U.S. patent application number 16/721356 was filed with the patent office on 2020-10-29 for modulation of t cells with bispecific antibodies and fc fusions.
The applicant listed for this patent is Xencor, Inc.. Invention is credited to Matthew Bernett, Seung Chu, John Desjarlais, Dilki Wickramarachichi.
Application Number | 20200339624 16/721356 |
Document ID | / |
Family ID | 1000004942432 |
Filed Date | 2020-10-29 |
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United States Patent
Application |
20200339624 |
Kind Code |
A1 |
Chu; Seung ; et al. |
October 29, 2020 |
MODULATION OF T CELLS WITH BISPECIFIC ANTIBODIES AND FC FUSIONS
Abstract
The present invention relates to methods and compositions for
modulating T cells. The modulation includes suppressing or inducing
regulatory T cells or cytotoxic T cells.
Inventors: |
Chu; Seung; (Upland, CA)
; Bernett; Matthew; (Monrovia, CA) ;
Wickramarachichi; Dilki; (Pasadena, CA) ; Desjarlais;
John; (Pasadena, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Xencor, Inc. |
Monrovia |
CA |
US |
|
|
Family ID: |
1000004942432 |
Appl. No.: |
16/721356 |
Filed: |
December 19, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14217166 |
Mar 17, 2014 |
10544187 |
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16721356 |
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61911438 |
Dec 3, 2013 |
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61800743 |
Mar 15, 2013 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/73 20130101;
C07K 2319/00 20130101; C07K 2317/74 20130101; C07K 1/18 20130101;
C07K 2317/31 20130101; A61K 2039/505 20130101; C07K 2317/60
20130101; C07K 2317/515 20130101; C07K 16/2866 20130101; C07K
2317/52 20130101; C07K 14/55 20130101; C07K 16/2815 20130101; C07K
16/2812 20130101 |
International
Class: |
C07K 1/18 20060101
C07K001/18; C07K 16/28 20060101 C07K016/28; C07K 14/55 20060101
C07K014/55 |
Claims
1.-69. (canceled)
70. A heterodimeric protein comprising: (a) a first monomer
comprising: (i) a first Fc domain; (ii) an IL-2 protein; and (b) a
second monomer comprising: (i) a second Fc domain.
71. The heterodimeric protein according to claim 70, wherein the
first and second Fc domains are variant Fc domains comprising amino
acid variants selected from the group consisting of: L368D/K370S
and S364K; L368D/K370S and S364K/E357L; L368D/K370S and
S364K/E357Q; T411E/K360E/Q362E and D401K; L368E/K370S and S364K;
and K370S and S364K/E357Q, wherein numbering is according to EU
index as in Kabat.
72. The heterodimeric protein according to claim 71, wherein the
first and/or second variant Fc domain further comprises an amino
acid variant independently selected from the group consisting of:
236R, 328R, 330L, 236R/328R, 239D/332E,
E233P/L234V/L235A/G236del/S239K,
E233P/L234V/L235A/G236del/S239K/A327G,
E233P/L234V/L235A/G236del/S267K/A327G, E233P/L234V/L235A/G236del,
and E233P/L234V/L235A/G236del/S267K, wherein numbering is according
to EU index as in Kabat.
73. The heterodimeric protein according to claim 70, wherein the
first Fc domain is a variant Fc domain comprising amino acid
variants E233P/L234V/L235A/G236del/S267K and S364K/E357Q, and
wherein the second Fc domain is a variant Fc domain comprising
amino acid variants E233P/L234V/L235A/G236del/S267K and
L368D/K370S, wherein numbering is according to EU index as in
Kabat.
74. The heterodimeric protein according to claim 70, wherein the
IL-2 protein is an IL-2 variant having reduced ability to bind to
IL-2R.beta., IL-2R.gamma., and/or IL-2R.alpha..
75. The heterodimeric protein according to claim 70, wherein the
IL-2 protein is an IL-2 variant having increased ability to bind to
IL-2R.alpha..
76. The heterodimeric protein according to claim 70, wherein the
IL-2 protein is an IL-2 variant having reduced ability to bind to
IL-2R.beta. and/or IL-2R.gamma. and increased ability to bind to
IL-2R.alpha..
77. A method of inducing T cells, the method comprising contacting
the T cells with a composition comprising a heterodimeric protein
according to claim 70.
78. The method according to claim 77, wherein the T cells are
regulatory T cells (Tregs).
79. A method of suppressing T cells, the method comprising
contacting the T cells with a composition comprising a
heterodimeric protein according to claim 70.
80. A method A method of treating an autoimmune disease in a
subject, the method comprising administering to the subject a
composition comprising a heterodimeric protein according to claim
70.
81. A method A method of treating a cancer in a subject, the method
comprising administering to the subject a composition comprising a
heterodimeric protein according to claim 70.
82. A nucleic acid composition encoding a heterodimeric protein,
the nucleic acid composition comprising: a) a first nucleic acid
encoding the first monomer of claim 70; and b) a second nucleic
acid encoding the second monomer of claim 70.
83. A host cell comprising the nucleic acid composition of claim
82.
84. A method of making a heterodimeric protein comprising culturing
a host cell according to claim 83 under conditions whereby the
heterodimeric protein is produced.
85. A method of purifying a heterodimeric protein according to
claim 70, the method comprising: (a) providing a composition
comprising the heterodimeric protein; (b) loading the composition
onto an ion exchange column; and (c) collecting a fraction
containing the heterodimeric protein.
86. An IL-2 Fc fusion comprising: (a) a first monomer comprising:
(i) a first Fc domain; (ii) a first IL-2 protein; and (b) a second
monomer comprising (i) a second Fc domain; and (ii) a second IL-2
protein.
87. The IL-2 Fc fusion according to claim 86, wherein the first
and/or second IL-2 protein is an IL-2 variant engineered to have
reduced ability to bind to IL-2R.beta., IL-2R.gamma., and/or
IL-2R.alpha..
88. A method of inducing T cells, the method comprising contacting
the T cells with an IL-2 Fc fusion according to claim 86.
89. The method according to claim 88, wherein the T cells are
regulatory T cells (Tregs).
90. A method of suppressing T cells, the method comprising
contacting the T cells with an IL-2 Fc fusion according to claim
86.
91. A method A method of treating an autoimmune disease in a
subject, the method comprising administering to the subject an IL-2
Fc fusion according to claim 86.
92. A method A method of treating a cancer in a subject, the method
comprising administering to the subject an IL-2 Fc fusion according
to claim 86.
93. A nucleic acid composition encoding an IL-2 fusion, the nucleic
acid composition comprising: a) a first nucleic acid encoding the
first monomer of claim 86; and b) a second nucleic acid encoding
the second monomer of claim 86.
94. A host cell comprising the one or more nucleic acids of claim
86.
95. A method of making an IL-2 Fc fusion, the method comprising
culturing a host cell according to claim 94 under conditions
whereby the IL-2 Fc fusion is produced.
96. A method of purifying an IL-2 Fc fusion according to claim 86,
the method comprising: (a) providing a composition comprising the
IL-2 Fc fusion; (b) loading the composition onto an ion exchange
column; and (c) collecting a fraction containing the IL-2 Fc
fusion.
Description
PRIORITY CLAIM
[0001] This application is a continuation of U.S. patent
application Ser. No. 14/217,166, filed Mar. 17, 2014, which claims
priority to U.S. Provisional Patent Application Nos. 61/800,743,
filed Mar. 15, 2013 and 61/911,438, filed Dec. 3, 2013, each of
which is expressly incorporated by reference in the entirety.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted electronically in ASCII format and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Dec. 19, 2019, is named 067461-5163-US01_ST25.txt and is 831,510
bytes in size.
RELATED APPLICATIONS
[0003] U.S. Ser. No. 14/216,705, filed Mar. 17, 2014; Ser. No.
13/194,904, filed Jul. 29, 2011; Ser. No. 14/205,248, filed Mar.
11, 2014; Ser. No. 12/875,015, filed Sep. 2, 2010; Ser. No.
13/568,028, filed Aug. 6, 2012; Ser. No. 13/887,234, filed May 3,
2013; Ser. No. 13/648,951, filed Oct. 10, 2012; 61/913,832, filed
Dec. 9, 2013, and 61/938,095, filed Feb. 10, 2014 are all expressly
incorporated by reference in their entirety, particularly for the
recitation of amino acid positions and substitutions, and all data,
figures and legends relating thereto.
TECHNICAL FIELD
[0004] The present disclosure relates to methods and compositions
for modulating T cells.
BACKGROUND OF THE INVENTION
[0005] Immune system homeostasis relies on a fine balance between a
variety of T cell populations, including effector CD8 and CD4 T
cells and regulatory T cells. In disease states however, such as
cancer and autoimmune disease, this balance can be perturbed. In
cancer, infiltrating anti-tumor cytotoxic T cells can be prevented
from attacking cancer cells by tumor-resident regulatory T cells.
This can be seen from analysis of most human tumors, in which there
is a significant correlation between immune infiltration by
cytotoxic T cells and improved outcome, whereas infiltration by
regulatory T cells is instead associated with a poor outcome.
Indeed, several studies have demonstrated prognostic significance
of the CD8/Treg tumor ratio. Numerous mouse models have shown that
depletion of Treg with anti-CD25 antibody before tumor implantation
can have a dramatic impact on prevention of tumor growth. In
autoimmune diseases, effector T cells remain unregulated and attack
the body's own tissues. A major premise in this regard is that
defects in Treg cell number or function are a contributing factor.
Therefore, the ability to alter the balance between cytotoxicity
and regulation by fine-tuning the T cell response has great
potential for the treatment of cancer, autoimmune, and other
diseases.
[0006] One approach to controlling the balance of effector to
regulatory T cells is to target the Treg population for direct
modulation. However, despite years of effort, the discovery of a
single Treg-specific surface marker has been elusive, frustrating
efforts to deplete them specifically with monoclonal
antibodies.
[0007] Effector versus regulatory T cells can be loosely identified
by their surface markers, which can change based on their
activation state. Cytotoxic T cells express CD8, which interacts
with class I MHC. Effector helper T cells express CD4, which
interacts with class II MHC on antigen-presenting cells. The
hallmark of Treg cells is constitutive expression of both CD4 and
CD25. CD25 is the alpha component of the IL2 receptor
(IL2R.alpha.), which, together with CD122 (IL2R.beta.) and the
common cytokine receptor .gamma.-chain(y.sub.c) (CD132) form the
trimeric high-affinity receptor for IL2. Several approaches have
attempted Treg-specific depletion by targeting the high-affinity
IL2 receptor CD25 (IL2R.alpha.) with anti-CD25 antibodies such as
daclizumab, or with IL2-diptheria toxin (IL2-DT) fusions. However,
CD25 alone is not an ideal target because it also expressed on CD8
and CD4 effector T cells upon activation. Thus, approaches that
target Treg CD25 by itself might defeat their own purpose by also
depleting the activated effector cells that could potentially
attack the tumor.
[0008] Because of the importance of IL2 for T cell proliferation
and homeostasis, a variety of approaches to T cell modulation have
utilized IL2 itself or blocking of its high-affinity receptor
component CD25. Systemic IL2 administration (Proleukin) is an
approved therapy for metastatic melanoma and metastatic renal cell
carcinoma based on its ability to promote expansion of effector T
cells. However, systemic IL2 administration is also expected to
promote the suppressive Treg population, potentially diminishing or
confounding the desired enhancement of cytotoxic T cells.
Furthermore, systemic IL2 administration is also associated with a
variety of toxicities. Patients receiving systemic IL2 treatment
frequently experience severe cardiovascular, pulmonary, renal,
hepatic, gastrointestinal, neurological, cutaneous, haematological
and systemic adverse events. The majority of these side effects can
be explained by the development of so-called vascular leak syndrome
(VLS), a pathological increase in vascular permeability leading to
pulmonary edema and other issues. There is no treatment of VLS
other than withdrawal of IL2. These problems have led to the
pursuit of IL2 variants that perturb its affinity for one or more
of its receptor subunits. Alternatively, anti-CD25 antibodies that
block IL2-mediated T cell expansion have been utilized to treat
various diseases. Zenapax (daclizumab) is an approved
immunosuppressant for organ transplantation and is being
investigated for the treatment of autoimmune diseases such as
multiple sclerosis. These uses were developed based on daclizumab's
presumed ability to reduce effector T cell responses. However, due
to the more recently recognized dependence of Treg on IL2 for
survival, daclizumab is now--somewhat paradoxically--being utilized
in efforts to reduce Treg numbers in oncology. Because of the
strong potential of either IL2 or anti-CD25 agents to promote or
reduce both effector T cells and Treg with limited selectivity,
there is a strong need in the field to create more selective Treg
modulators.
SUMMARY OF THE INVENTION
[0009] Accordingly, the present invention provides methods and
compositions for suppressing and inducing T cells. In preferred
aspects, the methods and compositions of the invention suppress or
induce specific T cell types with little or no impact on other T
cell types. In further embodiments, the methods and compositions of
the invention suppress or induce regulatory T cells with little or
no impact on other T cell types, including cytotoxic T cells. In
other embodiments, the methods and compositions of the invention
suppress or induce cytotoxic T cells with little or no impact on
other T cell types, including regulatory T cells.
[0010] In one aspect, the present invention provides a method for
suppressing T cells that includes the step of administering a
composition comprising a bispecific antibody, wherein that
bispecific antibody includes: (a) a first monomer that has (i) a
first heavy chain constant region with a first variant Fc domain;
and (ii) an anti-CD25 binding moiety; and (b) a second monomer that
has (i) a second heavy chain constant region with a second variant
Fc domain; and (ii) a member selected from the group: an anti-CD4
binding moiety, an anti-CD8 binding moiety, an anti-CCR4 moiety, an
anti-GITR binding moiety, and an anti-PD-1 binding moiety. In
specific embodiments, the first variant Fc domain has a different
amino acid sequence than the second variant Fc domain. The
administration of such a bispecific antibody serves to suppress the
T cells. Suppression can be measured using assays known in the art,
including cell proliferation assays. Suppression can be shown in
such assays by a decrease in cell proliferation and/or general T
cell number as compared to the proliferation and/or numbers seen in
the absence of the bispecific antibody of the invention.
[0011] In further embodiments and in accordance with the above, the
T cells suppressed by the methods of the invention are regulatory T
cells. In still further embodiments, the second monomer comprises
the anti-CD4 binding moiety, and the bispecific antibody
specifically targets regulatory T cells with limited to no impact
on other T cell types.
[0012] In still further embodiments and in accordance with any of
the above, the anti-CD25 binding moiety is an anti-CD25 scFV
sequence that is covalently attached to the first heavy chain
sequence.
[0013] In still further embodiments and in accordance with any of
the above, the T cells suppressed by the methods and compositions
of the invention are cytotoxic T-cells. In yet further embodiments,
the second monomer comprises said anti-CD8 binding moiety, and the
bispecific antibody specifically targets cytotoxic T cells with
limited to no impact on other T cell types. In yet further
embodiments, the anti-CD8 binding moiety comprises all or a portion
of an antigen binding region of an antibody selected from the group
consisting of MCD8, 3B5, Sk1, OKT-8, and DK-25.
[0014] In still further embodiments and in accordance with any of
the above, the first and second variant Fc domains include a set of
amino acid substitutions selected from those sets depicted in FIG.
33A-33C.
[0015] In still further embodiments and in accordance with any of
the above, the first and second variant Fc domains comprise a set
of amino acid substitutions selected from the group consisting of
those sets depicted in FIG. 34A-34C.
[0016] In still further embodiments and in accordance with any of
the above, the first and second variant Fc domains comprise a set
of amino acid substitutions selected from the group consisting of
those sets depicted in FIG. 35.
[0017] In still further embodiments and in accordance with any of
the above, the first and/or second variant Fc domain comprises an
amino acid variant selected from the group consisting of: 236R,
239D, 239E, 243L, M252Y, V259I, 267D, 267E, 298A, V308F, 328F,
328R, 330L, 332D, 332E, M428L, N434A, N434S, 236R/328R, 239D/332E,
M428L, 236R/328F, V259I/V308F, 267E/328F, M428L/N434S, Y436I/M428L,
Y436V/M428L, Y436I/N434S, Y436V/N434S, 239D/332E/330L,
M252Y/S254T/T256E, V259I/V308F/M428L, and
E233P/L234V/L235A/G236del/S267K.
[0018] In still further embodiments and in accordance with any of
the above, the bispecific antibody comprises a sequence selected
from the sequences depicted in FIGS. 30-31.
[0019] In still further embodiments and in accordance with any of
the above, the first monomer comprises a sequence according to the
sequence designated as
11209-OKT4A_H0L0_scFv_Anti-TAC_H1L1_scFv_GDQ-Fc(216)_IgG1_pI_ISO(-)/pI_IS-
O(+RR)_IgG1, Heavy chain 2 (Heavy chain 2
(Anti-TAC_H1L1_scFv_GDQ-Fc(216)_IgG1_pI_ISO(+RR)) in FIG.
30A-30FF.
[0020] In still further embodiments and in accordance with any of
the above, the second monomer comprises a sequence according to the
sequence designated as
11209-OKT4A_HOL0_scFv_Anti-TAC_H1L1_scFv_GDQ-Fc(216)_IgG1_pI_ISO(-)/pI_IS-
O(+RR)_IgG1, Heavy chain 1
(OKT4A_HOL0_scFv_GDQ-Fc(216)_IgG1_pI_ISO(-)) in FIG. 30A-30FF.
[0021] In still further embodiments and in accordance with any of
the above, the first monomer comprises a sequence according to the
sequence designated as
12143-OKT4A_HOL0_scFv_Anti-TAC_H1L1_scFv_-Fc(216)_IgG1_pI_ISO(-)/pI_ISO(+-
RR)_C220S/FcKO, Heavy chain 2
(Anti-TAC_H1L1_scFv_Fc(216)_IgG1_pI_ISO(+RR)_G236R/L328R) in FIG.
30A-30FF.
[0022] In still further embodiments and in accordance with any of
the above, the second monomer comprises a sequence according to the
sequence designated as
12143-OKT4A_H0L0_scFv_Anti-TAC_H1L1_scFv_-Fc(216)_IgG1_pI_ISO(-)/pI_ISO(+-
RR)_C220S/FcKO, Heavy chain 1
(OKT4A_HOL0_scFv_Fc(216)_IgG1_pI_ISO(-)_G236R/L328R) in FIG.
30A-30FF.
[0023] In still further embodiments and in accordance with any of
the above, the first monomer comprises a sequence according to the
sequence designated as
13531-OKT4A_H1L1_Fab-Anti-TAC_H1.8L1_scFv_Fc(216)_IgG1_pI_ISO
(-)-pI_ISO(+RR)_C220S_IgG1_G236R/L328R, Heavy chain 2
(Anti-TAC_H1.8L1_scFv_Fc(216)_IgG1_pI_ISO(+RR)_G236R/L328R) in FIG.
30A-30FF.
[0024] In still further embodiments and in accordance with any of
the above, the second monomer comprises a sequence according to the
sequence designated as
13531-OKT4A_H1L1_Fab-Anti-TAC_H1.8L1_scFv_Fc(216)_IgG1_pI_ISO
(-)-pI_ISO(+RR)_C220S_IgG1_G236R/L328R, Heavy chain 1
(OKT4A_H1_IgG1_pI_ISO(-)_G236R/L328R) in FIG. 30A-30FF.
[0025] In still further embodiments and in accordance with any of
the above, the second monomer further comprises a sequence
according to the sequence designated as
13531-OKT4A_H1L1_Fab-Anti-TAC_H1.8L1_scFv_Fc(216)_IgG1_pI_ISO
(-)-pI_ISO(+RR)_C220S_IgG1_G236R/L328R, Light chain (OKT4A_L1) in
FIG. 30A-30FF.
[0026] In a further aspect, the present invention provides a method
for stimulating T cells that includes administering a heterodimeric
protein, where the heterodimeric protein includes: (a) a first
monomer with (i) a first heavy chain constant region comprising a
first variant Fc domain; (ii) an IL-2 protein; and (b) a second
monomer with: (i) a second heavy chain constant region comprising a
second variant Fc domain; (ii) a member selected from the group
consisting of: an anti-CD4 binding moiety, an anti-CD8 binding
moiety, an anti-CTLA4 binding moiety, an anti-CCR4 binding moiety,
an anti-PD-1 binding moiety, and an anti-GITR binding moiety. In
further embodiments, the first variant Fc domain has a different
amino acid sequence than the second variant Fc domain.
Administration of this heterodimeric protein stimulates the T
cells. As will be appreciated, the IL2 protein may comprise a full
length protein or a portion of the full length IL2 protein. In
further embodiments, the full or portion of the IL2 protein that is
part of the heterodimeric protein comprises a human IL2 protein
sequence.
[0027] In a further embodiment and in accordance with the above,
the T cells are regulatory T cells and the second monomer is the
anti-CD4 binding moiety.
[0028] In still further embodiments and in accordance with any of
the above, the second monomer further includes: (a) the second
heavy chain constant region further having a heavy chain variable
domain, and (b) a light chain sequence, where the heavy chain
variable domain and the light chain sequence together form antigen
binding moiety, including without limitation the anti-CD4 binding
moiety.
[0029] In still further embodiments and in accordance with any of
the above, the stimulated T cells are cytotoxic T cells and the
second monomer comprises the anti-CD8 binding moiety. In yet
further embodiments, the anti-CD8 binding moiety comprises all or a
portion of an antigen binding region of an antibody selected from
the group consisting of MCD8, 3B5, Sk1, OKT-8, and DK-25.
[0030] In still further embodiments and in accordance with any of
the above, the first and second variant Fc domains include a set of
amino acid substitutions selected from the group consisting of
those sets depicted in FIG. 33, 34 or 35.
[0031] In still further embodiments and in accordance with any of
the above, the first and/or second variant Fc domain comprises an
amino acid variant selected from the group consisting of: 236R,
239D, 239E, 243L, M252Y, V259I, 267D, 267E, 298A, V308F, 328F,
328R, 330L, 332D, 332E, M428L, N434A, N434S, 236R/328R, 239D/332E,
M428L, 236R/328F, V259I/V308F, 267E/328F, M428L/N434S, Y436I/M428L,
Y436V/M428L, Y436I/N434S, Y436V/N434S, 239D/332E/330L,
M252Y/S254T/T256E, V259I/V308F/M428L, and
E233P/L234V/L235A/G236del/S267K.
[0032] In still further embodiments and in accordance with any of
the above, the heterodimeric protein comprises a sequence selected
from the sequences depicted in FIGS. 30-31.
[0033] In still further embodiments and in accordance with any of
the above, the first monomer comprises a sequence according to the
sequence designated as
13027-hIL2_OKT4A_H1L1_IgG1_pI_ISO(-/+RR)_C220S_G236R/L328R, Heavy
chain 1 (hIL2_IgG1_pI_ISO(-)_C220S/G236R/L328R) in FIG.
30A-30FF.
[0034] In still further embodiments and in accordance with any of
the above, the second monomer comprises a sequence according to the
sequence designated as
13027-hIL2_OKT4A_H1L1_IgG1_pI_ISO(-/+RR)_C220S_G236R/L328R, Heavy
chain 2 (OKT4A_H1_IgG1_pI_ISO(+RR)_G236R/L328R) in FIG.
30A-30FF.
[0035] In still further embodiments and in accordance with any of
the above, the second monomer further comprises a sequence
according to the sequence designated as
13027-hIL2_OKT4A_H1L1_IgG1_pI_ISO(-/+RR)_C220S_G236R/L328R, Light
chain (OKT4A_L1) in FIG. 30A-30FF.
[0036] In still further embodiments and in accordance with any of
the above, the second monomer comprises a sequence according to the
sequence designated as 13038-OKT4A_H1L1_IgG1_G236R/L328R_hIL2(2),
Heavy chain (OKT4A_H1_IgG1_G236R/L328R_hIL2) in FIG. 30A-30FF.
[0037] In still further embodiments and in accordance with any of
the above, the second monomer further comprises a sequence
according to the sequence designated as
13038-OKT4A_H1L1_IgG1_G236R/L328R_hIL2(2), Light chain (OKT4A_L1)
in FIG. 30A-30FF.
[0038] In further aspects and in accordance with any of the above,
the present invention provides a composition comprising a
heterodimeric antibody, where the heterodimeric antibody includes:
(a) a first monomer having (i) a first antigen-binding domain,
which is an anti-CD25 binding domain; (ii) a first heavy chain
sequence comprising a first variant Fc domain as compared to a
human Fc domain; and (b) a second monomer having (i) a second
antigen-binding domain that binds to a member selected from the
group consisting of: CD4, CD8, CCR4, GITR, and PD-1, and (ii) a
second heavy chain sequence comprising a second variant Fc domain
as compared to a human Fc domain. In further embodiments, the first
and second variant Fc domains have different amino acid
sequences.
[0039] In further embodiments and in accordance with the above, the
antigen-binding domain binds to CD4.
[0040] In still further embodiments and in accordance with any of
the above, the first and second variant Fc domain includes an amino
acid variant independently selected from the variants listed in
FIG. 33, 34, or 35.
[0041] In still further embodiments and in accordance with any of
the above, the first and second variant Fc domain comprises an
amino acid variant selected from the group consisting of:
L368D/K370S and S364K; L368D/K370S and S364K/E357L; L368D/K370S and
S364K/E357Q; T411E/K360E/Q362E and D401K; L368E/K370S and S364K;
K370S and S364K/E357Q; and K370S and S364K/E357Q.
[0042] In still further embodiments and in accordance with any of
the above, the first and/or second variant Fc domain further
comprises an amino acid variant selected from the group consisting
of: 236R, 239D, 239E, 243L, M252Y, V259I, 267D, 267E, 298A, V308F,
328F, 328R, 330L, 332D, 332E, M428L, N434A, N434S, 236R/328R,
239D/332E, M428L, 236R/328F, V259I/V308F, 267E/328F, M428L/N434S,
Y436I/M428L, Y436V/M428L, Y436I/N434S, Y436V/N434S, 239D/332E/330L,
M252Y/S254T/T256E, V259I/V308F/M428L, and
E233P/L234V/L235A/G236del/S267K.
