine at position 43 with valine is designated as "A1a43Va1" or -A43V".
Multiple
mutations are separated by forward slash marks (-/"), e.g., "N11D/S16G/N31S",
representing substitutions at positions 11, 16 and 31, respectively. . The
name, 3-letter
abbreviation, and 1-letter abbreviation for each of the 20 amino acids is
shown in Table 1.
1001201 Table 1. The name, 3-letter abbreviation, and 1-letter
abbreviation for each of
the 20 amino acids.
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Amino Acid 3-Letter 1-Letter
Code Code
Alanine Ala A
Cysteille ______________________________________________ Cys
Aspartic acid or aspartate Asp
Glutarnic acid or glutamate Gin
Phenylalanine Phe _
Glycine Gly
Histidine His
Is leucine Ile
Lysine Lys
Leucine Len
Methionine Met
Asparagine Asn N
Proline Pro
Glutamine Gin
Arginine Arg
Serine Ser
Threonine
Valine Val V
1
Tryptophan. Ti?
Tyrosine Tyr
1001211 The term -nucleic acid construct- refers to a nucleic
acid molecule, either
single-stranded or double-stranded, which is isolated from a naturally
occurring gene or is
modified to contain segments of nucleic acids in a manner that would not
otherwise exist in
nature or which is synthetic, and which comprises one or more control
sequences.
1001221 The term "operably linked" refers to a configuration in
which a control
sequence is placed at an appropriate position relative to the coding sequence
of a
polynucleotide such that the control sequence directs expression of the coding
sequence.
1001231 "Fc variant" or "variant Fc" as used herein is meant a
protein comprising at
least one amino acid sequence modification as compared to a parental Fc
domain. In some
embodiments, the parent Fc domain, is a human wild type Fc sequence, such as
the Fc
region from IgGl, IgG2, or IgG3. In some embodiments, the parent Fc domains
are human
Fc sequences with variants. For all positions discussed in the present
invention that relate to
the Fc domain of a human IgG, unless otherwise noted, amino acid position
numbering is
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according to the EU index. The modification can be an addition, deletion,
substitution or
any combination thereof as outlined herein. Alternatively, the variant Fe
domains can have
from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 11, 12, 13, 14,
15, 16, 17, 18, 19 or 20
amino acid modifications as compared to the parental Fc domain. Additionally,
as discussed
herein, the variant Fc domains herein still retain the ability to form a dimer
with another Fc
domain as well as bind to the FcRn receptor as measured using known techniques
as
described herein, such as non-denaturing gel electrophoresis.
[00124] The term "soluble BCMA- or "sBCMA- herein is meant a
soluble portion of
BCMA containing the extracellular domain (ECD) or a fragment or truncated
version
thereof, but not the entirety of the transmembrane domain or the cytoplasmic
(intracellular)
domain of BCMA. The ECD of human wild type sBCMA is shown as SEQ ID NO:l. In
some embodiments, the parent wild type sBCMA domain can have N-terminal and/or
C
terminal truncations as long as the truncated wild type sBCMA retains
biological activity,
e.g. binding to APRIL and/or BAFF, as discussed below.
[00125] The term "sBCMA variant" or "variant sBCMA" refers to a
variant of a parent
sBCMA protein by virtue of at least one amino acid sequence modification. In
some
embodiments, the parent protein is a human wild type sBCMA. In some
embodiments, the
sBCMA variant retains specific binding to TGF family member(s), such as APRIL
and/or
BAFF, but has amino acid sequence modifications, e.g. amino acid
substitutions, and can
have N- or C-terminal truncations as compared to wild type sBCMA. Specific
binding in
this case is determined by any appropriate binding assay, such as ELISA,
Biacore, Sapidyne
KinExA, or Flow Cytometry binding analysis, which assays can also be used to
determine
binding affinity as outlined below. As discussed herein, sBCMA variants may
have, in some
instances, increased binding affinity for TGF family members (e.g. APRIL
and/or BAFF) as
compared to wild type sBCMA.
[00126] The term "binding affinity" refers to the ability of a
ligand or variant thereof to
form coordinated bonds with a protein, e.g., a receptor or a variant thereof
The binding
affinity between a ligand and protein can be represented by an equilibrium
dissociation
constant (Kd), a ratio of koff/kon between the ligand and the protein (e.g.,
receptor or a
variant thereof). Kd and binding affinity are inversely related. For instance,
the Kd value
relates the concentration of the sBCMA variant needed to bind to a TGF family
member,
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and a lower Kd value (lower sBCMA variant concentration) corresponds to a
higher binding
affinity for the TGF family member. A high binding affinity corresponds to a
greater
intermolecular force between the ligand and the protein. A low binding
affinity corresponds
to a lower intermolecular force between the ligand and the protein. In some
cases, an
increase in ligand binding affinity can be represented as a decrease of the
off-rate by, for
example, at least 1.4-fold, at least 1.6-fold, at least 1.8-fold, at least 2-
fold, at least 3-fold, at
least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8-
fold, at least 9-fold, at
least 10-fold, at least 20-fold, at least 50-fold, at least 100-fold, at least
200-fold, at least
500-fold, or more.
[00127] "Specific binding" or "specifically binds to" or is
"specific for" a particular
ligand or variant thereof means binding that is measurably different from a
non-specific
interaction. Specific binding can be measured, for example, by determining
binding of a
molecule compared to binding of a control molecule, which generally is a
molecule of
similar structure that does not have binding activity. For example, specific
binding can be
determined by competition with a control molecule that is similar to the
target. In some
embodiments, the binding affinity is measured using any appropriate assay as
would be
understood by those skilled in the art as discussed above, such as a standard
Biacore assay.
[00128] Specific binding for a particular ligand or variant
thereof can be exhibited, for
example, by a protein having a Kd for another ligand protein of at least about
104 M, at least
about 10-5M, at least about 10-6M, at least about 10-7 M, at least about 10-
8M, at least about
10-9 M, alternatively at least about 10-10 M, at least about 10-11M, at least
about 10-12M_ at
least about 1015 M, or greater, where Kd refers to a dissociation rate of a
particular protein-
ligand interaction. In some embodiments, the variant sBCMA(s) of the present
invention
bind(s) a ligand with a binding affinity that is 1.5-, 2-, 3-, 4-, 5-, 6-, 7-,
8-, 9-, 10-, 15-, 20-,
50-, 100-, 200-, 500-, 1000-, 5,000-, 10,000- or more times greater as
compared with a
control molecule.
[00129] By "residue" as used herein is meant a position in a
protein and its associated
amino acid identity. For example, Asparagine 297 (also referred to as Asn297
or N297) is a
residue at position 297 in the human antibody IgGl.
[00130] By "hinge- or "hinge region- or "antibody hinge region-
or "hinge domain"
herein is meant the flexible polypeptide comprising the amino acids between
the first and
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second constant domains of an antibody. Structurally, the IgG CH1 domain ends
at EU
position 215, and the IgG CH2 domain begins at residue EU position 231. Thus
for IgG, the
antibody hinge is herein defined to include positions 216 (E216 in IgG1) to
230 (p230 in
IgG1), wherein the numbering is according to the EU index as in Kabat. In some
cases, a
"hinge fragment" is used, which contains fewer amino acids at either or both
of the N- and
C-termini of the hinge domain. As outlined herein, in some cases, Fe domains
inclusive of
the hinge are used, with the hinge generally being used as a flexible linker.
(Additionally, as
further described herein, additional flexible linker components can be used
either with or
without the hinge).
[00131] By "Fe" or "Fe region" or "Fe domain" as used herein is
meant the
polypeptide comprising the CH2-CH3 domains of an IgG molecule, and in some
cases,
inclusive of the hinge. In EU numbering for human IgGl, the CH2-CH3 domain
comprises
amino acids 231 to 447, and the hinge is 216 to 230. Thus the definition of
"Fc domain"
includes both amino acids 231-447 (CH2-CH3) or 216-447 (hinge-CH2-CH3), or
fragments
thereof Thus Fc refers to the last two constant region immunoglobulin domains
of IgA,
IgD, and IgG, the last three constant region immunoglobulin domains of IgE and
IgM, and
in some cases, includes the flexible hinge N-terminal to these domains. For
IgA and IgM,
Fc may include the J chain. For IgG, the Fc domain comprises immunoglobulin
domains
Cy2 and Cy3 and in some cases, includes the lower hinge region between Cyl and
C72. An
"Fe fragment" in this context may contain fewer amino acids from either or
both of the N-
and C-termini but still retains the ability to form a dimer with another Fe
domain or Fc
fragment as can be detected using standard methods, generally based on size
(e.g. non-
denaturing chromatography, size exclusion chromatography, etc). Human IgG Fc
domains
are of particular use in the present invention, and can be the Fc domain from
human IgGl,
IgG2, or IgG3. In general, IgG1 and IgG2 are used more frequently than IgG3.
In some
embodiments, amino acid sequence modifications are made to the Fc region, for
example to
alter binding to one or more FcyR receptors or to the FcRn receptor, and/or to
increase the
half-life in vivo.
[00132] By "IgG subclass modification" or -isotype
modification" as used herein is
meant an amino acid sequence modification that exchanges one amino acid of one
IgG
isotype to the corresponding amino acid in a different, aligned IgG isotype.
For example,
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because IgG1 comprises a tyrosine and IgG2 comprises a phenylalanine at EU
position 296,
a F296Y substitution in IgG2 is considered an IgG subclass modification.
Similarly, because
IgG1 has a proline at position 241 and IgG4 has a serine, an IgG4 molecule
with a S241P is
considered an IgG subclass modification. Note that subclass modifications are
considered
amino acid substitutions herein.
[00133] By "amino acid" and "amino acid identity" as used
herein is meant one of the
20 naturally occurring amino acids that are coded for by DNA and RNA.
[00134] By "effector function" as used herein is meant a
biochemical event that results
from the interaction of an antibody Fc region with an Fc receptor or ligand.
Effector
functions include but are not limited to antibody-dependent cellular
cytotoxicity (ADCC),
antibody-dependent cellular phagocytosis (ADCP), and complement-dependent
cytotoxicity
(CDC). In many cases, it is desirable to ablate most or all effector functions
using either
different IgG isotypes (e.g IgG4) or amino acid substitutions in the Fc
domain; however,
preserving binding to the FcRn receptor is desirable, as this contributes to
the half-life of the
fusion protein in human serum.
[00135] By "FcRn" or "neonatal Fc Receptor" as used herein is
meant a protein that
binds the IgG antibody Fc region and is encoded at least in part by an FcRn
gene.
[00136] By "target cell" as used herein is meant a cell that
expresses a target
polypeptide or protein.
[00137] By -host cell" in the context of producing the variant
sBCMA or the sBCMA
variant - Fc fusion proteins according to the invention herein is meant a cell
that contains
the exogenous nucleic acids encoding the components of the variant sBCMA or
the sBCMA
variant - Fc fusion protein, and is capable of expressing such variant sBCMA
or Fc fusion
protein under suitable conditions. Suitable host cells are described below.
[00138] By "improved activity" or "improved function" herein
meant a desirable
change of at least one biochemical property. An improved function in this
context can be
measured as a percentage increase or decrease of a particular activity, or as
a "fold" change,
with increases of desirable properties (e.g. increased binding affinity and/or
specificity for
APRIL and/or BAFF, increased protein stability of the, increased half-life in
vivo, etc.). In
general, percentage changes are used to describe changes in biochemical
activity of less than
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100%, and fold-changes are used to describe changes in biochemical activity of
greater than
100% (as compared to the parent protein). In the present invention, percentage
changes
(usually increases) of biochemical activity of at least about 10%, 20%, 30%,
40%, 50%,
60%, 70%, 80%, 90%, 95%, 98% and 99% can be accomplished. In the present
invention, a
"fold increase" (or decrease) is measured as compared to the parent protein.
In many
embodiments, the improvement is at least 1.4 fold, 1.5 fold, 1.6 fold, 1.8
fold, 2 fold, 3 fold,
4 fold, 5 fold, 6 fold, 7 fold, 8 fold, 9 fold, 10 fold, 50 fold, 100 fold,
200 fold or higher.
C. sBCMA Variant ¨ Fc Fusion Proteins
[00139] The sBCMA variant ¨ Fc fusion proteins of the present
invention include a
composition comprising a variant sBCMA domain, an Fc domain, and optionally a
linker
linking the variant sBCMA domain with the Fc domain.
[00140] In some embodiments, the present invention provides the
composition as
described herein, wherein said fusion protein comprises, from N- to C-
terminal:
a) said variant sBCMA domain;
b) said optional linker; and
c) said Fc domain.
1001411 In some embodiments, the present invention provides the
composition as
described herein, wherein said fusion protein comprises, from N- to C-
terminal:
a) said Fc domain;
b) said optional linker; and
c) said variant sBCMA domain.
D. Variant sBCMA Proteins and Domains
[00142] The invention provides variant sBCMA proteins both
independently and as
fusion protein constructs as an sBCMA domain fused with Fc domains. Variant
sBCMA
proteins of the present invention include at least a portion of the soluble
ECD of human
BCMA, generally the entire ECD domain (SEQ ID NO:1) as shown in Figure 30,
with
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amino acid variants. In some embodiments, variant sBCMA proteins or sBCMA
variants
exhibits increased binding affinity and/or specificity for APRIL and/or BAFF
as compared
to wild-type sBCMA as determined by binding affinity assays in the art and
discussed
below, such as Biacore or Octet assays.
[00143] In some embodiments, variant sBCMA proteins (either as
isolated proteins or
as sBCMA domains of the fusion proteins herein) are antagonists that bind to
APRIL and/or
BAFF to mitigate or to block their interaction with endogenous BCMA, BAFFR,
and TACT
receptors. Variant sBCMA proteins as antagonists can be used in treating
conditions
associated with altered signaling pathways through BCMA, BAFFR, TACT and/or
other
receptors that are activated through binding to APRIL and/or BAFF, in
particular tumor
therapy/chemotherapy, immunomodulatory and/or fibrotic diseases.
[00144] In an additional embodiment, variant sBCMA proteins
(either as isolated
proteins or as sBCMA domains of the fusion proteins herein) can be used in a
method of
inhibiting the activity of APRIL in a subject having an autoimmune disease
that expresses
APRIL, said method comprising administering to the subject a therapeutically
effective dose
of one or more said variant sBCMA proteins as disclosed herein.
[00145] In an additional embodiment, variant sBCMA proteins
(either as isolated
proteins or as sBCMA domains of the fusion proteins herein) can be used in a
method of
inhibiting the activity of APRIL in a subject having an autoimmune disease
that expresses
BCMA, TACT and/or other receptors that are activated through binding to APRIL,
said
method comprising administering to the subject a therapeutically effective
dose of one or
more said variant sBCMA proteins as disclosed herein.
[00146] In a further embodiment, variant sBCMA proteins (either
as isolated proteins
or as sBCMA domains of the fusion proteins herein) can be used in a method of
inhibiting
the activity of APRIL in a subject having fibrosis that expresses APRIL, said
method
comprising administering to the subject a therapeutically effective dose of
one or more said
variant sBCMA proteins as disclosed herein.
[00147] In a further embodiment, variant sBCMA proteins (either
as isolated proteins
or as sBCMA domains of the fusion proteins herein) can be used in a method of
inhibiting
the activity of APRIL in a subject having fibrosis that expresses BCMA, TACT
and/or other
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receptors that are activated through binding to APRIL, said method comprising
administering to the subject a therapeutically effective dose of one or more
said variant
sBCMA proteins as disclosed herein.
[00148] In an additional embodiment, variant sBCMA proteins
(either as isolated
proteins or as sBCMA domains of the fusion proteins herein) can be used in a
method of
inhibiting B-cell growth, immunoglobulin production, or both in a subject,
where the variant
sBCMA protein binds to BAFF, said method comprising administering to the
subject a
therapeutically effective dose of one or more said variant sBCMA proteins as
disclosed
herein.
[00149] In a further embodiment, variant sBCMA proteins (either
as isolated proteins
or as sBCMA domains of the fusion proteins herein) can be used in a method of
inhibiting
the activity of BAFF in a subject having B cell hyperplasia or an autoimmune
disease
expressing BCMA, BAFFR, TACT and/or other receptors that are activated through
binding
to BAFF, said method comprising administering to the subject a therapeutically
effective
dose of one or more said variant sBCMA proteins as disclosed herein.
[00150] In an additional embodiment, variant sBCMA proteins
(either as isolated
proteins or as sBCMA domains of the fusion proteins herein) can be used in a
method of
treating an autoimmune disease expressing at least one receptor selected from
the group
consisting of BCMA, BAFFR, TACT and other receptor(s) that are activated
through binding
to BAFF in a subject, said method comprising administering to the subject a
therapeutically
effective dose of one or more said variant sBCMA proteins as disclosed herein.
[00151] In a further embodiment, variant sBCMA proteins (either
as isolated proteins
or as sBCMA domains of the fusion proteins herein) can be used in a method of
treating an
autoimmune disease expressing BAFF and/or APRIL in a subject, said method
comprising
administering to the subject a therapeutically effective dose of one or more
said variant
sBCMA proteins as disclosed herein.
[00152] In an additional embodiment, variant sBCMA proteins
(either as isolated
proteins or as sBCMA domains of the fusion proteins herein) can be used in a
method of
treating fibrosis expressing BCMA, BAFFR and/or TACT in a subject, said method
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comprising administering to the subject a therapeutically effective dose of
one or more said
variant sBCMA proteins as disclosed herein.
1001531 In an additional embodiment, variant sBCMA proteins
(either as isolated
proteins or as sBCMA domains of the fusion proteins herein) can be used in a
method of
treating fibrosis expressing BAFF and/or APRIL in a subject, said method
comprising
administering to the subject a therapeutically effective dose of one or more
said variant
sBCMA proteins as disclosed herein.
[00154] In some embodiments, variant sBCMA proteins include
amino acid
substitutions, deletions or insertions or any combination thereof as compared
to the wild
type sBCMA, and increase their binding activity to either APRIL, BAFF or both
as
compared to the wild-type sBCMA.
[00155] The present disclosure provides variant sBCMA
protein(s) comprising at least
one amino acid substitution at one or more (e.g., 2, 3,4, 5, 6,7, 8,9 or 10)
positions as
compared to a parent sBCMA. In some embodiments, a variant sBCMA has at least
80%, at
least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least
98%, or at least
99%, but less than 100% sequence identity to the parent sBCMA. In some
embodiments, a
parent sBCMA domain is human wild-type sBCMA. In some embodiments, a parent
sBCMA domain has the amino acid sequence of SEQ ID NO: 1. In some embodiments,
a
variant sBCMA has at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at
least 95%, at least 96%, at least 97%, at least 98%, or at least 99%, but less
than 100%
sequence identity to SEQ ID NO: 1. In some embodiments, as noted herein, a
variant
sBCMA can have N-terminal and/or C terminal truncations compared to wild type
sBCMA
as long as the truncated variant sBCMA retains biological activity (e.g.
binding to APRIL
and/or BAFF), as measured by one of the binding assays outlined herein. To be
clear, the
variant BCMA of the present invention has at least one amino acid substitution
as compared
to SEQ ID NO:1, and thus is not SEQ ID NO:l.
[00156] In some embodiments, a variant sBCMA described herein
has a binding
affinity for TGF family member (i.e., APRIL and/or BAFF) that is stronger than
the wild-
type sBCMA polypeptide/domain. In some embodiments, the variant sBCMA has a
binding
affinity for APRIL and/or BAFF that is at least 1.4-fold, 1.5-fold, 1.6-fold,
1.8-fold, 2-fold,
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3-fold, 4-fold, 5-fold, 10-fold, 50-fold, 100-fold, 200-fold or greater than
that of the wild-
type sBCMA.
1001571 In certain embodiments, the binding affinity of the
variant sBCMA for APRIL
and/or BAFF is increased by at least about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%,
8%, 9%,
10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or higher as compared to that of
the
wild-type sBCMA. In other embodiments, the variant sBCMA proteins of the
present
invention have a Kd value of less than about 1 x 108M, 1 x 1(19M, 1 x 10-1
1\4,1 x 10-12m
or 1 x 10-15M for binding with APRIL and/or BAFF. In yet other embodiments,
sBCMA
variants inhibit or compete with wild-type sBCMA in binding to APRIL and/or
BAFF either
in vivo, in vitro or both.
1. Specific Variant sBCMA Proteins
[00158] The present invention provides a composition comprising
a variant sBCMA
comprising at least one amino acid substitution as compared to SEQ ID NO:1,
wherein said
amino acid substitution is at a position number selected from the group
consisting of 1, 2, 3,
4, 5, 6, 7, 9, 10, 11, 12, 14, 16, 19, 20, 22, 23, 25, 26, 29, 31, 32, 35, 36,
38, 39, 42, 43, 44,
45, 46, 47, 48, 49, 50, 51, 52, 53, and 54, wherein the numbering is according
to the EU
index.
[00159] In some embodiments, the variant sBCMA as described
herein has at least
80%, at least 85%, at least 90%, or at least 95% sequence identity to SEQ ID
NO:l.
[00160] In some embodiments, the variant sBCMA as described
herein comprises an
amino acid substitution of the methionine at position 1 with the position
numbering starting
from the mature region. In some embodiments, the substitution is with any
other of the 19
naturally occurring amino acids, serine, threonine, asparagine, glutamic acid,
glutamine,
aspartic acid, lysine, arginine, histidine, cysteine, glycine, alanine,
isoleucine, leucine,
proline, phenylalanine, tryptophan, valine and tyrosine, with some embodiments
not utilizing
proline (due to steric effects). In some embodiments, the amino acid
substitution is selected
from M1A, M1C, M1I, M1R, M1T, and M1V.
[00161] In some embodiments, the variant sBCMA as described
herein comprises an
amino acid substitution of the leucine at position 2 with the position
numbering starting from
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the mature region. In some embodiments, the substitution is with any other of
the 19 naturally
occun-ing amino acids, serine, threonine, asparagine, glutamic acid,
glutamine, aspartic acid,
lysine, arginine, histidine, cysteine, glycine, alanine, isoleucine,
methionine, proline,
phenylalanine, tryptophan, valine and tyrosine, with some embodiments not
utilizing proline
(due to steric effects). In some embodiments, the amino acid substitution is
L2C or L2S.
[00162] In some embodiments, the variant sBCMA as described
herein comprises an
amino acid substitution of the glutamine at position 3 with the position
numbering starting
from the mature region. In some embodiments, the substitution is with any
other of the 19
naturally occurring amino acids, serine, threonine, asparagine, glutamic acid,
aspartic acid,
lysine, arginine, histidine, cysteine, glycine, alanine, isoleucine, leucine,
methionine, proline,
phenylalanine, tryptophan, valine and tyrosine, with some embodiments not
utilizing cysteine
(due to possible disulfide formation). In some embodiments, the amino acid
substitution is
Q3P or Q3R.
[00163] In some embodiments, the variant sBCMA as described
herein comprises an
amino acid substitution of the methionine at position 4 with the position
numbering starting
from the mature region. In some embodiments, the substitution is with any
other of the 19
naturally occurring amino acids, serine, threonine, asparagine, glutamic acid,
glutamine,
aspartic acid, lysine, arginine, histidine, cysteine, glycine, alanine,
isoleucine, leucine,
methionine, proline, phenylalanine, tryptophan, valine and tyrosine, with some
embodiments
not utilizing cysteine (due to possible disulfide formation) or proline (due
to steric effects). In
some embodiments, the amino acid substitution is selected from M4E, M4I, M4T,
and M4V.
[00164] In some embodiments, the variant sBCMA as described
herein comprises an
amino acid substitution of the alanine at position 5 with the position
numbering starting from
the mature region. In some embodiments, the substitution is with any other of
the 19 naturally
occurring amino acids, serine, threonine, asparagine, glutamic acid,
glutamine, aspartic acid,
lysine, arginine, histidine, cysteine, glycine, isoleucine, leucine,
methionine, proline,
phenylalanine, tryptophan, valine and tyrosine, with some embodiments not
utilizing cysteine
(due to possible disulfide formation) or proline (due to steric effects). In
some embodiments,
the amino acid substitution is AST.
[00165] In some embodiments, the variant sBCMA as described
herein comprises an
amino acid substitution of the glycine at position 6 with the position
numbering starting from
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the mature region. In some embodiments, the substitution is with any other of
the 19 naturally
occun-ing amino acids, serine, threonine, asparagine, glutamic acid,
glutamine, aspartic acid,
lysine, arginine, histidine, cysteine, alanine, isoleucine, leucine,
methionine, proline,
phenylalanine, tryptophan, valine and tyrosine, with some embodiments not
utilizing cysteine
(due to possible disulfide formation) or proline (due to steric effects). In
some embodiments,
the amino acid substitution is G6E.
[00166] In some embodiments, the variant sBCMA as described
herein comprises an
amino acid substitution of the glutamine at position 7 with the position
numbering starting
from the mature region. In some embodiments, the substitution is with any
other of the 19
naturally occurring amino acids, serine, threonine, asparagine, glutamic acid,
aspartic acid,
lysine, arginine, histidine, cysteine, glycine, alanine, isoleucine, leucine,
methionine, proline,
phenylalanine, tryptophan, valine and tyrosine, with some embodiments not
utilizing cysteine
(due to possible disulfide formation) or proline (due to steric effects). In
some embodiments,
the amino acid substitution is Q7R.
[00167] In some embodiments, the variant sBCMA as described
herein comprises an
amino acid substitution of the serine at position 9 with the position
numbering starting from
the mature region. In some embodiments, the substitution is with any other of
the 19 naturally
occurring amino acids, threonine, asparagine, glutamic acid, glutamine,
aspartic acid, lysine,
arginine, histidine, cysteine, glycine, alanine, isoleucine, leucine,
methionine, proline,
phenylalanine, tryptophan, valine and tyrosine, with some embodiments not
utilizing cysteine
(due to possible disulfide formation). In some embodiments, the amino acid
substitution is
selected from S9A, S9F and S9P.
[00168] In some embodiments, the variant sBCMA as described
herein comprises an
amino acid substitution of the glutamine at position 10 with the position
numbering starting
from the mature region. In some embodiments, the substitution is with any
other of the 19
naturally occurring amino acids, serine, threonine, asparagine, glutamic acid,
aspartic acid,
lysine, arginine, histidine, cysteine, glycine, alanine, isoleucine, leucine,
methionine, proline,
phenylalanine, tryptophan, valine and tyrosine, with some embodiments not
utilizing cysteine
(due to possible disulfide formation). In some embodiments, the amino acid
substitution is
selected from Q10H, Q1OP and Q10R.
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[00169] In some embodiments, the variant sBCMA as described
herein comprises an
amino acid substitution of the asparagine at position 11 with the position
numbering starting
from the mature region. In some embodiments, the substitution is with any
other of the 19
naturally occurring amino acids, serine, threonine, glutamic acid, glutamine,
aspartic acid,
lysine, arginine, histidine, cysteine, glycine, alanine, isoleucine, leucine,
methionine, proline,
phenylalanine, tryptophan, valine and tyrosine, with some embodiments not
utilizing cysteine
(due to possible disulfide formation) or proline (due to steric effects). In
some embodiments,
the amino acid substitution is N11D or N11S.
[00170] In some embodiments, the variant sBCMA as described
herein comprises an
amino acid substitution of the glutamic acid at position 12 with the position
numbering
starting from the mature region. In some embodiments, the substitution is with
any other of
the 19 naturally occurring amino acids, serine, threonine, asparagine,
glutamine, aspartic
acid, lysine, arginine, histidine, cysteine, glycine, alanine, isoleucine,
leucine, methionine,
proline, phenylalanine, tryptophan, valine and tyrosine, with some embodiments
not utilizing
cysteine (due to possible disulfide formation) or proline (due to steric
effects). In some
embodiments, the amino acid substitution is E12K.
[00171] In some embodiments, the variant sBCMA as described
herein comprises an
amino acid substitution of the phenylalanine at position 14 with the position
numbering
starting from the mature region. In some embodiments, the substitution is with
any other of
the 19 naturally occurring amino acids, serine, threonine, asparagine,
glutamic acid,
glutamine_ aspartic acid, lysine, arginine, histidine, cysteine, glycine,
alanine, isoleucine,
leucine, methionine, proline, tryptophan, valine and tyrosine, with some
embodiments not
utilizing cysteine (due to possible disulfide formation) or proline (due to
steric effects). In
some embodiments, the amino acid substitution is F14L.
[00172] In some embodiments, the variant sBCMA as described
herein comprises an
amino acid substitution of the serine at position 16 with the position
numbering starting from
the mature region. In some embodiments, the substitution is with any other of
the 19 naturally
occurring amino acids, threonine, asparagine, glutamic acid, glutamine,
aspartic acid, lysine,
arginine, histidine, cysteine, glycine, alanine, isoleucine, leucine,
methionine, proline,
phenylalanine, tryptophan, valine and tyrosine, with some embodiments not
utilizing cysteine
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(due to possible disulfide formation) or proline (due to steric effects). In
some embodiments,
the amino acid substitution is selected from S16G, S16N, and S16R.
1001731 In some embodiments, the variant sBCMA as described
herein comprises an
amino acid substitution of the histidine at position 19 with the position
numbering starting
from the mature region. In some embodiments, the substitution is with any
other of the 19
naturally occurring amino acids, serine, threonine, asparagine, glutamic acid,
glutamine,
aspartic acid, lysine, arginine, cysteine, glycine, alanine, isoleucine,
leucine, methionine,
proline, phenylalanine, tryptophan, valine and tyrosine, with some embodiments
not utilizing
cysteine (due to possible disulfide formation) or proline (due to steric
effects). In some
embodiments, the amino acid substitution is H19L or H19Y.
[00174] In some embodiments, the variant sBCMA as described
herein comprises an
amino acid substitution of the alanine at position 20 with the position
numbering starting
from the mature region. In some embodiments, the substitution is with any
other of the 19
naturally occurring amino acids, serine, threonine, asparagine, glutamic acid,
glutamine,
aspartic acid, lysine, arginine, histidine, cysteine, glycine, isoleucine,
leucine, methionine,
proline, phenylalanine, tryptophan, valine and tyrosine, with some embodiments
not utilizing
cysteine (due to possible disulfide formation) or proline (due to steric
effects). In some
embodiments, the amino acid substitution is A20Vor A20T.
1001751 In some embodiments, the variant sBCMA as described
herein comprises an
amino acid substitution of the isoleucine at position 22 with the position
numbering starting
from the mature region. In some embodiments, the substitution is with any
other of the 19
naturally occurring amino acids, serine, threonine, asparagine, glutamic acid,
glutamine,
aspartic acid, lysine, arginine, histidine, cysteine, glycine, alanine,
leucine, methionine,
proline, phenylalanine, tryptophan, valine and tyrosine, with some embodiments
not utilizing
cysteine (due to possible disulfide formation) or proline (due to steric
effects). In some
embodiments, the amino acid substitution is I22M or I22V.
[00176] In some embodiments, the variant sBCMA as described
herein comprises an
amino acid substitution of the proline at position 23 with the position
numbering starting
from the mature region. In some embodiments, the substitution is with any
other of the 19
naturally occurring amino acids, serine, threonine, asparagine, glutamic acid,
glutamine,
aspartic acid, lysine, arginine, histidine, cysteine, glycine, alanine,
isoleucine, leucine,
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methionine, phenylalanine, tryptophan, valine and tyrosine, with some
embodiments not
utilizing cysteine (due to possible disulfide formation) or proline (due to
steric effects). In
some embodiments, the amino acid substitution is P23S.
[00177] In some embodiments, the variant sBCMA as described
herein comprises an
amino acid substitution of the glutamine at position 25 with the position
numbering starting
from the mature region. In some embodiments, the substitution is with any
other of the 19
naturally occurring amino acids, serine, threonine, asparagine, glutamic acid,
aspartic acid,
lysine, arginine, histidine, cysteine, glycine, alanine, isoleucine, leucine,
methionine, proline,
phenylalanine, tryptophan, valine and tyrosine, with some embodiments not
utilizing cysteine
(due to possible disulfide formation) or proline (due to steric effects). In
some embodiments,
the amino acid substitution is Q25R.
[00178] In some embodiments, the variant sBCMA as described
herein comprises an
amino acid substitution of the leucine at position 26 with the position
numbering starting
from the mature region. In some embodiments, the substitution is with any
other of the 19
naturally occurring amino acids, serine, threonine, asparagine, glutamic acid,
glutamine,
aspartic acid, lysine, arginine, histidine, cysteine, glycine, alanine,
isoleucine, methionine,
proline, phenylalanine, tryptophan, valine and tyrosine, with some embodiments
not utilizing
cysteine (due to possible disulfide formation) or proline (due to steric
effects). In some
embodiments, the amino acid substitution is L26F.
[00179] In some embodiments, the variant sBCMA as described
herein comprises an
amino acid substitution of the serine at position 29 with the position
numbering starting from
the mature region. In some embodiments, the substitution is with any other of
the 19 naturally
occurring amino acids, threonine, asparagine, glutamic acid, glutamine,
aspartic acid, lysine,
arginine, histidine, cysteine, glycine, alanine, isoleucine, leucine,
methionine, proline,
phenylalanine, tryptophan, valine and tyrosine, with some embodiments not
utilizing cysteine
(due to possible disulfide formation) or proline (due to steric effects). In
some embodiments,
the amino acid substitution is S29A.
[00180] In some embodiments, the variant sBCMA as described
herein comprises an
amino acid substitution of the asparagine at position 31 with the position
numbering starting
from the mature region. In some embodiments, the substitution is with any
other of the 19
naturally occurring amino acids, serine, threonine, glutamic acid, glutamine,
aspartic acid,
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lysine, arginine, histidine, cysteine, glycine, alanine, isoleucine, leucine,
methionine, proline,
phenylalanine, tryptophan, valine and tyrosine, with some embodiments not
utilizing cysteine
(due to possible disulfide formation) or proline (due to steric effects). In
some embodiments,
the amino acid substitution is N31D or N31S.