[0043] In still further embodiments and in accordance with any of
the above, the anti-CD25 binding domain is an anti-CD25 scFv
sequence and is covalently attached to said first heavy chain
sequence.
[0044] In still further embodiments and in accordance with any of
the above, the second antigen-binding domain comprises an scFv
sequence.
[0045] In still further embodiments and in accordance with any of
the above, the second monomer further has the second heavy chain
sequence further comprising a heavy chain variable domain, and a
light chain sequence, where the heavy chain variable domain and the
light chain sequence form said second antigen-binding domain.
[0046] In still further embodiments and in accordance with any of
the above, the composition comprises a format in accordance with a
format as depicted in FIG. 3 or FIGS. 36A-37U.
[0047] In a further aspect, the present invention provides a
composition comprising a heterodimeric protein that has: (a) a
first monomer comprising: (i) a first protein comprising a cell
marker; (ii) a first heavy chain sequence with a first variant Fc
domain as compared to a human Fc domain; and (b) a second monomer
comprising: (i) an antigen-binding domain that binds to a member
selected from the group consisting of: CD4, CD8, CTLA-4, CCR4, and
PD-1, and (ii) a second heavy chain sequence comprising a second
variant Fc domain as compared to a human Fc domain. In further
embodiments, the first and second variant Fc domains have different
amino acid sequences.
[0048] In still further embodiments and in accordance with any of
the above, the protein of the first monomer comprises a regulatory
T cell marker selected from the group listed in FIG. 32. In other
embodiments, the protein of the first monomer comprises a cytokine.
In yet further embodiments, the cytokine is IL2.
[0049] In still further embodiments and in accordance with any of
the above, the first and second variant Fc domain comprises an
amino acid variant independently selected from the variants listed
in FIG. 33, 34 or 35.
[0050] In still further embodiments and in accordance with any of
the above, the first and second variant Fc domain includes an amino
acid variant independently selected from the group consisting of:
L368D/K370S and S364K; L368D/K370S and S364K/E357L; L368D/K370S and
S364K/E357Q; T411E/K360E/Q362E and D401K; L368E/K370S and S364K;
K370S and S364K/E357Q; and K370S and S364K/E357Q.
[0051] In still further embodiments and in accordance with any of
the above, the first and/or second variant Fc domain further
includes an amino acid variant selected from the group consisting
of: 236R, 239D, 239E, 243L, M252Y, V259I, 267D, 267E, 298A, V308F,
328F, 328R, 330L, 332D, 332E, M428L, N434A, N434S, 236R/328R,
239D/332E, M428L, 236R/328F, V259I/V308F, 267E/328F, M428L/N434S,
Y436I/M428L, Y436V/M428L, Y436I/N434S, Y436V/N434S, 239D/332E/330L,
M252Y/S254T/T256E, V259I/V308F/M428L, and
E233P/L234V/L235A/G236del/S267K.
[0052] In still further embodiments and in accordance with any of
the above, the antigen-binding domain is an scFv sequence that is
covalently attached to said second heavy chain sequence.
[0053] In still further embodiments and in accordance with any of
the above, the second monomer further includes: (a) the second
heavy chain sequence further comprising a heavy chain variable
domain, and (b) a light chain sequence, wherein said heavy chain
variable domain and said light chain sequence form said second
antigen-binding domain.
[0054] In still further embodiments and in accordance with any of
the above, the heterodimeric protein comprises a sequence as listed
in FIGS. 30 and 31.
[0055] In still further embodiments and in accordance with any of
the above, the invention provides one or more nucleic acids
encoding a composition according to any of the compositions
described above. In yet further embodiments, the invention includes
a host cell expressing those one or more nucleic acids. In yet
further embodiments, the present invention provides a method of
making any of the compositions described herein, the method
including the step of culturing a host cell or more nucleic acids
encoding a composition according to any of the compositions
described above under conditions whereby the composition is
produced.
[0056] In further aspects, the present invention provides a method
of purifying a heterodimeric protein or bispecific antibody in
accordance with any of the above, the method including: (a)
providing a composition in accordance with any of the above, (b)
loading the composition onto an ion exchange column; and (c)
collecting a fraction containing the heterodimeric protein or
bispecific antibody, thus purifying the protein or antibody.
[0057] In a further aspect, the present invention provides a method
of treating cancer in a subject, the method comprising
administering to said subject a composition comprising a bispecific
antibody, where the bispecific antibody includes: (a) a first
monomer that has (i) a first heavy chain constant region with a
first variant Fc domain; and (ii) an anti-CD25 binding moiety; and
(b) a second monomer that has (i) a second heavy chain constant
region with a second variant Fc domain; and (ii) a member selected
from the group: an anti-CD4 binding moiety, an anti-CD8 binding
moiety, an anti-CCR4 moiety, an anti-GITR binding moiety, and an
anti-PD-1 binding moiety. In specific embodiments, the first
variant Fc domain has a different amino acid sequence than the
second variant Fc domain. The administration of such a bispecific
antibody serves to suppress the T cells. Suppression can be
measured using assays known in the art, including cell
proliferation assays. Suppression can be shown in such assays by a
decrease in cell proliferation and/or general T cell number as
compared to the proliferation and/or numbers seen in the absence of
the bispecific antibody of the invention.
[0058] In further embodiments and in accordance with the above, the
T cells suppressed by the methods of the invention are regulatory T
cells. In still further embodiments, the second monomer comprises
the anti-CD4 binding moiety, and the bispecific antibody
specifically targets regulatory T cells with limited to no impact
on other T cell types.
[0059] In still further embodiments and in accordance with any of
the above, the anti-CD25 binding moiety is an anti-CD25 scFV
sequence that is covalently attached to the first heavy chain
sequence.
[0060] In still further embodiments and in accordance with any of
the above, the T cells suppressed by the methods and compositions
of the invention are cytotoxic T-cells. In yet further embodiments,
the second monomer comprises said anti-CD8 binding moiety, and the
bispecific antibody specifically targets cytotoxic T cells with
limited to no impact on other T cell types. In yet further
embodiments, the anti-CD8 binding moiety comprises all or a portion
of an antigen binding region of an antibody selected from the group
consisting of MCD8, 3B5, Sk1, OKT-8, and DK-25.
[0061] In still further embodiments and in accordance with any of
the above, the first and second variant Fc domains include a set of
amino acid substitutions selected from those sets depicted in FIG.
33.
[0062] In still further embodiments and in accordance with any of
the above, the first and second variant Fc domains comprise a set
of amino acid substitutions selected from the group consisting of
those sets depicted in FIG. 34A-34C.
[0063] In still further embodiments and in accordance with any of
the above, the first and second variant Fc domains comprise a set
of amino acid substitutions selected from the group consisting of
those sets depicted in FIG. 35.
[0064] In still further embodiments and in accordance with any of
the above, the first and/or second variant Fc domain comprises an
amino acid variant selected from the group consisting of: 236R,
239D, 239E, 243L, M252Y, V259I, 267D, 267E, 298A, V308F, 328F,
328R, 330L, 332D, 332E, M428L, N434A, N434S, 236R/328R, 239D/332E,
M428L, 236R/328F, V259I/V308F, 267E/328F, M428L/N434S, Y436I/M428L,
Y436V/M428L, Y436I/N434S, Y436V/N434S, 239D/332E/330L,
M252Y/S254T/T256E, V259I/V308F/M428L, and
E233P/L234V/L235A/G236del/S267K.
[0065] In still further embodiments and in accordance with any of
the above, the bispecific antibody comprises a sequence selected
from the sequences depicted in FIGS. 30-31.
[0066] In still further embodiments and in accordance with any of
the above, the first monomer comprises a sequence according to the
sequence designated as
11209-OKT4A_HOL0_scFv_Anti-TAC_H1L1_scFv_GDQ-Fc(216)_IgG1_pI_ISO(-)/pI_IS-
O(+RR)_IgG1, Heavy chain 2 (Heavy chain 2
(Anti-TAC_H1L1_scFv_GDQ-Fc(216)_IgG1_pI_ISO(+RR)) in FIG.
30A-30FF.
[0067] In still further embodiments and in accordance with any of
the above, the second monomer comprises a sequence according to the
sequence designated as
11209-OKT4A_HOL0_scFv_Anti-TAC_H1L1_scFv_GDQ-Fc(216)_IgG1_pI_ISO(-)/pI_IS-
O(+RR)_IgG1, Heavy chain 1
(OKT4A_HOL0_scFv_GDQ-Fc(216)_IgG1_pI_ISO(-)) in FIG. 30A-30FF.
[0068] In still further embodiments and in accordance with any of
the above, the first monomer comprises a sequence according to the
sequence designated as
12143-OKT4A_HOL0_scFv_Anti-TAC_H1L1_scFv_-Fc(216)_IgG1_pI_ISO(-)/pI_ISO(+-
RR)_C220S/FcKO, Heavy chain 2
(Anti-TAC_H1L1_scFv_Fc(216)_IgG1_pI_ISO(+RR)_G236R/L328R) in FIG.
30A-30FF.
[0069] In still further embodiments and in accordance with any of
the above, the second monomer comprises a sequence according to the
sequence designated as
12143-OKT4A_HOL0_scFv_Anti-TAC_H1L1_scFv_-Fc(216)_IgG1_pI_ISO(-)/pI_ISO(+-
RR)_C220S/FcKO, Heavy chain 1
(OKT4A_HOL0_scFv_Fc(216)_IgG1_pI_ISO(-)_G236R/L328R) in FIG.
30A-30FF.
[0070] In still further embodiments and in accordance with any of
the above, the first monomer comprises a sequence according to the
sequence designated as
13531-OKT4A_H1L1_Fab-Anti-TAC_H1.8L1_scFv_Fc(216)_IgG1_pI_ISO
(-)-pI_ISO(+RR)_C220S_IgG1_G236R/L328R, Heavy chain 2
(Anti-TAC_H1.8L1_scFv_Fc(216)_IgG1_pI_ISO(+RR)_G236R/L328R) in FIG.
30A-30FF.
[0071] In still further embodiments and in accordance with any of
the above, the second monomer comprises a sequence according to the
sequence designated as
13531-OKT4A_H1L1_Fab-Anti-TAC_H1.8L1_scFv_Fc(216)_IgG1_pI_ISO
(-)-pI_ISO(+RR)_C220S_IgG1_G236R/L328R, Heavy chain 1
(OKT4A_H1_IgG1_pI_ISO(-)_G236R/L328R) in FIG. 30A-30FF.
[0072] In still further embodiments and in accordance with any of
the above, the second monomer further comprises a sequence
according to the sequence designated as
13531-OKT4A_H1L1_Fab-Anti-TAC_H1.8L1_scFv_Fc(216)_IgG1_pI_ISO
(-)-pI_ISO(+RR)_C220S_IgG1_G236R/L328R, Light chain (OKT4A_L1) in
FIG. 30A-30FF.
[0073] In a further aspect, the present invention provides a method
for treating autoimmune disease in a subject that includes
administering a heterodimeric protein to the subject, where the
heterodimeric protein includes: (a) a first monomer with (i) a
first heavy chain constant region comprising a first variant Fc
domain; (ii) an IL-2 protein; and (b) a second monomer with: (i) a
second heavy chain constant region comprising a second variant Fc
domain; (ii) a member selected from the group consisting of: an
anti-CD4 binding moiety, an anti-CD8 binding moiety, an anti-CTLA4
binding moiety, an anti-CCR4 binding moiety, an anti-PD-1 binding
moiety, and an anti-GITR binding moiety. In further embodiments,
the first variant Fc domain has a different amino acid sequence
than the second variant Fc domain. Administration of this
heterodimeric protein stimulates the T cells. As will be
appreciated, the IL2 protein may comprise a full length protein or
a portion of the full length IL2 protein. In further embodiments,
the full or portion of the IL2 protein that is part of the
heterodimeric protein comprises a human IL2 protein sequence.
[0074] In a further embodiment and in accordance with the above,
the T cells are regulatory T cells and the second monomer is the
anti-CD4 binding moiety.
[0075] In still further embodiments and in accordance with any of
the above, the second monomer further includes: (a) the second
heavy chain constant region further having a heavy chain variable
domain, and (b) a light chain sequence, where the heavy chain
variable domain and the light chain sequence together form antigen
binding moiety, including without limitation the anti-CD4 binding
moiety.
[0076] In still further embodiments and in accordance with any of
the above, the stimulated T cells are cytotoxic T cells and the
second monomer comprises the anti-CD8 binding moiety. In yet
further embodiments, the anti-CD8 binding moiety comprises all or a
portion of an antigen binding region of an antibody selected from
the group consisting of MCD8, 3B5, Sk1, OKT-8, and DK-25.
[0077] In still further embodiments and in accordance with any of
the above, the first and second variant Fc domains include a set of
amino acid substitutions selected from the group consisting of
those sets depicted in FIG. 33, 34 or 35.
[0078] In still further embodiments and in accordance with any of
the above, the first and/or second variant Fc domain comprises an
amino acid variant selected from the group consisting of: 236R,
239D, 239E, 243L, M252Y, V259I, 267D, 267E, 298A, V308F, 328F,
328R, 330L, 332D, 332E, M428L, N434A, N434S, 236R/328R, 239D/332E,
M428L, 236R/328F, V259I/V308F, 267E/328F, M428L/N434S, Y436I/M428L,
Y436V/M428L, Y436I/N434S, Y436V/N434S, 239D/332E/330L,
M252Y/S254T/T256E, V259I/V308F/M428L, and
E233P/L234V/L235A/G236del/S267K.
[0079] In still further embodiments and in accordance with any of
the above, the heterodimeric protein comprise a sequence selected
from the sequences depicted in FIGS. 30A-31OOO.
[0080] In still further embodiments and in accordance with any of
the above, the first monomer comprises a sequence according to the
sequence designated as
13027-hIL2_OKT4A_H1L1_IgG1_pI_ISO(-/+RR)_C220S_G236R/L328R, Heavy
chain 1 (hIL2_IgG1_pI_ISO(-)_C220S/G236R/L328R) in FIG.
30A-30FF.
[0081] In still further embodiments and in accordance with any of
the above, the second monomer comprises a sequence according to the
sequence designated as
13027-hIL2_OKT4A_H1L1_IgG1_pI_ISO(-/+RR)_C220S_G236R/L328R, Heavy
chain 2 (OKT4A_H1_IgG1_pI_ISO(+RR)_G236R/L328R) in FIG.
30A-30FF.
[0082] In still further embodiments and in accordance with any of
the above, the second monomer further comprises a sequence
according to the sequence designated as
13027-hIL2_OKT4A_H1L1_IgG1_pI_ISO(-/+RR)_C220S_G236R/L328R, Light
chain (OKT4A_L1) in FIG. 30A-30FF.
[0083] In still further embodiments and in accordance with any of
the above, the second monomer comprises a sequence according to the
sequence designated as 13038-OKT4A_H1L1_IgG1_G236R/L328R_hIL2(2),
Heavy chain (OKT4A_H1_IgG1_G236R/L328R_hIL2) in FIG. 30A-30FF.
[0084] In still further embodiments and in accordance with any of
the above, the second monomer further comprises a sequence
according to the sequence designated as
13038-OKT4A_H1L1_IgG1_G236R/L328R_hIL2(2), Light chain (OKT4A_L1)
in FIG. 30A-30FF.
BRIEF DESCRIPTION OF THE DRAWINGS
[0085] FIG. 1. Diagram illustrating suppression of Treg cells with
anti-CD4.times.anti-CD25 bispecifics.
[0086] FIG. 2. Evaluation of the ability of various anti-CD25 heavy
chains to pair with anti-CD4 light chains and anti-CD4 heavy chains
to pair with anti-CD25 light chains. Biacore was used to examine
binding of the various pairs to both CD4 and CD25 and the results
tabulated. The HuMax-TAC anti-CD25 heavy chain has the unique
ability to pair with the anti-CD4 lights chains of OKT4A and
zanolimumab, with the HuMax-TAC/OKT4A pair showing the strongest
binding.
[0087] FIG. 3. Diagram showing three exemplary
anti-CD4.times.anti-CD25 bispecific formats. Common light-chain,
dual scFv, and Fab/scFv-Fc formats are shown. Purification of
heterodimer formats is accomplished utilizing Protein A and IEX
chromatography. IgG1, FcR enhanced, and/or FcR knockout Fc regions
may be used in further embodiments of the invention.
[0088] FIG. 4. Dual scFv-Fc bispecific antibody
OKT4A_HOL0_scFv_Anti-TAC_H1L1_scFv_GDQ-Fc(216)_IgG1_pI_ISO(-)/pI_ISO(+RR)-
_IgG1_S239D/1332E can bind to CD25 and CD4 simultaneously. The
antibody was bound to a CD25 chip on Biacore followed by binding of
CD4. As a control, anti-CD25 antibody Anti-TAC_H1L1_IgG1 does not
bind CD4.
[0089] FIG. 5. Suppression of Treg cells by
anti-CD4.times.anti-CD25 bispecifics. Proliferation of Tregs was
assayed using CFSE in the presence of bispecific or control
antibodies with 15 U/mL IL2.
[0090] FIG. 6. Effect of anti-CD4.times.anti-CD25 bispecifics on
helper (CD4.sup.+CD25.sup.+) and cytotoxic (CD8.sup.+CD25.sup.+) T
cell populations.
[0091] FIG. 7. Effect of bispecific antibodies and control
anti-CD25 and anti-CD4 antibodies on cell proliferation of Tregs,
CD4+ T-effectors, and CD8+ T-effectors. Bispecific antibody 12143
has higher potency on Tregs compared to controls as well as lower
potency compared to controls on unwanted suppression of T effector
cells.
[0092] FIG. 8. Direct binding of anti-CD4.times.anti-CD25
bispecifics and control antibodies on Treg cells.
[0093] FIG. 9. Direct binding of anti-CD4.times.anti-CD25
bispecifics and control antibodies on Treg cells. Variants
engineered for altered CD25 affinity are shown.
[0094] FIG. 10. Direct binding of anti-CD4.times.anti-CD25
bispecifics and control antibodies on Treg cells. Variants
containing the ibalizumab anti-CD4 Fv are shown.
[0095] FIG. 11. Direct binding of anti-CD4.times.anti-CD25
bispecifics and control antibodies on Treg cells.
[0096] FIG. 12. Suppression of Treg cells by
anti-CD4.times.anti-CD25 bispecifics. Proliferation of Tregs was
assayed using CFSE in the presence of bispecific or control
antibodies with 15 U/mL IL2.
[0097] FIG. 13. Suppression of Treg cells by
anti-CD4.times.anti-CD25 bispecifics. Proliferation of Tregs was
assayed using Alamar Blue in the presence of bispecific or control
antibodies with 15 U/mL IL2.
[0098] FIG. 14. Binding of anti-CD4.times.anti-CD25 bispecifics and
control antibodies to purified naive human CD4+ T cells.
[0099] FIG. 15. Effect of altering CD25 binding affinity on
suppression of Treg cells by anti-CD4.times.anti-CD25 bispecifics.
Proliferation of Tregs was assayed using CFSE in the presence of
bispecific or control antibodies with 15 U/mL IL2.
[0100] FIG. 16. Direct binding of altered CD25 affinity
anti-CD4.times.anti-CD25 bispecifics to purified naive human CD4+ T
cells.
[0101] FIG. 17. Direct binding of anti-CD4.times.anti-CD25
bispecifics and controls to activated T effector cells
(CD4+CD25+).
[0102] FIG. 18. Direct binding of altered CD25 affinity
anti-CD4.times.anti-CD25 bispecifics to activated T effector cells
(CD4+CD25+).
[0103] FIG. 19. Direct binding of anti-CD4.times.anti-CD25
bispecifics and controls to activated T effector cells
(CD4+CD25+).
[0104] FIG. 20. Direct binding of anti-CD4.times.anti-CD25
bispecifics and controls to activated T effector cells
(CD8+CD25+).
[0105] FIG. 21. Direct binding of altered CD25 affinity
anti-CD4.times.anti-CD25 bispecifics to activated T effector cells
(CD8+CD25+).
[0106] FIG. 22. Direct binding of anti-CD4.times.anti-CD25
bispecifics and controls to activated T effector cells
(CD8+CD25+).
[0107] FIG. 23. Summary of IL2 variants that can be used for
suppression or induction of Tregs.
[0108] FIG. 24. Diagram illustrating induction of Treg cells with
anti-CD4.times.IL2 Fc-fusions. An example construct is also
shown.
[0109] FIG. 25. Exemplary IL2 Fc-fusions and bispecific
antibody-IL2 Fc-fusions for induction of Tregs.
[0110] FIG. 26. Purification and analysis of anti-CD4.times.IL2
Fc-fusions. Fc-fusions are purified by Protein A and IEX
chromatography, and purity assessed by SEC and SDS-PAGE.
[0111] FIG. 27. Induction of regulatory T cells (Tregs) by
anti-CD4.times.IL2 Fc-fusions. Induction of Tregs was assayed using
the alamar blue cell viability assay in the presence of
anti-CD4.times.IL2 Fc-fusions or control antibodies.
[0112] FIG. 28. Diagram illustrating suppression of activated
cytotoxic (CD8+CD25+) T cells by anti-CD8.times.anti-CD25
bispecific antibodies.
[0113] FIG. 29. Diagram illustrating induction of naive and
activated cytotoxic (CD8.sup.+CD25.sup.+) T cells by
anti-CD8.times.IL2 Fc-fusions.
[0114] FIG. 30A-30FF. Sequences of anti-CD4.times.anti-CD25
bispecifics, anti-CD4.times.IL2 Fc-fusions, and control
antibodies.
[0115] FIG. 31A-31OOO. Sequences of T cell modulating bispecifics,
Fc-fusions, and control antibodies.
[0116] FIG. 32. Table of exemplary Treg markers for use in
embodiments of the invention.
[0117] FIG. 33A-33C. Table of exemplary amino acid variants for
embodiments of heterodimeric proteins of the invention.
[0118] FIG. 34A-34C. Table of exemplary amino acid variants for
embodiments of heterodimeric proteins of the invention.
[0119] FIG. 35. Table of exemplary amino acid variants for
embodiments of heterodimeric proteins of the invention.
[0120] FIG. 36A-36M. Illustration of a number of heterodimeric
protein formats, including heterodimeric Fc fusion proteins as well
as heterodimeric antibodies. FIG. 36A shows the basic concept of a
dimeric Fc region with four possible fusion partners A, B, C and D.
A, B, C and D are optionally and independently selected from
immunoglobulin domain(s) (e.g. Fab, vH, vL, scFv, scFv2, scFab,
dAb, etc.), peptide(s), cytokines (e.g. IL-2, IL-10, IL-12, GCSF,
GM-CSF, etc.), chemokine(s) (e.g. RANTES, CXCL9, CXCL10, CXCL12,
etc.), hormone(s) (e.g. FSH, growth hormone), immune receptor(s)
(e.g. CTLA-4, TNFR1, TNFRII, other TNFSF, other TNFRSF, etc.) and
blood factor(s) (e.g. Factor VII, Factor VIII, Factor IX, etc.).
Domains filled with solid white or solid black are engineered with
heterodimerization variants as outlined herein. FIG. 36B depicts
the "triple F" format (sometimes also referred to as the
"bottle-opener" configuration as discussed below). FIG. 36C shows a
"triple F" configuration with another scFv attached to the Fab
monomer (this one, along with FIG. 36F, has a greater molecular
weight differential as well). FIG. 36D depicts a "triple F" with
another scFv attached to the scFv monomer. FIG. 36E depicts a
"three scFv" format. FIG. 36F depicts an additional Fab attached to
the Fab monomer. FIG. 36G depicts a Fab hooked to one of the scFv
monomers. FIGS. 1H-1L show additional varieties of "higher
multispecificity" embodiments of the "triple F" format, all with
one monomer comprising an scFv (and all of which have molecular
weight differentials which can be exploited for purification of the
heterodimers). FIG. 36H shows a "Fab-Fv" format with binding to two
different antigens, with FIG. 361 depicting the "Fab-Fv" format
with binding to a single antigen (e.g. bivalent binding to antigen
1). FIGS. 36J and 36K depicts a "Fv-Fab" format with similar
bivalent or monovalent additional antigen binding. FIG. 36L depicts
one monomer with a CH1-CL attached to the second scFv. FIG. 36M
depicts a dual scFv format.
[0121] FIG. 37A-37U. Depicts a wide variety of the multispecific
(e.g. heterodimerization) formats and the combinations of different
types of heterodimerization variants that can be used in the
present invention (these are sometimes referred to herein as
"heterodimeric scaffolds"). Note in addition that all of these
formats can include addition variants in the Fc region, as more
fully discussed below, including "ablation" or "knock out" variants
(FIG. 39), Fc variants to alter Fc.gamma.R binding (Fc.gamma.RIIb,
Fc.gamma.RIIIa, etc.), Fc variants to alter binding to FcRn
receptor, etc. FIG. 37A shows a dual scFv-Fc format, that, as for
all heterodimerization formats herein can include
heterodimerization variants such as pI variants, knobs in holes
(KIH, also referred to herein as steric variants or "skew"
variants), charge pairs (a subset of steric variants), isosteric
variants, and SEED body ("strand-exchange engineered domain"; see
Klein et al., mAbs 4:6 653-663 (2012) and Davis et al, Protein Eng
Des Sel 2010 23:195-202) which rely on the fact that the CH3
domains of human IgG and IgA do not bind to each other. FIG. 37B
depicts a bispecific IgG, again with the option of a variety of
heterodimerization variants. FIG. 37C depicts the "one armed"
version of DVD-Ig which utilizes two different variable heavy and
variable light domains. FIG. 37D is similar, except that rather
than an "empty arm", the variable heavy and light chains are on
opposite heavy chains. FIG. 37E is generally referred to as
"mAb-Fv". FIG. 37F depicts a multi-scFv format; as will be
appreciated by those in the art, similar to the "A, B, C, D"
formats discussed herein, there may be any number of associated
scFvs (or, for that matter, any other binding ligands or
functionalities). Thus, FIG. 37F could have 1, 2, 3 or 4 scFvs
(e.g. for bispecifics, the scFv could be "cis" or "trans", or both
on one "end" of the molecule). FIG. 37G depicts a heterodimeric
FabFc with the Fab being formed by two different heavy chains one
containing heavy chain Fab sequences and the other containing light
chain Fab sequences. FIG. 37H depicts the "one armed Fab-Fc", where
one heavy chain comprises the Fab. FIG. 37I depicts a "one armed
scFv-Fc", wherein one heavy chain Fc comprises an scFv and the
other heavy chain is "empty". FIG. 37J shows a scFv-CH3, wherein
only heavy chain CH3 regions are used, each with their own scFv.