[00181] In some embodiments, the variant sBCMA as described
herein comprises an
amino acid substitution of the threonine at position 32 with the position
numbering starting
from the mature region. In some embodiments, the substitution is with any
other of the 19
naturally occurring amino acids, serine, asparagine, glutamic acid, glutamine,
aspartic acid,
lysine, arginine, histidine, cysteine, glycine, alanine, isoleucine, leucine,
methionine, proline,
phenylalanine, tryptophan, valine and tyrosine, with some embodiments not
utilizing cysteine
(due to possible disulfide formation). In some embodiments, the amino acid
substitution is
selected from T32A, T32I and T32P.
[00182] In some embodiments, the variant sBCMA as described
herein comprises an
amino acid substitution of the leucine at position 35 with the position
numbering starting
from the mature region. In some embodiments, the substitution is with any
other of the 19
naturally occurring amino acids, serine, threonine, asparagine, glutamic acid,
glutamine,
aspartic acid, lysine, arginine, histidine, cysteine, glycine, alanine,
isoleucine, methionine,
proline, phenylalanine, tryptophan, valine and tyrosine, with some embodiments
not utilizing
cysteine (due to possible disulfide formation). In some embodiments, the amino
acid
substitution is L35S or L35P.
[00183] In some embodiments, the variant sBCMA as described
herein comprises an
amino acid substitution of the threonine at position 36 with the position
numbering starting
from the mature region. In some embodiments, the substitution is with any
other of the 19
naturally occurring amino acids, serine, asparagine, glutamic acid, glutamine,
aspartic acid,
lysine, arginine, histidine, cysteine, glycine, alanine, isoleucine, leucine,
methionine, proline,
phenylalanine, tryptophan, valine and tyrosine, with some embodiments not
utilizing cysteine
(due to possible disulfide formation). In some embodiments, the amino acid
substitution is
selected from T36A, T36I, and T36P.
[00184] In some embodiments, the variant sBCMA as described
herein comprises an
amino acid substitution of the glutamine at position 38 with the position
numbering starting
from the mature region. In some embodiments, the substitution is with any
other of the 19
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naturally occurring amino acids, serine, threonine, asparagine, glutamic acid,
aspartic acid,
lysine, arginine, histidine, cysteine, glycine, alanine, isoleucine, leucine,
methionine, proline,
phenylalanine, typtophan, valine and tyrosine, with some embodiments not
utilizing cysteine
(due to possible disulfide formation) or proline (due to steric effects). In
some embodiments,
the amino acid substitution is Q38R.
[00185] In some embodiments, the variant sBCMA as described
herein comprises an
amino acid substitution of the arginine at position 39 with the position
numbering starting
from the mature region. In some embodiments, the substitution is with any
other of the 19
naturally occurring amino acids, serine, threonine, asparagine, glutamic acid,
glutamine,
aspartic acid, lysine, histidine, cysteine, glycine, alanine, isoleucine,
leucine, methionine,
proline, phenylalanine, tryptophan, valine and tyrosine, with some embodiments
not utilizing
cysteine (due to possible disulfide formation) or proline (due to steric
effects). In some
embodiments, the amino acid substitution is R39H.
[00186] In some embodiments, the variant sBCMA as described
herein comprises an
amino acid substitution of the asparagine at position 42 with the position
numbering starting
from the mature region. In some embodiments, the substitution is with any
other of the 19
naturally occurring amino acids, serine, threonine, glutamic acid, glutamine,
aspartic acid,
lysine, arginine, histidine, cysteine, glycine, alanine, isoleucine, leucine,
methionine, proline,
phenylalanine, typtophan, valine and tyrosine, with some embodiments not
utilizing cysteine
(due to possible disulfide formation) or proline (due to steric effects). In
some embodiments,
the amino acid substitution is selected from N42D, N42R and N42S.
[00187] In some embodiments, the variant sBCMA as described
herein comprises an
amino acid substitution of the alanine at position 43 with the position
numbering starting
from the mature region. In some embodiments, the substitution is with any
other of the 19
naturally occurring amino acids, serine, threonine, asparagine, glutamic acid,
glutamine,
aspartic acid, lysine, arginine, histidine, cysteine, glycine, isoleucine,
leucine, methionine,
proline, phenylalanine, tryptophan, valine and tyrosine, with some embodiments
not utilizing
cysteine (due to possible disulfide formation) or proline (due to steric
effects). In some
embodiments, the amino acid substitution is A43T or A43V.
[00188] In some embodiments, the variant sBCMA as described
herein comprises an
amino acid substitution of the serine at position 44 with the position
numbering starting from
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the mature region. In some embodiments, the substitution is with any other of
the 19 naturally
occurring amino acids, threonine, asparagine, glutamic acid, glutamine,
aspartic acid, lysine,
arginine, histidine, cysteine, glycine, alanine, isoleucine, leucine,
methionine, proline,
phenylalanine, tryptophan, valine and tyrosine, with some embodiments not
utilizing cysteine
(due to possible disulfide formation) or proline (due to steric effects). In
some embodiments,
the amino acid substitution is selected from S44D, S44G, S44N and S44R.
[00189] In some embodiments, the variant sBCMA as described
herein comprises an
amino acid substitution of the valine at position 45 with the position
numbering starting from
the mature region. In some embodiments, the substitution is with any other of
the 19 naturally
occurring amino acids, serine, threonine, asparagine, glutamic acid,
glutamine, aspartic acid,
lysine, arginine, histidine, cysteine, glycine, alanine, isoleucine, leucine,
methionine, proline,
phenylalanine, tryptophan and tyrosine, with some embodiments not utilizing
cysteine (due to
possible disulfide formation) or proline (due to steric effects). In some
embodiments, the
amino acid substitution is V45A or V45M.
[00190] In some embodiments, the variant sBCMA as described
herein comprises an
amino acid substitution of the threonine at position 46 with the position
numbering starting
from the mature region. In some embodiments, the substitution is with any
other of the 19
naturally occurring amino acids, serine, asparagine, glutamic acid, glutamine,
aspartic acid,
lysine, arginine, histidine, cysteine, glycine, alanine, isoleucine, leucine,
methionine, proline,
phenylalanine, tryptophan, valine and tyrosine, with some embodiments not
utilizing cysteine
(due to possible disulfide formation) or proline (due to steric effects). In
some embodiments,
the amino acid substitution is T46A or T46I.
[00191] In some embodiments, the variant sBCMA as described
herein comprises an
amino acid substitution of the asparagine at position 47 with the position
numbering starting
from the mature region. In some embodiments, the substitution is with any
other of the 19
naturally occurring amino acids, serine, threonine, glutamic acid, glutamine,
aspartic acid,
lysine, arginine, histidine, cysteine, glycine, alanine, isoleucine, leucine,
methionine, proline,
phenylalanine, tryptophan, valine and tyrosine, with some embodiments not
utilizing cysteine
(due to possible disulfide formation) or proline (due to steric effects). In
some embodiments,
the amino acid substitution is selected from N47D, N47K, N47R and N47S.
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[00192] In some embodiments, the variant sBCMA as described
herein comprises an
amino acid substitution of the serine at position 48 with the position
numbering starting from
the mature region. In some embodiments, the substitution is with any other of
the 19 naturally
occurring amino acids, threonine, asparagine, glutamic acid, glutamine,
aspartic acid, lysine,
arginine, histidine, cysteine, glycine, alanine, isoleucine, leucine,
methionine, proline,
phenylalanine, tryptophan, valine and tyrosine, with some embodiments not
utilizing cysteine
(due to possible disulfide formation). In some embodiments, the amino acid
substitution is
selected from S48L, S48P and S48T.
[00193] In some embodiments, the variant sBCMA as described
herein comprises an
amino acid substitution of the valine at position 49 with the position
numbering starting from
the mature region. In some embodiments, the substitution is with any other of
the 19 naturally
occurring amino acids, serine, threonine, asparagine, glutamic acid,
glutamine, aspartic acid,
lysine, arginine, histidine, cysteine, glycine, alanine, isoleucine, leucine,
methionine, proline,
phenylalanine, tryptophan, and tyrosine, with some embodiments not utilizing
cysteine (due
to possible disulfide formation) or proline (due to steric effects). In some
embodiments, the
amino acid substitution is V49A or V49M.
[00194] In some embodiments, the variant sBCMA as described
herein comprises an
amino acid substitution of the lysine at position 50 with the position
numbering starting from
the mature region. In some embodiments, the substitution is with any other of
the 19 naturally
occurring amino acids, serine, threonine, asparagine, glutamic acid,
glutamine, aspartic acid,
arginine, histidine, cysteine, glycine, alanine, isoleucine, leucine,
methionine, proline,
phenylalanine, tryptophan, valine and tyrosine, with some embodiments not
utilizing cysteine
(due to possible disulfide formation) or proline (due to steric effects). In
some embodiments,
the amino acid substitution is selected from K50E, K50G, K5OR and K50T.
[00195] In some embodiments, the variant sBCMA as described
herein comprises an
amino acid substitution of the glycine at position 51 with the position
numbering starting
from the mature region. In some embodiments, the substitution is with any
other of the 19
naturally occurring amino acids, serine, threonine, asparagine, glutamic acid,
glutamine,
aspartic acid, lysine, arginine, histidine, cysteine, alanine, isoleucine,
leucine, methionine,
proline, phenylalanine, tryptophan, valine and tyrosine, with some embodiments
not utilizing
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cysteine (due to possible disulfide formation) or proline (due to steric
effects). In some
embodiments, the amino acid substitution is G51E.
1001961 In some embodiments, the variant sBCMA as described
herein comprises an
amino acid substitution of the threonine at position 52 with the position
numbering starting
from the mature region. In some embodiments, the substitution is with any
other of the 19
naturally occurring amino acids, serine, asparagine, glutamic acid, glutamine,
aspartic acid,
lysine, arginine, histidine, cysteine, glycine, alanine, isoleucine, leucine,
methionine, proline,
phenylalanine, tryptophan, valine and tyrosine, with some embodiments not
utilizing cysteine
(due to possible disulfide formation) or proline (due to steric effects). In
some embodiments,
the amino acid substitution is T52A or T52M.
[00197] In some embodiments, the variant sBCMA as described
herein comprises an
amino acid substitution of the asparagine at position 53 with the position
numbering starting
from the mature region. In some embodiments, the substitution is with any
other of the 19
naturally occurring amino acids, serine, threonine, glutamic acid, glutamine,
aspartic acid,
lysine, arginine, histidine, cysteine, glycine, alanine, isoleucine, leucine,
methionine, proline,
phenylalanine, tryptophan, valine and tyrosine, with some embodiments not
utilizing cysteine
(due to possible disulfide formation) or proline (due to steric effects). In
some embodiments,
the amino acid substitution is selected from N53D, N53K and N53S.
1001981 In some embodiments, the variant sBCMA as described
herein comprises an
amino acid substitution of the alanine at position 54 with the position
numbering starting
from the mature region. In some embodiments, the substitution is with any
other of the 19
naturally occurring amino acids, serine, threonine, asparagine, glutamic acid,
glutamine,
aspartic acid, lysine, arginine, histidine, cysteine, glycine, isoleucine,
leucine, methionine,
proline, phenylalanine, tryptophan, valine and tyrosine, with some embodiments
not utilizing
cysteine (due to possible disulfide formation) or proline (due to steric
effects). In some
embodiments, the amino acid substitution is A54V or A54T.
[00199] In some embodiments, the variant sBCMA as described
herein comprises
amino acid substitution(s) selected from the group consisting of M1A, M1C,
M11, M1R,
M1T, M1V, L2C, L2S, Q3P, Q3R, M4E, M4I, M4T, M4V, A5T, G6E, Q7R, S9A, S9F,
S9P, Q10H, Q10P, Q10R,N11D, N11S, E12K, F14L, S16G, S16N, S16R, H19L, H19Y,
A20V, A20T, I22M, I22V, P23S, Q25R, L26F, S29A, N31D, N31S, T32A, T32I, T32P,
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L35S, L35P, T36A, T36I, T36P, Q38R, R39H, N42D, N42R, N42S, A43T, A43V, S44D,
S44G, S44N, S44R, V45A, V45M, T46A, T46I, N47D, N47K, N47R, N47S, S48L, S48P,
S48T, V49A, V49M, K50E, K50G, K5OR, K50T, G51E, T52A, T52M, N53D, N53K,
N53S, A54V, and A54T.
[00200] In some embodiments, the variant sBCMA as described
herein comprises
amino acid substitution(s) selected from the group consisting of M1V, L2S,
Q3P, M4T, S9P,
N11D, Si 6G, Hi 9Y, N315, N31D, T32I, T36A, R39H, N475, K50E, and N53E.
[00201] In some embodiments, the variant sBCMA as described
herein comprises
amino acid substitution(s) selected from the group consisting of 516G, H19Y
and T36A.
1002021 In some embodiments, the variant sBCMA as described
herein comprises
amino acid substitutions selected from the group consisting of
L2S/S9P/E12K/N31D/T36A/N425/N53S, M1V/T32P/T36A/T461/N53D/A54V,
Q3R/S16N/T36A/A43T, F 1 4L/S16G/T36AN45A/N47D,
MIT/M4V/S9F/S16G/T32A/Q38R, M1A/S9A/Q38R, G6E/Q25R/Q38R,
M1V/M4I/G6E/S9P/N11DN49M/T52M/A54V, N11D/S16G/N3 is,
N11D/H19Y/122M/T32P/N47S/N53S, G6E/Q7R/H19Y/L35S, H19Y/N42D/S48P/T52A,
M1VN31D/T321/T36A, M1V/A5T/H19L/T36A,
M1T/N31D/T32A/136A/Q38R/S44D/V49A/K50E, M1V/T36A/Q38R/A43V,
M1V/L2S/S9P/Q10H/T36A/Q38R/K50G, T36A/Q38R/N53S,
M1T/L2S/L35P/T36A/Q38R/T46A/K5OR, A5T/A20V/T36A/Q38R,
M1T/S16G/122V/T36A/S44G/T46AN49A, Sl6G/T36A,
M1I/N11D/S16G/122M/S29A/T36A/S44G/K5 OR, M1C/L2C/Q3R/M4E/N11D/S16G/T36P,
M1I/N11D/S16G/122M/S29A/T36A/S44G/K5OR,
N11D/N31D/T321/T36A/S44N/N47D/N53D, M1R/L2C/Q3R, H19Y/T36A/S44G,
H19Y/T321/T36A/V49A, H19Y/N31S/T36A/V45A, H19Y/N31S/T36A, H19Y/T36P/T52A,
H19Y/N31D/T52M, M1V/H19YN45M, Sl6G/H19Y/N47D, Sl6G/H19Y/K50T,
S16G/H19Y/S44N/K5OR, N11D/H19Y/S48T,
S9P/N11D/S16R/T32A/Q38R/S44G/T461/T52A/N53D/A54T, N11D/S16G/S44R,
H19L/T32A/S44G/G51E/T52A, Sl6N/H19Y/T36A/K5OR, M1V/H19Y/T36A/R39H/T46A,
M1V/H19Y/T36A, H19Y/T36A/N42D/N47S/S48P, M1V/H19Y/T36A/S44G/N47D,
MIV/H19Y/T36A/N42R/N53S, H19Y/L35P/T36A/N42D/T461N49A,
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Q3P/S9P/H19Y/N31S/T36A/R39H/N47R/K50E, M1V/H19Y/T36A/N42R/N53S,
M1T/H19Y/T36A, M1V/S16N/H19Y/122M/T36A,
M1T/N11D/H19Y/T36A/N42S1V45A/N53S, N11D/S16G/H19Y/T36A/N47S/N53D,
M1V/S9P/Q10P/S16G/H19Y/L26F/T36A/A43V/N53D, Sl6G/H19Y/T36A/V49A/N53D,
Sl6G/T36A/A43T/S44GN45M, M4V/S9P/S16G/T36A/Q38R,
S9P/N1 is/Si 6G/T36A/Q38R, N1 1 DIE12K/S16R/T36A/T52M,
M4V/T32I/T36A/Q38R/A43TN45A/548P, S9P/N1 1 D/S 1 6G/Q25R,
M1T/A5T/S9P/S16G/Q25R/N31DN49M,
L2S/S9P/S16G/A20T/1321/Q38R/N42D/T46A/S48L, Si 6G/Q25R/146A,
G6E/S9A/S16G/Q25R/N31D/N47S/T52M, H19Y/Q38R/T52M,
Ni 1D/H19Y/122M/T32P/N47S/N53S, Sl6G/H19Y/T36A, Sl6G/H19Y/T36A/N53D,
S9P/N11D/516G/H19Y/T36A/N475/N53D,
Q3P/S 9P/H19Y/N315/T36A/R39H/N47R/K5 OE, M1 V/L2S/M4T/N11D/H19Y/T36A,
M1V/L2S/M4T/N11D/T36A, M1V/L2S/M4T/H19Y/T36IN45A/V49M,
M1V/L2S/M4T/N11D/H19Y/T36A, M1V/L2S/M4T/S9P/Q10R/H19Y/T36A/T46A/N47S,
M1V/L25/M4T/516G/N31D/T321/T36A, M1V/M4T/T36A/Q38R/N53K,
M1T/N11D/H19Y/T36A/N42SN45A/N53S,
M1T/N31D/T32A/T36A/A38R/S44DN49A/K50E,
M1T/S9P/P23S/Q38R/N425/S48PN49A/A54V, H19Y/T36A/S44G, H19Y/T36A, and
M4T/T36A/Q38R/N42S/S44G/T46A/N47K/S48P/T52A.
[00203] In some embodiments, the variant sBCMA as described
herein comprises
amino acid substitutions Sl6G/H19Y/T36A, and at least one further amino acid
substitution
selected from the group consisting of MIA, M1C, M1I, MIR, MIT, M1V, L2C, L2S,
Q3P,
Q3R, M4E, M4I, M4T, M4V, MT, G6E, Q7R, S9A, S9F, S9P, Q10H, Ql0P, Ql0R, N1 1D,
NI IS, El2K, Fl4L, Sl6N, Sl6R, H19L, A20V, A20T, I22M, I22V, P23S, Q25R, L26F,
529A, N31D, N31S, T32A, T32I, T32P, L355, L35P, T36I, T36P, Q38R, R39H, N42D,
N42R, N425, A43T, A43V, 544D, 544G, 544N, S44R, V45A, V45M, T46A, T46I, N47D,
N47K, N47R, N47S, S48L, S48P, S48T, V49A, V49M, K50E, K50G, K5OR, K50T, G51E,
T52A, T52M, N53D, N53K, N53S, A54V, and A54T.
[00204] In some embodiments, the variant sBCMA as described
herein comprises
amino acid substitutions Sl6G/H19Y/T36A/N53D, and at least one further amino
acid
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substitution selected from the group consisting of M1A, M1C, M1I, M1R, M1T,
M1V, L2C,
L2S, Q3P, Q3R, M4E, M4I, M4T, M4V, A5T, G6E, Q7R, S9A, S9F, S9P, Q10H, Q10P,
Q10R, N11D, N11S, E12K, F14L, S16N, S16R, H19L, A20V, A20T, I22M, I22V, P23S,
Q25R, L26F, S29A, N31D, N31S, T32A, T32I, T32P, L35S, L35P, T36I, T36P, Q38R,
R39H, N42D, N42R, N42S, A43T, A43V, S44D, S44G, S44N, S44R, V45A, V45M, T46A,
T46I, N47D, N47K, N47R, N47S, S48L, S48P, S48T, V49A, V49M, K50E, K50G, K5OR,
K50T, G51E, T52A, T52M, N53K, N53S, A54V, and A54T.
[00205] In some embodiments, the variant sBCMA as described
herein comprises
amino acid substitutions S9P/N11D/S16G/H19Y/T36A/N47S/N53D, and at least one
further
amino acid substitution selected from the group consisting of M1A, M1C, M1I,
M1R, M1T,
M1V, L2C, L2S, Q3P, Q3R, M4E, M4I, M4T, M4V, A5T, G6E, Q7R, S9A, S9F, Q10H,
Q10P, Q10R, N11S, E12K, F14L, S16N, S16R, H19L, A20V, A20T, I22M, I22V, P23S,
Q25R, L26F, S29A, N31D, N31S, T32A, T32I, T32P, L35S, L35P, T36I, T36P, Q38R,
R39H, N42D, N42R, N42S, A43T, A43V, S44D, S44G, S44N, S44R, V45A, V45M, T46A,
T46I, N47D, N47K, N47R, S48L, S48P, S48T, V49A, V49M, K50E, K50G, K5OR, K50T,
G51E, T52A, T52M, N53K, N53S, A54V, and A54T.
[00206] In some embodiments, the variant sBCMA as described
herein comprises
amino acid substitutions Q3P/S9P/H19Y/N31S/T36A/R39H/N47R/K50E, and at least
one
further amino acid substitution selected from the group consisting of M1A,
M1C, M1I, M1R,
M1T, M1V, L2C, L2S, Q3R, M4E, M4I, M4T, M4V, A5T, G6E, Q7R, S9A, S9F, Q10H,
Q10P, Q10R, Ni 1D, N11S, E12K, F14L, Sl6G, Sl6N, S16R, H19L, A20V, A20T, I22M,
I22V, P23S, Q25R, L26F, S29A, N31D, T32A, T32I, T32P, L35S, L35P, T36I, T36P,
Q38R,
N42D, N42R, N42S, A43T, A43V, S44D, S44G, S44N, S44R, V45A, V45M, T46A, T46I,
N47D, N47K, N47S, S48L, S48P, S48T, V49A, V49M, K50G, K5OR, K50T, G51E, T52A,
152M, N53D, N53K, N53S, A54V, and A54T.
[00207] In some embodiments, the variant sBCMA as described
herein comprises
amino acid substitutions M1V/L2S/M4T/S16G/N31D/1321/T36A, and at least one
further
amino acid substitution selected from the group consisting of M1A, M1C, M1I,
M1R, M1T,
L2C, Q3P, Q3R, M4E, M4I, M4V, A5T, G6E, Q7R, S9A, S9F, S9P, Q10H, Q10P, Q10R,
N11D, N11S, E12K, F14L, S16N, S16R, H19L, H19Y, A20V, A20T, I22M, I22V, P23S,
Q25R, L26F, S29A, N31S, T32A, T32P, L35S, L35P, T36I, T36P, Q38R, R39H, N42D,
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N42R, N42S, A43T, A43V, S44D, S44G, S44N, S44R, V45A, V45M, T46A, T46I, N47D,
N47K, N47R, N47S, S48L, S48P, S48T, V49A, V49M, K50E, K50G, K5OR, K50T, G51E,
T52A, T52M, N53D, N53K, N53S, A54V, and A54T.
[00208] In some embodiments, the variant sBCMA as described
herein has at least
90% sequence identity to SEQ ID NO: 67.
[00209] In some embodiments, the variant sBCMA as described
herein has at least
90% sequence identity to SEQ ID NO: 68.
[00210] In some embodiments, the variant sBCMA as described
herein has at least
90% sequence identity to SEQ ID NO: 69.
[00211] In some embodiments, the variant sBCMA as described
herein has at least
90% sequence identity to SEQ ID NO: 49.
[00212] In some embodiments, the variant sBCMA as described
herein has at least
90% sequence identity to SEQ ID NO: 74.
[00213] In some embodiments, the variant sBCMA as described
herein has the amino
acid sequence of SEQ ID NO: 67.
[00214] In some embodiments, the variant sBCMA as described
herein has the amino
acid sequence of SEQ ID NO: 68.
[00215] In some embodiments, the variant sBCMA as described
herein has the amino
acid sequence of SEQ ID NO: 69.
[00216] In some embodiments the variant sBCMA as described
herein has the amino
acid sequence of SEQ ID NO: 49.
[00217] In some embodiments, the variant sBCMA as described
herein has the amino
acid sequence of SEQ ID NO: 74.
[00218] The clone Nos., amino acid substitutions as compared to
the amino acid
sequence of SEQ ID NO:1, and assigned SEQ ID NOs of exemplary variant sBCMA
proteins
are shown in Table 2.
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1002191 Table 2: The clone numbers, amino acid substitutions as
compared to the
amino acid sequence of SEQ ID NO:1, and the assigned SEQ ID NOs. of exemplary
variant
sBCMA proteins/domains.
Clone Nos. Amino Acid Substitutions as compared to SEQ ID NO:1 SEQ
ID
Nos.
# 1 L2S/S9P/E12K/N31D/T36A/N42S/N53S SEQ
ID
NO: 2
# 2 M1V/T32P/T36A/T46I/N53D/A54V SEQ
ID
NO: 3
#3 Q3R/S16N/T36A/A43T SEQ
ID
NO: 4
# 4 F 1 4L/S 16G/T36A/V45A/N47D SEQ
ID
NO: 5
#5 M1T/M4V/S9F/S16G/T32A/Q3812 SEQ
ID
NO: 6
#6 M1A/S9A/Q38R SEQ
ID
NO: 7
#7 G6E/Q25R/Q38R SEQ
ID
NO: 8
# 8 M1V/M4I/G6E/S9P/N11DN49M/T52M/A54V SEQ
ID
NO: 9
#9 N11D/S16G/N31S SEQ
ID
NO: 10
# 10 and 70 N11D/H19Y/I22M/T32P/N475/N535 SEQ
ID
NO: 11
14 11 G6E/Q7R/H19Y/L35S SEQ
ID
NO: 12
# 12 H19Y/N42D/S48P/T52A SEQ
ID
NO: 13
# 13 M1V/N31D/T32I/T36A SEQ
ID
NO: 14
# 14 M1V/A5T/H19L/T36A SEQ
ID
NO: 15
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# 15 M1T/N31D/T32A/T36A/Q38R/S44DN49A/K5 OE SEQ
ID
NO: 16
# 16 M1V/T36A/Q38R/A43V SEQ
ID
NO: 17
# 17 M1V/L2S/S9P/Q1 OH/T36A/Q38R/K5OG SEQ
ID
NO: 18
#18 T36A/Q38R/N53S SEQ
ID
NO: 19
# 19 M1T/L2S/L35P/T36A/Q38R/T46A/K5OR SEQ
ID
NO: 20
#20 A5T/A20V/T36A/Q38R SEQ
ID
NO: 21
# 21 M1T/S16G/I22V/T36A/544G/T46AN49A SEQ
ID
NO: 22
# 22 & 73 S16G/T36A SEQ
ID
NO: 23
# 23 and 25 M1I/N11D/S16G/I22M/S29A/T36A/S44G/K5OR SEQ
ID
NO: 24
#24 M1C/L2C/Q3R/M4E/N11D/S16G/T36P SEQ
ID
NO: 25
#26 N11D/N31D/T32I/T36A/S44N/N47D/N53D SEQ
ID
NO: 26
# 27 M1R/L2C/Q3R/N11D/H19Y/T36A/N42SN45A/N53S SEQ
ID
NO: 27
# 28 and H19Y/T36A/544G SEQ
ID
116 NO:
28
# 29 H19Y/T32I/T36A/V49A SEQ
ID
NO: 29
# 30 H19Y/N31S/T36A/V45A SEQ
ID
NO: 30
#31 H19Y/N31S/T36A SEQ
ID
NO: 31
# 32 H19Y/T36P/152A SEQ
ID
NO: 32
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#33 H19Y/N31D/T52M
SEQ ID
NO: 33
#34 M1V/H19YN45M
SEQ ID
NO: 34
#35 S 1 6G/H19Y/N47D
SEQ ID
NO: 35
#36 S 1 6G/H19Y/K5OT
SEQ ID
NO: 36
#37 S 1 6G/H19Y/S44N/K5OR
SEQ ID
NO: 37
#38 N11D/H19Y/S48T
SEQ ID
NO: 38
#39
S9P/N11D/S16R/T32A/Q38R/S44G/T46I/T52A/N53D/A54T SEQ ID
NO: 39
#40 N11D/S16G/544R
SEQ ID
NO: 40
#41 H19L/T32A/S44G/G51E/T52A
SEQ ID
NO: 41
#42 S16N/H19Y/T36A/K5OR
SEQ ID
NO: 42
#43 M1V/H19Y/T36A/R39H/T46A
SEQ ID
NO: 43
#44 M1V/H19Y/T36A
SEQ ID
NO: 44
# 45 H19Y/T36A/N42D/N47S/S48P
SEQ ID
NO: 45
# 46 M1V/H19Y/T36A/S44G/N47D
SEQ ID
NO: 46
# 47 and 50 M1V/H19Y/T36A/N42R/N53S
SEQ ID
NO: 47
# 48 H19Y/L35P/T36A/N42D/T46IN49A
SEQ ID
NO: 48
# 49, 75-94 Q3P/S9P/H19Y/N31S/136A/R39H/N47R/K5OE
SEQ ID
NO: 49
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#51 M1T/H19Y/T36A SEQ
ID
NO: 50
# 52 M1V/516N/H19Y/122M/T36A SEQ
ID
NO: 51
# 53, 54 and M1T/N11D/H19Y/T36A/N425N45A/N535 SEQ
ID
113 NO:
52
#55 and 56 N11D/S16G/H19Y/T36A/N475/N53D SEQ
ID
NO: 53
# 57 M1V/S9P/Q10P/S16G/H19Y/L26F/T36A/A43V/N53D SEQ
ID
NO: 54
# 58 S16G/H19Y/T36A/V49A/N53D SEQ
ID
NO: 55
# 59 S 1 6G/T36A/A43T/S44GN45M SEQ
ID
NO: 56
# 60 M4V/S9P/516G/T36A/Q38R SEQ
ID
NO: 57
#61 S9P/N11S/S16G/T36A/Q38R SEQ
ID
NO: 58
#62 N11D/E12K/S16R/T36A/T52M SEQ
ID
NO: 59
# 63 M4V/T32I/T36A/Q38R/A43TN45A/S 48P SEQ
ID
NO: 60
#64 S9P/N11D/S16G/Q25R SEQ
ID
NO: 61
# 65 M1T/A5T/S9P/S16G/Q25R/N31DN49M SEQ
ID
NO: 62
# 66 L2S/S9P/S16G/A20T/T321/Q38R/N42D/T46A/S48L SEQ
ID
NO: 63
# 67 S 1 6G/Q25R/T46A SEQ
ID
NO: 64
# 68 G6E/S9A/516G/Q25R/N31D/N475/T52M SEQ
ID
NO: 65
#69 H19Y/Q38R/152M SEQ
ID
NO: 66
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#71 S 1 6G/H19Y/T36A/N53D
SEQ ID
NO: 67
#72 516G/H19Y/T36A
SEQ ID
NO: 68
#74 S9P/N11D/516G/H19Y/T36A/N475/N53D
SEQ ID
NO: 69
# 95, and M1V/L2S/M4T/N11D/H19Y/T36A
SEQ ID
101 -103
NO: 70
# 96 -99 M1V/L2S/M4T/N11D/T36A
SEQ ID
NO: 71
# 100 M1V/L2S/M4T/H19Y/T361/V45A/V49M
SEQ ID
NO: 72
# 104 and M1V/L2S/M4T/S9P/Q10R/H19Y/T36A/T46A/N475
SEQ ID
105
NO: 73
# 106- 111 M1V/L2S/M4T/S16G/N31D/T321/T36A
SEQ ID
NO: 74
# 112 M1V/M4T/T36A/Q38R/N53K
SEQ ID
NO: 75
# 114 M1T/N31D/T32A/T36A/A38R/S44DN49A/K5OE
SEQ ID
NO: 76
# 115 M1T/S9P/P23S/Q38R/N42S/S48PN49A/A54V
SEQ ID
NO: 77
#117 H19Y/T36A
SEQ ID
NO: 78
# 118 M4T/T36A/Q38R/N42S/S44G/T46A/N47K/S48P/T52A
SEQ ID
NO: 79
[00220] In some embodiments, the variant sBCMA as described
herein exhibits
enhanced binding affinity for APRIL or BAFF as compared to SEQ ID NO: 1.
[00221] In some embodiments, the variant sBCMA as described
herein exhibits
enhanced binding affinity for APRIL and BAFF as compared to SEQ ID NO: 1.
[00222]
In some embodiments, the sBCMA variant ¨ Fc fusion protein as described
herein has the amino acid sequence of SEQ ID NO: 80.
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[00223] In some embodiments, the sBCMA variant ¨ Fe fusion
protein as described
herein has the amino acid sequence of SEQ ID NO: 81
1002241 In some embodiments, the sBCMA variant ¨ Fe fusion
protein as described
herein has the amino acid sequence of SEQ ID NO: 82.
[00225] In some embodiments, the sBCMA variant ¨ Fc fusion
protein as described
herein has the amino acid sequence of SEQ ID NO:83.
[00226] In some embodiments, the sBCMA variant ¨ Fe fusion
protein as described
herein has the amino acid sequence of SEQ ID NO: 84,
2. Assays to Measure Binding Affinity
[00227] As outlined herein, the present invention provides
sBCMA variants and fusion
proteins comprising these variants that exhibit increased binding affinity for
either or both of
human APRIL and/or human BAFF. In this context, increased binding affinity is
compared
to the human wild type BCMA or SEQ ID NO:1 in vitro or ex vivo studies as
outlined
below. In some embodiments, the variant sBCMA domain as described herein has a
binding
affinity for TGF family member (e.g., APRIL and/or BAFF) that is stronger than
the wild-
type sBCMA polypeptide/domain and/or SEQ ID NO: 1. In some embodiments, the
variant
sBCMA domain has a binding affinity for APRIL and/or BAFF that is at least 1.4-
fold, 1.5-
fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 3-fold, 4-fold, 5-fold,
10-fold, 50-fold,
100-fold, 200-fold or greater than that of the wild-type sBCMA and/or SEQ ID
NO:l.
[00228] The ability of an sBCMA variant to bind to APRIL and/or
BAFF can be
determined, for example, by the ability of the putative ligand to bind to
APRIL and/or BAFF
coated on an assay plate. Alternatively, binding affinity of an sBCMA
(variant) for APRIL
and/or BAFF can be determined by displaying the sBCMA (variant) on a microbial
cell
surface, e.g., a yeast cell surface and detecting the bound complex by, for
example, flow
cytometry (see, Example 3). The binding affinity of sBCMA (variant) for APRIL
and/or
BAFF can be measured using any appropriate method as would be understood by
those
skilled in the art including, but not limited to, radioactive ligand binding
assays, non-
radioactive (fluorescent) ligand binding assays, surface plasmon resonance
(SPR), such as
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BiacoreTM, OctetTM, plasmon-waveguide resonance (PWR), thermodynamic binding
assays,
whole cell ligand-binding assays, and structure-based ligand binding assays.