FIG. 37K depicts a mAb-scFv, wherein one end of the molecule
engages an antigen bivalently with a monovalent engagement using an
scFv on one of the heavy chains. FIG. 37L depicts the same
structure except that both heavy chains comprise an additional
scFv, which can either bind the same antigen or different antigens.
FIG. 37M shows the "CrossMab" structure, where the problem of
multiplex formation due to two different light chains is addressed
by switching sequences in the Fab portion. FIG. 37N depicts an
scFv, FIG. 370 is a "BITE" or scFv-scFv linked by a linker as
outlined herein, FIG. 37P depicts a DART, FIG. 37Q depicts a
TandAb, and FIG. 37R shows a diabody. FIGS. 37S, 37T and 37U depict
additional alternative scaffold formats that find use in the
present invention.
[0122] FIG. 38. Depicts a list of isotypic and isosteric variant
antibody constant regions and their respective substitutions.
pI_(-) indicates lower pI variants, while pI_(+) indicates higher
pI variants. These can be optionally and independently combined
with other heterodimerization variants of the invention.
[0123] FIG. 39. Depicts a number of suitable "knock out" ("KO")
variants to reduce binding to some or all of the Fc.gamma.R
receptors. As is true for many if not all variants herein, these KO
variants can be independently and optionally combined, both within
the set described in FIG. 39 and with any heterodimerization
variants outlined herein, including steric and pI variants. For
example, E233P/L234V/L235A/G236del can be combined with any other
single or double variant from the list. In addition, while it is
preferred in some embodiments that both monomers contain the same
KO variants, it is possible to combine different KO variants on
different monomers, as well as have only one monomer comprise the
KO variant(s). Reference is also made to the Figures and Legends of
U.S. Ser. No. 61/913,870, all of which is expressly incorporated by
reference in its entirety as it relates to "knock out" or
"ablation" variants.
[0124] FIG. 40. Depicts a number of charged scFv linkers that find
use in increasing or decreasing the pI of heterodimeric proteins
that utilize one or more scFv as a component. A single prior art
scFv linker with a single charge is referenced as "Whitlow", from
Whitlow et al., Protein Engineering 6(8):989-995 (1993). It should
be noted that this linker was used for reducing aggregation and
enhancing proteolytic stability in scFvs.
[0125] FIG. 41A-41B. FIGS. 41A and 41B provides an additional list
of potential heterodimerization variants for use in the present
invention, including isotypic variants.
[0126] FIG. 42A-42J. Depicts additional exemplary
heterodimerization variant pairs for use in heterodimeric proteins
of the invention.
[0127] FIG. 43. Depicts amino acid sequences of wild-type constant
regions used in the invention.
[0128] FIG. 44. Depicts two different Triple F embodiments for
bispecific antibodies of the invention.
[0129] FIG. 45. Literature pIs of the 20 amino acids. It should be
noted that the listed pIs are calculated as free amino acids; the
actual pI of any side chain in the context of a protein is
different, and thus this list is used to show pI trends and not
absolute numbers for the purposes of the invention.
[0130] FIG. 46A-46C. List of all possible reduced pI variants
created from isotypic substitutions of IgG1-4. Shown are the pI
values for the three expected species as well as the average delta
pI between the heterodimer and the two homodimer species present
when the variant heavy chain is transfected with IgG1-WT heavy
chain.
[0131] FIG. 47. List of all possible increased pI variants created
from isotypic substitutions of IgG1-4. Shown are the pI values for
the three expected species as well as the average delta pI between
the heterodimer and the two homodimer species present when the
variant heavy chain is transfected with IgG1-WT heavy chain.
[0132] FIG. 48A-48B. Matrix of possible combinations of first and
second monomers for heterodimeric proteins of the invention, FcRn
variants, Scaffolds, Fc variants and combinations, with each
variant being independently and optionally combined from the
appropriate source. Note that the target antigens for the first and
second monomer are each independently selected from the list
provided in the first column. Legend: Legend A are suitable FcRn
variants: 434A, 434S, 428L, 308F, 259I, 428L/434S, 259I/308F,
436I/428L, 436I or V/434S, 436V/428L, 252Y, 252Y/254T/256E,
259I/308F/428L. Legend B are suitable scaffolds and include IgG1,
IgG2, IgG3, IgG4, and IgG1/2. Sequences for such scaffolds can be
found for example in US Patent Publication No. 2012/0128663,
published on May 24, 2012, which is hereby incorporated by
reference in its entirety for all purposes and in particular for
all teachings, figures and legends related to scaffolds and their
sequences. Legend C are suitable Fc variants: 236A, 239D, 239E,
332E, 332D, 239D/332E, 267D, 267E, 328F, 267E/328F, 236A/332E,
239D/332E/330Y, 239D, 332E/330L, 236R, 328R, 236R/328R, 236N/267E,
243L, 298A and 299T. (Note, additional suitable Fc variants are
found in FIG. 41 of US 2006/0024298, the figure and legend of which
are hereby incorporated by reference in their entirety). Legend D
reflects the following possible combinations, again, with each
variant being independently and optionally combined from the
appropriate source Legend: 1) Monomer targets (each independently
selected from the list in the first column) plus FcRn variants; 2)
Monomer targets (each independently selected from the list in the
first column) plus FcRn variants plus Scaffold; 3) Monomer targets
(each independently selected from the list in the first column)
plus FcRn variants plus Scaffold plus Fc variants; 4) Monomer
targets (each independently selected from the list in the first
column) plus Scaffold 5) Monomer targets (each independently
selected from the list in the first column) plus Fc variants; 6)
FcRn variants plus Scaffold; 7) Monomer targets (each independently
selected from the list in the first column) plus Fc variants; 8)
Scaffold plus Fc variants; 9) Monomer targets (each independently
selected from the list in the first column) plus Scaffold plus Fc
variants; and 10) Monomer targets (each independently selected from
the list in the first column) plus FcRn variants plus Fc
variants.
DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS
Overview of Invention
[0133] The present invention provides methods and compositions for
modulating T cells by administering heterodimeric proteins.
Heterodimeric proteins include without limitation heterodimeric
antibodies (such as bispecific antibodies) and heterodimeric fusion
proteins. By "modulating T cells" as discussed herein is meant
suppressing or stimulating T cells. In general, the heterodimeric
proteins of the invention are specific for their target T cell,
meaning that the heterodimeric proteins primarily affect one type
of T cell with little or no impact on other T cell types. For
example (and as is described in further detail herein), methods and
compositions of the present invention for suppression or induction
of regulatory T cells (also referred to herein as "Tregs") have
little or no impact on other T cell types. Similarly, methods and
compositions of the invention for suppressing or inducing other T
cell types, such as cytotoxic T cells, primarily affect the
cytotoxic T cells with little or no impact on other T cell
types.
[0134] By "suppressing T cells" as used herein refers to decreasing
any aspect of T cell expression or function as compared to
expression or function in the absence of the administered
heterodimeric protein. For example, suppression of regulatory T
cells by admiinistering a heterodimeric protein includes
suppression of the proliferation of regulatory T cells as compared
to proliferation in the absence of the administered heterodimeric
protein. "Inducing T cells" as used herein refers to increasing any
aspect of T cell expression or function as compared to expression
or function in the absence of the administered heterodimeric
protein, including stimulation of the proliferation of the target T
cell.
[0135] As discussed in further detail herein, suppression or
induction of T cells can be measured with assays to quantify T cell
numbers. For example, cell proliferation assays can be used to
detect and quantitate T cells. Other methods of quantifying T
cells, particularly Tregs, may also be used, including methods
utilizing qPCR to measure the amount of demethylated FOXP3 (a Treg
marker) that is present. Such assays are described for example in
Wieczorek et al., 2009, Cancer Res, 69(2): 599-608, Vries et al.,
2011, Clin Cancer Res, 17:841-848, and Baron et al., 2007, Eur. J.
Immunol., 37:2378-2389, each of which is hereby incorporated by
reference in its entirety for all purposes and in particular for
all teachings, figures and legends related to assays for FOXP3,
demethylated FOXP3, and quantification of Tregs.
[0136] Suppression of T cells of a particular type is generally
accomplished by administering a heterodimeric protein that targets
antigens specific for that T cell type. For example, for regulatory
T cells, administering a heterodimeric protein that targets both
CD4 and CD25 reduces regulatory T cell proliferation with minimal
to no effect on other T cells, such as CD4+CD25- T effector cells
or CD8+CD25+ cyotoxic T cells. For suppression of T cells, such a
heterodimeric protein is generally a bispecific antibody, although
other multispecific antibodies and other heterodimeric proteins
such as fusion proteins are also contemplated. In certain
instances, suppression of regulatory T cells is accomplished by
administering a bispecific antibody that targets both CD4 and CD25;
in other words, the bispecific antibody comprises two monomers in
which one monomer comprises an anti-CD4 binding domain and the
other monomer comprises an anti-CD25 binding domain ("binding
domain" and "binding moiety" are used interchangeably herein).
"Anti-X binding domain" refers to a domain of the monomer that
binds to X (i.e., an anti-CD25 binding domain is a part of the
monomer that binds to CD25).
[0137] Other bispecific antibodies that suppress Tregs include
without limitation anti-CD25.times.anti-CTLA4,
anti-CD25.times.anti-PD-1, anti-CD25.times.anti-CCR4, and
anti-CD25.times.anti-G ITR antibodies.
[0138] Bispecific antibodies can also be used to suppress other T
cell types, such as cytotoxic T cells. In some instances, one
monomer of the bispecific antibody comprises an anti-CD8 domain,
including without limitation domains from anti-CD8 antibodies such
as MCD8, 3B5, SK1, OKT-8, 51.1 and DK-25. The monomer with the
anti-CD8 domain can be combined with a monomer comprising an
anti-CD25 binding domain to produce a bispecific antibody for
suppression of cytoxic T cells.
[0139] Generally, the antigen binding domains of bispecific
antibodies of the invention are part of monomers that further
comprise at least a heavy chain constant region that contains a
variant Fc domain as compared to a parent Fc domain.
[0140] In some situations, anti-CD4 and anti-CD8 targeting agents
may further be utilized in combination with T cell cytokines,
including without limitation IL-7, IL-12, IL-15, and IL-17.
[0141] Fc fusion proteins may also be used in accordance with the
invention to suppress T cells. For example, a fusion protein
comprising an IL2 protein on one arm can be engineered to have
reduced ability to bind to IL2R.beta., IL2R.gamma., and or
IL2R.alpha. in order to ablate IL2 receptor signaling. When coupled
with an anti-CD4 antibody (or any other Treg surface marker
antibody), this results in an anti-CD4.times.IL2 Fc-fusion capable
of suppressing Treg cells through targeted binding to CD4 and CD25,
but without the ability to induce Treg proliferation. In one
non-limiting theory, the mechanism of action for this fusion may be
that it blocks endogenous IL2 from binding to receptor, thus
preventing Treg proliferation. Exemplary embodiments of such fusion
proteins are provided in FIG. 23.
[0142] Heterodimeric proteins may also be used to induce T cells.
As with methods and compositions for suppressing T cells, induction
of T cells in accordance with the present invention is generally
accomplished by administering a heterodimeric protein that targets
antigens and proteins specific for that T cell type. In specific
instances, the present invention provides Fc fusion proteins
comprising one monomer with a binding domain that targets a T cell
marker and a second monomer comprising an IL2 protein. Examples of
fusion proteins of use in the present invention for inducing T
cells include without limitation fusion proteins that comprise IL2
on one monomer and one of the following binding domains on the
other monomer: anti-CD4, anti-CCR4, anti-PD-1, anti-CD8, LAG3, and
anti-CTLA4. In some situations, potency of the fusion proteins is
increased by engineering the IL2 arm to increase the affinity of
IL2 for IL2R.alpha.. Exemplary variants of IL2 of use in the
present invention are listed in FIG. 23. Other cytokines that may
be used in Fc fusion proteins of the invention include without
limitation IL-7, IL-12, IL-15, and IL-17.
[0143] As will be appreciated and as is described in further detail
herein, the heterodimeric proteins discussed herein may comprise a
variety of formats, including those described herein (see for
example FIGS. 3, 25, 36 and 37) and those described in the art (see
for example Kontermann et al., 2012, Landes Bioscience, which is
incorporated herein by reference for all purposes and in particular
for all teachings related to heterodimeric proteins such as
bispecific antibodies). In some situations, bispecific antibodies
may have one heavy chain containing a single chain Fv ("scFv", as
defined herein) and the other heavy chain is a "regular" FAb
format, comprising a variable heavy chain and a light chain. This
structure is sometimes referred to herein as "triple F" format
(scFv-FAb-Fc) or the "bottle-opener" format, due to a rough visual
similarity to a bottle-opener, as described for example in U.S.
Ser. No. 14/205,248, filed Mar. 11, 2014, which is hereby
incorporated by reference for all purposes and in particular for
all teachings related to the triple F or bottle opener format. In
some situations, both of the heavy chains of the bispecific
antibodies described herein contain scFvs. Similarly, for any of
the fusion proteins described herein, the antibody arm of the
fusion protein may be in the scFv or regular FAb format.
[0144] As is discussed in further herein, the heterodimeric
proteins of the present invention may further include one or more
amino acid substitutions in the Fc region that have the effect of
increasing serum half-life, ablating binding to Fc.quadrature.R,
and/or increasing ADCC. The heterodimeric proteins of the invention
may also further include "heterodimerization variants" that, as is
also described in further detail herein, promote heterodimeric
formation and/or allow for ease of purification of heterodimers
over the homodimers. In certain situations, the heterodimeric
proteins of the invention comprise one or more variant Fc domains
comprising an amino acid variant selected from among the variants
listed in FIGS. 33 and 34. In some situations, the amino acid
variants may further comprise variants selected from the group:
236R; 239D; 239E; 243L; M252Y; V259I; 267D; 267E; 298A; V308F;
328F; 328R; 330L; 332D; 332E; M428L; N434A; N434S; 236R/328R;
239D/332E; M428L; 236R/328F; V259I/V308F; 267E/328F; M428L/N434S;
Y436I/M428L; Y436V/M428L; Y436I/N434S; Y436V/N434S; 239D/332E/330L;
M252Y/S254T/T256E; V259I/V308F/M428L; and
E233P/L234V/L235A/G236del/S267K.
[0145] The methods and compositions of the present invention
further include methods for treating and/or alleviating the
symptoms of diseases and disorders affected by T cells, including
without limitation cancer and autoimmune disease. In particular,
methods and compositions of the present invention for the
suppression of T cells, particularly Tregs, can be used to treat
cancer. In addition, methods and compositions of the present
invention for stimulation of T cells can be used to treat
autoimmune disease.
[0146] As will be appreciated, suppression of T cells in accordance
with the present invention may be used to treat any type of cancer.
Bispecific antibodies targeting both CD4 and CD25 (or any other
combination of T cell markers as described herein and listed in
FIG. 32) may in certain further embodiments be beneficial for the
treatment of adult T cell leukemia (ATL), a rare disease associated
with human T cell lymphotrophic virus (HTLV-1). Diseased cells from
ATL patients function as regulatory cells and may arise from Treg
cells, since these cells display a CD4.sup.+CD25.sup.+ phenotype
consistent with that of Treg cells. Depletion of tumor cells with
anti-CD4/CD25 bispecific antibodies coupled to an enhanced effector
function Fc domain may be a viable treatment option.
[0147] As discussed above, the balance of Treg versus effector T
cells can be disregulated in autoimmune disease, and therapeutic
approaches to favor higher Treg ratios utilizing methods and
compositions of the invention can be of use for treating such
diseases. Induction and promotion of T cells in accordance with the
methods described herein can also be used to treat (i.e., suppress)
anti-graft responses in organ transplant and graft-vs-host disease
after allogeneic stem cell or bone marrow transplant. Fc-fusion
molecules, which in one non-limiting mechanism may selectively
`feed` IL2 to Treg, promote the survival and expansion. Such
agents, which should alter the balance in favor of Treg vs effector
T cells, may provide a viable treatment option for controlling
autoimmune disease, organ transplant rejection, and graft-vs-host
disease. In general, such treatments include the use of
antibody-IL2 fusion proteins, in particular wherein a single IL2
protein is coupled with an anti-CD4 (or other Treg marker) moiety
to provide selectivity for Treg versus effector T cells through the
requirement for simultaneous engagement of CD4 and the
high-affinity IL-2 receptor CD25.
[0148] Treatment of cancer, autoimmune disease or any other T cell
associated disease or disorder in accordance with the present
invention generally involves administering a composition containing
a heterodimeric protein of the invention (antibody or Fc fusion) to
a patient in need thereof.
Definitions
[0149] In order that the application may be more completely
understood, several definitions are set forth below. Such
definitions are meant to encompass grammatical equivalents.
[0150] By "ablation" herein is meant a decrease or removal of
activity. Thus for example, "ablating Fc.gamma.R binding" means the
Fc region amino acid variant has less than 50% starting binding as
compared to an Fc region not containing the specific variant, with
less than 70-80-90-95-98% loss of activity being preferred, and in
general, with the activity being below the level of detectable
binding in a Biacore assay. Of particular use in the ablation of
Fc.gamma.R binding are those shown in FIG. 7.
[0151] By "ADCC" or "antibody dependent cell-mediated cytotoxicity"
as used herein is meant the cell-mediated reaction wherein
nonspecific cytotoxic cells that express Fc.gamma.Rs recognize
bound antibody on a target cell and subsequently cause lysis of the
target cell. ADCC is correlated with binding to Fc.gamma.RIIIa;
increased binding to Fc.gamma.RIIIa leads to an increase in ADCC
activity.
[0152] By "ADCP" or antibody dependent cell-mediated phagocytosis
as used herein is meant the cell-mediated reaction wherein
nonspecific cytotoxic cells that express Fc.gamma.Rs recognize
bound antibody on a target cell and subsequently cause phagocytosis
of the target cell.
[0153] By "modification" herein is meant an amino acid
substitution, insertion, and/or deletion in a polypeptide sequence
or an alteration to a moiety chemically linked to a protein. For
example, a modification may be an altered carbohydrate or PEG
structure attached to a protein. By "amino acid modification"
herein is meant an amino acid substitution, insertion, and/or
deletion in a polypeptide sequence. For clarity, unless otherwise
noted, the amino acid modification is always to an amino acid coded
for by DNA, e.g. the 20 amino acids that have codons in DNA and
RNA.
[0154] By "amino acid substitution" or "substitution" herein is
meant the replacement of an amino acid at a particular position in
a parent polypeptide sequence with a different amino acid. In
particular, in some embodiments, the substitution is to an amino
acid that is not naturally occurring at the particular position,
either not naturally occurring within the organism or in any
organism. For example, the substitution E272Y refers to a variant
polypeptide, in this case an Fc variant, in which the glutamic acid
at position 272 is replaced with tyrosine. For clarity, a protein
which has been engineered to change the nucleic acid coding
sequence but not change the starting amino acid (for example
exchanging CGG (encoding arginine) to CGA (still encoding arginine)
to increase host organism expression levels) is not an "amino acid
substitution"; that is, despite the creation of a new gene encoding
the same protein, if the protein has the same amino acid at the
particular position that it started with, it is not an amino acid
substitution.
[0155] By "amino acid insertion" or "insertion" as used herein is
meant the addition of an amino acid sequence at a particular
position in a parent polypeptide sequence. For example, -233E or
233E designates an insertion of glutamic acid after position 233
and before position 234. Additionally, -233ADE or A233ADE
designates an insertion of AlaAspGlu after position 233 and before
position 234.
[0156] By "amino acid deletion" or "deletion" as used herein is
meant the removal of an amino acid sequence at a particular
position in a parent polypeptide sequence. For example, E233- or
E233# or E233( ) designates a deletion of glutamic acid at position
233. Additionally, EDA233- or EDA233# designates a deletion of the
sequence GluAspAla that begins at position 233.
[0157] By "variant protein" or "protein variant", or "variant" as
used herein is meant a protein that differs from that of a parent
protein by virtue of at least one amino acid modification. Protein
variant may refer to the protein itself, a composition comprising
the protein, or the amino sequence that encodes it. Preferably, the
protein variant has at least one amino acid modification compared
to the parent protein, e.g. from about one to about seventy amino
acid modifications, and preferably from about one to about five
amino acid modifications compared to the parent. As described
below, in some embodiments the parent polypeptide, for example an
Fc parent polypeptide, is a human wild type sequence, such as the
Fc region from IgG1, IgG2, IgG3 or IgG4, although human sequences
with variants can also serve as "parent polypeptides", for example
the IgG1/2 hybrid of FIG. 13. The protein variant sequence herein
will preferably possess at least about 80% identity with a parent
protein sequence, and most preferably at least about 90% identity,
more preferably at least about 95-98-99% identity. Variant protein
can refer to the variant protein itself, compositions comprising
the protein variant, or the DNA sequence that encodes it.
Accordingly, by "antibody variant" or "variant antibody" as used
herein is meant an antibody that differs from a parent antibody by
virtue of at least one amino acid modification, "IgG variant" or
"variant IgG" as used herein is meant an antibody that differs from
a parent IgG (again, in many cases, from a human IgG sequence) by
virtue of at least one amino acid modification, and "immunoglobulin
variant" or "variant immunoglobulin" as used herein is meant an
immunoglobulin sequence that differs from that of a parent
immunoglobulin sequence by virtue of at least one amino acid
modification. "Fc variant" or "variant Fc" as used herein is meant
a protein comprising an amino acid modification in an Fc domain.
The Fc variants of the present invention are defined according to
the amino acid modifications that compose them. Thus, for example,
N434S or 434S is an Fc variant with the substitution serine at
position 434 relative to the parent Fc polypeptide, wherein the
numbering is according to the EU index. Likewise, M428L/N434S
defines an Fc variant with the substitutions M428L and N434S
relative to the parent Fc polypeptide. The identity of the WT amino
acid may be unspecified, in which case the aforementioned variant
is referred to as 428L/434S. It is noted that the order in which
substitutions are provided is arbitrary, that is to say that, for
example, 428L/434S is the same Fc variant as M428L/N434S, and so
on. For all positions discussed in the present invention that
relate to antibodies, unless otherwise noted, amino acid position
numbering is according to the EU index. The EU index or EU index as
in Kabat or EU numbering scheme refers to the numbering of the EU
antibody (Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85,
hereby entirely incorporated by reference.) The modification can be
an addition, deletion, or substitution. Substitutions can include
naturally occurring amino acids and, in some cases, synthetic amino
acids. Examples include U.S. Pat. No. 6,586,207; WO 98/48032; WO
03/073238; US2004-0214988A1; WO 05/35727A2; WO 05/74524A2; J. W.
Chin et al., (2002), Journal of the American Chemical Society
124:9026-9027; J. W. Chin, & P. G. Schultz, (2002), ChemBioChem
11:1135-1137; J. W. Chin, et al., (2002), PICAS United States of
America 99:11020-11024; and, L. Wang, & P. G. Schultz, (2002),
Chem. 1-10, all entirely incorporated by reference.
[0158] As used herein, "protein" herein is meant at least two
covalently attached amino acids, which includes proteins,
polypeptides, oligopeptides and peptides. The peptidyl group may
comprise naturally occurring amino acids and peptide bonds, or
synthetic peptidomimetic structures, i.e. "analogs", such as
peptoids (see Simon et al., PNAS USA 89(20):9367 (1992), entirely
incorporated by reference). The amino acids may either be naturally
occurring or synthetic (e.g. not an amino acid that is coded for by
DNA); as will be appreciated by those in the art. For example,
homo-phenylalanine, citrulline, ornithine and noreleucine are
considered synthetic amino acids for the purposes of the invention,
and both D- and L-(R or S) configured amino acids may be utilized.
The variants of the present invention may comprise modifications
that include the use of synthetic amino acids incorporated using,
for example, the technologies developed by Schultz and colleagues,
including but not limited to methods described by Cropp &
Shultz, 2004, Trends Genet. 20(12):625-30, Anderson et al., 2004,
Proc Natl Acad Sci USA 101 (2):7566-71, Zhang et al., 2003,
303(5656):371-3, and Chin et al., 2003, Science 301(5635):964-7,
all entirely incorporated by reference. In addition, polypeptides
may include synthetic derivatization of one or more side chains or
termini, glycosylation, PEGylation, circular permutation,
cyclization, linkers to other molecules, fusion to proteins or
protein domains, and addition of peptide tags or labels.
[0159] 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 IgG1.
[0160] By "Fab" or "Fab region" as used herein is meant the
polypeptide that comprises the VH, CH1, VL, and CL immunoglobulin
domains. Fab may refer to this region in isolation, or this region
in the context of a full length antibody, antibody fragment or Fab
fusion protein. By "Fv" or "Fv fragment" or "Fv region" as used
herein is meant a polypeptide that comprises the VL and VH domains
of a single antibody.
[0161] By "IgG subclass modification" or "isotype modification" as
used herein is meant an amino acid modification that converts one
amino acid of one IgG isotype to the corresponding amino acid in a
different, aligned IgG isotype. For example, because IgG1 comprises
a tyrosine and IgG2 a phenylalanine at EU position 296, a F296Y
substitution in IgG2 is considered an IgG subclass
modification.
[0162] By "non-naturally occurring modification" as used herein is
meant an amino acid modification that is not isotypic. For example,
because none of the IgGs comprise a serine at position 434, the
substitution 434S in IgG1, IgG2, IgG3, or IgG4 (or hybrids thereof)
is considered a non-naturally occurring modification.
[0163] 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.
[0164] 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 ADCC, ADCP, and CDC.
[0165] By "IgG Fc ligand" as used herein is meant a molecule,
preferably a polypeptide, from any organism that binds to the Fc
region of an IgG antibody to form an Fc/Fc ligand complex. Fc
ligands include but are not limited to Fc.gamma.RIs, Fc.gamma.RIIs,
Fc.gamma.RIIIs, FcRn, C1q, C3, mannan binding lectin, mannose
receptor, staphylococcal protein A, streptococcal protein G, and
viral Fc.gamma.R. Fc ligands also include Fc receptor homologs
(FcRH), which are a family of Fc receptors that are homologous to
the Fc.gamma.Rs (Davis et al., 2002, Immunological Reviews
190:123-136, entirely incorporated by reference). Fc ligands may
include undiscovered molecules that bind Fc. Particular IgG Fc
ligands are FcRn and Fc gamma receptors. By "Fc ligand" as used
herein is meant a molecule, preferably a polypeptide, from any
organism that binds to the Fc region of an antibody to form an
Fc/Fc ligand complex.
[0166] By "Fc gamma receptor, "Fc.gamma.R" or "FcqammaR" as used
herein is meant any member of the family of proteins that bind the
IgG antibody Fc region and is encoded by an Fc.gamma.R gene. In
humans this family includes but is not limited to Fc.gamma.RI
(CD64), including isoforms Fc.gamma.RIa, Fc.gamma.RIb, and
Fc.gamma.RIc; Fc.gamma.RII (CD32), including isoforms Fc.gamma.RIIa
(including allotypes H131 and R131), Fc.gamma.RIIb (including
Fc.gamma.RIIb-1 and Fc.gamma.RIIb-2), and Fc.gamma.RIIc; and
Fc.gamma.RIII (CD16), including isoforms Fc.gamma.RIIIa (including
allotypes V158 and F158) and Fc.gamma.RIIIb (including allotypes
Fc.gamma.RIIb-NA1 and Fc.gamma.RIIb-NA2) (Jefferis et al., 2002,
Immunol Lett 82:57-65, entirely incorporated by reference), as well
as any undiscovered human Fc.gamma.Rs or Fc.gamma.R isoforms or
allotypes. An Fc.gamma.R may be from any organism, including but
not limited to humans, mice, rats, rabbits, and monkeys. Mouse
Fc.gamma.Rs include but are not limited to Fc.gamma.RI (CD64),
Fc.gamma.RII (CD32), Fc.gamma.RIII (CD16), and Fc.gamma.RIII-2
(CD16-2), as well as any undiscovered mouse Fc.gamma.Rs or
Fc.gamma.R isoforms or allotypes.
[0167] 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. The FcRn may be from any organism,
including but not limited to humans, mice, rats, rabbits, and
monkeys. As is known in the art, the functional FcRn protein
comprises two polypeptides, often referred to as the heavy chain
and light chain. The light chain is beta-2-microglobulin and the
heavy chain is encoded by the FcRn gene. Unless otherwise noted
herein, FcRn or an FcRn protein refers to the complex of FcRn heavy
chain with beta-2-microglobulin. A variety of FcRn variants used to
increase binding to the FcRn receptor, and in some cases, to
increase serum half-life, are shown in paragraph [0320] of this
specification.
[0168] By "parent polypeptide" as used herein is meant a starting
polypeptide that is subsequently modified to generate a variant.
The parent polypeptide may be a naturally occurring polypeptide, or
a variant or engineered version of a naturally occurring
polypeptide. Parent polypeptide may refer to the polypeptide
itself, compositions that comprise the parent polypeptide, or the
amino acid sequence that encodes it. Accordingly, by "parent
immunoglobulin" as used herein is meant an unmodified
immunoglobulin polypeptide that is modified to generate a variant,
and by "parent antibody" as used herein is meant an unmodified
antibody that is modified to generate a variant antibody. It should
be noted that "parent antibody" includes known commercial,
recombinantly produced antibodies as outlined below.
[0169] By "Fc fusion protein" or "immunoadhesin" herein is meant a
protein comprising an Fc region, generally linked (optionally
through a linker moiety, as described herein) to a different
protein, such as a binding moiety to a target protein, as described
herein. In some cases, one monomer of the heterodimeric protein
comprises an antibody heavy chain (either including an scFv or
further including a light chain) and the other monomer is a Fc
fusion, comprising a variant Fc domain and a ligand. In some
embodiments, these "half antibody-half fusion proteins" are
referred to as "Fusionbodies".
[0170] By "position" as used herein is meant a location in the
sequence of a protein. Positions may be numbered sequentially, or
according to an established format, for example the EU index for
antibody numbering.
[0171] By "target antigen" as used herein is meant the molecule
that is bound specifically by the variable region of a given
antibody. A target antigen may be a protein, carbohydrate, lipid,
or other chemical compound. A wide number of suitable target
antigens are described below.
[0172] By "strandedness" in the context of the monomers of the
heterodimeric proteins of the invention herein is meant that,
similar to the two strands of DNA that "match", heterodimerization
variants are incorporated into each monomer so as to preserve the
ability to "match" to form heterodimers. For example, if some pI
variants are engineered into monomer A (e.g. making the pI higher)
then steric variants that are "charge pairs" that can be utilized
as well do not interfere with the pI variants, e.g. the charge
variants that make a pI higher are put on the same "strand" or
"monomer" to preserve both functionalities.
[0173] By "target cell" as used herein is meant a cell that
expresses a target antigen.
[0174] By "variable region" as used herein is meant the region of
an immunoglobulin that comprises one or more Ig domains
substantially encoded by any of the V.kappa., V.lamda., and/or VH
genes that make up the kappa, lambda, and heavy chain
immunoglobulin genetic loci respectively.
[0175] 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 amino acid sequence or a nucleotide
sequence that has not been intentionally modified.
[0176] The antibodies of the present invention are generally
isolated or recombinant. "Isolated," when used to describe the
various polypeptides disclosed herein, means a polypeptide that has
been identified and separated and/or recovered from a cell or cell
culture from which it was expressed. Ordinarily, an isolated
polypeptide will be prepared by at least one purification step. An
"isolated antibody," refers to an antibody which is substantially
free of other antibodies having different antigenic
specificities.
[0177] "Specific binding" or "specifically binds to" or is
"specific for" a particular antigen or an epitope 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.
[0178] Specific binding for a particular antigen or an epitope can
be exhibited, for example, by an antibody having a KD for an
antigen or epitope of at least about 10-4 M, at least about 10-5 M,
at least about 10-6 M, at least about 10-7 M, at least about 10-8
M, at least about 10-9 M, alternatively at least about 10-10 M, at
least about 10-11 M, at least about 10-12 M, or greater, where KD
refers to a dissociation rate of a particular antibody-antigen
interaction. Typically, an antibody that specifically binds an
antigen will have a KD that is 20-, 50-, 100-, 500-, 1000-, 5,000-,
10,000- or more times greater for a control molecule relative to
the antigen or epitope.
[0179] Also, specific binding for a particular antigen or an
epitope can be exhibited, for example, by an antibody having a KA
or Ka for an antigen or epitope of at least 20-, 50-, 100-, 500-,
1000-, 5,000-, 10,000- or more times greater for the epitope
relative to a control, where KA or Ka refers to an association rate
of a particular antibody-antigen interaction.
Methods and Compositions for Suppressing T Cells
[0180] In one aspect, the present invention provides methods and
compositions for suppressing T cells. In preferred embodiments, the
methods and compositions for suppressing T cells are specific for
one type of T cell with limited to no impact on other T cells. In
further embodiments, the methods and compositions of the present
invention suppress Tregs with limited to no impact on other T cell
types. In other embodiments, the methods and compositions of the
present invention suppress cytotoxic T cells with limited to no
impact on other T cell types.
[0181] In one aspect, the methods and compositions of the present
invention suppress T cells by administration of heterodimeric
proteins. Such heterodimeric proteins include without limitation
bispecific (although trispecific, tetraspecific and higher order
specificities are also contemplated) antibodies and fusion
proteins.
[0182] In certain embodiments, suppression of T cells by methods
and compositions of the invention serve to increase the numbers
and/or proliferation as compared to T cells that were not treated
in accordance with the present invention. In further embodiments,
administration of any of the heterodimeric proteins discussed
herein serves to increase the numbers and/or proliferation of T
cells over that seen without the administration of the
heterodimeric protein (or that seen with administration of a
control protein) by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,
80%, 90%, 100%, 150%, 200%. In yet further embodiments, the
increase in the is about 10-20%, 10-50%, 20-90%, 30-80%, 40-70%,
50-60%. In further embodiments, an increase in numbers and/or
proliferation is measured in comparison for the targeted T cell
type against the non-targeted type. For example, in embodiments in
which the administered heterodimeric protein suppresses regulatory
T cells, the increase in cell number and/or proliferation of
regulatory T cells is measured in comparison to that of other T
cell types. In still further embodiments, this comparative increase
is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%,
150%, 200% as compared to the non-targeted T cell type. In yet
further embodiments, the comparative increase in the targeted T
cell type is about 10-20%, 10-50%, 20-90%, 30-80%, 40-70%, 50-60%
over that of the non-targeted T cell type.
[0183] In general, the heterodimeric proteins of use for
suppression of T cells in accordance with the present invention
comprise two monomers, and each monomer comprises a heavy chain
constant region with a variant Fc domain as compared to a parent Fc
domain and an antigen binding moiety. In certain embodiments, the
variant Fc domain of one of the heavy chain constant region of one
of the monomers is different than the heavy chain constant region
of the other monomer.
[0184] In certain aspects, the heterodimeric proteins of the
invention for suppression of T cells comprise bispecific antibodies
or Fc fusion proteins. The bispecific antibodies of the invention
can take any format described herein and known in the art,
including those pictured in FIG. 3 and FIGS. 36 and 37. The antigen
binding domains of these bispecific antibodies will generally
comprise an anti-CD25 binding domain on one monomer and a binding
domain for a T cell marker on another arm. As will be appreciated,
however, any combination of proteins on T cells, including those
listed in FIG. 32 can be targets in any combination for bispecific
antibodies of the invention. In other words, bispecific antibodies
of the invention for suppression of T cells may target any two T
cell markers, including any two of those listed in FIG. 32.
[0185] In further exemplary embodiments, bispecific antibodies of
the invention comprise an anti-CD25 binding domain on one monomer
and an anti-CD4 binding domain on the other monomer (such
antibodies are also designated herein as anti-CD25.times.anti-CD4
bispecific antibodies). In further embodiments, the bispecific
antibodies of the invention comprise the following combinations of
antigen binding domains: anti-CD25.times.anti-CTLA4,
anti-CD25.times.anti-PD-1, anti-CD25.times.anti-CCR4,
Anti-CD4.times.Anti-CTLA4 and anti-CD4.times.Anti-CCR4 and
anti-CD25.times.anti-GITR antibodies.
[0186] Treg cells express CD4 and CD25 simultaneously, and
targeting both antigens with a bispecific antibody could in one
non-limiting theory be a powerful mechanism to selectively suppress
Treg cells and allow the immune system to mount a response against
tumor cells. Thus, a bispecific antibody allowing for simultaneous
avid targeting of CD4 and CD25 (or any other combination of
antigens as discussed above) may in certain embodiments reduce Treg
cell proliferation, either via cytotoxic depletion or by
interfering with IL2-dependent proliferation. Such an approach will
in further embodiments have little or no effect on unactivated
CD4+CD25.sup.- T effector cells or CD8+CD25.sup.- cytotoxic T
cells. Although it may exhibit some suppression of activated
CD4+CD25+ effector T cells, Tregs are reported to have
significantly higher dependence on IL-2 for survival (Malek and
Bayer Nature 2004, hereby incorporated by reference in its entirety
for all purposes and in particular for all teachings related to T
cells), providing additional selectivity of this approach for Treg
vs CD4 effector T cells.
[0187] Bispecific antibodies can also be used to suppress other T
cell types, such as cytotoxic T cells. In some instances, one
monomer of the bispecific antibody comprises an anti-CD8 domain,
including without limitation domains from anti-CD8 antibodies such
as MCD8, 3B5, SK1, OKT-8, 51.1 and DK-25. The monomer with the
anti-CD8 domain can be combined with a monomer comprising an
anti-CD25 binding domain to produce a bispecific antibody for
suppression of cytoxic T cells.
[0188] Generally, the antigen binding domains of bispecific
antibodies of the invention are part of monomers that further
comprise at least a heavy chain constant region that contains a
variant Fc domain as compared to a parent Fc domain.
[0189] Fc fusion proteins may also be used in accordance with the
invention to suppress T cells. For example, a fusion protein
comprising an IL2 protein on one arm can be engineered to have
reduced ability to bind to IL2R.beta., IL2R.gamma., and or
IL2R.alpha. in order to ablate IL2 receptor signaling. When coupled
with an anti-CD4 antibody (or any other Treg surface marker
antibody), this results in an anti-CD4.times.IL2 Fc-fusion capable
of suppressing Treg cells through targeted binding to CD4 and CD25,
but without the ability to induce Treg proliferation. In one
non-limiting theory, the mechanism of action for this fusion may be
that it blocks endogenous IL2 from binding to receptor, thus
preventing Treg proliferation. Exemplary embodiments of such fusion
proteins are provided in FIG. 23.
[0190] Any of the above described heterodimeric antibodies and
fusion proteins for suppressing T cells may further include
additional amino acid substitutions in the Fc domain. Such
substitutions may include one or any combination of substitutions
that affect heterodimer formation, serum half-life and/or binding
to FcRn (also referred to herein as "Fc variants"), binding to Fc
receptors, or ADCC. Exemplary further substitutions of use in any
of the heterodimeric proteins discussed herein for suppression of T
cells are listed in FIGS. 38-42 and 48.
Methods and Compositions for Inducing T Cells
[0191] In one aspect, the present invention provides methods and
compositions for inducing T cells. In preferred embodiments, the
methods and compositions for inducing T cells are specific for one
type of T cell with limited to no impact on other T cells. In
further embodiments, the methods and compositions of the present
invention induce Tregs with limited to no impact on other T cell
types. In other embodiments, the methods and compositions of the
present invention induce cytotoxic T cells with limited to no
impact on other T cell types.
[0192] "Inducing T cells" as used herein refers to increasing any
aspect of T cell expression or function as compared to expression
or function in the absence of the administered heterodimeric
protein, including stimulation of the proliferation of the target T
cell.
[0193] As discussed herein and understood in the art, induction (as
well as suppression) of T cells can be measured with assays to
quantify T cell numbers. For example, cell proliferation assays can
be used to detect and quantitate T cells. Other methods of
quantifying T cells, particularly Tregs, may also be used,
including methods utilizing qPCR to measure the amount of
demethylated FOXP3 (a Treg marker) that is present. Such assays are
described for example in Wieczorek et al., 2009, Cancer Res, 69(2):
599-608, Vries et al., 2011, Clin Cancer Res, 17:841-848, and Baron
et al., 2007, Eur. J. Immunol., 37:2378-2389, each of which is
hereby incorporated by reference in its entirety for all purposes
and in particular for all teachings, figures and legends related to
assays for FOXP3, demethylated FOXP3, and quantification of Tregs.
Such assays can also be used to quantify the specificity of
induction by providing quantitative measurements of numbers of T
cells of one type that are induced as compared to other types of T
cells (for example, numbers of Tregs induced as compared to
cytotoxic T cells).
[0194] In certain embodiments, induction of T cells by methods and
compositions of the invention serve to increase the numbers and/or
proliferation as compared to T cells that were not treated in
accordance with the present invention. In further embodiments,
administration of any of the heterodimeric proteins discussed
herein serves to increase the numbers and/or proliferation of T
cells over that seen without the administration of the
heterodimeric protein (or that seen with administration of a
control protein) by at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%,
80%, 90%, 100%, 150%, 200%. In yet further embodiments, the
increase in the is about 10-20%, 10-50%, 20-90%, 30-80%, 40-70%,
50-60%. In further embodiments, an increase in numbers and/or
proliferation is measured in comparison for the targeted T cell
type against the non-targeted type. For example, in embodiments in
which the administered heterodimeric protein induces regulatory T
cells, the increase in cell number and/or proliferation of
regulatory T cells is measured in comparison to that of other T
cell types. In still further embodiments, this comparative increase
is at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%,
150%, 200% as compared to the non-targeted T cell type. In yet
further embodiments, the comparative increase in the targeted T
cell type is about 10-20%, 10-50%, 20-90%, 30-80%, 40-70%, 50-60%
over that of the non-targeted T cell type.
[0195] In further aspects, induction of T cells is accomplished in
accordance with the present invention using heterodimeric Fc fusion
proteins. Such fusion proteins are also referred to herein as
"fusionbodies" because they generally comprise two monomers, in
which one monomer is an Fc domain fused to a ligand, such as IL2,
and the other monomer is a FAb monomer comprising a heavy chain and
a light chain.
[0196] In certain embodiments, Fc fusion proteins of the invention
include one monomer that comprises a T cell protein, including
without limitation those proteins listed in FIG. 32. In further
embodiments, the monomer comprises all or a portion of an IL2
protein. As will be appreciated, the IL2 protein may comprise an
IL2 protein from any source, including any mammalian species. In
preferred embodiments, the IL2 portion of the monomer comprises a
sequence from human IL2. Variants of IL2 may also be used in Fc
fusion proteins of the invention, including without limitation
variants such as those listed in FIG. 23.
[0197] In further embodiments, the Fc fusion proteins comprise a
second monomer that comprises a T cell protein, including without
limitation any of the proteins listed in FIG. 32. In exemplary
embodiments, the fusionbodies of the present invention for
induction of T cells have one monomer that is an Fc domain fused to
all or part of an IL2 protein and the second monomer comprises an
antigen binding domain that targets one of the following: CD4, CD8,
CTLA-4, CCR4, and PD-1. In still further embodiments, the second
monomer comprises both a heavy chain and a light chain sequence,
and the variable domains of those heavy and light chain sequences
form the antigen-binding domain.
Methods of Making Compositions of the Invention
[0198] Any of the heterodimeric proteins discussed herein,
including bispecific antibodies and heterodimeric Fc fusion
proteins, can be made using methods known in the art and methods
described in further detail herein.
[0199] In certain aspects, the invention provides one or more
nucleic acids encoding a composition according to any of the
compositions described herein. As will be appreciated, different
monomers of the heterodimeric proteins of the invention may be
expressed using nucleic acids encoding all or a portion of one or
more of the monomers of the protein. Thus, for example, for a
bispecific antibody in which one monomer targets CD4 and the other
monomer target CD25, the present invention further provides a
nucleic acid encoding the first and second monomers as separate
molecules that are then assembled together by co-expression in the
same host cell. In other embodiments, the two monomers may be
encoded in the same nucleic acid, in some embodiments within the
same vector. In embodiments in which one or both of the monomers
comprise both heavy and light chain sequences, those sequences may
also be encoded by one or by multiple nucleic acids.
[0200] In further embodiments, the invention further provides host
cells expressing the one or more nucleic acids encoding the one or
more monomers of heterodimeric proteins of the invention. As will
be appreciated and as is discussed above, the heterodimeric
proteins of the present invention may be encoded by one or more
nucleic acids. These one or more nucleic acids may be expressed in
a single host cell or in separate host cells. For example, for
heterodimeric proteins that are in the bottle-opener format in
which one of the monomers is an scFv and the other monomer is a
FAb, there may be three nucleic acids encoding this protein: one
for the scFv, one for the heavy chain sequence of the FAb, and one
for the light chain sequence of the FAb. These three nucleic acids
will in general be expressed in the same host cell in order to
produce the heterodimeric protein, although expression in separate
host cells is also contemplated.
[0201] In yet further embodiments, and in accordance with any of
the above, the present invention provides a method of making any of
the compositions described herein, the method including the step of
culturing a host cell or more nucleic acids encoding a
heterodimeric protein of the invention, including any of the
bispecific antibodies or Fc fusion proteins described herein.
[0202] In further aspects, the present invention provides a method
of purifying a heterodimeric protein or bispecific antibody in
accordance with any of the above, the method including: (a)
providing a composition in accordance with any of the heterodimeric
proteins described herein, (b) loading the composition onto an ion
exchange column; and (c) collecting a fraction containing the
heterodimeric protein or bispecific antibody, thus purifying the
protein or antibody.
Heterodimeric Proteins Overview
[0203] The present invention is directed to methods of modulating T
cells using novel constructs to provide heterodimeric proteins that
allow binding to more than one antigen or ligand, e.g. to allow for
multispecific binding. The heterodimeric protein constructs are
based on the self-assembling nature of the two Fc domains of the
heavy chains of antibodies, e.g. two "monomers" that assemble into
a "dimer". Heterodimeric proteins are made by altering the amino
acid sequence of each monomer as more fully discussed below. Thus,
the present invention is generally directed to the creation of
heterodimeric proteins including antibodies, which can co-engage
antigens in several ways, relying on amino acid variants in the
constant regions that are different on each chain to promote
heterodimeric formation and/or allow for ease of purification of
heterodimers over the homodimers. As discussed more fully below,
the heterodimeric proteins can be antibody variants or based on Fc
fusion proteins. Although much of the following discussion is in
terms of heterodimeric antibodies, it will be appreciated by those
in the art and more fully described below, the discussion applies
equally to heterodimeric proteins that are based on Fc fusion
proteins (also referred to herein as fusionbodies).
[0204] Thus, the present invention provides bispecific antibodies
(or, as discussed below, trispecific or tetraspecific antibodies
can also be made). An ongoing problem in antibody technologies is
the desire for "bispecific" (and/or multispecific) antibodies that
bind to two (or more) different antigens simultaneously, in general
thus allowing the different antigens to be brought into proximity
and resulting in new functionalities and new therapies. In general,
these antibodies are made by including genes for each heavy and
light chain into the host cells. This generally results in the
formation of the desired heterodimer (A-B), as well as the two
homodimers (A-A and B-B). However, a major obstacle in the
formation of multispecific antibodies is the difficulty in
purifying the heterodimeric antibodies away from the homodimeric
antibodies and/or biasing the formation of the heterodimer over the
formation of the homodimers.
[0205] There are a number of mechanisms that can be used to
generate the heterodimers of the present invention. In addition, as
will be appreciated by those in the art, these mechanisms can be
combined to ensure high heterodimerization. Thus, amino acid
variants that lead to the production of heterodimers are referred
to as "heterodimerization variants". As discussed below,
heterodimerization variants can include steric variants (e.g. the
"knobs and holes" or "skew" variants described below and the
"charge pairs" variants described below) as well as "pI variants",
which allows purification of homodimers away from heterodimers.
[0206] One mechanism is generally referred to in the art as "knobs
and holes" ("KIH") or sometimes herein as "skew" variants,
referring to amino acid engineering that creates steric influences
to favor heterodimeric formation and disfavor homodimeric formation
can also optionally be used; this is sometimes referred to as
"knobs and holes"; as described in U.S. Ser. No. 61/596,846 and
U.S. Ser. No. 12/875,0015, Ridgway et al., Protein Engineering
9(7):617 (1996); Atwell et al., J. Mol. Biol. 1997 270:26; U.S.
Pat. No. 8,216,805, US 2012/0149876, all of which are hereby
incorporated by reference in their entirety. The Figures identify a
number of "monomer A-monomer B" pairs that include "knobs and
holes" amino acid substitutions. In addition, as described in
Merchant et al., Nature Biotech. 16:677 (1998), these "knobs and
hole" mutations can be combined with disulfide bonds to skew
formation to heterodimerization.
[0207] An additional mechanism that finds use in the generation of
heterodimers is sometimes referred to as "electrostatic steering"
or "charge pairs" as described in Gunasekaran et al., J. Biol.
Chem. 285(25):19637 (2010), hereby incorporated by reference in its
entirety. This is sometimes referred to herein as "charge pairs".
In this embodiment, electrostatics are used to skew the formation
towards heterodimerization. As those in the art will appreciate,
these may also have an effect on pI, and thus on purification, and
thus could in some cases also be considered pI variants. However,
as these were generated to force heterodimerization and were not
used as purification tools, they are classified as "steric
variants". These include, but are not limited to, D221E/P228E/L368E
paired with D221R/P228R/K409R (e.g. these are "monomer
corresponding sets) and C220E/P228E/368E paired with
C220R/E224R/P228R/K409R and others shown in the Figures.
[0208] In the present invention, in some embodiments, pI variants
are used to alter the pI of one or both of the monomers and thus
allowing the isoelectric purification of A-A, A-B and B-B dimeric
proteins.
[0209] In the present invention, there are several basic mechanisms
that can lead to ease of purifying heterodimeric proteins; one
relies on the use of pI variants, such that each monomer has a
different pI, thus allowing the isoelectric purification of A-A,
A-B and B-B dimeric proteins. Alternatively, some scaffold formats,
such as the "triple F" format, also allows separation on the basis
of size. As is further outlined below, it is also possible to
"skew" the formation of heterodimers over homodimers. Thus, a
combination of steric heterodimerization variants and pI or charge
pair variants find particular use in the invention. Additionally,
as more fully outlined below, scaffolds that utilize scFv(s) such
as the Triple F format can include charged scFv linkers (either
positive or negative), that give a further pI boost for
purification purposes. As will be appreciated by those in the art,
some Triple F formats are useful with just charged scFv linkers and
no additional pI adjustments, although the invention does provide
the use of skew variants with charged scFv linkers as well (and
combinations of Fc, FcRn and KO variants).