3. Formats of the Fusion Proteins
[00229] As described herein, the format of the fusion protein
can take on several
configurations, with the component domains switching order in the protein
(from N- to C-
terminal). In one embodiment, a fusion protein comprises, from N- to C-
terminus, a variant
sBCMA domain-domain linker-Fc domain. In some embodiments, a fusion protein
comprises, from N- to C-terminus, Fc domain-domain linker- variant sBCMA
domain. In
some embodiments, a linker is not used, in which case the fusion protein
comprises from N-
to C-terminus, either variant sBCMA domain-Fc domain or Fe domain- variant
sBCMA
domain. Note that in some cases, the same fusion protein can be labeled
somewhat
differently. For example, in the case in which the Fc domain includes a hinge
domain, a
fusion protein comprising variant sBCMA domain-Fc domain still includes a
linker in the
form of the hinge domain. Alternatively, this same protein may not have the
hinge domain
included in the Fc domain, in which case the fusion protein comprises variant
sBCMA
domain-CH2-CH3.
[00230] Thus, in some embodiments, the present disclosure
provides a variant sBCMA
¨ Fc fusion protein as described herein, where the Fc domain comprises a hinge
domain and
the variant sBCMA domain is linked with the Fc domain by the hinge domain:
variant
sBCMA domain-hinge domain-CH2-CH3.
[00231] In some embodiments, the present disclosure provides a
variant sBCMA ¨ Fc
fusion proteins as described above, where the Fc domain comprises a hinge
domain and the
variant sBCMA domain is linked with the Fc domain by an additional linker as
described
herein. That is, the fusion protein can be, from N- to C-terminal: variant
sBCMA domain-
domain linker-hinge domain-CH2-CH3; variant sBCMA domain-domain linker-CH2-
CH3;
hinge domain-CH2-CH3-domain linker-variant sBCMA domain or CH2-CH3-domain
linker-
variant sBCMA domain.
[00232] In some embodiments, the present disclosure provides
variant sBCMA ¨ Fc
fusion proteins as described above, where the Fc domain does not comprise a
hinge domain
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and the variant sBCMA domain is linked with the Fc domain by a domain linker
(e.g. non-
hinge) as described herein.
1002331 In some embodiments, the present disclosure provides a
composition
comprising an sBCMA variant - Fc fusion protein comprising:
a) a variant sBCMA domain comprising at least one amino acid substitution as
compared to SEQ ID NO:1, wherein said amino acid substitution is at a position
number
selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 14,
16, 19, 20, 22, 23,
25, 26, 29, 31, 32, 35, 36, 38, 39, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51,
52, 53, and 54,
wherein the numbering is according to the EU index;
b) an optional linker; and
c) an Fc domain.
[00234] In some embodiments, the sBCMA variant - Fc fusion
protein as described
herein comprises, from N- to C-terminal:
a) said variant sBCMA domain;
b) said optional linker; and
c) said Fc domain.
[00235] In some embodiments, the sBCMA variant - Fc fusion
protein as described
herein comprises, from N- to C-terminal:
a) said Fc domain;
b) said optional linker; and
c) said variant sBCMA domain.
[00236] In some embodiments, a variant sBCMA domain of the
sBCMA variant- Fc
fusion protein as described herein serves to increase the binding affinity for
APRIL and/or
BAFF. In various embodiments, a (variant) Fc domain of the sBCMA variant - Fc
fusion
protein as described herein increases the half-life of the fusion protein. In
a number of
embodiments, fusion proteins are used to treat fibrosis and/or
immunomodulatory diseases.
[00237] The names of the designated proteins/protein domains
and corresponding
amino acid sequences are listed in Table 3, respectively.
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Table 3. SEQ ID numbers, descriptions and corresponding amino acid sequences.
SEQ ID NO Amino Acid Sequence
(Description)
SEQ ID NO:1 MLQMAGQCSQNEYFDSLLHACIPCQLRCSSNTPPLTCQRY
C
(sBCMA WT ECD) NASVTNSVKGTNA
SEQ ID NO:87 IEGRMD
(domain linker)
SEQ ID NO:88 GGGGS
(domain linker)
1002381 In some embodiments, the variant sBCMA domain as
described herein
includes amino acid substitution(s), deletion(s) or insertion(s) or any
combination thereof to
the amino acid sequence of SEQ ID NO:1 that increases its binding activity to
either APRIL,
BAFF or both as compared to wild-type sBCMA.
[00239] The present disclosure provides variant sBCMA domains
comprising at least
one amino acid substitution at one or more (e.g., 2, 3,4, 5, 6,7, 8,9 or 10)
positions as
compared to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the
variant
sBCMA domain has at least 80%, at least 85%, at least 90%, at least 95%, at
least 96%, at
least 97%, at least 98%, or at least 99%, but less than 100% sequence identity
to the parent
sBCMA domain. In some embodiments, a parent sBCMA domain has the amino acid
sequence of SEQ ID NO: 1. In some embodiments, a variant sBCMA domain has at
least
90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at
least 96%, at
least 97%, at least 98%, or at least 99%, but less than 100% sequence identity
to SEQ ID
NO: 1. In some embodiments, as noted herein, a variant sBCMA domain can have N-
terminal and/or C terminal truncations compared to wild type sBCMA as long as
the
truncated variant sBCMA retains biological activity (e.g. binding to APRIL
and/or BAFF),
as measured by one of the binding assays outlined herein. To be clear, the
variant BCMA
domain of the present invention has at least one amino acid substitution and
thus is not the
amino acid sequence of SEQ ID NO: 1.
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[00240] In some embodiments, the variant sBCMA domain as
described herein has
amino acid substitution(s) at one position, two positions, three positions,
four positions, five
positions, six positions, seven positions, eight positions, nine positions, or
ten positions.
[00241] In certain embodiments, the binding affinity of the
variant sBCMA domain as
described herein for APRIL and/or BAFF is increased by at least about 0.4%,
0.5%, 1%, 2%,
3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50% or
higher
as compared to that of the wild-type sBCMA. In other embodiments, variant BCMA
domains of the present invention have a binding affinity of less than about 1
x 10-8M, 1 x
10-9M, 1 x 10-1 M, 1 x 10-12M or 1 x 10-1' M for APRIL and/or BAFF. In yet
other
embodiments, variant BCMA domains as described herein inhibit or compete with
wild-type
sBCMA binding to APRIL and/or BAFF either in vivo, in vitro or both.
[00242] In some embodiments, the variant sBCMA domain as
described herein
comprises at least one amino acid substitution as compared to SEQ ID NO:1,
wherein said
amino acid substitution is at a position number selected from the group
consisting of 1, 2, 3,
4, 5, 6, 7, 9, 10, 11, 12, 14, 16, 19, 20, 22, 23, 25, 26, 29, 31, 32, 35, 36,
38, 39, 42, 43, 44,
45, 46, 47, 48, 49, 50, 51, 52, 53, and 54, wherein the numbering is according
to the EU
index.
[00243] In some embodiments, the variant sBCMA domain as
described herein has at
least 80%, at least 85%, at least 90%, or at least 95% sequence identity to
SEQ ID NO: 1.
[00244] In some embodiments, the variant sBCMA domain as
described herein
comprises amino acid substitution(s) selected from the group consisting of
M1A, M1C, NHL
M1R, M1T, M1V, L2C, L2S, Q3P, Q3R, M4E, M4I, M4T, M4V, AST, G6E, Q7R, S9A,
S9F, S9P, Q10H, Q10P, Q10R,N11D, N11S, E12K, F14L, S16G, S16N, S16R, H19L,
H19Y, A20V, A20T, I22M, I22V, P23S, Q25R. L26F, S29A, N31D, N3IS, T32A, T32I,
T32P, L35S, L35P, T36A, T36I, T36P, Q38R, R39H, N42D, N42R, N425, A43T, A43V,
544D, 544G, 544N, 544R, V45A, V45M, T46A, T46I, N47D, N47K, N47R, N475, 548L,
S48P, S48T, V49A, V49M, K50E, K50G, K5OR, K50T, G51E, T52A, T52M, N53D, N53K,
N53S, A54V, and A54T.
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[00245] In some embodiments, the variant sBCMA domain as
described herein
comprises amino acid substitution(s) selected from the group consisting of
MIV, L2S, Q3P,
M4T, S9P, NI ID, Sl6G, H19Y, N31S, N31D, T32I, T36A, R39H, N47S, K50E, and
N53E.
[00246] In some embodiments, the variant sBCMA domain as
described herein
comprises amino acid substitution(s) selected from the group consisting of Si
6G, H19Y and
T36A.
[00247] In some embodiments, the variant sBCMA domain as
described herein
comprises amino acid substitutions selected from the group consisting of
L2S/S9P/E12K/N31D/T36A/N42S/N53S, M1V/T32P/T36A/T461/N53D/A54V,
Q3R/S16N/T36A/A43T, F 1 4L/S16G/T36AN45A/N47D,
M1T/M4V/S9F/S16G/T32A/Q38R, M1A/S9A/Q38R, G6E/Q25R/Q38R,
M1V/M41/G6E/S9P/N11D/V49M/T52M/A54V, N11D/S16G/N31 is,
Ni 1D/H19Y/122M/T32P/N47S/N53S, G6E/Q7R/H19Y/L35S, H19Y/N42D/S48P/T52A,
M1V/N31D/T32I/T36A, MI V/A5T/H19L/T36A,
M1T/N31D/T32A/T36A/Q38R/S44DN49A/K50E, M1V/T36A/Q38R/A43V,
M1V/L2S/S9P/Q10H/T36A/Q38R/K50G, T36A/Q38R/N53S,
M1T/L2S/L35P/T36A/Q38R/T46A/K5OR, A5T/A20V/T36A/Q38R,
M1T/S16G/122V/T36A/S44G/T46AN49A, Sl6G/T36A,
M1I/N11D/S16G/122M/S29A/T36A/S44G/K5 OR, M1C/L2C/Q3R/M4E/N11D/S16G/T36P,
M1I/N11D/S16G/122M/S29A/T36A/S44G/K5OR,
N11D/N31D/T321/T36A/S44N/N47D/N53D, M1R/L2C/Q3R, H19Y/T36A/S44G,
H19Y/T321/T36AN49A, H19Y/N31S/T36AN45A, H19Y/N31S/T36A, H19Y/T36P/T52A,
H19Y/N31D/T52M, M1V/H19YN45M, Sl6G/H19Y/N47D, Sl6G/H19Y/K50T,
S 16G/H19Y/S44N/K5OR, N11D/H19Y/S48T,
S9P/NIID/S16R/132A/Q38R/S44G/1461/152A/N53D/A54'1', NIID/S16G/S44R,
H19L/T32A/S44G/G51E/T52A, Sl6N/H19Y/T36A/K5OR, M1V/H19Y/T36A/R39H/T46A,
M1V/H19Y/T36A, H19Y/T36A/N42D/N47S/S48P, M1V/H19Y/T36A/S44G/N47D,
M1V/H19Y/T36A/N42R/N53S, H19Y/L35P/T36A/N42D/T461N49A,
Q3P/S 9P/H19Y/N31S/T36A/R39H/N47R/K5 OE, MI V/H19Y/T36A/N42R/N53S,
M1T/H19Y/T36A, M1V/S16N/H19Y/122M/T36A,
MI T/N11D/H19Y/T36A/N42S/V45A/N53 S, N11D/S16G/H19Y/T36A/N47S/N53D,
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M1V/S9P/Q10P/S16G/H19Y/L26F/T36A/A43V/N53D, Sl6G/H19Y/T36AN49A/N53D,
Sl6G/T36A/A43T/S44GN45M, M4V/S9P/S16G/T36A/Q38R,
S9P/N1 1 S/S 1 6G/T36A/Q38R, N11D/E12K/S16R/T36A/T52M,
M4V/T32I/T36A/Q38R/A43T/V45A/S48P, S9P/N11D/S16G/Q25R,
M1T/A5T/S9P/S16G/Q25R/N31DN49M,
L25/59P/S16G/A20T/T321/Q38R/N42D/T46A/548L, Sl6G/Q25R/T46A,
G6E/59A/S16G/Q25R/N31D/N47S/T52M, H19Y/Q38R/T52M,
NI 1D/H19Y/122M/T32P/N47S/N53S, Sl6G/H19Y/T36A, Sl6G/H19Y/T36A/N53D,
S9P/NI1D/S16G/H19Y/136A/N47S/N53D,
Q3P/S 9P/H19Y/N31S/T36A/R39H/N47R/K5 OE, M1V/L2S/M4T/N11D/H19Y/T36A,
M1V/L2S/M4T/N11D/T36A, M1V/L2S/M4T/H19Y/T36IN45A/V49M,
M1V/L2S/M4T/N11D/H19Y/T36A, M1V/L2S/M4T/S9P/Q10R/H19Y/T36A/T46A/N47S,
M1V/L2S/M4T/S16G/N31D/T321/T36A, M1V/M4T/T36A/Q38R/N53K,
M1T/N11D/H19Y/T36A/N42SN45A/N53S,
M1T/N31D/T32A/T36A/A38R/S44DN49A/K50E,
M1T/S9P/P23S/Q38R/N42S/S48PN49A/A54V, H19Y/T36A/S44G, H19Y/T36A, and
M4T/T36A/Q38R/N425/544G/T46A/N47K/S48P/T52A.
[00248] In some embodiments, the variant sBCMA domain as
described herein
comprises amino acid substitutions SI6G/H19Y/T36A, and at least one further
amino acid
substitution selected from the group consisting of M1A, MIC, M1I, MIR, MIT,
M1V, L2C,
L2S, Q3P, Q3R, M4E, M4I, M4T, M4V, A5T, G6E, Q7R, S9A, S9F, S9P, QI OH, Q10P,
Ql0R, NI ID, NI IS, El2K, Fl4L, Sl6N, Sl6R, H19L, A20V, A20T, I22M, I22V,
P23S,
Q25R, L26F, S29A, N31D, N31S, T32A, T321, T32P, L35S, L35P, T361, T36P, Q38R,
R39H, N42D, N42R, N425, A43T, A43V, 544D, 544G, 544N, 544R, V45A, V45M, T46A,
T46I, N47D, N47K, N47R, N475, 548L, 548P, 548T, V49A, V49M, K50E, K50G, K5OR,
K50T, G51E, T52A, T52M, N53D, N53K, N535, A54V, and A54T.
[00249] In some embodiments, the variant sBCMA domain as
described herein
comprises amino acid substitutions Sl6G/H19Y/T36A/N53D, and at least one
further amino
acid substitution selected from the group consisting of M1A, M1C, M11, M1R,
M1T, M1V,
L2C, L25, Q3P, Q3R, M4E, M4I, M4T, M4V, A5T, G6E, Q7R, 59A, 59F, 59P, Q10H,
Ql0P, Ql0R, N11D, N11S, E12K, F14L, Sl6N, Sl6R, H19L, A20V, A20T, 122M, 122V,
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P23S, Q25R, L26F, S29A, N31D, N31S, T32A, T32I, T32P, L35S, L35P, T36I, T36P,
Q38R, R39H, N42D, N42R, N42S, A43T, A43V, S44D, S44G, S44N, S44R, V45A, V45M,
T46A, T46I, N47D, N47K, N47R, N47S, S48L, S48P, S48T, V49A, V49M, K50E, K50G,
K5OR, K50T, G51E, T52A, T52M, N53K, N53S, A54V, and A54T.
[00250] In some embodiments, the variant sBCMA domain as
described herein
comprises amino acid substitutions S9P/N11D/S16G/H19Y/T36A/N47S/N53D, and at
least
one further amino acid substitution selected from the group consisting of M1A,
M1C, MIL
M1R, M1T, M1V, L2C, L2S, Q3P, Q3R, M4E, M4T, M4T, M4V, A5T, G6E, Q7R, S9A,
S9F, Q10H, Q10P, Q10R, N11S, E12K, F14L, S 16N, S 16R, H19L, A20V, A20T, I22M,
I22V, P23S, Q25R, L26F, S29A, N31D, N31S, T32A, T32I, T32P, L35S, L35P, T36I,
T36P,
Q38R, R39H, N42D, N42R, N42S, A431, A43V, S44D, S44G, S44N, S44R, V45A, V45M,
T46A, T46I, N47D, N47K, N47R, S48L, S48P, S48T, V49A, V49M, K50E, K50G, K5OR,
K50T, G51E, T52A, T52M, N53K, N53S, A54V, and A54T.
[00251] In some embodiments, the variant sBCMA domain as
described herein
comprises amino acid substitutions Q3P/S9P/H19Y/N31S/T36A/R39H/N47R/K50E, and
at
least one further amino acid substitution selected from the group consisting
of M1A, M1C,
M1I, M1R, M1T, M1V, L2C, L2S, Q3R, M4E, M4T, M4T, M4V, A5T, G6E, Q7R, S9A,
S9F, Q10H, Q10P, Q10R, N11D, N11S, E12K, F14L, S16G, S16N, S16R, H19L, A20V,
A20T, I22M, I22V, P23S, Q25R, L26F, S29A, N31D, T32A, T32I, T32P, L35S, L35P,
T36I,
T36P, Q38R, N42D, N42R, N42S, A43T, A43V, S44D, S44G, S44N, S44R, V45A, V45M,
T46A, T46I, N47D, N47K, N47S, S48L, S48P, S48T, V49A, V49M, K50G, K5OR, K50T,
G51E, T52A, T52M, N53D, N53K, N53S, A54V, and A54T.