[0210] In the present invention that utilizes pI as a separation
mechanism to allow the purification of heterodimeric proteins,
amino acid variants can be introduced into one or both of the
monomer polypeptides; that is, the pI of one of the monomers
(referred to herein for simplicity as "monomer A") can be
engineered away from monomer B, or both monomer A and B change be
changed, with the pI of monomer A increasing and the pI of monomer
B decreasing. As is outlined more fully below, the pI changes of
either or both monomers can be done by removing or adding a charged
residue (e.g. a neutral amino acid is replaced by a positively or
negatively charged amino acid residue, e.g. glycine to glutamic
acid), changing a charged residue from positive or negative to the
opposite charge (aspartic acid to lysine) or changing a charged
residue to a neutral residue (e.g. loss of a charge; lysine to
serine.). A number of these variants are shown in the Figures.
[0211] Accordingly, in this embodiment of the present invention
provides for creating a sufficient change in pI in at least one of
the monomers such that heterodimers can be separated from
homodimers. As will be appreciated by those in the art, and as
discussed further below, this can be done by using a "wild type"
heavy chain constant region and a variant region that has been
engineered to either increase or decrease its pI (wt A-+B or wt
A--B), or by increasing one region and decreasing the other region
(A+-B- or A-B+).
[0212] Thus, in general, a component of some embodiments of the
present invention are amino acid variants in the constant regions
of antibodies that are directed to altering the isoelectric point
(pI) of at least one, if not both, of the monomers of a dimeric
protein to form "pI heterodimers" (when the protein is an antibody,
these are referred to as "pI antibodies") by incorporating amino
acid substitutions ("pI variants" or "pI substitutions") into one
or both of the monomers. As shown herein, the separation of the
heterodimers from the two homodimers can be accomplished if the pIs
of the two monomers differ by as little as 0.1 pH unit, with 0.2,
0.3, 0.4 and 0.5 or greater all finding use in the present
invention.
[0213] As will be appreciated by those in the art, the number of pI
variants to be included on each or both monomer(s) to get good
separation will depend in part on the starting pI of the scFv and
Fab of interest. That is, to determine which monomer to engineer or
in which "direction" (e.g. more positive or more negative), the Fv
sequences of the two target antigens are calculated and a decision
is made from there. As is known in the art, different Fvs will have
different starting pIs which are exploited in the present
invention. In general, as outlined herein, the pIs are engineered
to result in a total pI difference of each monomer of at least
about 0.1 logs, with 0.2 to 0.5 being preferred as outlined
herein.
[0214] Furthermore, as will be appreciated by those in the art and
outlined herein, heterodimers can be separated from homodimers on
the basis of size. For example, as shown in FIGS. 36 and 37,
heterodimers with two scFvs can be separated by those of the
"triple F" format and a bispecific mAb. This can be further
exploited in higher valency with additional antigen binding sites
being utilized. For example, as additionally shown, one monomer
will have two Fab fragments and the other will have one scFv,
resulting in a differential in size and thus molecular weight.
[0215] In addition, as will be appreciated by those in the art and
outlined herein, the format outlined herein can be expanded to
provide trispecific and tetraspecific antibodies as well. In this
embodiment, some variations of which are depicted in the FIG.
36A36M, it will be recognized that it is possible that some
antigens are bound divalently (e.g. two antigen binding sites to a
single antigen; for example, A and B could be part of a typical
bivalent association and C and D can be optionally present and
optionally the same or different). As will be appreciated, any
combination of Fab and scFvs can be utilized to achieve the desired
result and combinations.
[0216] In the case where pI variants are used to achieve
heterodimerization, by using the constant region(s) of the heavy
chain(s), a more modular approach to designing and purifying
multispecific proteins, including antibodies, is provided. Thus, in
some embodiments, heterodimerization variants (including skew and
purification heterodimerization variants) are not included in the
variable regions, such that each individual antibody must be
engineered. In addition, in some embodiments, the possibility of
immunogenicity resulting from the pI variants is significantly
reduced by importing pI variants from different IgG isotypes such
that pI is changed without introducing significant immunogenicity.
Thus, an additional problem to be solved is the elucidation of low
pI constant domains with high human sequence content, e.g. the
minimization or avoidance of non-human residues at any particular
position.
[0217] A side benefit that can occur with this pI engineering is
also the extension of serum half-life and increased FcRn binding.
That is, as described in U.S. Ser. No. 13/194,904 (incorporated by
reference in its entirety), lowering the pI of antibody constant
domains (including those found in antibodies and Fc fusions) can
lead to longer serum retention in vivo. These pI variants for
increased serum half life also facilitate pI changes for
purification.
[0218] In addition, it should be noted that the pI variants of the
heterodimerization variants give an additional benefit for the
analytics and quality control process of bispecific antibodies, the
ability to eliminate, minimize and/or distinguish when homodimers
are present is significant. Similarly, the ability to reliably test
the reproducibility of the heterodimeric protein production is
important.
[0219] In addition to all or part of a variant heavy constant
domain, one or both of the monomers may contain one or two fusion
partners, such that the heterodimers form multivalent proteins. As
is generally depicted in the Figures, and specifically FIG. 36A,
the fusion partners are depicted as A, B, C and D, with all
combinations possible. In general, A, B, C and D are selected such
that the heterodimer is at least bispecific or bivalent in its
ability to interact with additional proteins.
[0220] As will be appreciated by those in the art and discussed
more fully below, the heterodimeric fusion proteins of the present
invention can take on a wide variety of configurations, as are
generally depicted in FIGS. 36 and 37. Some figures depict "single
ended" configurations, where there is one type of specificity on
one "arm" of the molecule and a different specificity on the other
"arm". Other figures depict "dual ended" configurations, where
there is at least one type of specificity at the "top" of the
molecule and one or more different specificities at the "bottom" of
the molecule. Furthermore as is shown, these two configurations can
be combined, where there can be triple or quadruple specificities
based on the particular combination. Thus, the present invention
provides "multispecific" binding proteins, including multispecific
antibodies. Thus, the present invention is directed to novel
immunoglobulin compositions that co-engage at least a first and a
second antigen. First and second antigens of the invention are
herein referred to as antigen-1 and antigen-2 respectively.
[0221] One heterodimeric scaffold that finds particular use in the
present invention is the "triple F" or "bottle opener" scaffold
format. In this embodiment, one heavy chain of the antibody
contains an single chain Fv ("scFv", as defined below) and the
other heavy chain is a "regular" FAb format, comprising a variable
heavy chain and a light chain. This structure is sometimes referred
to herein as "triple F" format (scFv-FAb-Fc) or the "bottle-opener"
format, due to a rough visual similarity to a bottle-opener (see
FIG. 36B). The two chains are brought together by the use of amino
acid variants in the constant regions (e.g. the Fc domain and/or
the hinge region) that promote the formation of heterodimeric
antibodies as is described more fully below.
[0222] There are several distinct advantages to the present "triple
F" format. As is known in the art, antibody analogs relying on two
scFv constructs often have stability and aggregation problems,
which can be alleviated in the present invention by the addition of
a "regular" heavy and light chain pairing. In addition, as opposed
to formats that rely on two heavy chains and two light chains,
there is no issue with the incorrect pairing of heavy and light
chains (e.g. heavy 1 pairing with light 2, etc.)
[0223] In addition to all or part of a variant heavy constant
domain, one or both of the monomers may contain one or two fusion
partners, such that the heterodimers form multivalent proteins. As
is generally depicted in the FIG. 64 of U.S. Ser. No. 13/648,951,
hereby incorporated by reference with its accompanying legend, the
fusion partners are depicted as A, B, C and D, with all
combinations possible. In general, A, B, C and D are selected such
that the heterodimer is at least bispecific or bivalent in its
ability to interact with additional proteins. In the context of the
present "triple F" format, generally A and B are an scFv and a Fv
(as will be appreciated, either monomer can contain the scFv and
the other the Fv/Fab) and then optionally one or two additional
fusion partners.
[0224] Furthermore, as outlined herein, additional amino acid
variants may be introduced into the bispecific antibodies of the
invention, to add additional functionalities. For example, amino
acid changes within the Fc region can be added (either to one
monomer or both) to facilitate increased ADCC or CDC (e.g. altered
binding to Fc.gamma. receptors); to allow or increase yield of the
addition of toxins and drugs (e.g. for ADC), as well as to increase
binding to FcRn and/or increase serum half-life of the resulting
molecules. As is further described herein and as will be
appreciated by those in the art, any and all of the variants
outlined herein can be optionally and independently combined with
other variants.
[0225] Similarly, another category of functional variants are
"Fc.gamma. ablation variants" or "Fc knock out (FcKO or KO)
variants. In these embodiments, for some therapeutic applications,
it is desirable to reduce or remove the normal binding of the Fc
domain to one or more or all of the Fc.gamma. receptors (e.g.
Fc.gamma.RI, Fc.gamma.RIIa, Fc.gamma.RIIb, Fc.gamma.RIIIa, etc.) to
avoid additional mechanisms of action. That is, for example, in
many embodiments, particularly in the use of bispecific antibodies
of the invention, it is generally desirable to ablate
Fc.gamma.RIIIa binding to eliminate or significantly reduce ADCC
activity.
Antibodies
[0226] The present invention relates to the generation of
multispecific antibodies, generally therapeutic antibodies. As is
discussed below, the term "antibody" is used generally. Antibodies
that find use in the present invention can take on a number of
formats as described herein, including traditional antibodies as
well as antibody derivatives, fragments and mimetics, described
below. In general, the term "antibody" includes any polypeptide
that includes at least one constant domain, including, but not
limited to, CH1, CH2, CH3 and CL.
[0227] Traditional antibody structural units typically comprise a
tetramer. Each tetramer is typically composed of two identical
pairs of polypeptide chains, each pair having one "light"
(typically having a molecular weight of about 25 kDa) and one
"heavy" chain (typically having a molecular weight of about 50-70
kDa). Human light chains are classified as kappa and lambda light
chains. The present invention is directed to the IgG class, which
has several subclasses, including, but not limited to IgG1, IgG2,
IgG3, and IgG4. Thus, "isotype" as used herein is meant any of the
subclasses of immunoglobulins defined by the chemical and antigenic
characteristics of their constant regions. It should be understood
that therapeutic antibodies can also comprise hybrids of isotypes
and/or subclasses. For example, as shown in US Publication
2009/0163699, incorporated by reference, the present invention
covers pI engineering of IgG1/G2 hybrids.
[0228] The amino-terminal portion of each chain includes a variable
region of about 100 to 110 or more amino acids primarily
responsible for antigen recognition, generally referred to in the
art and herein as the "Fv domain" or "Fv region". In the variable
region, three loops are gathered for each of the V domains of the
heavy chain and light chain to form an antigen-binding site. Each
of the loops is referred to as a complementarity-determining region
(hereinafter referred to as a "CDR"), in which the variation in the
amino acid sequence is most significant. "Variable" refers to the
fact that certain segments of the variable region differ
extensively in sequence among antibodies. Variability within the
variable region is not evenly distributed. Instead, the V regions
consist of relatively invariant stretches called framework regions
(FRs) of 15-30 amino acids separated by shorter regions of extreme
variability called "hypervariable regions" that are each 9-15 amino
acids long or longer.
[0229] Each VH and VL is composed of three hypervariable regions
("complementary determining regions," "CDRs") and four FRs,
arranged from amino-terminus to carboxy-terminus in the following
order: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.
[0230] The hypervariable region generally encompasses amino acid
residues from about amino acid residues 24-34 (LCDR1; "L" denotes
light chain), 50-56 (LCDR2) and 89-97 (LCDR3) in the light chain
variable region and around about 31-35B (HCDR1; "H" denotes heavy
chain), 50-65 (HCDR2), and 95-102 (HCDR3) in the heavy chain
variable region; Kabat et al., SEQUENCES OF PROTEINS OF
IMMUNOLOGICAL INTEREST, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md. (1991) and/or those residues
forming a hypervariable loop (e.g. residues 26-32 (LCDR1), 50-52
(LCDR2) and 91-96 (LCDR3) in the light chain variable region and
26-32 (HCDR1), 53-55 (HCDR2) and 96-101 (HCDR3) in the heavy chain
variable region; Chothia and Lesk (1987) J. Mol. Biol. 196:901-917.
Specific CDRs of the invention are described below.
[0231] Throughout the present specification, the Kabat numbering
system is generally used when referring to a residue in the
variable domain (approximately, residues 1-107 of the light chain
variable region and residues 1-113 of the heavy chain variable
region) and the EU numbering system for Fc regions (e.g, Kabat et
al., supra (1991)).
[0232] The CDRs contribute to the formation of the antigen-binding,
or more specifically, epitope binding site of antibodies. "Epitope"
refers to a determinant that interacts with a specific antigen
binding site in the variable region of an antibody molecule known
as a paratope. Epitopes are groupings of molecules such as amino
acids or sugar side chains and usually have specific structural
characteristics, as well as specific charge characteristics. A
single antigen may have more than one epitope.
[0233] The epitope may comprise amino acid residues directly
involved in the binding (also called immunodominant component of
the epitope) and other amino acid residues, which are not directly
involved in the binding, such as amino acid residues which are
effectively blocked by the specifically antigen binding peptide; in
other words, the amino acid residue is within the footprint of the
specifically antigen binding peptide.
[0234] Epitopes may be either conformational or linear. A
conformational epitope is produced by spatially juxtaposed amino
acids from different segments of the linear polypeptide chain. A
linear epitope is one produced by adjacent amino acid residues in a
polypeptide chain. Conformational and nonconformational epitopes
may be distinguished in that the binding to the former but not the
latter is lost in the presence of denaturing solvents.
[0235] An epitope typically includes at least 3, and more usually,
at least 5 or 8-10 amino acids in a unique spatial conformation.
Antibodies that recognize the same epitope can be verified in a
simple immunoassay showing the ability of one antibody to block the
binding of another antibody to a target antigen, for example
"binning."
[0236] The carboxy-terminal portion of each chain defines a
constant region primarily responsible for effector function. Kabat
et al. collected numerous primary sequences of the variable regions
of heavy chains and light chains. Based on the degree of
conservation of the sequences, they classified individual primary
sequences into the CDR and the framework and made a list thereof
(see SEQUENCES OF IMMUNOLOGICAL INTEREST, 5th edition, NIH
publication, No. 91-3242, E. A. Kabat et al., entirely incorporated
by reference).
[0237] In the IgG subclass of immunoglobulins, there are several
immunoglobulin domains in the heavy chain. By "immunoglobulin (Ig)
domain" herein is meant a region of an immunoglobulin having a
distinct tertiary structure. Of interest in the present invention
are the heavy chain domains, including, the constant heavy (CH)
domains and the hinge domains. In the context of IgG antibodies,
the IgG isotypes each have three CH regions. Accordingly, "CH"
domains in the context of IgG are as follows: "CH1" refers to
positions 118-220 according to the EU index as in Kabat. "CH2"
refers to positions 237-340 according to the EU index as in Kabat,
and "CH3" refers to positions 341-447 according to the EU index as
in Kabat. As shown herein and described below, the pI variants can
be in one or more of the CH regions, as well as the hinge region,
discussed below.
[0238] It should be noted that for the IgG sequences depicted
herein start at the CH1 region, position 118; the variable regions
are not included except as noted. For example, the first amino
acid, while designated as position "1" in the sequence listing,
corresponds to position 118 of the CH1 region, according to EU
numbering.
[0239] Another type of Ig domain of the heavy chain is the hinge
region. By "hinge" or "hinge region" or "antibody hinge region" or
"immunoglobulin hinge region" herein is meant the flexible
polypeptide comprising the amino acids between the first and second
constant domains of an antibody. Structurally, the IgG CH1 domain
ends at EU position 220, and the IgG CH2 domain begins at residue
EU position 237. Thus for IgG the antibody hinge is herein defined
to include positions 221 (D221 in IgG1) to 236 (G236 in IgG1),
wherein the numbering is according to the EU index as in Kabat. In
some embodiments, for example in the context of an Fc region, the
lower hinge is included, with the "lower hinge" generally referring
to positions 226 or 230. As noted herein, pI variants can be made
in the hinge region as well.
[0240] The light chain generally comprises two domains, the
variable light domain (containing the light chain CDRs and together
with the variable heavy domains forming the Fv region), and a
constant light chain region (often referred to as CL or CK).
[0241] Another region of interest for additional substitutions,
outlined below, is the Fc region. By "Fc" or "Fc region" or "Fc
domain" as used herein is meant the polypeptide comprising the
constant region of an antibody excluding the first constant region
immunoglobulin domain and in some cases, part of the hinge. 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 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 C.gamma.2 and C.gamma.3
(C.gamma.2 and C.gamma.3) and the lower hinge region between
C.gamma.1 (C.gamma.1) and C.gamma.2 (C.gamma.2). Although the
boundaries of the Fc region may vary, the human IgG heavy chain Fc
region is usually defined to include residues C226 or P230 to its
carboxyl-terminus, wherein the numbering is according to the EU
index as in Kabat. In some embodiments, as is more fully described
below, amino acid modifications are made to the Fc region, for
example to alter binding to one or more Fc.gamma.R receptors or to
the FcRn receptor.
[0242] Accordingly, in some embodiments the present invention
provides heterodimeric antibodies that rely on the use of two
different heavy chain variant Fc domains that will self-assemble to
form heterodimeric antibodies.
[0243] In some embodiments, the antibodies are full length. By
"full length antibody" herein is meant the structure that
constitutes the natural biological form of an antibody, including
variable and constant regions, including one or more modifications
as outlined herein, particularly in the Fc domains to allow either
heterodimerization formation or the purification of heterodimers
away from homodimers. A full length heterodimeric antibody is two
heavy chains with different Fc domains and either two light chains
or a common light chain.
[0244] Alternatively, the antibodies can include a variety of
structures as are generally shown in the Figures, including, but
not limited to, antibody fragments, monoclonal antibodies,
bispecific antibodies, minibodies, domain antibodies, synthetic
antibodies (sometimes referred to herein as "antibody mimetics"),
chimeric antibodies, humanized antibodies, antibody fusions
(sometimes referred to as "antibody conjugates"), and fragments of
each, respectively.
[0245] In one embodiment, the antibody is an antibody fragment, as
long as it contains at least one constant domain which can be
engineered to produce heterodimers, such as pI engineering. Other
antibody fragments that can be used include fragments that contain
one or more of the CH1, CH2, CH3, hinge and CL domains of the
invention that have been pI engineered. For example, Fc fusions are
fusions of the Fc region (CH2 and CH3, optionally with the hinge
region) fused to another protein. A number of Fc fusions are known
the art and can be improved by the addition of the
heterodimerization variants of the invention. In the present case,
antibody fusions can be made comprising CH1; CH1, CH2 and CH3; CH2;
CH3; CH2 and CH3; CH1 and CH3, any or all of which can be made
optionally with the hinge region, utilizing any combination of
heterodimerization variants described herein.
scFv Embodiments
[0246] In some embodiments of the present invention, one monomer
comprises a heavy chain comprises a scFV linked to an Fc domain,
and the other monomer comprises a heavy chain comprising a Fab
linked to an Fc domain, e.g. a "typical" heavy chain, and a light
chain. By "Fab" or "Fab region" as used herein is meant the
polypeptide that comprises the VH, CH1, VL, and CL immunoglobulin
domains. Fab may refer to this region in isolation, or this region
in the context of a full length antibody, antibody fragment or Fab
fusion protein. By "Fv" or "Fv fragment" or "Fv region" as used
herein is meant a polypeptide that comprises the VL and VH domains
of a single antibody.
[0247] Several of the heterodimeric antibody embodiments described
herein rely on the use of one or more scFv domains, comprising the
variable heavy and variable light chains, covalently linked using a
linker, forming an antigen binding domain. Some embodiments herein
use "standard" linkers, usually linkers of glycine and serine, as
is well known in the art.
[0248] The present invention further provides charged scFv linkers,
to facilitate the separation in pI between a first and a second
monomer. That is, by incorporating a charged scFv linker, either
positive or negative (or both, in the case of scaffolds that use
scFvs on different monomers), this allows the monomer comprising
the charged linker to alter the pI without making further changes
in the Fc domains. These charged linkers can be substituted into
any scFv containing standard linkers. Again, as will be appreciated
by those in the art, charged scFv linkers are used on the correct
"strand" or monomer, according to the desired changes in pI. For
example, as discussed herein, to make triple F format heterodimeric
antibody, the original pI of the Fv region for each of the desired
antigen binding domains are calculated, and one is chosen to make
an scFv, and depending on the pI, either positive or negative
linkers are chosen.
[0249] In addition, disulfide bonds can be engineered into the
variable heavy and variable light chains to give additional
stability.
Chimeric and Humanized Antibodies
[0250] In some embodiments, the antibody can be a mixture from
different species, e.g. a chimeric antibody and/or a humanized
antibody. In general, both "chimeric antibodies" and "humanized
antibodies" refer to antibodies that combine regions from more than
one species. For example, "chimeric antibodies" traditionally
comprise variable region(s) from a mouse (or rat, in some cases)
and the constant region(s) from a human. "Humanized antibodies"
generally refer to non-human antibodies that have had the
variable-domain framework regions swapped for sequences found in
human antibodies. Generally, in a humanized antibody, the entire
antibody, except the CDRs, is encoded by a polynucleotide of human
origin or is identical to such an antibody except within its CDRs.
The CDRs, some or all of which are encoded by nucleic acids
originating in a non-human organism, are grafted into the
beta-sheet framework of a human antibody variable region to create
an antibody, the specificity of which is determined by the
engrafted CDRs. The creation of such antibodies is described in,
e.g., WO 92/11018, Jones, 1986, Nature 321:522-525, Verhoeyen et
al., 1988, Science 239:1534-1536, all entirely incorporated by
reference. "Backmutation" of selected acceptor framework residues
to the corresponding donor residues is often required to regain
affinity that is lost in the initial grafted construct (U.S. Pat.
Nos. 5,530,101; 5,585,089; 5,693,761; 5,693,762; 6,180,370;
5,859,205; 5,821,337; 6,054,297; 6,407,213, all entirely
incorporated by reference). The humanized antibody optimally also
will comprise at least a portion of an immunoglobulin constant
region, typically that of a human immunoglobulin, and thus will
typically comprise a human Fc region. Humanized antibodies can also
be generated using mice with a genetically engineered immune
system. Roque et al., 2004, Biotechnol. Prog. 20:639-654, entirely
incorporated by reference. A variety of techniques and methods for
humanizing and reshaping non-human antibodies are well known in the
art (See Tsurushita & Vasquez, 2004, Humanization of Monoclonal
Antibodies, Molecular Biology of B Cells, 533-545, Elsevier Science
(USA), and references cited therein, all entirely incorporated by
reference). Humanization methods include but are not limited to
methods described in Jones et al., 1986, Nature 321:522-525;
Riechmann et al., 1988; Nature 332:323-329; Verhoeyen et al., 1988,
Science, 239:1534-1536; Queen et al., 1989, Proc Natl Acad Sci, USA
86:10029-33; He et al., 1998, J. Immunol. 160: 1029-1035; Carter et
al., 1992, Proc Natl Acad Sci USA 89:4285-9, Presta et al., 1997,
Cancer Res. 57(20):4593-9; Gorman et al., 1991, Proc. Natl. Acad.
Sci. USA 88:4181-4185; O'Connor et al., 1998, Protein Eng 11:321-8,
all entirely incorporated by reference. Humanization or other
methods of reducing the immunogenicity of nonhuman antibody
variable regions may include resurfacing methods, as described for
example in Roguska et al., 1994, Proc. Natl. Acad. Sci. USA
91:969-973, entirely incorporated by reference. In one embodiment,
the parent antibody has been affinity matured, as is known in the
art. Structure-based methods may be employed for humanization and
affinity maturation, for example as described in U.S. Ser. No.
11/004,590. Selection based methods may be employed to humanize
and/or affinity mature antibody variable regions, including but not
limited to methods described in Wu et al., 1999, J. Mol. Biol.
294:151-162; Baca et al., 1997, J. Biol. Chem. 272(16):10678-10684;
Rosok et al., 1996, J. Biol. Chem. 271(37): 22611-22618; Rader et
al., 1998, Proc. Natl. Acad. Sci. USA 95: 8910-8915; Krauss et al.,
2003, Protein Engineering 16(10):753-759, all entirely incorporated
by reference. Other humanization methods may involve the grafting
of only parts of the CDRs, including but not limited to methods
described in U.S. Ser. No. 09/810,510; Tan et al., 2002, J.
Immunol. 169:1119-1125; De Pascalis et al., 2002, J. Immunol.
169:3076-3084, all entirely incorporated by reference.
Heterodimeric Heavy Chain Constant Regions
[0251] Accordingly, the present invention provides heterodimeric
proteins based on the use of monomers containing variant heavy
chain constant regions, and specifically the Fc domains, as a first
domain. By "monomer" herein is meant one half of the heterodimeric
protein. It should be noted that traditional antibodies are
actually tetrameric (two heavy chains and two light chains). In the
context of the present invention, one pair of heavy-light chains
(if applicable, e.g. if the monomer comprises an Fab) is considered
a "monomer". Similarly, a heavy chain region comprising the scFv is
considered a monomer. In the case where an Fv region is one fusion
partner (e.g. heavy and light variable domains) and a non-antibody
protein is another fusion partner, each "half" is considered a
monomer. Essentially, each monomer comprises sufficient heavy chain
constant region to allow heterodimerization engineering, whether
that be all the constant region, e.g. Ch1-hinge-CH2-CH3, the Fc
region (CH2-CH3), or just the CH3 domain.
[0252] The variant heavy chain constant regions can comprise all or
part of the heavy chain constant region, including the full length
construct, CH1-hinge-CH2-CH3, or portions thereof, including for
example CH2-CH3 or CH3 alone. In addition, the heavy chain region
of each monomer can be the same backbone (CH1-hinge-CH2-CH3 or
CH2-CH3) or different. N- and C-terminal truncations and additions
are also included within the definition; for example, some pI
variants include the addition of charged amino acids to the
C-terminus of the heavy chain domain.
[0253] Thus, in general, one monomer of the present "triple F"
construct is a scFv region-hinge-Fc domain) and the other is
(VH-CH1-hinge-CH2-CH3 plus associated light chain), with
heterodimerization variants, including steric, isotypic, charge
steering, and pI variants, Fc and FcRn variants, ablation variants,
and additional antigen binding domains (with optional linkers)
included in these regions.
[0254] In addition to the heterodimerization variants (e.g. steric
and pI variants) outlined herein, the heavy chain regions may also
contain additional amino acid substitutions, including changes for
altering Fc.gamma.R and FcRn binding as discussed below.
[0255] In addition, some monomers can utilize linkers between the
variant heavy chain constant region and the fusion partner. For the
scFv portion of the "bottle-opener", standard linkers as are known
in the art can be used, or the charged scFv linkers described
herein. In the case where additional fusion partners are made (e.g.
FIGS. 1 and 2), traditional peptide linkers can be used, including
flexible linkers of glycine and serine, or the charged linkers of
FIG. 9. In some cases, the linkers for use as components of the
monomer are different from those defined below for the ADC
constructs, and are in many embodiments not cleavable linkers (such
as those susceptible to proteases), although cleavable linkers may
find use in some embodiments.
[0256] The heterodimerization variants include a number of
different types of variants, including, but not limited to, steric
variants (including charge variants) and pI variants, that can be
optionally and independently combined with any other variants. In
these embodiments, it is important to match "monomer A" with
"monomer B"; that is, if a heterodimeric protein relies on both
steric variants and pI variants, these need to be correctly matched
to each monomer: e.g. the set of steric variants that work (1 set
on monomer A, 1 set on monomer B) is combined with pI variant sets
(1 set on monomer A, 1 set on monomer B), such that the variants on
each monomer are designed to achieve the desired function, keeping
in mind the pI "strandedness" such that steric variants that may
alter pI are put on the appropriate monomer.
[0257] It is important to note that the heterodimerization variants
outlined herein (for example, including but not limited to those
variants shown in FIGS. 3 and 12), can be optionally and
independently combined with any other variants, and on any other
monomer. That is, what is important for the heterodimerization is
that there are "sets" of variants, one set for one monomer and one
set for the other. Whether these are combined from the FIGS. 1 to 1
(e.g. monomer 1 listings can go together) or switched (monomer 1 pI
variants with monomer 2 steric variants) is irrelevant. However, as
noted herein, "strandedness" should be preserved when combinations
are made as outlined above. Furthermore, for the additional Fc
variants (such as for Fc.gamma.R binding, FcRn binding, etc.),
either monomer, or both monomers, can include any of the listed
variants, independently and optionally. In some cases, both
monomers have the additional variants and in some only one monomer
has the additional variants, or they can be combined.
Heterodimerization Variants
[0258] The present invention provides heterodimeric proteins,
including heterodimeric antibodies in a variety of formats, which
utilize heterodimeric variants to allow for heterodimeric formation
and/or purification away from homodimers.
Steric Variants
[0259] In some embodiments, the formation of heterodimers can be
facilitated by the addition of steric variants. That is, by
changing amino acids in each heavy chain, different heavy chains
are more likely to associate to form the heterodimeric structure
than to form homodimers with the same Fc amino acid sequences.
Suitable steric variants are included in FIG. 41A-41B.
[0260] One mechanism is generally referred to in the art as "knobs
and holes", referring to amino acid engineering that creates steric
influences to favor heterodimeric formation and disfavor
homodimeric formation can also optionally be used; this is
sometimes referred to as "knobs and holes", as described in U.S.
Ser. No. 61/596,846, Ridgway et al., Protein Engineering 9(7):617
(1996); Atwell et al., J. Mol. Biol. 1997 270:26; U.S. Pat. No.
8,216,805, all of which are hereby incorporated by reference in
their entirety. The Figures identify a number of "monomer A-monomer
B" pairs that rely on "knobs and holes". In addition, as described
in Merchant et al., Nature Biotech. 16:677 (1998), these "knobs and
hole" mutations can be combined with disulfide bonds to skew
formation to heterodimerization.
[0261] An additional mechanism that finds use in the generation of
heterodimers is sometimes referred to as "electrostatic steering"
as described in Gunasekaran et al., J. Biol. Chem. 285(25):19637
(2010), hereby incorporated by reference in its entirety. This is
sometimes referred to herein as "charge pairs". In this embodiment,
electrostatics are used to skew the formation towards
heterodimerization. As those in the art will appreciate, these may
also have an effect on pI, and thus on purification, and thus could
in some cases also be considered pI variants. However, as these
were generated to force heterodimerization and were not used as
purification tools, they are classified as "steric variants". These
include, but are not limited to, D221E/P228E/L368E paired with
D221R/P228R/K409R (e.g. these are "monomer corresponding sets) and
C220E/P228E/368E paired with C220R/E224R/P228R/K409R.
[0262] Additional monomer A and monomer B variants that can be
combined with other variants, optionally and independently in any
amount, such as pI variants outlined herein or other steric
variants that are shown in FIG. 37 of US 2012/0149876, the figure
and legend and SEQ ID NOs of which are incorporated expressly by
reference herein.
[0263] In some embodiments, the steric variants outlined herein can
be optionally and independently incorporated with any pI variant
(or other variants such as Fc variants, FcRn variants, etc.) into
one or both monomers, and can be independently and optionally
included or excluded from the proteins of the invention.
pI (Isoelectric Point) Variants for Heterodimers
[0264] In general, as will be appreciated by those in the art,
there are two general categories of pI variants: those that
increase the pI of the protein (basic changes) and those that
decrease the pI of the protein (acidic changes). As described
herein, all combinations of these variants can be done: one monomer
may be wild type, or a variant that does not display a
significantly different pI from wild-type, and the other can be
either more basic or more acidic. Alternatively, each monomer is
changed, one to more basic and one to more acidic.
[0265] Combinations of pI variants are shown in the figures.
Heavy Chain pI Changes
[0266] As outlined herein and shown in the figures, PI variants are
shown relative to IgG1, but all isotypes can be altered this way,
as well as isotype hybrids. In the case where the heavy chain
constant domain is from IgG2-4, R133E and R133Q can also be
used.
Antibody Heterodimers Light Chain Variants
[0267] In the case of antibody based heterodimers, e.g. where at
least one of the monomers comprises a light chain in addition to
the heavy chain domain, pI variants can also be made in the light
chain. Amino acid substitutions for lowering the pI of the light
chain include, but are not limited to, K126E, K126Q, K145E, K145Q,
N152D, S156E, K169E, S202E, K207E and adding peptide DEDE at the
c-terminus of the light chain. Changes in this category based on
the constant lambda light chain include one or more substitutions
at R108Q, Q124E, K126Q, N138D, K145T and Q199E. In addition,
increasing the pI of the light chains can also be done.
Isotypic Variants
[0268] In addition, many embodiments of the invention rely on the
"importation" of pI amino acids at particular positions from one
IgG isotype into another, thus reducing or eliminating the
possibility of unwanted immunogenicity being introduced into the
variants. A number of these are shown in FIGS. 10A and 10B. That
is, IgG1 is a common isotype for therapeutic antibodies for a
variety of reasons, including high effector function. However, the
heavy constant region of IgG1 has a higher pI than that of IgG2
(8.10 versus 7.31). By introducing IgG2 residues at particular
positions into the IgG1 backbone, the pI of the resulting monomer
is lowered (or increased) and additionally exhibits longer serum
half-life. For example, IgG1 has a glycine (pI 5.97) at position
137, and IgG2 has a glutamic acid (pI 3.22); importing the glutamic
acid will affect the pI of the resulting protein. As is described
below, a number of amino acid substitutions are generally required
to significant affect the pI of the variant antibody. However, it
should be noted as discussed below that even changes in IgG2
molecules allow for increased serum half-life.
[0269] In other embodiments, non-isotypic amino acid changes are
made, either to reduce the overall charge state of the resulting
protein (e.g. by changing a higher pI amino acid to a lower pI
amino acid), or to allow accommodations in structure for stability,
etc. as is more further described below.
[0270] In addition, by pI engineering both the heavy and light
constant domains, significant changes in each monomer of the
heterodimer can be seen. As discussed herein, having the pIs of the
two monomers differ by at least 0.5 can allow separation by ion
exchange chromatography or isoelectric focusing, or other methods
sensitive to isoelectric point.
Calculating pI
[0271] The pI of each monomer can depend on the pI of the variant
heavy chain constant domain and the pI of the total monomer,
including the variant heavy chain constant domain and the fusion
partner. Thus, in some embodiments, the change in pI is calculated
on the basis of the variant heavy chain constant domain, using the
chart in FIGS. 46A-46C and 47. As discussed herein, which monomer
to engineer is generally decided by the inherent pI of the Fv and
scaffold regions. Alternatively, the pI of each monomer can be
compared.
[0272] Heterodimeric Fc Fusion Proteins
[0273] In addition to heterodimeric antibodies, the invention
provides heterodimeric proteins that comprise a first monomer
comprising a variant Fc region and a first fusion partner and a
second monomer, also comprising a variant Fc region and a second
fusion partner. The variant Fc regions are engineered as herein for
antibodies, and are thus different, and in general the first and
second fusion partners are different as well. In some cases, where
one monomer is antibody based (e.g. either comprising a standard
heavy and light chain or a Fc domain with an scFv) and the other is
an Fc fusion protein, the resulting heterodimeric protein is called
a "fusionbody".
pI Variants that Also Confer Better FcRn In Vivo Binding
[0274] In the case where the pI variant decreases the pI of the
monomer, they can have the added benefit of improving serum
retention in vivo.
[0275] Although still under examination, Fc regions are believed to
have longer half-lives in vivo, because binding to FcRn at pH 6 in
an endosome sequesters the Fc (Ghetie and Ward, 1997 Immunol Today.
18(12): 592-598, entirely incorporated by reference). The endosomal
compartment then recycles the Fc to the cell surface. Once the
compartment opens to the extracellular space, the higher pH,
.about.7.4, induces the release of Fc back into the blood. In mice,
Dall' Acqua et al. showed that Fc mutants with increased FcRn
binding at pH 6 and pH 7.4 actually had reduced serum
concentrations and the same half life as wild-type Fc (Dall' Acqua
et al. 2002, J. Immunol. 169:5171-5180, entirely incorporated by
reference). The increased affinity of Fc for FcRn at pH 7.4 is
thought to forbid the release of the Fc back into the blood.
Therefore, the Fc mutations that will increase Fc's half-life in
vivo will ideally increase FcRn binding at the lower pH while still
allowing release of Fc at higher pH. The amino acid histidine
changes its charge state in the pH range of 6.0 to 7.4. Therefore,
it is not surprising to find His residues at important positions in
the Fc/FcRn complex.
[0276] Recently it has been suggested that antibodies with variable
regions that have lower isoelectric points may also have longer
serum half-lives (Igawa et al., 2010 PEDS. 23(5): 385-392, entirely
incorporated by reference). However, the mechanism of this is still
poorly understood. Moreover, variable regions differ from antibody
to antibody. Constant region variants with reduced pI and extended
half-life would provide a more modular approach to improving the
pharmacokinetic properties of antibodies, as described herein.
[0277] pI variants that find use in this embodiment, as well as
their use for purification optimization, are disclosed in FIG.
20.
Combination of Heterodimeric Variants
[0278] As will be appreciated by those in the art, all of the
recited heterodimerization variants can be optionally and
independently combined in any way, as long as they retain their
"strandedness" or "monomer partition". In addition, all of these
variants can be combined into any of the heterodimerization
formats.
[0279] In the case of pI variants, while embodiments finding
particular use are shown in the Figures, other combinations can be
generated, following the basic rule of altering the pI difference
between two monomers to facilitate purification.
Nucleic Acids of the Invention
[0280] As discussed above regarding methods of making compositions
of the present invention, the invention further provides nucleic
acid compositions encoding the heterodimeric proteins of the
invention. As will be appreciated by those in the art, the nucleic
acid compositions will depend on the format and scaffold of the
heterodimeric protein. Thus, for example, when the format requires
three amino acid sequences, such as for the triple F format (e.g. a
first amino acid monomer comprising an Fc domain and a scFv, a
second amino acid monomer comprising a heavy chain and a light
chain), three nucleic acid sequences can be incorporated into one
or more expression vectors for expression. Similarly, some formats
(e.g. dual scFv formats such as disclosed in FIG. 36M) only two
nucleic acids are needed; again, they can be put into one or two
expression vectors.
Target Antigens
[0281] The heterodimeric proteins of the invention may target
virtually any antigens. The "triple F" format is particularly
beneficial for targeting two (or more) distinct antigens. (As
outlined herein, this targeting can be any combination of
monovalent and divalent binding, depending on the format). Thus the
immunoglobulins herein preferably co-engage two target antigens,
although in some cases, three or four antigens can be monovalently
engaged. Each monomer's specificity can be selected from the lists
below. While the triple F immunoglobulins described herein are
particularly beneficial for targeting distinct antigens, in some
cases it may be beneficial to target only one antigen. That is,
each monomer may have specificity for the same antigen.
[0282] Particular suitable applications of the heterodimeric
proteins herein are co-target pairs for which it is beneficial or
critical to engage each target antigen monovalently. Such antigens
may be, for example, immune receptors that are activated upon
immune complexation. Cellular activation of many immune receptors
occurs only by cross-linking, achieved typically by
antibody/antigen immune complexes, or via effector cell to target
cell engagement. For some immune receptors, activation only upon
engagement with co-engaged target is critical, as nonspecific
cross-linking in a clinical setting can elicit a cytokine storm and
toxicity. Therapeutically, by engaging such antigens monovalently
rather than multivalently, using the immunoglobulins herein, such
activation occurs only in response to cross-linking only in the
microenvironment of the primary target antigen. The ability to
target two different antigens with different valencies is a novel
and useful aspect of the present invention. Examples of target
antigens for which it may be therapeutically beneficial or
necessary to co-engage monovalently include but are not limited to
immune activating receptors such as CD3, Fc.gamma.Rs, toll-like
receptors (TLRs) such as TLR4 and TLR9, cytokine, chemokine,
cytokine receptors, and chemokine receptors. In many embodiments,
one of the antigen binding sites binds to CD3, and in some
embodiments it is the scFv-containing monomer.
[0283] Virtually any antigen may be targeted by the immunoglobulins
herein, including but not limited to proteins, subunits, domains,
motifs, and/or epitopes belonging to the following list of target
antigens, which includes both soluble factors such as cytokines and
membrane-bound factors, including transmembrane receptors: 17-IA,
4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 Adenosine
Receptor, A33, ACE, ACE-2, Activin, Activin A, Activin AB, Activin
B, Activin C, Activin RIA, Activin RIA ALK-2, Activin RIB ALK-4,
Activin RIIA, Activin RIIB, ADAM, ADAM10, ADAM12, ADAM15,
ADAM17/TACE, ADAMS, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, Addressins,
aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha-1-antitrypsin, alpha-V/beta-1
antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, ARC, ART,
Artemin, anti-Id, ASPARTIC, Atrial natriuretic factor, av/b3
integrin, Axl, b2M, B7-1, B7-2, B7-H, B-lymphocyte Stimulator
(BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1,
BCAM, Bcl, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BIM, BLC, BL-CAM,
BLK, BMP, BMP-2 BMP-2a, BMP-3 Osteogenin, BMP-4 BMP-2b, BMP-5,
BMP-6 Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2), BMPR, BMPR-IA
(ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMPs,
b-NGF, BOK, Bombesin, Bone-derived neurotrophic factor, BPDE,
BPDE-DNA, BTC, complement factor 3 (C3), C3a, C4, C5, C5a, C10,
CA125, CAD-8, Calcitonin, cAMP, carcinoembryonic antigen (CEA),
carcinoma-associated antigen, Cathepsin A, Cathepsin B, Cathepsin
C/DPPI, Cathepsin D, Cathepsin E, Cathepsin H, Cathepsin L,
Cathepsin O, Cathepsin S, Cathepsin V, Cathepsin X/Z/P, CBL, CCI,
CCK2, CCL, CCL1, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17,
CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25,
CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9/10,
CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8,
CCR9, CD1, CD2, CD3, CD3E, CD4, CD5, CD6, CD7, CD8, CD10, CD11a,
CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22,
CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33 (p67
proteins), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52,
CD54, CD55, CD56, CD61, CD64, CD66e, CD74, CD80 (B7-1), CD89, CD95,
CD123, CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164,
CEACAM5, CFTR, cGMP, CINC, Clostridium botulinum toxin, Clostridium
perfringens toxin, CKb8-1, CLC, CMV, CMV UL, CNTF, CNTN-1, COX,
C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1, CX3CR1, CXCL,
CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9,
CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR,
CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, cytokeratin
tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, Decay
accelerating factor, des(1-3)-IGF-1 (brain IGF-1), Dhh, digoxin,
DNAM-1, Dnase, Dpp, DPPIV/CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2,
EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, ENA, endothelin receptor,
Enkephalinase, eNOS, Eot, eotaxin1, EpCAM, Ephrin B2/EphB4, EPO,
ERCC, E-selectin, ET-1, Factor IIa, Factor VII, Factor VIIIc,
Factor IX, fibroblast activation protein (FAP), Fas, FcR1, FEN-1,
Ferritin, FGF, FGF-19, FGF-2, FGF3, FGF-8, FGFR, FGFR-3, Fibrin,
FL, FLIP, Flt-3, Flt-4, Follicle stimulating hormone, Fractalkine,
FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250,
Gas 6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1, GDF-3 (Vgr-2), GDF-5
(BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3),
GDF-8 (Myostatin), GDF-9, GDF-15 (MIC-1), GDNF, GDNF, GFAP, GFRa-1,
GFR-alpha1, GFR-alpha2, GFR-alpha3, GITR, Glucagon, Glut 4,
glycoprotein IIb/IIIa (GP IIb/IIIa), GM-CSF, gp130, gp72, GRO,
Growth hormone releasing factor, Hapten (NP-cap or NIP-cap),
HB-EGF, HCC, HCMV gB envelope glycoprotein, HCMV) gH envelope
glycoprotein, HCMV UL, Hemopoietic growth factor (HGF), Hep B
gp120, heparanase, Her2, Her2/neu (ErbB-2), Her3 (ErbB-3), Her4
(ErbB-4), herpes simplex virus (HSV) gB glycoprotein, HSV gD
glycoprotein, HGFA, High molecular weight melanoma-associated
antigen (HMW-MAA), HIV gp120, HIV IIIB gp 120 V3 loop, HLA, HLA-DR,
HM1.24, HMFG PEM, HRG, Hrk, human cardiac myosin, human
cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, I-309,
IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE,
IGF, IGF binding proteins, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1,
IL-1R, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8,
IL-9, IL-10, IL-12, IL-13, IL-15, IL-18, IL-18R, IL-23, interferon
(INF)-alpha, INF-beta, INF-gamma, Inhibin, iNOS, Insulin A-chain,
Insulin B-chain, Insulin-like growth factor 1, integrin alpha2,
integrin alpha3, integrin alpha4, integrin alpha4/beta1, integrin
alpha4/beta7, integrin alpha5 (alphaV), integrin alpha5/beta1,
integrin alpha5/beta3, integrin alpha6, integrin beta1, integrin
beta2, interferon gamma, IP-10, I-TAC, JE, Kallikrein 2, Kallikrein
5, Kallikrein 6, Kallikrein 11, Kallikrein 12, Kallikrein 14,
Kallikrein 15, Kallikrein L1, Kallikrein L2, Kallikrein L3,
Kallikrein L4, KC, KDR, Keratinocyte Growth Factor (KGF), laminin
5, LAMP, LAP, LAP (TGF-1), Latent TGF-1, Latent TGF-1 bp1, LBP,
LDGF, LECT2, Lefty, Lewis-Y antigen, Lewis-Y related antigen,
LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoproteins, LIX, LKN, Lptn,
L-Selectin, LT-a, LT-b, LTB4, LTBP-1, Lung surfactant, Luteinizing
hormone, Lymphotoxin Beta Receptor, Mac-1, MAdCAM, MAG, MAP2, MARC,
MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, METALLOPROTEASES, MGDF
receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-alpha, MK,
MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15,
MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP,
mucin (Muc1), MUC18, Muellerian-inhibitin substance, Mug, MuSK,
NAIP, NAP, NCAD, N-Cadherin, NCA 90, NCAM, NCAM, Neprilysin,
Neurotrophin-3,-4, or -6, Neurturin, Neuronal growth factor (NGF),
NGFR, NGF-beta, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG,
OPN, OSM, OX40L, OX40R, p150, p95, PADPr, Parathyroid hormone,
PARC, PARP, PBR, PBSF, PCAD, P-Cadherin, PCNA, PDGF, PDGF, PDK-1,
PECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental
alkaline phosphatase (PLAP), PIGF, PLP, PP14, Proinsulin,
Prorelaxin, Protein C, PS, PSA, PSCA, prostate specific membrane
antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES,
RANTES, Relaxin A-chain, Relaxin B-chain, renin, respiratory
syncytial virus (RSV) F, RSV Fgp, Ret, Rheumatoid factors, RLIP76,
RPA2, RSK, S100, SCF/KL, SDF-1, SERINE, Serum albumin, sFRP-3, Shh,
SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat,
STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated
glycoprotein-72), TARC, TCA-3, T-cell receptors (e.g., T-cell
receptor alpha/beta), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT,
testicular PLAP-like alkaline phosphatase, TfR, TGF, TGF-alpha,
TGF-beta, TGF-beta Pan Specific, TGF-beta RI (ALK-5), TGF-beta RII,
TGF-beta RIIb, TGF-beta RIII, TGF-beta1, TGF-beta2, TGF-beta3,
TGF-beta4, TGF-beta5, Thrombin, Thymus Ck-1, Thyroid stimulating
hormone, Tie, TIMP, TIQ, Tissue Factor, TMEFF2, Tmpo, TMPRSS2, TNF,
TNF-alpha, TNF-alpha beta, TNF-beta2, TNFc, TNF-RI, TNF-RII,
TNFRSF10A (TRAIL R1 Apo-2, DR4), TNFRSF10B (TRAIL R2 DR5, KILLER,
TRICK-2A, TRICK-B), TNFRSF10C (TRAIL R3 DcR1, LIT, TRID), TNFRSF10D
(TRAIL R4 DcR2, TRUNDD), TNFRSF11A (RANK ODF R, TRANCE R),
TNFRSF11B (OPG OCIF, TR1), TNFRSF12 (TWEAK R FN14), TNFRSF13B
(TACI), TNFRSF13C (BAFF R), TNFRSF14 (HVEM ATAR, HveA, LIGHT R,
TR2), TNFRSF16 (NGFR p75NTR), TNFRSF17 (BCMA), TNFRSF18 (GITR
AITR), TNFRSF19 (TROY TAJ, TRADE), TNFRSF19L (RELT), TNFRSF1A (TNF
RI CD120a, p55-60), TNFRSF1B (TNF RII CD120b, p75-80), TNFRSF26
(TNFRH3), TNFRSF3 (LTbR TNF RIII, TNFC R), TNFRSF4 (OX40 ACT35,
TXGP1 R), TNFRSF5 (CD40 p50), TNFRSF6 (Fas Apo-1, APT1, CD95),
TNFRSF6B (DcR3 M68, TR6), TNFRSF7 (CD27), TNFRSF8 (CD30), TNFRSF9
(4-1BB CD137, ILA), TNFRSF21 (DR6), TNFRSF22 (DcTRAIL R2 TNFRH2),
TNFRST23 (DcTRAIL R1 TNFRH1), TNFRSF25 (DR3 Apo-3, LARD, TR-3,
TRAMP, WSL-1), TNFSF10 (TRAIL Apo-2 Ligand, TL2), TNFSF11
(TRANCE/RANK Ligand ODF, OPG Ligand), TNFSF12 (TWEAK Apo-3 Ligand,
DR3 Ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS, TALL1,
THANK, TNFSF20), TNFSF14 (LIGHT HVEM Ligand, LTg), TNFSF15
(TL1A/VEGI), TNFSF18 (GITR Ligand AITR Ligand, TL6), TNFSF1A
(TNF-.alpha. Conectin, DIF, TNFSF2), TNFSF1B (TNF-b LTa, TNFSF1),
TNFSF3 (LTb TNFC, p33), TNFSF4 (OX40 Ligand gp34, TXGP1), TNFSF5
(CD40 Ligand CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas Ligand
Apo-1 Ligand, APT1 Ligand), TNFSF7 (CD27 Ligand CD70), TNFSF8 (CD30
Ligand CD153), TNFSF9 (4-1BB Ligand CD137 Ligand), TP-1, t-PA, Tpo,
TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transferring receptor,
TRF, Trk, TROP-2, TSG, TSLP, tumor-associated antigen CA 125,
tumor-associated antigen expressing Lewis Y related carbohydrate,
TWEAK, TXB2, Ung, uPAR, uPAR-1, Urokinase, VCAM, VCAM-1, VECAD,
VE-Cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3
(flt-4), VEGI, VIM, Viral antigens, VLA, VLA-1, VLA-4, VNR
integrin, von Willebrands factor, WIF-1, WNT1, WNT2, WNT2B/13,
WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B,
WNT9A, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2,
XCR1, XCR1, XEDAR, XIAP, XPD, and receptors for hormones and growth
factors. To form the bispecific or trispecific antibodies of the
invention, antibodies to any combination of these antigens can be
made; that is, each of these antigens can be optionally and
independently included or excluded from a multispecific antibody
according to the present invention.
[0284] Exemplary antigens that may be targeted specifically by the
immunoglobulins of the invention include but are not limited to:
CD20, CD19, Her2, EGFR, EpCAM, CD3, Fc.gamma.RIIIa (CD16),
Fc.gamma.RIIa (CD32a), Fc.gamma.RIIb (CD32b), Fc.gamma.RI (CD64),
Toll-like receptors (TLRs) such as TLR4 and TLR9, cytokines such as
IL-2, IL-5, IL-13, IL-12, IL-23, and TNF.alpha., cytokine receptors
such as IL-2R, chemokines, chemokine receptors, growth factors such
as VEGF and HGF, and the like. To form the multispecific antibodies
of the invention, antibodies to any combination of these antigens
can be made; that is, each of these antigens can be optionally and
independently included or excluded from a multispecific antibody
according to the present invention.