[00252] In some embodiments, the variant sBCMA domain as
described herein
comprises amino acid substitutions M1V/L2S/M4T/S16G/N31D/T321/T36A, and at
least one
further amino acid substitution selected from the group consisting of M1A,
M1C, M1I, M1R,
M1T, L2C, Q3P, Q3R, M4E, M4T, M4V, A5T, G6E, Q7R, S9A, S9F, S9P, Q10H, Q10P,
Q10R, N11D, N11S, E12K, F14L, S 16N, S 16R, H19L, H19Y, A20V, A20T, 122M,
I22V,
P23S, Q25R, L26F, S29A, N31S, T32A, T32P, L35S, L35P, T36I, T36P, Q38R, R39H,
N42D, N42R, N42S, A43T, A43V, S44D, S44G, S44N, S44R, V45A, V45M, T46A, T46I,
N47D, N47K, N47R, N47S, S48L, S48P, S48T, V49A, V49M, K50E, K50G, K5OR, K50T,
G51E, T52A, T52M, N53D, N53K, N53S, A54V, and A54T.
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[00253] In some embodiments, the variant sBCMA domain as
described herein has at
least 90% sequence identity to SEQ ID NO: 67.
1002541 In some embodiments, the variant sBCMA domain as
described herein has at
least 90% sequence identity to SEQ ID NO: 68.
[00255] In some embodiments, the variant sBCMA domain as
described herein has at
least 90% sequence identity to SEQ ID NO: 69.
[00256] In some embodiments, the variant sBCMA domain as
described herein has at
least 90% sequence identity to SEQ ID NO: 49.
[00257] In some embodiments, the variant sBCMA domain as
described herein has at
least 90% sequence identity to SEQ ID NO: 74.
[00258] In some embodiments, the variant sBCMA domain as
described herein has
SEQ ID NO: 67.
[00259] In some embodiments, the variant sBCMA domain as
described herein has
SEQ ID NO: 68.
[00260] In some embodiments, the variant sBCMA domain as
described herein has
SEQ ID NO: 69.
[00261] In some embodiments, the variant sBCMA domain as
described herein has
SEQ ID NO: 49.
[00262] In some embodiments, the variant sBCMA domain as
described herein has
SEQ ID NO: 74.
[00263] The Clone Nos., amino acid substitutions as compared to
the amino acid
sequence of SEQ ID NO:1 and assigned SEQ ID NOs of exemplary variant sBCMA
domains
are shown in Table 2.
[00264] In some embodiments, the variant sBCMA domain as
described herein
exhibits enhanced binding affinity for APRIL as compared to SEQ ID NO: 1.
[00265] In some embodiments, the variant sBCMA domain as
described herein
exhibits enhanced binding affinity for BAFF as compared to SEQ ID NO: 1.
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[00266] In some embodiments, the variant sBCMA domain as
described herein
exhibits enhanced binding affinity for APRIL and BAFF as compared to SEQ ID
NO: 1.
4. Fc Domains
[00267] As discussed herein, in addition to sBCMA variant
domains described above,
the fusion proteins of the invention also include Fc domains of antibodies
that generally are
based on the IgG class, which has several subclasses, including, but not
limited to IgGI,
IgG2, and IgG3. As described herein, an Fc domain optionally includes the
hinge domain of
an IgG antibody.
[00268] Human IgG Fc domains are of particular use in the
present invention, and can
be derived from the Fc domain from human IgGl, IgG2, or IgG3. In general, IgG1
and
IgG2 are used more frequently than IgG3.
[00269] An Fc domain of a human IgG protein included in the
fusion protein of the
present invention can confer a significant increase in half-life of the fusion
protein, and can
provide additional binding or interaction with the Ig molecules. In some
embodiments, an
sBCMA variant ¨ Fc fusion protein can facilitate purification,
multimerization, binding and
neutralizing other molecules as compared to a monomeric variant sBCMA
polypeptide.
[00270] Fc domains can also contain Fc variants to alter
function as needed. However,
in accordance with many embodiments, Fc variants generally need to retain both
the ability
to form dimers as well as the ability to bind FcRn. Thus, while many of the
embodiments
herein rely on the use of a human IgG1 domain, Fc variants can be made to
augment or
abrogate function in other IgG domains. Thus, for example, ablation variants
that reduce or
eliminate effector function in IgG1 or IgG2 can be used, and/or FcRn variants
that confer
tighter binding to the FcRn receptor can be used, as will be appreciated by
those in the art.
[00271] In one embodiment, an Fc domain is a human IgG Fc
domain or a variant
human IgG Fc domain.
[00272] In another embodiment, an Fc domain is human IgG1 Fc
domain.
[00273] In a further embodiment, an Fc domain comprises the
hinge-CH2-CH3 of
human IgGl.
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[00274] In another embodiment, an Fe domain comprises the CH2-
CH3 of human
IgGl.
1002751 In some embodiments, Fe domains can be the Fe domains
from other IgGs
than IgGl, such as human IgG2 or IgG3. In general, IgG2 is used more
frequently than
IgG3.
[00276] In an additional embodiment, an Fe domain is a variant
human IgG Fe
domain. However, the variant Fe domains herein still retain the ability to
form a dimer with
another Fe domain as measured using known, as well as the ability to bind to
FcRn, as this
contributes significantly to the increase in serum half life of the fusion
proteins herein.
1002771 The variant IgG Fe domain can include an addition,
deletion, substitution or
any combination thereof compared with the parent human IgG Fe domain.
1002781 In some embodiments, variant human IgG Fe domains of
the present invention
can have at least about 80%, 85%, 90%, 95%, 95%, 97%, 98% or 99% identity to
the
corresponding parental human IgG Fe domain (using the identity algorithms
discussed
above, with one embodiment utilizing the BLAST algorithm as is known in the
art, using
default parameters).
[00279] In some embodiments, variant human IgG Fe domains of
the present invention
can have from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 11, 12,
13, 14, 15, 16, 17, 18,
19 or 20 amino acid sequence modifications as compared to the parental human
IgG Fe
domains.
[00280] In some embodiments, the Fe domain as described herein
is a human IgG Fe
domain or a variant human IgG Fe domain.
[00281] In some embodiments, the Fe domain as described herein
comprises the hinge-
CH2-CH3 of human IgGl.
[00282] In some embodiments, the Fe domain as described herein
is a variant human
IgG Fe domain.
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5. Linkers
[00283] The fusion proteins of the invention can include
optional linkers to connect the
sBCMA domain to the Fc domain.
[00284] By "linker" or "linker peptide" as used herein have a
length that is adequate to
link two molecules in such a way that they assume the correct conformation
relative to one
another so that they retain the desired activity. In one embodiment, the
linker is from about 1
to 20 amino acids in length, preferably about 1 to 10 amino acids in length.
In one
embodiment, linkers of 4 to 10 amino acids in length may be used. Useful
linkers include
IEGRIVID or glycine- serine polymers, including for example (GS)n, (GSGGS)n,
(GGGGS)n, and (GGGS)n, where n is an integer of at least one (and generally
from 3 to 4),
glycine-alanine polymers, alanine-serine polymers, and other flexible linkers.
Alternatively,
a variety of nonproteinaceous polymers, including but not limited to
polyethylene glycol
(PEG), polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene
glycol and
polypropylene glycol, may find use as linkers.
[00285] In some embodiments, the linker is a "domain linker",
used to link any two
domains as outlined herein together, such as to link the variant sBCMA domain
with Fc
domain. As discussed above, many suitable linkers can be used to allow for
recombinant
attachment of the two domains with sufficient length and flexibility to allow
each domain to
retain its biological function. As discussed herein, a particularly useful
domain linker is an
IEGRMD linker joined to the hinge domain of IgGl.
[00286] In various embodiments, two domains (e.g. the sBCMA
variant domain and
the Fc domain) are generally linked using a domain linker as described herein.
In many
embodiments, two domains are attached using a flexible linker in such a way
that the two
domains can act independently. Flexible linkage can be accomplished in a
variety of ways,
using traditional linkers and/or the hinge linker.
[00287] In some embodiments, the linker as described herein is
1EGRMD (SEQ. ID
NO. 87).
[00288] In some embodiments, the linker as described herein is
GGGGS (SEQ. ID
NO. 88).
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[00289] In some embodiments, a hinge domain of a human IgG
antibody is used. In
some cases, a hinge domain can contain amino acid substitutions as well.
1002901 In some embodiments, a domain linker is a combination
of a hinge domain
and a flexible linker, such as an IgG1 hinge with an IEGRMD linker.
[00291] In one embodiment, a linker is from about 1 to 50 amino
acids in length,
preferably about 1 to 30 amino acids in length and more preferably about 4 to
10 amino
acids.
6. Particular Embodiments of the Invention
[00292] In some embodiments, an sBCMA variant ¨ Fc fusion
protein exhibits at least
90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:80.
[00293] In some embodiments, an sBCMA variant ¨ Fc fusion
protein exhibits at least
90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:81.
[00294] In some embodiments, an sBCMA variant Fc fusion protein
exhibits at least
90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:82.
[00295] In some embodiments, an sBCMA variant ¨ Fc fusion
protein exhibits at least
90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:83.
1002961 In some embodiments, an sBCMA variant ¨ Fc fusion
protein exhibits at least
90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:84.
[00297] In some embodiments, an sBCMA variant ¨ Fc fusion
protein has the amino
acid sequence as set forth in SEQ ID NO:80.
[00298] In some embodiments, an sBCMA variant ¨ Fc fusion
protein has the amino
acid sequence as set forth in SEQ ID NO:81.
[00299] In some embodiments, an sBCMA variant ¨ Fc fusion
protein has the amino
acid sequence as set forth in SEQ ID NO:82.
[00300] In some embodiments, an sBCMA variant ¨ Fc fusion
protein has the amino
acid sequence as set forth in SEQ ID NO:83.
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[00301] In some embodiments, an sBCMA variant ¨ Fe fusion
protein has the amino
acid sequence as set forth in SEQ ID NO:84.
E. Methods of Treatment
1. Subjects amenable to treatment
[00302] Various embodiments are directed to methods comprising
administering to a
subject in need of treatment a therapeutically effective amount of one or more
variant
sBCMA proteins as described herein.
[00303] Various embodiments are directed to methods comprising
administering to a
subject in need of treatment a therapeutically effective amount of one or more
sBCMA
variant ¨ Fe fusion proteins as described herein.
[00304] In some embodiments, the present invention provides
methods of reducing
immunoglobulin production in subjects diagnosed with autoimmune disease(s)
and/or
fibrosis comprising administering to the subjects a therapeutically effective
dose of sBCMA
variant protein(s).
[00305] In some embodiments, the present invention provides
methods of reducing
production of IgA, IgM and/or IgG in subjects diagnosed with autoimmune
disease(s) and/or
fibrosis comprising administering to the subjects a therapeutically effective
dose of sBCMA
variant protein(s).
[00306] In some embodiments, the present invention provides
methods of reducing
production of IgA, IgM or both in subjects diagnosed with autoimmune
disease(s) and/or
fibrosis comprising administering to the subjects a therapeutically effective
dose of sBCMA
variant protein(s). In some embodiments, the present invention provides
methods of
reducing production of IgA, IgG or both in subjects diagnosed with autoimmune
disease(s)
and/or fibrosis comprising administering to the subjects a therapeutically
effective dose of
sBCMA variant protein(s). In some embodiments, the present invention provides
methods of
reducing production of IgG, IgM or both in subjects diagnosed with autoimmune
disease(s)
and/or fibrosis comprising administering to the subjects a therapeutically
effective dose of
sBCMA variant protein(s).
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[00307] In some embodiments, the present invention provides
methods of reducing
production of IgA in subjects diagnosed with autoimmune disease(s) and/or
fibrosis
comprising administering to the subjects a therapeutically effective dose of
sBCMA variant
protein(s). In some embodiments, the present invention provides methods of
reducing
production of IgM in subjects diagnosed with autoimmune disease(s) and/or
fibrosis
comprising administering to the subjects a therapeutically effective dose of
sBCMA variant
protein(s). In some embodiments, the present invention provides methods of
reducing
production of IgG in subjects diagnosed with autoimmune disease(s) and/or
fibrosis
comprising administering to the subjects a therapeutically effective dose of
sBCMA variant
protein(s).
[00308] In some embodiments, the present invention provides
methods of reducing
production of IgA and IgM in subjects diagnosed with autoimmune disease(s)
and/or fibrosis
comprising administering to the subjects a therapeutically effective dose of
sBCMA variant
protein(s). In some embodiments, the present invention provides methods of
reducing
production of IgA and IgG in subjects diagnosed with autoimmune disease(s)
and/or fibrosis
comprising administering to the subjects a therapeutically effective dose of
sBCMA variant
protein(s). In some embodiments, the present invention provides methods of
reducing
production of IgG and IgM in subjects diagnosed with autoimmune disease(s)
and/or fibrosis
comprising administering to the subjects a therapeutically effective dose of
sBCMA variant
protein(s).
[00309] In some embodiments, the present invention provides
methods of reducing
production of IgA, IgM and IgG in subjects diagnosed with autoimmune
disease(s) and/or
fibrosis comprising administering to the subjects a therapeutically effective
dose of sBCMA
variant protein(s).
[00310] In some embodiments, the method as disclosed herein
does not affect normal
B cell viability. In some embodiments, the subject is diagnosed with an
autoimmune disease.
In some embodiments, the subject is diagnosed with fibrosis. In some
embodiments, the
subject is human.
1003H1 In some embodiments, the present invention provides
methods of reducing
immunoglobulin production in subjects diagnosed with autoimmune disease(s)
and/or
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fibrosis comprising administering to the subjects a therapeutically effective
dose of sBCMA
variant ¨ Fc fusion protein(s).
1003121 In some embodiments, the present invention provides
methods of reducing
production of IgA, IgM and/or IgG in subjects diagnosed with autoimmune
disease(s) and/or
fibrosis comprising administering to the subjects a therapeutically effective
dose of sBCMA
variant ¨ Fc fusion protein(s).
[00313] In some embodiments, the present invention provides
methods of reducing
production of IgA, IgM or both in subjects diagnosed with autoimmune
disease(s) and/or
fibrosis comprising administering to the subjects a therapeutically effective
dose of sBCMA
variant ¨ Fc fusion protein(s). In some embodiments, the present invention
provides methods
of reducing production of IgA, IgG or both in subjects diagnosed with
autoimmune
disease(s) and/or fibrosis comprising administering to the subjects a
therapeutically effective
dose of sBCMA variant ¨ Fc fusion protein(s). In some embodiments, the present
invention
provides methods of reducing production of IgG, IgM or both in subjects
diagnosed with
autoimmune disease(s) and/or fibrosis comprising administering to the subjects
a
therapeutically effective dose of sBCMA variant ¨ Fc fusion protein(s).
[00314] In some embodiments, the present invention provides
methods of reducing
production of IgA in subjects diagnosed with autoimmune disease(s) and/or
fibrosis
comprising administering to the subjects a therapeutically effective dose of
sBCMA variant
¨ Fc fusion protein(s). In some embodiments, the present invention provides
methods of
reducing production of IgM in subjects diagnosed with autoimmune disease(s)
and/or
fibrosis comprising administering to the subjects a therapeutically effective
dose of sBCMA
variant ¨ Fc fusion protein(s). In some embodiments, the present invention
provides methods
of reducing production of IgG in subjects diagnosed with autoimmune disease(s)
and/or
fibrosis comprising administering to the subjects a therapeutically effective
dose of sBCMA
variant ¨ Fc fusion protein(s).
[00315] In some embodiments, the present invention provides
methods of reducing
production of IgA and IgM in subjects diagnosed with autoimmune disease(s)
and/or fibrosis
comprising administering to the subjects a therapeutically effective dose of
sBCMA variant
¨ Fc fusion protein(s). In some embodiments, the present invention provides
methods of
reducing production of IgA and IgG in subjects diagnosed with autoimmune
disease(s)
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and/or fibrosis comprising administering to the subjects a therapeutically
effective dose of
sBCMA variant ¨ Fc fusion protein(s). In some embodiments, the present
invention provides
methods of reducing production of IgG and IgM in subjects diagnosed with
autoimmune
disease(s) and/or fibrosis comprising administering to the subjects a
therapeutically effective
dose of sBCMA variant ¨ Fc fusion protein(s).
[00316] In some embodiments, the present invention provides
methods of reducing
production of IgA, IgM and IgG in subjects diagnosed with autoimmune
disease(s) and/or
fibrosis comprising administering to the subjects a therapeutically effective
dose of sBCMA
variant ¨ Fc fusion protein(s).
[00317] In some embodiments, the method as disclosed herein
does not affect normal
B cell viability. In some embodiments, the subject is diagnosed with an
autoimmune disease.
In some embodiments, the subject is diagnosed with fibrosis. In some
embodiments, the
subject is human.