[0285] The choice of suitable target antigens and co-targets
depends on the desired therapeutic application. Some targets that
have proven especially amenable to antibody therapy are those with
signaling functions. Other therapeutic antibodies exert their
effects by blocking signaling of the receptor by inhibiting the
binding between a receptor and its cognate ligand. Another
mechanism of action of therapeutic antibodies is to cause receptor
down regulation. Other antibodies do not work by signaling through
their target antigen. The choice of co-targets will depend on the
detailed biology underlying the pathology of the indication that is
being treated.
[0286] Monoclonal antibody therapy has emerged as an important
therapeutic modality for cancer (Weiner et al., 2010, Nature
Reviews Immunology 10:317-327; Reichert et al., 2005, Nature
Biotechnology 23[9]:1073-1078; herein expressly incorporated by
reference). For anti-cancer treatment it may be desirable to target
one antigen (antigen-1) whose expression is restricted to the
cancerous cells while co-targeting a second antigen (antigen-2)
that mediates some immunological killing activity. For other
treatments, it may be beneficial to co-target two antigens, for
example two angiogenic factors or two growth factors, that are each
known to play some role in proliferation of the tumor. Exemplary
co-targets for oncology include but are not limited to HGF and
VEGF, IGF-1R and VEGF, Her2 and VEGF, CD19 and CD3, CD20 and CD3,
Her2 and CD3, CD19 and Fc.gamma.RIIIa, CD20 and Fc.gamma.RIIIa,
Her2 and Fc.gamma.RIIIa. An immunoglobulin of the invention may be
capable of binding VEGF and phosphatidylserine; VEGF and ErbB3;
VEGF and PLGF; VEGF and ROBO4; VEGF and BSG2; VEGF and CDCP1; VEGF
and ANPEP; VEGF and c-MET; HER-2 and ERB3; HER-2 and BSG2; HER-2
and CDCP1; HER-2 and ANPEP; EGFR and CD64; EGFR and BSG2; EGFR and
CDCP1; EGFR and ANPEP; IGF1R and PDGFR; IGF1R and VEGF; IGF1R and
CD20; CD20 and CD74; CD20 and CD30; CD20 and DR4; CD20 and VEGFR2;
CD20 and CD52; CD20 and CD4; HGF and c-MET; HGF and NRP1; HGF and
phosphatidylserine; ErbB3 and IGF1R; ErbB3 and IGF1,2; c-Met and
Her-2; c-Met and NRP1; c-Met and IGF1R; IGF1,2 and PDGFR; IGF1,2
and CD20; IGF1,2 and IGF1R; IGF2 and EGFR; IGF2 and HER2; IGF2 and
CD20; IGF2 and VEGF; IGF2 and IGF1R; IGF1 and IGF2; PDGFRa and
VEGFR2; PDGFRa and PLGF; PDGFRa and VEGF; PDGFRa and c-Met; PDGFRa
and EGFR; PDGFRb and VEGFR2; PDGFRb and c-Met; PDGFRb and EGFR; RON
and c-Met; RON and MTSP1; RON and MSP; RON and CDCP1; VGFR1 and
PLGF; VGFR1 and RON; VGFR1 and EGFR; VEGFR2 and PLGF; VEGFR2 and
NRP1; VEGFR2 and RON; VEGFR2 and DLL4; VEGFR2 and EGFR; VEGFR2 and
ROBO4; VEGFR2 and CD55; LPA and S1P; EPHB2 and RON; CTLA4 and VEGF;
CD3 and EPCAM; CD40 and IL6; CD40 and IGF; CD40 and CD56; CD40 and
CD70; CD40 and VEGFR1; CD40 and DR5; CD40 and DR4; CD40 and APRIL;
CD40 and BCMA; CD40 and RANKL; CD28 and MAPG; CD80 and CD40; CD80
and CD30; CD80 and CD33; CD80 and CD74; CD80 and CD2; CD80 and CD3;
CD80 and CD19; CD80 and CD4; CD80 and CD52; CD80 and VEGF; CD80 and
DR5; CD80 and VEGFR2; CD22 and CD20; CD22 and CD80; CD22 and CD40;
CD22 and CD23; CD22 and CD33; CD22 and CD74; CD22 and CD19; CD22
and DR5; CD22 and DR4; CD22 and VEGF; CD22 and CD52; CD30 and CD20;
CD30 and CD22; CD30 and CD23; CD30 and CD40; CD30 and VEGF; CD30
and CD74; CD30 and CD19; CD30 and DR5; CD30 and DR4; CD30 and
VEGFR2; CD30 and CD52; CD30 and CD4; CD138 and RANKL; CD33 and
FTL3; CD33 and VEGF; CD33 and VEGFR2; CD33 and CD44; CD33 and DR4;
CD33 and DR5; DR4 and CD137; DR4 and IGF1,2; DR4 and IGF1R; DR4 and
DR5; DR5 and CD40; DR5 and CD137; DR5 and CD20; DR5 and EGFR; DR5
and IGF1,2; DR5 and IGFR, DR5 and HER-2, and EGFR and DLL4. Other
target combinations include one or more members of the
EGF/erb-2/erb-3 family.
[0287] Other targets (one or more) involved in oncological diseases
that the immunoglobulins herein may bind include, but are not
limited to those selected from the group consisting of: CD52, CD20,
CD19, CD3, CD4, CD8, BMP6, IL12A, IL1A, IL1B, 1L2, IL24, INHA, TNF,
TNFSF10, BMP6, EGF, FGF1, FGF10, FGF11, FGF12, FGF13, FGF14, FGF16,
FGF17, FGF18, FGF19, FGF2, FGF20, FGF21, FGF22, FGF23, FGF3, FGF4,
FGF5, FGF6, FGF7, FGF8, FGF9, GRP, IGF1, IGF2, IL12A, IL1A, IL1B,
1L2, INHA, TGFA, TGFB1, TGFB2, TGFB3, VEGF, CDK2, FGF10, FGF18,
FGF2, FGF4, FGF7, IGF1R, IL2, BCL2, CD164, CDKN1A, CDKN1B, CDKN1C,
CDKN2A, CDKN2B, CDKN2C, CDKN3, GNRH1, IGFBP6, IL1A, IL1B, ODZ1,
PAWR, PLG, TGFB1I1, AR, BRCA1, CDK3, CDK4, CDK5, CDK6, CDK7, CDK9,
E2F1, EGFR, ENO1, ERBB2, ESR1, ESR2, IGFBP3, IGFBP6, IL2, INSL4,
MYC, NOX5, NR6A1, PAP, PCNA, PRKCQ, PRKD1, PRL, TP53, FGF22, FGF23,
FGF9, IGFBP3, IL2, INHA, KLK6, TP53, CHGB, GNRH1, IGF1, IGF2, INHA,
INSL3, INSL4, PRL, KLK6, SHBG, NR1D1, NR1H3, NR113, NR2F6, NR4A3,
ESR1, ESR2, NROB1, NROB2, NR1D2, NR1H2, NR1H4, NR112, NR2C1, NR2C2,
NR2E1, NR2E3, NR2F1, NR2F2, NR3C1, NR3C2, NR4A1, NR4A2, NR5A1,
NR5A2, NR6 p1, PGR, RARB, FGF1, FGF2, FGF6, KLK3, KRT1, APOC1,
BRCA1, CHGA, CHGB, CLU, COL1A1, COL6A1, EGF, ERBB2, ERK8, FGF1,
FGF10, FGF11, FGF13, FGF14, FGF16, FGF17, FGF18, FGF2, FGF20,
FGF21, FGF22, FGF23, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9,
GNRH1, IGF1, IGF2, IGFBP3, IGFBP6, IL12A, IL1A, IL1B, 1L2, IL24,
INHA, INSL3, INSL4, KLK10, KLK12, KLK13, KLK14, KLK15, KLK3, KLK4,
KLK5, KLK6, KLK9, MMP2, MMP9, MSMB, NTN4, ODZ1, PAP, PLAU, PRL,
PSAP, SERPINA3, SHBG, TGFA, TIMP3, CD44, CDH1, CDH10, CDH19, CDH20,
CDH7, CDH9, CDH1, CDH10, CDH13, CDH18, CDH19, CDH2O, CDH7, CDH8,
CDH9, ROBO2, CD44, ILK, ITGA1, APC, CD164, COL6A1, MTSS1, PAP,
TGFB111, AGR2, AIG1, AKAP1, AKAP2, CANT1, CAV1, CDH12, CLDN3, CLN3,
CYB5, CYC1, DAB21P, DES, DNCL1, ELAC2, ENO2, ENO3, FASN, FLJ12584,
FLJ25530, GAGEB1, GAGEC1, GGT1, GSTP1, HIP1, HUMCYT2A, IL29, K6HF,
KAI1, KRT2A, MIB1, PART1, PATE, PCA3, PIAS2, PIK3CG, PPID, PR1,
PSCA, SLC2A2, SLC33 pI, SLC43 pI, STEAP, STEAP2, TPM1, TPM2, TRPC6,
ANGPT1, ANGPT2, ANPEP, ECGF1, EREG, FGF1, FGF2, FIGF, FLT1, JAG1,
KDR, LAMA5, NRP1, NRP2, PGF, PLXDC1, STAB 1, VEGF, VEGFC, ANGPTL3,
BAI1, COL4A3, IL8, LAMA5, NRP1, NRP2, STAB 1, ANGPTL4, PECAM1, PF4,
PROK2, SERPINF1, TNFAIP2, CCL11, CCL2, CXCL1, CXCL10, CXCL3, CXCL5,
CXCL6, CXCL9, IFNA1, IFNB1, IFNG, IL1B, IL6, MDK, EDG1, EFNA1,
EFNA3, EFNB2, EGF, EPHB4, FGFR3, HGF, IGF1, ITGB3, PDGFA, TEK,
TGFA, TGFB1, TGFB2, TGFBR1, CCL2, CDH5, COL1A1, EDG1, ENG, ITGAV,
ITGB3, THBS1, THBS2, BAD, BAG1, BCL2, CCNA1, CCNA2, CCND1, CCNE1,
CCNE2, CDH1 (E-cadherin), CDKN1B (p27Kip1), CDKN2A (p161NK4a),
COL6A1, CTNNB1 (b-catenin), CTSB (cathepsin B), ERBB2 (Her-2),
ESR1, ESR2, F3 (TF), FOSL1 (FRA-1), GATA3, GSN (Gelsolin), IGFBP2,
IL2RA, IL6, IL6R, IL6ST (glycoprotein 130), ITGA6 (a6 integrin),
JUN, KLK5, KRT19, MAP2K7 (c-Jun), MKI67 (Ki-67), NGFB (GF), NGFR,
NME1 (M23A), PGR, PLAU (uPA), PTEN, SERPINB5 (maspin), SERPINE1
(PAI-1), TGFA, THBS1 (thrombospondin-1), TIE (Tie-1), TNFRSF6
(Fas), TNFSF6 (FasL), TOP2A (topoisomerase Iia), TP53, AZGP1
(zinc-a-glycoprotein), BPAG1 (plectin), CDKN1A (p21Wap1/Cip1),
CLDN7 (claudin-7), CLU (clusterin), ERBB2 (Her-2), FGF1, FLRT1
(fibronectin), GABRP (GABAa), GNAS1, ID2, ITGA6 (a6 integrin),
ITGB4 (b 4 integrin), KLF5 (GC Box BP), KRT19 (Keratin 19), KRTHB6
(hair-specific type II keratin), MACMARCKS, MT3
(metallothionectin-III), MUC1 (mucin), PTGS2 (COX-2), RAC2
(p21Rac2), S100A2, SCGB1D2 (lipophilin B), SCGB2A1 (mammaglobin 2),
SCGB2A2 (mammaglobin 1), SPRR1B (Spr1), THBS1, THBS2, THBS4, and
TNFAIP2 (B94), RON, c-Met, CD64, DLL4, PLGF, CTLA4,
phophatidylserine, ROBO4, CD80, CD22, CD40, CD23, CD28, CD80, CD55,
CD38, CD70, CD74, CD30, CD138, CD56, CD33, CD2, CD137, DR4, DR5,
RANKL, VEGFR2, PDGFR, VEGFR1, MTSP1, MSP, EPHB2, EPHA1, EPHA2,
EpCAM, PGE2, NKG2D, LPA, SIP, APRIL, BCMA, MAPG, FLT3, PDGFR alpha,
PDGFR beta, ROR1, PSMA, PSCA, SCD1, and CD59. To form the
bispecific or trispecific antibodies of the invention, antibodies
to any combination of these antigens can be made; that is, each of
these antigens can be optionally and independently included or
excluded from a multispecific antibody according to the present
invention.
[0288] Monoclonal antibody therapy has become an important
therapeutic modality for treating autoimmune and inflammatory
disorders (Chan & Carter, 2010, Nature Reviews Immunology
10:301-316; Reichert et al., 2005, Nature Biotechnology
23[9]:1073-1078; herein expressly incorporated by reference). Many
proteins have been implicated in general autoimmune and
inflammatory responses, and thus may be targeted by the
immunoglobulins of the invention. Autoimmune and inflammatory
targets include but are not limited to C5, CCL1 (1-309), CCL11
(eotaxin), CCL13 (mcp-4), CCL15 (MIP-1d), CCL16 (HCC-4), CCL17
(TARC), CCL18 (PARC), CCL19, CCL2 (mcp-1), CCL20 (MIP-3a), CCL21
(MIP-2), CCL23 (MPIF-1), CCL24 (MPIF-2/eotaxin-2), CCL25 (TECK),
CCL26, CCL3 (MIP-1a), CCL4 (MIP-1b), CCL5 (RANTES), CCL7 (mcp-3),
CCL8 (mcp-2), CXCL1, CXCL10 (IP-10), CXCL11 (1-TAC/IP-9), CXCL12
(SDF1), CXCL13, CXCL14, CXCL2, CXCL3, CXCL5 (ENA-78/LIX), CXCL6
(GCP-2), CXCL9, 1L13, IL8, CCL13 (mcp-4), CCR1, CCR2, CCR3, CCR4,
CCR5, CCR6, CCR7, CCR8, CCR9, CX3CR1, IL8RA, XCR1 (CCXCR1), IFNA2,
IL10, 1L13, IL17C, IL1A, IL1B, IL1F10, IL1F5, IL1F6, IL1F7, IL1F8,
IL1F9, IL22, IL5, IL8, IL9, LTA, LTB, MIF, SCYE1 (endothelial
Monocyte-activating cytokine), SPP1, TNF, TNFSF5, IFNA2, IL10RA,
IL10RB, IL13, IL13RA1, IL5RA, IL9, IL9R, ABCF1, BCL6, C3, C4A,
CEBPB, CRP, ICEBERG, IL1R1, IL1RN, IL8RB, LTB4R, TOLLIP, FADD,
IRAK1, IRAK2, MYD88, NCK2, TNFAIP3, TRADD, TRAF1, TRAF2, TRAF3,
TRAF4, TRAF5, TRAF6, ACVR1, ACVR1B, ACVR2, ACVR2B, ACVRL1, CD28,
CD3E, CD3G, CD3Z, CD69, CD80, CD86, CNR1, CTLA4, CYSLTR1, FCER1A,
FCER2, FCGR3A, GPR44, HAVCR2, OPRD1, P2RX7, TLR2, TLR3, TLR4, TLR5,
TLR6, TLR7, TLR8, TLR9, TLR10, BLR1, CCL1, CCL2, CCL3, CCL4, CCL5,
CCL7, CCL8, CCL11, CCL13, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20,
CCL21, CCL22, CCL23, CCL24, CCL25, CCR1, CCR2, CCR3, CCR4, CCR5,
CCR6, CCR7, CCR8, CCR9, CX3CL1, CX3CR1, CXCL1, CXCL2, CXCL3, CXCL5,
CXCL6, CXCL10, CXCL11, CXCL12, CXCL13, CXCR4, GPR2, SCYE1, SDF2,
XCL1, XCL2, XCR1, AMH, AMHR2, BMPR1A, BMPR1B, BMPR2, C19orf10
(IL27w), CER1, CSF1, CSF2, CSF3, DKFZp451J0118, FGF2, GFI1, IFNA1,
IFNB1, IFNG, IGF1, IL1A, IL1B, IL1R1, IL1R2, IL2, IL2RA, IL2RB,
IL2RG, IL3, IL4, IL4R, IL5, IL5RA, IL6, IL6R, IL6ST, IL7, IL8,
IL8RA, IL8RB, IL9, IL9R, IL10, MORA, IL10RB, IL11, IL12RA, IL12A,
IL12B, IL12RB1, IL12RB2, 1L13, IL13RA1, IL13RA2, 1L15, IL15RA,
IL16, 1L17, IL17R, IL18, IL18R1, 1L19, IL20, KITLG, LEP, LTA, LTB,
LTB4R, LTB4R2, LTBR, MIF, NPPB, PDGFB, TBX21, TDGF1, TGFA, TGFB1,
TGFB111, TGFB2, TGFB3, TGFB1, TGFBR1, TGFBR2, TGFBR3, TH1L, TNF,
TNFRSF1A, TNFRSF1B, TNFRSF7, TNFRSF8, TNFRSF9, TNFRSF11A, TNFRSF21,
TNFSF4, TNFSF5, TNFSF6, TNFSF11, VEGF, ZFPM2, and RNF110 (ZNF144).
To form the bispecific or trispecific antibodies of the invention,
antibodies to any combination of these antigens can be made; that
is, each of these antigens can be optionally and independently
included or excluded from a multispecific antibody according to the
present invention.
[0289] Exemplary co-targets for autoimmune and inflammatory
disorders include but are not limited to IL-1 and TNFalpha, IL-6
and TNFalpha, IL-6 and IL-1, IgE and IL-13, IL-1 and IL-13, IL-4
and IL-13, IL-5 and IL-13, IL-9 and IL-13, CD19 and Fc.gamma.RIIb,
and CD79 and Fc.gamma.RIIb.
[0290] Immunoglobulins of the invention with specificity for the
following pairs of targets to treat inflammatory disease are
contemplated: TNF and IL-17A; TNF and RANKL; TNF and VEGF; TNF and
SOST; TNF and DKK; TNF and alphaVbeta3; TNF and NGF; TNF and
IL-23p19; TNF and IL-6; TNF and SOST; TNF and IL-6R; TNF and CD-20;
IgE and IL-13; IL-13 and IL23p19; IgE and IL-4; IgE and IL-9; IgE
and IL-9; IgE and IL-13; IL-13 and IL-9; IL-13 and IL-4; IL-13 and
IL-9; IL-13 and IL-9; IL-13 and IL-4; IL-13 and IL-23p19; IL-13 and
IL-9; IL-6R and VEGF; IL-6R and IL-17A; IL-6R and RANKL; IL-17A and
IL-1beta; IL-1beta and RANKL; IL-1beta and VEGF; RANKL and CD-20;
IL-1alpha and IL-1beta; IL-1alpha and IL-1beta.
[0291] Pairs of targets that the immunoglobulins described herein
can bind and be useful to treat asthma may be determined. In an
embodiment, such targets include, but are not limited to, IL-13 and
IL-1beta, since IL-1beta is also implicated in inflammatory
response in asthma; IL-13 and cytokines and chemokines that are
involved in inflammation, such as IL-13 and IL-9; IL-13 and IL-4;
IL-13 and IL-5; IL-13 and IL-25; IL-13 and TARC; IL-13 and MDC;
IL-13 and MIF; IL-13 and TGF-.beta.; IL-13 and LHR agonist; IL-13
and CL25; IL-13 and SPRR2a; IL-13 and SPRR2b; and IL-13 and ADAMS.
The immunoglobulins herein may have specificity for one or more
targets involved in asthma selected from the group consisting of
CSF1 (MCSF), CSF2 (GM-CSF), CSF3 (GCSF), FGF2, IFNA1, IFNB1, IFNG,
histamine and histamine receptors, IL1A, IL1B, IL2, IL3, IL4, IL5,
IL6, IL7, IL8, IL9, IL10, IL11, IL12A, IL12B, IL13, IL14, IL15,
IL16, IL17, IL18, IL19, KITLG, PDGFB, IL2RA, IL4R, IL5RA, IL8RA,
IL8RB, IL12RB1, IL12RB2, IL13RA1, IL13RA2, IL18R1, TSLP, CCLi,
CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL13, CCL17, CCL18, CCL19,
CCL20, CCL22, CCL24, CX3CL1, CXCL1, CXCL2, CXCL3, XCLi, CCR2, CCR3,
CCR4, CCR5, CCR6, CCR7, CCR8, CX3CR1, GPR2, XCR1, FOS, GATA3, JAK1,
JAK3, STATE, TBX21, TGFB1, TNF, TNFSF6, YY1, CYSLTR1, FCER1A,
FCER2, LTB4R, TB4R2, LTBR, and Chitinase. To form the bispecific or
trispecific antibodies of the invention, antibodies to any
combination of these antigens can be made; that is, each of these
antigens can be optionally and independently included or excluded
from a multispecific antibody according to the present
invention.
[0292] Pairs of targets involved in rheumatoid arthritis (RA) may
be co-targeted by the invention, including but not limited to TNF
and IL-18; TNF and IL-12; TNF and IL-23; TNF and IL-1beta; TNF and
MIF; TNF and IL-17; and TNF and IL-15.
[0293] Antigens that may be targeted in order to treat systemic
lupus erythematosus (SLE) by the immunoglobulins herein include but
are not limited to CD-20, CD-22, CD-19, CD28, CD4, CD80, HLA-DRA,
IL10, IL2, IL4, TNFRSF5, TNFRSF6, TNFSF5, TNFSF6, BLR1, HDAC4,
HDAC5, HDAC7A, HDAC9, ICOSL, IGBP1, MS4A1, RGSI, SLA2, CD81, IFNB1,
IL10, TNFRSF5, TNFRSF7, TNFSF5, AICDA, BLNK, GALNAC4S-6ST, HDAC4,
HDAC5, HDAC7A, HDAC9, IL10, IL11, IL4, INHA, INHBA, KLF6, TNFRSF7,
CD28, CD38, CD69, CD80, CD83, CD86, DPP4, FCER2, IL2RA, TNFRSF8,
TNFSF7, CD24, CD37, CD40, CD72, CD74, CD79A, CD79B, CR2, ILIR2,
ITGA2, ITGA3, MS4A1, ST6GALI, CDIC, CHSTIO, HLA-A, HLA-DRA, and
NT5E.; CTLA4, B7.1, B7.2, BlyS, BAFF, C5, IL-4, IL-6, IL-10,
IFN-.alpha., and TNF-.alpha.. To form the bispecific or trispecific
antibodies of the invention, antibodies to any combination of these
antigens can be made; that is, each of these antigens can be
optionally and independently included or excluded from a
multispecific antibody according to the present invention.
[0294] The immunoglobulins herein may target antigens for the
treatment of multiple sclerosis (MS), including but not limited to
IL-12, TWEAK, IL-23, CXCL13, CD40, CD40L, IL-18, VEGF, VLA-4, TNF,
CD45RB, CD200, IFNgamma, GM-CSF, FGF, C5, CD52, and CCR2. An
embodiment includes co-engagement of anti-IL-12 and TWEAK for the
treatment of MS.
[0295] One aspect of the invention pertains to immunoglobulins
capable of binding one or more targets involved in sepsis, in an
embodiment two targets, selected from the group consisting TNF,
IL-1, MIF, IL-6, IL-8, IL-18, IL-12, IL-23, FasL, LPS, Toll-like
receptors, TLR-4, tissue factor, MIP-2, ADORA2A, CASP1, CASP4,
IL-10, IL-1B, NFKB1, PROC, TNFRSFIA, CSF3, CCR3, ILIRN, MIF, NFKB1,
PTAFR, TLR2, TLR4, GPR44, HMOX1, midkine, IRAK1, NFKB2, SERPINA1,
SERPINE1, and TREM1. To form the bispecific or trispecific
antibodies of the invention, antibodies to any combination of these
antigens can be made; that is, each of these antigens can be
optionally and independently included or excluded from a
multispecific antibody according to the present invention.
[0296] In some cases, immunoglobulins herein may be directed
against antigens for the treatment of infectious diseases.
Antigen Binding Domains
[0297] As will be appreciated by those in the art, there are two
basic types of antigen binding domains, those that resemble
antibody antigen binding domains (e.g. comprising a set of 6 CDRs)
and those that can be ligands or receptors, for example, that bind
to targets without the use of CDRs.
Modified Antibodies
[0298] In addition to the modifications outlined above, other
modifications can be made. For example, the molecules may be
stabilized by the incorporation of disulphide bridges linking the
VH and VL domains (Reiter et al., 1996, Nature Biotech.
14:1239-1245, entirely incorporated by reference). In addition,
there are a variety of covalent modifications of antibodies that
can be made as outlined below.
[0299] Covalent modifications of antibodies are included within the
scope of this invention, and are generally, but not always, done
post-translationally. For example, several types of covalent
modifications of the antibody are introduced into the molecule by
reacting specific amino acid residues of the antibody with an
organic derivatizing agent that is capable of reacting with
selected side chains or the N- or C-terminal residues.
[0300] Cysteinyl residues most commonly are reacted with
.alpha.-haloacetates (and corresponding amines), such as
chloroacetic acid or chloroacetamide, to give carboxymethyl or
carboxyamidomethyl derivatives. Cysteinyl residues may also be
derivatized by reaction with bromotrifluoroacetone,
.alpha.-bromo-.beta.-(5-imidozoyl)propionic acid, chloroacetyl
phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl
2-pyridyl disulfide, p-chloromercuribenzoate,
2-chloromercuri-4-nitrophenol, or
chloro-7-nitrobenzo-2-oxa-1,3-diazole and the like.