[00318] The present invention provides inter alia, a method of
reducing production of
IgA, IgM, and/or IgG in a subject diagnosed with an autoimmune disease and/or
fibrosis,
said method comprising administering to the subject a therapeutically
effective dose of an
sBCMA variant protein and/or sBCMA variant -Fc fusion protein. In some
embodiments,
the present invention provides inter alia, a method of reducing production of
IgA, IgM,
and/or IgG in a subject diagnosed with an autoimmune disease, said method
comprising
administering to the subject a therapeutically effective dose of an sBCMA
variant protein. In
some embodiments, the present invention provides inter al/a, a method of
reducing
production of IgA, IgM, and/or IgG in a subject diagnosed with an autoimmune
disease, said
method comprising administering to the subject a therapeutically effective
dose of an
sBCMA variant -Fc fusion protein. In some embodiments, the present invention
provides
inter alia, a method of reducing production of IgA, IgM, and/or IgG in a
subject diagnosed
with fibrosis, said method comprising administering to the subject a
therapeutically effective
dose of an sBCMA variant protein. In some embodiments, the present invention
provides
inter alia, a method of reducing production of IgA, IgM, and/or IgG in a
subject diagnosed
with fibrosis, said method comprising administering to the subject a
therapeutically effective
dose of an sBCMA variant -Fc fusion protein.
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[00319] In some embodiments, the invention provides a method of
reducing
production of IgA, IgM, and/or IgG in a subject diagnosed with an autoimmune
disease or
fibrosis, said method comprising administering to the subject a
therapeutically effective dose
of an sBCMA variant-Fc fusion protein, wherein the sBCMA variant-Fc fusion
protein
comprises:
a) a variant sBCMA domain comprising at least one amino acid substitution as
compared to SEQ ID NO:1, wherein said amino acid substitution is at a position
number selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 9, 10, 11,
12, 14, 16,
19, 20, 22, 23, 25, 26, 29, 31, 32, 35, 36, 38, 39, 42, 43, 44, 45, 46, 47,
48, 49, 50, 51,
52, 53, and 54, wherein the numbering is according to the EU index;
b) an optional linker; and
c) an Fc domain.
[00320] In some embodiments, the invention provides the method
as disclosed herein,
wherein normal B cell viability is not altered.
[00321] In some embodiments, the invention provides the method
as disclosed herein,
wherein the subject is diagnosed with the autoimmune disease.
[00322] In some embodiments, the invention provides the method
as disclosed herein,
wherein the method reduces production of IgA.
[00323] In some embodiments, the invention provides the method
as disclosed herein,
wherein the method reduces production of IgG.
[00324] In some embodiments, the invention provides the method
as disclosed herein,
wherein the method reduces production of IgM.
[00325] In some embodiments, the invention provides the method
as disclosed herein,
wherein the method reduces production of both IgA and IgM.
[00326] In some embodiments, the invention provides the method
as disclosed herein,
wherein the method reduces production of both IgA and IgG.
[00327] In some embodiments, the invention provides the method
as disclosed herein,
wherein the method reduces production of IgM and IgG.
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[00328] In some embodiments, the invention provides the method
as disclosed herein,
wherein the method reduces production of both IgA, IgM and IgG.
1003291 In some embodiments, the invention provides the method
as disclosed herein,
wherein the autoimmune disease is selected from the group consisting of IgA
Nephropathy,
Systemic Lupus Erythematosus, Churg-Strauss Syndrome, Myasthenia Gravis,
Multiple
Sclerosis, and rheumatoid arthritis.
[00330] In some embodiments, the invention provides the method
as disclosed herein,
wherein the subject is diagnosed with the fibrosis.
[00331] In some embodiments, the invention provides the method
as disclosed herein,
wherein the fibrosis is selected from the group consisting of idiopathic
pulmonary fibrosis,
non-alcoholic steatohepatitis, scleroderma, and kidney fibrosis.
1003321 In some embodiments, the invention provides the method
as disclosed herein,
wherein said fusion protein comprises, from N- to C-terminal:
a) said variant sBCMA domain;
b) said optional linker; and
c) said Fc domain.
[00333] In some embodiments, the invention provides the method
as disclosed herein,
wherein said fusion protein comprises, from N- to C-terminal:
a) said Fc domain;
b) said optional linker; and
c) said variant sBCMA domain.
[00334] In some embodiments, the invention provides the method
as disclosed herein,
wherein said variant sBCMA domain has at least 80%, at least 85%, at least
90%, or at least
95% sequence identity to SEQ ID NO:l.
[00335] In some embodiments, the invention provides the method
as disclosed herein,
wherein said amino acid substitution(s) occur at one of said positions, two of
said positions,
three of said positions, four of said positions, five of said positions, six
of said positions,
seven of said positions, eight of said positions, or nine of said positions.
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[00336] In some embodiments, the invention provides the method
as disclosed herein,
wherein said amino acid substitution(s) is selected from the group consisting
of M1A, MIC,
M1I, MIR, MIT, MIV, L2C, L2S, Q3P, Q3R, M4E, M4I, M4T, M4V, A5T, G6E, Q7R,
S9A, S9F, S9P, Q10H, Q10P, Q10R.N11D. N11S, E12K, F14L, S16G, S16N, S16R,
H19L,
H19Y, A20V, A20T, I22M, I22V, P23S, Q25R, L26F, S29A, N31D, N31S, T32A, T32I,
T32P, L35S, L35P, T36A, T36I, T36P, Q38R, R39H, N42D, N42R, N42S, A43T, A43V,
S44D, S44G, S44N, S44R, V45A, V45M, T46A, T46I, N47D, N47K, N47R, N47S, S48L,
S48P, S48T, V49A, V49M, K50E, K50G, K5OR, K50T, G51E, T52A, T52M, N53D, N53K,
N53S, A54V, and A541.
[00337] In some embodiments, the invention provides the method
as disclosed herein,
wherein said amino acid substitution(s) is selected from the group consisting
of M1V, L2S,
Q3P, M4T, S9P, NIID, Sl6G, H19Y, N31S, N31D, T32I, T36A, R39H, N47S, K50E, and
N53E.
[00338] In some embodiments, the invention provides the method
as disclosed herein,
wherein said amino acid substitution(s) is selected from the group consisting
of Sl6G, H19Y
and T36A.
[00339] In some embodiments, the invention provides the method
as disclosed herein,
wherein said amino acid substitutions are selected from the group consisting
of
L2S/S9P/E12K/N31D/T36A/N42S/N535, M1V/T32P/T36A/T461/N53D/A54V,
Q3R/S16N/T36A/A43T, F 1 4L/S16G/T36AN45A/N47D,
M1T/M4V/S9F/S16G/T32A/Q38R, M1A/S9A/Q38R, G6E/Q25R/Q38R,
M1V/M4I/G6E/S9P/N11DN49M/T52M/A54V, N11D/S16G/N31S,
N11D/H19Y/122M/T32P/N47S/N53S, G6E/Q7R/H19Y/L35S, H19Y/N42D/S48P/T52A,
M1V/N31D/T32I/T36A, M1V/A5T/H19L/T36A,
M1T/N31D/T32A/T36A/Q38R/S44DN49A/K50E, M1V/T36A/Q38R/A43V,
M1V/L2S/S9P/Q10H/T36A/Q38R/K50G, T36A/Q38R/N53S,
M1T/L2S/L35P/T36A/Q38R/T46A/K5OR, A5T/A20V/136A/Q38R,
M1T/S16G/122V/T36A/S44G/T46AN49A, Sl6G/T36A,
M1I/N11D/S16G/122M/S29A/T36A/S44G/K5 OR, M1C/L2C/Q3R/M4E/N11D/S16G/T36P,
MH/N11D/S16G/122M/S29A/T36A/S44G/K5OR,
NI1D/N31D/T321/T36A/S44N/N47D/N53D, M1R/L2C/Q3R, H19Y/T36A/S44G,
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H19Y/T321/T36AN49A, H19Y/N31S/T36AN45A, H19Y/N31S/T36A, H19Y/T36P/T52A,
H19Y/N31D/T52M, M1V/H19YN45M, Sl6G/H19Y/N47D, Sl6G/H19Y/K50T,
S 1 6G/H19Y/S44N/K5OR, Ni 1D/H19Y/S48T,
S9P/N11D/S16R/T32A/Q38R/S44G/T461/T52A/N53D/A54T, N11D/S16G/S44R,
H19L/T32A/S44G/G51E/T52A, Sl6N/H19Y/T36A/K5OR, M1V/H19Y/T36A/R39H/T46A,
M1V/H19Y/T36A, H19Y/T36A/N42D/N47S/S48P, M1V/H19Y/T36A/S44G/N47D,
M1V/H19Y/T36A/N42R/N53S, H19Y/L35P/T36A/N42D/T461N49A,
Q3P/S 9P/H19Y/N3 1S/T36A/R39H/N47R/K5 OE, M1V/H19Y/T36A/N42R/N53S,
MIT/H19Y/T36A, MIV/S16N/H19Y/122M/T36A,
M1T/N11D/H19Y/T36A/N42SN45A/N53S, N11D/S16G/H19Y/T36A/N47S/N53D,
M1V/S9P/Q10P/S16G/H19Y/L26F/T36A/A43V/N53D, 5l6G/H19Y/T36A/V49A/N53D,
Sl6G/T36A/A43T/S44GN45M, M4V/S9P/S16G/T36A/Q38R,
S9P/N11S/S16G/136A/Q38R, N11D/E12K/S16R/T36A/T52M,
M4V/T32I/T36A/Q38R/A43TN45A/S 48P, S9P/N11D/S16G/Q25R,
M1T/A5T/S9P/S16G/Q25R/N31DN49M,
L2S/S9P/S16G/A20T/T321/Q38R/N42D/T46A/S48L, Sl6G/Q25R/T46A,
G6E/S9A/S16G/Q25R/N31D/N47S/T52M, H19Y/Q38R/T52M,
Ni 1 D/H19Y/122M/T32P/N47S/N53 S, Si 6G/H19Y/T36A, Si 6G/H19Y/T36A/N53D,
S9P/N11D/S16G/H19Y/T36A/N47S/N53D,
Q3P/S 9P/H19Y/N31S/T36A/R39H/N47R/K5 OE, M1V/L2S/M4T/N11D/H19Y/T36A,
M1V/L2S/M4T/N11D/T36A, M1V/L2S/M4T/H19Y/T361/V45A/V49M,
M1V/L2S/M4T/N11D/H19Y/T36A, M1V/L2S/M4T/S9P/Q10R/H19Y/T36A/T46A/N47S,
M1V/L2S/M4T/S16G/N31D/T321/T36A, M1V/M4T/T36A/Q38R/N53K,
M1T/N11D/H19Y/T36A/N42SN45A/N53S,
M1T/N31D/T32A/T36A/A38R/S44DN49A/K50E,
M1T/S9P/P23S/Q38R/N42S/S48P/V49A/A54V, H19Y/T36A/S44G, H19Y/T36A, and
M4T/T36A/Q38R/N42S/S44G/T46A/N47K/S48P/T52A.
[00340] In some embodiments, the invention provides the method
as disclosed herein,
wherein said variant sBCMA domain comprises the amino acid substitutions
S 1 6G/H19Y/T36A, and at least one further amino acid substitution selected
from the group
consisting of MIA, MIC, M11, MIR, MIT, MIV, L2C, L2S, Q3P, Q3R, M4E, M41, M4T,
M4V, A5T, G6E, Q7R, S9A, S9F, S9P, QI01-1, QI0P, QIOR, NI ID, NI IS, El2K,
FI4L,
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S16N, S16R, H19L, A20V, A20T, I22M, I22V, P23S, Q25R, L26F, S29A, N31D, N31S,
T32A, T32I, T32P, L35S, L35P, T36I, T36P, Q38R, R39H, N42D, N42R, N42S, A43T,
A43V, S44D, S44G, S44N, S44R, V45A, V45M, T46A, T46I, N47D, N47K, N47R, N47S,
S48L. S48P, S48T, V49A, V49M, K50E, K50G, K5OR, K50T, G51E, T52A, T52M, N53D,
N53K, N53S, A54V, and A54T.
[00341] In some embodiments, the invention provides the method
as disclosed herein,
wherein said variant sBCMA domain comprises the amino acid substitutions
S16G/H19Y/T36A/N53D, and at least one further amino acid substitution selected
from the
group consisting of MIA, MIC, MH, MIR, MIT, MIV, L2C, L2S, Q3P, Q3R, M4E, M4I,
M4T, M4V, A5T, G6E, Q7R, S9A, S9F, S9P, Q10H, Q10P, Q10R, N11D, Ni IS, E12K,
Fl4L, Sl6N, Sl6R, H19L, A20V, A20T, I22M, I22V, P23S, Q25R, L26F, S29A, N31D,
N31S, T32A, T32I, T32P, L35S, L35P, T36I, T36P, Q38R, R39H, N42D, N42R, N42S,
A43T, A43V, S44D, S44G, S44N, S44R, V45A, V45M, T46A, T46I, N47D, N47K, N47R,
N47S, S48L, S48P, S48T, V49A, V49M, K50E, K50G, K5OR, K50T, G51E, T52A, T52M,
N53K, N53S, A54V, and A54T.
[00342] In some embodiments, the invention provides the method
as disclosed herein,
wherein said variant sBCMA domain comprises the amino acid substitutions
S9P/N11D/S16G/H19Y/T36A/N47S/N53D, and at least one further amino acid
substitution
selected from the group consisting of M1A, MIC, M1I, MIR, MIT, MIV, L2C, L2S,
Q3P,
Q3R, M4E, M4I, M4T, M4V, A5T, G6E, Q7R, S9A, S9F, Ql0H, Ql0P, Ql0R, N11S,
E12K, F14L, S16N, S16R, H19L, A20V, A20T, I22M, I22V, P23S, Q25R, L26F, S29A,
N31D, N31S, T32A, T32I, T32P, L35S, L35P, T36I, T36P, Q38R, R39H, N42D, N42R,
N42S, A43T, A43V, S44D, S44G, S44N, S44R, V45A, V45M, T46A, T46I, N47D, N47K,
N47R, S48L, S48P, S48T, V49A, V49M, K50E, K50G, K5OR, K50T, G51E, T52A, T52M,
N53K, N53S, A54V, and A54'I'.
[00343] In some embodiments, the invention provides the method
as disclosed herein,
wherein said variant sBCMA domain comprises the amino acid substitutions
Q3P/S9P/H19Y/N31S/T36A/R39H/N47R/K50E, and at least one further amino acid
substitution selected from the group consisting of MIA, MIC, M1I, MIR, MIT,
MIV, L2C,
L2S, Q3R, M4E, M4I, M4T, M4V, A5T, G6E, Q7R, S9A, S9F, Ql0H, Ql0P, Q10R, N1
1D,
NI IS, E12K, F14L, S16G, S I6N, S16R, H19L, A20V, A20T, I22M, I22V, P23S,
Q25R,
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L26F, S29A, N31D, T32A, T32I, T32P, L35S, L35P, T36I, T36P, Q38R, N42D, N42R,
N42S, A43T, A43V, S44D, S44G, S44N, S44R, V45A, V45M, T46A, T46I, N47D, N47K,
N47S, S48L, S48P, S48T, V49A, V49M, K50G, K5OR, K50T, G51E, T52A, T52M, N53D,
N53K, N53S, A54V, and A54T.
[00344] In some embodiments, the invention provides the method
as disclosed herein,
wherein said variant sBCMA domain comprises the amino acid substitutions
M1V/L2S/M4T/S16G/N31D/T321/T36A, and at least one further amino acid
substitution
selected from the group consisting of M1A, M1C, M1I, M1R, M1T, L2C, Q3P, Q3R,
M4E,
M4I, M4V, A5T, G6E, Q7R, S9A, S9F, S9P, Q10H, Q10P, Q10R, N11D, N11S, E12K,
F14L, S16N, S16R, H19L, H19Y, A20V, A20T, I22M, I22V, P23S, Q25R, L26F, S29A,
N31S, T32A, T32P, L35S, L35P, T36I, T36P, Q38R, R39H, N42D, N42R, N42S, A43T,
A43V, S44D, S44G, S44N, S44R, V45A, V45M, T46A, T46I, N47D, N47K, N47R, N47S,
S48L, S48P, S48T, V49A, V49M, K50E, K50G, K5OR, K50T, G51E, T52A, T52M, N53D,
N53K, N53S, A54V, and A54T.
[00345] In some embodiments, the invention provides the method
as disclosed herein,
wherein said variant sBCMA domain has at least 90% sequence identity to SEQ ID
NO: 67.
[00346] In some embodiments, the invention provides the method
as disclosed herein,
wherein said variant sBCMA domain has at least 90% sequence identity to SEQ ID
NO: 68.
[00347] In some embodiments, the invention provides the method
as disclosed herein,
wherein said variant sBCMA domain has at least 90% sequence identity to SEQ ID
NO: 69.
[00348] In some embodiments, the invention provides the method
as disclosed herein,
wherein said variant sBCMA domain has at least 90% sequence identity to SEQ ID
NO: 49.
[00349] In some embodiments, the invention provides the method
as disclosed herein,
wherein said variant sBCMA domain has at least 90% sequence identity to SEQ ID
NO: 74.
[00350] In some embodiments, the invention provides the method
as disclosed herein,
wherein said variant sBCMA domain has the amino acid sequence of SEQ ID NO:
67.
[00351] In some embodiments, the invention provides the method
as disclosed herein,
wherein said variant sBCMA domain has the amino acid sequence of SEQ ID NO:
68.
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[00352] In some embodiments, the invention provides the method
as disclosed herein,
wherein said variant sBCMA domain has the amino acid sequence of SEQ ID NO:
69.
1003531 In some embodiments, the invention provides the method
as disclosed herein,
wherein said variant sBCMA domain has the amino acid sequence of SEQ ID NO:
49.
[00354] In some embodiments, the invention provides the method
as disclosed herein,
wherein said variant sBCMA domain has the amino acid sequence of SEQ ID NO:
74.
[00355] In some embodiments, the invention provides the method
as disclosed herein,
wherein said Fc domain is a human IgG Fc domain or a variant human IgG Fe
domain.
[00356] In some embodiments, the invention provides the method
as disclosed herein,
wherein said human IgG Fc domain comprises the hinge-CH2-CH3 of human IgG1 .
[00357] In some embodiments, the invention provides the method
as disclosed herein,
wherein said Fc domain is a variant human IgG Fc domain.
[00358] In some embodiments, the invention provides the method
as disclosed herein,
wherein said Fc domain is a human IgG1 Fc domain.
[00359] In some embodiments, the invention provides the method
as disclosed herein,
wherein said linker is SEQ ID NO:87.
[00360] In some embodiments, the invention provides the method
as disclosed herein,
wherein said linker is selected from the group consisting of (GS)n, (GSGGS)n,
(GGGGS)n,
and (GGGS)n, wherein n is selected from the group consisting of 1, 2, 3, 4 and
5.
[00361] In some embodiments, the invention provides the method
as disclosed herein,
wherein said linker is SEQ ID NO:88.
[00362] In some embodiments, the invention provides the method
as disclosed herein,
wherein the sBCMA variant ¨ Fc fusion protein comprises the amino acid
sequence of SEQ
ID NO:80.
[00363] In some embodiments, the invention provides the method
as disclosed herein,
wherein the sBCMA variant ¨ Fc fusion protein comprises the amino acid
sequence of SEQ
Ill NO:81.
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[00364] In some embodiments, the invention provides the method
as disclosed herein,
wherein the sBCMA variant ¨ Fc fusion protein comprises the amino acid
sequence of SEQ
ID NO:82.
[00365] In some embodiments, the invention provides the method
as disclosed herein,
wherein the sBCMA variant ¨ Fc fusion protein comprises the amino acid
sequence of SEQ
ID NO:83.
[00366] In some embodiments, the invention provides the method
as disclosed herein,
wherein the sBCMA variant ¨ Fc fusion protein comprises the amino acid
sequence of SEQ
ID NO:84.
2. Therapeutic administration
[00367] In certain embodiments, a therapeutically effective
composition or formulation
having one or more variant sBCMA proteins may be administered systemically to
the
individual or via any other route of administration known in the art.
[00368] In certain embodiments, a therapeutically effective
composition or formulation
having one or more sBCMA variant ¨ Fc fusion proteins may be administered
systemically
to the individual or via any other route of administration known in the art.
3. Dosing
[00369] In some embodiments, an effective dose of the
therapeutic entity of the present
invention, e.g. for the treatment of fibrotic and/or immunomodulatory
disorders, varies
depending upon many different factors, including means of administration,
target site,
physiological state of the patient, whether the patient is human or an animal,
other
medications administered, and whether treatment is prophylactic or
therapeutic. Treatment
dosages can be titrated to optimize safety and efficacy.
VI. EXAMPLES
A. EXAMPLE 1: Cynomolgus Monkey Single Dose Toxicity Study
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[00370] The purposes of this study were to evaluate the acute
toxicity after single
administration of ABOO1 (sBCMA clone #71-Fc fusion protein) via intravenous
infusion in
cynomolgus monkeys, to provide the maximum tolerated dose (MTD) as reference
for the
design of subsequent toxicity studies and clinical trials, and to characterize
the toxicokinetics
and immunogenicity. A total of ten Cynomolgus monkeys (1 animal/gender/group)
were
assigned into 5 groups and given a single intravenous infusion of sBCMA
variant - Fc fusion
protein (0.1, 1, 10 and 100 mg/kg) or vehicle and observed for 6 weeks. The
dose volume
was adjusted as per the latest body weight of animals. The actual infusion
duration did not
exceed 5% of the nominal infusion duration (Figure 1).
[00371] Parameters evaluated included clinical observations,
body weights, food
consumption, hematology, coagulation, plasma chemistry, lymphocyte
immunophenotype,
immunoglobulin,cytokines and gross pathology. Clinical observations were
conducted daily
from the day next to the randomization to experimental completion according to
the
frequencies listed. Non-dosing day: once in the morning and once in the
afternoon. Dosing
days: once at pre-dose, once within lh and 3h to 6h post-dose. Body weight
monitored
during Pre-dose phase: once on Day -13 and Day -6, respectively, followed by
once on days
1, 2, 7, 14, 21, 28, 35 and 42 post-dose. Blood collection were performed
during pre-dose
phase: Day -13, Day -6, Day -3 (M1305, M1407), Day 1 (pre-dose) and dosing
phase: 4
times, Days 2, 7, 14 and 42, among which coagulation wasnot included on Days 7
and 14.
The animals were necropsied at the end of observation period (Day 43), Animals
were fasted
overnight (no longer than 24 hours) before necropsy (Figure 1). In conclusion,
no drug-
related abnormalities of body weight were observed in the animals of each
groupduring the
observation period (Figure 4). Hematology findings include: 100 mg/kg: A
decrease (by
56%) of LYMP was noted in the male animal (Figure 5A) on Day 2 and in the
female
animal on Day 7 (by 57%) (Figure 5B) when comparing with the pre-dose value on
Day 1.
mg/kg: A decrease (by 31%) of LYMP was noted in the female animal on Day 7
when
comparing with the pre-dose value on Day 1 (Figure 5B). No significant
abnormalities were
noted in the male animal (Figure 5A). 1 mg/kg: A decrease (by 26%) of LYMP was
noted
in the female animal on Day 7 when comparing with the pre-dose value on Day 1
(Figure
5B). No significant abnormalities were noted in the male animal. 0.1 mg/kg: No
significant
abnormalities were noted in the female or male animals (Figure 5). Declines of
RBC, HGB,
and HCT were observed in the female and male animals of each group on Day 2,
Day 7,
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and/or Day 14. Considering that much blood were sampled during the experiment,
it was
considered that the decreases in RBC, HGB, and HCT might be related to the
blood
sampling (Figures 8 to 11). No abnormalities of coagulation parameters were
noted in the
female or male animals of each group (Figures 12 to 13). No abnormalities of
plasma
chemistry parameters were noted in the female or male animals of each group
(Figures 14 to
19). No abnormalities of immunophenotype were noted in the female or male
animals of
each group. No pathological gross abnormalities were present in any animal
(Figures 2 and
3). Cytokine analysis showed at 100 mg/kg, an increase of IL-10, IFN-y, IL-17A
were noted
in the male animal on Day 1, Day 2or Day 3 when comparing with the pre-dose
value or the
vehicle (Figures 20 and 21). No abnormalities of cytokines were noted in the
female animal
(Figures 22 and 23). No abnormalities of cytokines were noted in the female or
male
animals of other groups. Importantly, differences were noted in immunoglobulin
levels in
both female and male treated groups. Specifically, 100 mg/kg: Decreases of
IgA, IgM,
and/or IgG were noted in the female and/or male animals during Day 2 to Day 42
when
comparing with the pre-dose value. 10 mg/kg: Decreases of IgA, IgM, and/or IgG
were
noted in the female and/or male animal during Day 7 to Day 42 when comparing
with the
pre-dose value. 1 mg/kg: Decreases of IgA and IgM were noted in the female
and/or male
animals on Day 7and Day 14 when comparing with the pre-dose value. 0.1 mg/kg:
No
abnormalities of immunoglobulin were noted in the female or male animals
(Figures 6 and
7, Figures 16 and 19).
B. EXAMPLE 2: Cell Development and Clone Selection
[00372] CHO-K1-C6-4G5 host cells thawed from CHO-K1-C6-4G5 SCB
cell bank,
has been maintained in exponential phase with HyCell TranFx-C medium in
several
passages. On the day of transfection, cells were adjusted to viable cell
density 1E+06
cells/mL in 27 mL cell culture (125 mL shake flask). Transfection mixtures was
prepared by
diluting 50 lig of linearized expression plasmid in 2.5 mL OptiPRO SFM. The
FreeStyle
MAX solution was then mixed with the DNA solution and left at room temperature
for
10-20 minutes. After incubation, the solution was added into the CHO-K1 -C6
culture (25
mL in a 125 mL shake flask). Transfected cells were incubated in a 130 rpm, 37
C, 5% CO2
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incubator. One portion of transfected cells were used for stable pools
generation post
transfection 48 hours.
1003731 48 hours post transfection, transfected cells were
subjected to drug selection.
Cells were seeded at density of 5 E+05 cells/mL in 30 mL medium (HyCell
TransFx-C
containing 4 mM L-glutamine and 0.1% F-68) in a 150T Flask with selection
drugs (15
g/mL Puromycin + 800 nM MTX). Cells were incubated in a 37 C, 8% CO2 static
incubator for Day 0.
[00374] After 5 to 7 days, cell viability would drop to 15 to
25%. Cell cultures were
centrifugation at 200 g, 5 minutes, 22 C. Cell culture media were removed and
cell pellets
were re-suspended in 10 mL fresh selective medium in a 75T Flask, incubated in
a 37 C, 8%
CO2 static incubator. In another 10 to 15 days, cells would then gradually
recovered to 40 to
60% viability and kept in a 10 mL culture in a 75T Flask. Once cell viability
achieved more
than 50-60%, cells would be expanded to 20-25 mL culture in a 125 mL shake
flask,
incubated in a 130 rpm, 37 C, 5% CO2 incubator. In another 8 to 10 days, each
pool would
recovery to about 90% viability. Once each pool reached 90% viability by the
subsequent
culture, cryopreservation was performed for at least 2 vials per pool.
[00375] Upon all pools recovered to 90% viability and
cryopreservation was done,
each pool would be thawed and evaluated by 11 days Fed-batch culture
experiment.
Inoculating each pool of cells at viable cell density of ¨5.5E+05 cells/mL in
two different 10
mL medium HyCell CHO or BalanCD CHO in a 50mL Spin tube, incubated in a 180
rpm,
37 C, 5%CO2 incubator as Day0. Viable cell density and viability were recorded
by
BIORAD TC20 cell counter (Figure 24A and B).
[00376] Cell culture fluids were harvested on day 11 and
followed by centrifugation at
2000 g, 20 minutes, 22 C. The culture supernatants were passed through a
0.221.im filter
and ready for titer determination. A portion of filtered supernatants was the
source materials
for the purification of sBCMA variant ¨ Fc fusion protein clones using GE
Protein A HP
SpinTrap column. ProA purified materials were subjected to non-reduced and
reduced SDS-
PAGE (Figure 25A and B).
[00377] Two pools of sBCMA variant ¨ Fc fusion proteins were
choosen based on
their titer and performance were thawed and adapted to HyCell CHO medium
containing 4
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mIVI L-glutamine with 15 ug/mL Puromycin and 800 nM MTX or 60 ug/mL Puromycin
and
3000 nM MTX about a week. Pools were kept at the exponential phase, incubated
in a 130
rpm, 37 C and 5% CO2 incubator. One day before the day of SCC experiment,
cells were
seeded at viable cell density 5-7E+05 cells/mL with selective drugs. On the
day of SCC
experiment, the condition of the cells should achieve viable cell density ¨
1.4E+06 cells/mL,
viability >= 90%. 200 uL cloning medium was dispensed into each well of the 96
well plate.
There were 4 plates for each pool. Each plate was labeled with a barcode.
Using a cell
strainer such as 40 um nylon mesh cell strainer to obtain a uniform single
cell suspension at
least 3 mL. Cells were then adjusted to viable cell density ¨7E+05 cells/mL in
HyCell CHO
medium with 4 mM L-glutamine. Load 70 uL of the cells into the cartridge of
SCPTm. The
parameter of single cell printer was set to dispense single cell into each
well in a 96 well
plate. The plates were then centrifugation at 200 g, 22 C for 5 minutes.
Images were taken
by CloneSelect Imager (CSI) under high resolution mode for day 0, 1 and 2. The
plates were
then incubated in a 37 C, 8% CO2 static incubator. On day 14 or 18, images
were taken by
CSI again.
[00378] There were 35 and 22 clones picked from 96 well plates
of two sBCMA
variant ¨ Fc fusion protein pools respectively. Some clones were not be able
to grow as the
concentration of selection drugs was gradually increased back to 100%
selection pressure
during a scale-up process. Ultimately, clones that were able to grow in a 50
mL Spin tube
and eventually achieved >= 95% viability and the viable cell density >= 1E+06
cells/mL
would be cryopreserved. There were 40 clones cryopreserved.
[00379] 40 clones from two pools were screened by the
experiment of 12 day Fed
batch culture. Cells from each clone were cultured at viable cell density
5.5E+05 cells/mL in
a total 10 mL No selective drug medium (HyCell CHO containing 6 mM L-gln) in a
50 mL
Spin tube, incubated in a 180 rpm, 37 C, 5% CO2 incubator. Cell culture fluids
were
harvested on day 12 and followed by centrifugation at 2000 g, 20 minutes, 22
C. The
culture supernatants were passed through a 0.22 [tm filter and ready for titer
determination
using ProA-HPLC. A portion of filtered supernatants was the source materials
for the
purification of JHL9931 antibodies using GE Protein A HP SpinTrap column. Top
clones
were chosen based on ProA-HPLC titer and Day12 cell viability (Figure 26). In
addition,
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Purified ProA materials from top clones were subjected to UPLC-FLR analysis
for N-glycan
profiles (Figure 27).
1003801 The examples set forth above are provided to give those
of ordinary skill in the
art a complete disclosure and description of how to make and use the
embodiments of the
compositions, systems and methods of the invention, and are not intended to
limit the scope
of what the inventors regard as their invention. Modifications of the above-
described modes
for carrying out the invention that are obvious to persons of skill in the art
are intended to be
within the scope of the following claims. All patents and publications
mentioned in the
specification are indicative of the levels of skill of those skilled in the
art to which the
invention pertains. All references cited in this disclosure are incorporated
by reference to the
same extent as if each reference had been incorporated by reference in its
entirety
individually.
[00381] All headings and section designations are used for
clarity and reference
purposes only and are not to be considered limiting in any way. For example,
those of skill
in the art will appreciate the usefulness of combining various aspects from
different headings
and sections as appropriate according to the spirit and scope of the invention
described
herein.
[00382] All references cited herein are hereby incorporated by
reference herein in their
entireties and for all purposes to the same extent as if each individual
publication or patent
or patent application was specifically and individually indicated to be
incorporated by
reference in its entirety for all purposes.
[00383] Many modifications and variations of this application
can be made without
departing from its spirit and scope, as will be apparent to those skilled in
the art. The specific
embodiments and examples described herein are offered by way of example only.
C. EXAMPLE 3: Evaluation of sBCMA Variants on lupus model
in NZBWF13
mice + Pristane
[00384] The effect of test articles on the Lupus model in
NZBWF1/J mice with an
intraperitoneal injection of pristane was evaluated. NZBWF1/J mice of 11 - 12
weeks were
randomly assigned into 4 groups, and animals in the model groups received
pristane.
C57BL/6 mouse was used as normal control.
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[00385] The NZB/W Fl mice is a classical lupus model generated
by the Fl hybrid
between the NZB and NZW strains. These hybrid mice develop severe lupus-like
phenotypes, similar to that in lupus patients. These lupus-like phenotypes
include
lymphadenopathy, splenomegaly, elevated serum antinuclear autoantibodies (ANA)
including anti-dsDNA IgG, and immune complex-mediated glomerulonephritis (GN).
As for
SLE patients, disease in the NZB/W Fl strain is strongly biased towards
females, which is in
part due to estrogen levels.
[00386] Intraperitoneal injection of pristane stimulates the
formation of lupus-
associated autoantibodies against multiple nuclear antigens. This leads to
chronic
inflammation with the development of lupus-like autoimmunity, which
particularly includes
the formation of antibodies characteristic of SLE as well as immune-complex
nephritis with
a high degree of similarity to human SLE.
[00387] 48 NZBWF1/J mice was randomized into 4 groups, 12 in
each group based on
body weight and urine protein level before the experiment. All NZBWF1/J mice
received
pristane in a volume of 0..5mL by intraperitoneal injection. Group 1 received
saline vehicle
control with pristane. Group 2 received 10mg/kg Telitacicept twice a week with
pristane.
Group 3 received 1 mg/kg sBCMA variant twice a week with pristane. Group 4
received
10mg/kg sBCMA variant twice a week with pristane.
1003881 Level of proteinuria (mg/ml) was measured on day 14
post treatment, a
reduction in proteinuria level was observed in treatment groups 2, 3 and 4
(Figure 31).
Lymph node swelling score was recorded (Figure 32). Vehicle treated group was
presented
with highest score of lymph node swelling compared to other treatment groups
and animals
treated with 10mg/kg of sBCMA variant observed no lymph node swelling.
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