[0301] In addition, modifications at cysteines are particularly
useful in antibody-drug conjugate (ADC) applications, further
described below. In some embodiments, the constant region of the
antibodies can be engineered to contain one or more cysteines that
are particularly "thiol reactive", so as to allow more specific and
controlled placement of the drug moiety. See for example U.S. Pat.
No. 7,521,541, incorporated by reference in its entirety
herein.
[0302] Histidyl residues are derivatized by reaction with
diethylpyrocarbonate at pH 5.5-7.0 because this agent is relatively
specific for the histidyl side chain. Para-bromophenacyl bromide
also is useful; the reaction is preferably performed in 0.1M sodium
cacodylate at pH 6.0.
[0303] Lysinyl and amino terminal residues are reacted with
succinic or other carboxylic acid anhydrides. Derivatization with
these agents has the effect of reversing the charge of the lysinyl
residues. Other suitable reagents for derivatizing
alpha-amino-containing residues include imidoesters such as methyl
picolinimidate; pyridoxal phosphate; pyridoxal; chloroborohydride;
trinitrobenzenesulfonic acid; O-methylisourea; 2,4-pentanedione;
and transaminase-catalyzed reaction with glyoxylate.
[0304] Arginyl residues are modified by reaction with one or
several conventional reagents, among them phenylglyoxal,
2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin.
Derivatization of arginine residues requires that the reaction be
performed in alkaline conditions because of the high pKa of the
guanidine functional group. Furthermore, these reagents may react
with the groups of lysine as well as the arginine epsilon-amino
group.
[0305] The specific modification of tyrosyl residues may be made,
with particular interest in introducing spectral labels into
tyrosyl residues by reaction with aromatic diazonium compounds or
tetranitromethane. Most commonly, N-acetylimidizole and
tetranitromethane are used to form O-acetyl tyrosyl species and
3-nitro derivatives, respectively. Tyrosyl residues are iodinated
using 125I or 131I to prepare labeled proteins for use in
radioimmunoassay, the chloramine T method described above being
suitable.
[0306] Carboxyl side groups (aspartyl or glutamyl) are selectively
modified by reaction with carbodiimides (R'--N.dbd.C.dbd.N--R'),
where R and R' are optionally different alkyl groups, such as
1-cyclohexyl-3-(2-morpholinyl-4-ethyl) carbodiimide or
1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore,
aspartyl and glutamyl residues are converted to asparaginyl and
glutaminyl residues by reaction with ammonium ions.
[0307] Derivatization with bifunctional agents is useful for
crosslinking antibodies to a water-insoluble support matrix or
surface for use in a variety of methods, in addition to methods
described below. Commonly used crosslinking agents include, e.g.,
1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,
N-hydroxysuccinimide esters, for example, esters with
4-azidosalicylic acid, homobifunctional imidoesters, including
disuccinimidyl esters such as 3,3'-dithiobis
(succinimidylpropionate), and bifunctional maleimides such as
bis-N-maleimido-1,8-octane. Derivatizing agents such as
methyl-3-[(p-azidophenyl)dithio]propioimidate yield
photoactivatable intermediates that are capable of forming
crosslinks in the presence of light. Alternatively, reactive
water-insoluble matrices such as cynomolgusogen bromide-activated
carbohydrates and the reactive substrates described in U.S. Pat.
Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and
4,330,440, all entirely incorporated by reference, are employed for
protein immobilization.
[0308] Glutaminyl and asparaginyl residues are frequently
deamidated to the corresponding glutamyl and aspartyl residues,
respectively. Alternatively, these residues are deamidated under
mildly acidic conditions. Either form of these residues falls
within the scope of this invention.
[0309] Other modifications include hydroxylation of proline and
lysine, phosphorylation of hydroxyl groups of seryl or threonyl
residues, methylation of the .alpha.-amino groups of lysine,
arginine, and histidine side chains (T. E. Creighton, Proteins:
Structure and Molecular Properties, W. H. Freeman & Co., San
Francisco, pp. 79-86 [1983], entirely incorporated by reference),
acetylation of the N-terminal amine, and amidation of any
C-terminal carboxyl group.
[0310] In addition, as will be appreciated by those in the art,
labels (including fluorescent, enzymatic, magnetic, radioactive,
etc. can all be added to the antibodies (as well as the other
compositions of the invention).
Glycosylation
[0311] Another type of covalent modification is alterations in
glycosylation. In another embodiment, the antibodies disclosed
herein can be modified to include one or more engineered
glycoforms. By "engineered glycoform" as used herein is meant a
carbohydrate composition that is covalently attached to the
antibody, wherein said carbohydrate composition differs chemically
from that of a parent antibody. Engineered glycoforms may be useful
for a variety of purposes, including but not limited to enhancing
or reducing effector function. A preferred form of engineered
glycoform is afucosylation, which has been shown to be correlated
to an increase in ADCC function, presumably through tighter binding
to the Fc.gamma.RIIIa receptor. In this context, "afucosylation"
means that the majority of the antibody produced in the host cells
is substantially devoid of fucose, e.g. 90-95-98% of the generated
antibodies do not have appreciable fucose as a component of the
carbohydrate moiety of the antibody (generally attached at N297 in
the Fc region). Defined functionally, afucosylated antibodies
generally exhibit at least a 50% or higher affinity to the
Fc.gamma.RIIIa receptor.
[0312] Engineered glycoforms may be generated by a variety of
methods known in the art (Uma a et al., 1999, Nat Biotechnol
17:176-180; Davies et al., 2001, Biotechnol Bioeng 74:288-294;
Shields et al., 2002, J Biol Chem 277:26733-26740; Shinkawa et al.,
2003, J Biol Chem 278:3466-3473; U.S. Pat. No. 6,602,684; U.S. Ser.
No. 10/277,370; U.S. Ser. No. 10/113,929; PCT WO 00/61739A1; PCT WO
01/29246A1; PCT WO 02/31140A1; PCT WO 02/30954A1, all entirely
incorporated by reference; (Potelligent.RTM. technology [Biowa,
Inc., Princeton, N.J.]; GlycoMAb.RTM. glycosylation engineering
technology [Glycart Biotechnology AG, Zurich, Switzerland]). Many
of these techniques are based on controlling the level of
fucosylated and/or bisecting oligosaccharides that are covalently
attached to the Fc region, for example by expressing an IgG in
various organisms or cell lines, engineered or otherwise (for
example Lec-13 CHO cells or rat hybridoma YB2/0 cells, by
regulating enzymes involved in the glycosylation pathway (for
example FUT8 [.alpha.1,6-fucosyltranserase] and/or
.beta.1-4-N-acetylglucosaminyltransferase III [GnTIII]), or by
modifying carbohydrate(s) after the IgG has been expressed. For
example, the "sugar engineered antibody" or "SEA technology" of
Seattle Genetics functions by adding modified saccharides that
inhibit fucosylation during production; see for example
20090317869, hereby incorporated by reference in its entirety.
Engineered glycoform typically refers to the different carbohydrate
or oligosaccharide; thus an antibody can include an engineered
glycoform.
[0313] Alternatively, engineered glycoform may refer to the IgG
variant that comprises the different carbohydrate or
oligosaccharide. As is known in the art, glycosylation patterns can
depend on both the sequence of the protein (e.g., the presence or
absence of particular glycosylation amino acid residues, discussed
below), or the host cell or organism in which the protein is
produced. Particular expression systems are discussed below.
[0314] Glycosylation of polypeptides is typically either N-linked
or O-linked. N-linked refers to the attachment of the carbohydrate
moiety to the side chain of an asparagine residue. The tri-peptide
sequences asparagine-X-serine and asparagine-X-threonine, where X
is any amino acid except proline, are the recognition sequences for
enzymatic attachment of the carbohydrate moiety to the asparagine
side chain. Thus, the presence of either of these tri-peptide
sequences in a polypeptide creates a potential glycosylation site.
O-linked glycosylation refers to the attachment of one of the
sugars N-acetylgalactosamine, galactose, or xylose, to a
hydroxyamino acid, most commonly serine or threonine, although
5-hydroxyproline or 5-hydroxylysine may also be used.
[0315] Addition of glycosylation sites to the antibody is
conveniently accomplished by altering the amino acid sequence such
that it contains one or more of the above-described tri-peptide
sequences (for N-linked glycosylation sites). The alteration may
also be made by the addition of, or substitution by, one or more
serine or threonine residues to the starting sequence (for O-linked
glycosylation sites). For ease, the antibody amino acid sequence is
preferably altered through changes at the DNA level, particularly
by mutating the DNA encoding the target polypeptide at preselected
bases such that codons are generated that will translate into the
desired amino acids.
[0316] Another means of increasing the number of carbohydrate
moieties on the antibody is by chemical or enzymatic coupling of
glycosides to the protein. These procedures are advantageous in
that they do not require production of the protein in a host cell
that has glycosylation capabilities for N- and O-linked
glycosylation. Depending on the coupling mode used, the sugar(s)
may be attached to (a) arginine and histidine, (b) free carboxyl
groups, (c) free sulfhydryl groups such as those of cysteine, (d)
free hydroxyl groups such as those of serine, threonine, or
hydroxyproline, (e) aromatic residues such as those of
phenylalanine, tyrosine, or tryptophan, or (f) the amide group of
glutamine. These methods are described in WO 87/05330 and in Aplin
and Wriston, 1981, CRC Crit. Rev. Biochem., pp. 259-306, both
entirely incorporated by reference.
[0317] Removal of carbohydrate moieties present on the starting
antibody (e.g. post-translationally) may be accomplished chemically
or enzymatically. Chemical deglycosylation requires exposure of the
protein to the compound trifluoromethanesulfonic acid, or an
equivalent compound. This treatment results in the cleavage of most
or all sugars except the linking sugar (N-acetylglucosamine or
N-acetylgalactosamine), while leaving the polypeptide intact.
Chemical deglycosylation is described by Hakimuddin et al., 1987,
Arch. Biochem. Biophys. 259:52 and by Edge et al., 1981, Anal.
Biochem. 118:131, both entirely incorporated by reference.
Enzymatic cleavage of carbohydrate moieties on polypeptides can be
achieved by the use of a variety of endo- and exo-glycosidases as
described by Thotakura et al., 1987, Meth. Enzymol. 138:350,
entirely incorporated by reference. Glycosylation at potential
glycosylation sites may be prevented by the use of the compound
tunicamycin as described by Duskin et al., 1982, J. Biol. Chem.
257:3105, entirely incorporated by reference. Tunicamycin blocks
the formation of protein-N-glycoside linkages.
[0318] Another type of covalent modification of the antibody
comprises linking the antibody to various nonproteinaceous
polymers, including, but not limited to, various polyols such as
polyethylene glycol, polypropylene glycol or polyoxyalkylenes, in
the manner set forth in, for example, 2005-2006 PEG Catalog from
Nektar Therapeutics (available at the Nektar website) U.S. Pat.
Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192 or
4,179,337, all entirely incorporated by reference. In addition, as
is known in the art, amino acid substitutions may be made in
various positions within the antibody to facilitate the addition of
polymers such as PEG. See for example, U.S. Publication No.
2005/0114037A1, entirely incorporated by reference.
Additional Fc Variants for Additional Functionality
[0319] In addition to pI amino acid variants, there are a number of
useful Fc amino acid modification that can be made for a variety of
reasons, including, but not limited to, altering binding to one or
more Fc.gamma.R receptors, altered binding to FcRn receptors,
etc.
[0320] Accordingly, the proteins of the invention can include amino
acid modifications, including the heterodimerization variants
outlined herein, which includes the pI variants and steric
variants. Each set of variants can be independently and optionally
included or excluded from any particular heterodimeric protein.
Fc.gamma.R Variants
[0321] Accordingly, there are a number of useful Fc substitutions
that can be made to alter binding to one or more of the Fc.gamma.R
receptors. Substitutions that result in increased binding as well
as decreased binding can be useful. For example, it is known that
increased binding to Fc.quadrature.RIIIa generally results in
increased ADCC (antibody dependent cell-mediated cytotoxicity; the
cell-mediated reaction wherein nonspecific cytotoxic cells that
express Fc.gamma.Rs recognize bound antibody on a target cell and
subsequently cause lysis of the target cell). Similarly, decreased
binding to Fc.gamma.RIIb (an inhibitory receptor) can be beneficial
as well in some circumstances. Amino acid substitutions that find
use in the present invention include those listed in U.S. Ser. No.
11/124,620 (particularly FIG. 41), Ser. Nos. 11/174,287,
11/396,495, 11/538,406, all of which are expressly incorporated
herein by reference in their entirety and specifically for the
variants disclosed therein. Particular variants that find use
include, but are not limited to, 236A, 239D, 239E, 332E, 332D,
239D/332E, 267D, 267E, 328F, 267E/328F, 236A/332E, 239D/332E/330Y,
239D, 332E/330L, 243A, 243L, 264A, 264V and 299T.
[0322] In addition, there are additional Fc substitutions that find
use in increased binding to the FcRn receptor and increased serum
half life, as specifically disclosed in U.S. Ser. No. 12/341,769,
hereby incorporated by reference in its entirety, including, but
not limited to, 434S, 434A, 428L, 308F, 259I, 428L/434S, 259I/308F,
436I/428L, 436I or V/434S, 436V/428L and 259I/308F/428L.
Linkers
[0323] The present invention optionally provides linkers as needed,
for example in the addition of additional antigen binding sites, as
depicted for example in FIG. 2, where "the other end" of the
molecule contains additional antigen binding components. In
addition, as outlined below, linkers are optionally also used in
antibody drug conjugate (ADC) systems. When used to join the
components of the central mAb-Fv constructs, the linker is
generally a polypeptide comprising two or more amino acid residues
joined by peptide bonds and are used to link one or more of the
components of the present invention. Such linker polypeptides are
well known in the art (see e.g., Holliger, P., et al. (1993) Proc.
Natl. Acad. Sci. USA 90:6444-6448; Poljak, R. J., et al. (1994)
Structure 2:1121-1123). A variety of linkers may find use in some
embodiments described herein. As will be appreciated by those in
the art, there are at least three different linker types used in
the present invention.
[0324] "Linker" herein is also referred to as "linker sequence",
"spacer", "tethering sequence" or grammatical equivalents thereof.
Homo- or hetero-bifunctional linkers as are well known (see, 1994
Pierce Chemical Company catalog, technical section on
cross-linkers, pages 155-200, incorporated entirely by reference).
A number of strategies may be used to covalently link molecules
together. These include, but are not limited to polypeptide
linkages between N- and C-termini of proteins or protein domains,
linkage via disulfide bonds, and linkage via chemical cross-linking
reagents. In one aspect of this embodiment, the linker is a peptide
bond, generated by recombinant techniques or peptide synthesis. The
linker peptide may predominantly include the following amino acid
residues: Gly, Ser, Ala, or Thr. The linker peptide should 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 50 amino acids in length, preferably
about 1 to 30 amino acids in length. In one embodiment, linkers of
1 to 20 amino acids in length may be used. Useful linkers include
glycine-serine polymers, including for example (GS)n, (GSGGS)n,
(GGGGS)n, and (GGGS)n, where n is an integer of at least one,
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, that is may find use as linkers.
[0325] Other linker sequences may include any sequence of any
length of CL/CH1 domain but not all residues of CL/CH1 domain; for
example the first 5-12 amino acid residues of the CL/CH1 domains.
Linkers can be derived from immunoglobulin light chain, for example
C.kappa. or C.lamda. Linkers can be derived from immunoglobulin
heavy chains of any isotype, including for example C.gamma.1,
C.gamma.2, C.gamma.3, C.gamma.4, C.alpha.1, C.alpha.2, C.delta.,
C.epsilon., and C.mu.. Linker sequences may also be derived from
other proteins such as Ig-like proteins (e.g. TCR, FcR, KIR), hinge
region-derived sequences, and other natural sequences from other
proteins.
Antibody-Drug Conjugates
[0326] In some embodiments, the multispecific antibodies of the
invention are conjugated with drugs to form antibody-drug
conjugates (ADCs). In general, ADCs are used in oncology
applications, where the use of antibody-drug conjugates for the
local delivery of cytotoxic or cytostatic agents allows for the
targeted delivery of the drug moiety to tumors, which can allow
higher efficacy, lower toxicity, etc. An overview of this
technology is provided in Ducry et al., Bioconjugate Chem., 21:5-13
(2010), Carter et al., Cancer J. 14(3):154 (2008) and Senter,
Current Opin. Chem. Biol. 13:235-244 (2009), all of which are
hereby incorporated by reference in their entirety.
[0327] Thus the invention provides multispecific antibodies
conjugated to drugs. Generally, conjugation is done by covalent
attachment to the antibody, as further described below, and
generally relies on a linker, often a peptide linkage (which, as
described below, may be designed to be sensitive to cleavage by
proteases at the target site or not). In addition, as described
above, linkage of the linker-drug unit (LU-D) can be done by
attachment to cysteines within the antibody. As will be appreciated
by those in the art, the number of drug moieties per antibody can
change, depending on the conditions of the reaction, and can vary
from 1:1 to 10:1 drug:antibody. As will be appreciated by those in
the art, the actual number is an average.
[0328] Thus the invention provides multispecific antibodies
conjugated to drugs. As described below, the drug of the ADC can be
any number of agents, including but not limited to cytotoxic agents
such as chemotherapeutic agents, growth inhibitory agents, toxins
(for example, an enzymatically active toxin of bacterial, fungal,
plant, or animal origin, or fragments thereof), or a radioactive
isotope (that is, a radioconjugate) are provided. In other
embodiments, the invention further provides methods of using the
ADCs.
[0329] Drugs for use in the present invention include cytotoxic
drugs, particularly those which are used for cancer therapy. Such
drugs include, in general, DNA damaging agents, anti-metabolites,
natural products and their analogs. Exemplary classes of cytotoxic
agents include the enzyme inhibitors such as dihydrofolate
reductase inhibitors, and thymidylate synthase inhibitors, DNA
intercalators, DNA cleavers, topoisomerase inhibitors, the
anthracycline family of drugs, the vinca drugs, the mitomycins, the
bleomycins, the cytotoxic nucleosides, the pteridine family of
drugs, diynenes, the podophyllotoxins, dolastatins, maytansinoids,
differentiation inducers, and taxols.
[0330] Members of these classes include, for example, methotrexate,
methopterin, dichloromethotrexate, 5-fluorouracil,
6-mercaptopurine, cytosine arabinoside, melphalan, leurosine,
leurosideine, actinomycin, daunorubicin, doxorubicin, mitomycin C,
mitomycin A, caminomycin, aminopterin, tallysomycin,
podophyllotoxin and podophyllotoxin derivatives such as etoposide
or etoposide phosphate, vinblastine, vincristine, vindesine,
taxanes including taxol, taxotere retinoic acid, butyric acid,
N8-acetyl spermidine, camptothecin, calicheamicin, esperamicin,
ene-diynes, duocarmycin A, duocarmycin SA, calicheamicin,
camptothecin, maytansinoids (including DM1), monomethylauristatin E
(MMAE), monomethylauristatin F (MMAF), and maytansinoids (DM4) and
their analogues.
[0331] Toxins may be used as antibody-toxin conjugates and include
bacterial toxins such as diphtheria toxin, plant toxins such as
ricin, small molecule toxins such as geldanamycin (Mandler et al
(2000) J. Nat. Cancer Inst. 92(19):1573-1581; Mandler et al (2000)
Bioorganic & Med. Chem. Letters 10:1025-1028; Mandler et al
(2002) Bioconjugate Chem. 13:786-791), maytansinoids (EP 1391213;
Liu et al., (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623), and
calicheamicin (Lode et al (1998) Cancer Res. 58:2928; Hinman et al
(1993) Cancer Res. 53:3336-3342). Toxins may exert their cytotoxic
and cytostatic effects by mechanisms including tubulin binding, DNA
binding, or topoisomerase inhibition.
[0332] Conjugates of a multispecific antibody and one or more small
molecule toxins, such as a maytansinoids, dolastatins, auristatins,
a trichothecene, calicheamicin, and CC1065, and the derivatives of
these toxins that have toxin activity, are contemplated.
Maytansinoids
[0333] Maytansine compounds suitable for use as maytansinoid drug
moieties are well known in the art, and can be isolated from
natural sources according to known methods, produced using genetic
engineering techniques (see Yu et al (2002) PNAS 99:7968-7973), or
maytansinol and maytansinol analogues prepared synthetically
according to known methods. As described below, drugs may be
modified by the incorporation of a functionally active group such
as a thiol or amine group for conjugation to the antibody.
[0334] Exemplary maytansinoid drug moieties include those having a
modified aromatic ring, such as: C-19-dechloro (U.S. Pat. No.
4,256,746) (prepared by lithium aluminum hydride reduction of
ansamytocin P2); C-20-hydroxy (or C-20-demethyl)+/-C-19-dechloro
(U.S. Pat. Nos. 4,361,650 and 4,307,016) (prepared by demethylation
using Streptomyces or Actinomyces or dechlorination using LAH); and
C-20-demethoxy, C-20-acyloxy (--OCOR), +/-dechloro (U.S. Pat. No.
4,294,757) (prepared by acylation using acyl chlorides) and those
having modifications at other positions
[0335] Exemplary maytansinoid drug moieties also include those
having modifications such as: C-9-SH (U.S. Pat. No. 4,424,219)
(prepared by the reaction of maytansinol with H2S or P2S5);
C-14-alkoxymethyl(demethoxy/CH2OR) (U.S. Pat. No. 4,331,598);
C-14-hydroxymethyl or acyloxymethyl (CH2OH or CH2OAc) (U.S. Pat.
No. 4,450,254) (prepared from Nocardia); C-15-hydroxy/acyloxy (U.S.
Pat. No. 4,364,866) (prepared by the conversion of maytansinol by
Streptomyces); C-15-methoxy (U.S. Pat. Nos. 4,313,946 and
4,315,929) (isolated from Trewia nudlflora); C-18-N-demethyl (U.S.
Pat. Nos. 4,362,663 and 4,322,348) (prepared by the demethylation
of maytansinol by Streptomyces); and 4,5-deoxy (U.S. Pat. No.
4,371,533) (prepared by the titanium trichloride/LAH reduction of
maytansinol).
[0336] Of particular use are DM1 (disclosed in U.S. Pat. No.
5,208,020, incorporated by reference) and DM4 (disclosed in U.S.
Pat. No. 7,276,497, incorporated by reference). See also a number
of additional maytansinoid derivatives and methods in 5,416,064,
WO/01/24763, 7,303,749, 7,601,354, U.S. Ser. No. 12/631,508,
WO02/098883, 6,441,163, 7,368,565, WO02/16368 and WO04/1033272, all
of which are expressly incorporated by reference in their
entirety.
[0337] ADCs containing maytansinoids, methods of making same, and
their therapeutic use are disclosed, for example, in U.S. Pat. Nos.
5,208,020; 5,416,064; 6,441,163 and European Patent EP 0 425 235
B1, the disclosures of which are hereby expressly incorporated by
reference. Liu et al., Proc. Natl. Acad. Sci. USA 93:8618-8623
(1996) described ADCs comprising a maytansinoid designated DM1
linked to the monoclonal antibody C242 directed against human
colorectal cancer. The conjugate was found to be highly cytotoxic
towards cultured colon cancer cells, and showed antitumor activity
in an in vivo tumor growth assay.
[0338] Chari et al., Cancer Research 52:127-131 (1992) describe
ADCs in which a maytansinoid was conjugated via a disulfide linker
to the murine antibody A7 binding to an antigen on human colon
cancer cell lines, or to another murine monoclonal antibody TA.1
that binds the HER-2/neu oncogene. The cytotoxicity of the
TA.1-maytansonoid conjugate was tested in vitro on the human breast
cancer cell line SK-BR-3, which expresses 3.times.105 HER-2 surface
antigens per cell. The drug conjugate achieved a degree of
cytotoxicity similar to the free maytansinoid drug, which could be
increased by increasing the number of maytansinoid molecules per
antibody molecule. The A7-maytansinoid conjugate showed low
systemic cytotoxicity in mice.
Auristatins and Dolastatins
[0339] In some embodiments, the ADC comprises a multispecific
antibody conjugated to dolastatins or dolostatin peptidic analogs
and derivatives, the auristatins (U.S. Pat. Nos. 5,635,483;
5,780,588). Dolastatins and auristatins have been shown to
interfere with microtubule dynamics, GTP hydrolysis, and nuclear
and cellular division (Woyke et al (2001) Antimicrob. Agents and
Chemother. 45(12):3580-3584) and have anticancer (U.S. Pat. No.
5,663,149) and antifungal activity (Pettit et al (1998) Antimicrob.
Agents Chemother. 42:2961-2965). The dolastatin or auristatin drug
moiety may be attached to the antibody through the N (amino)
terminus or the C (carboxyl) terminus of the peptidic drug moiety
(WO 02/088172).
[0340] Exemplary auristatin embodiments include the N-terminus
linked monomethylauristatin drug moieties DE and DF, disclosed in
"Senter et al, Proceedings of the American Association for Cancer
Research, Volume 45, Abstract Number 623, presented Mar. 28, 2004
and described in United States Patent Publication No. 2005/0238648,
the disclosure of which is expressly incorporated by reference in
its entirety.
[0341] An exemplary auristatin embodiment is MMAE (see U.S. Pat.
No. 6,884,869 expressly incorporated by reference in its
entirety).
[0342] Another exemplary auristatin embodiment is MMAF (see US
2005/0238649, 5,767,237 and 6,124,431, expressly incorporated by
reference in their entirety).
[0343] Additional exemplary embodiments comprising MMAE or MMAF and
various linker components (described further herein) have the
following structures and abbreviations (wherein Ab means antibody
and p is 1 to about 8):
[0344] Typically, peptide-based drug moieties can be prepared by
forming a peptide bond between two or more amino acids and/or
peptide fragments. Such peptide bonds can be prepared, for example,
according to the liquid phase synthesis method (see E. Schroder and
K. Lubke, "The Peptides", volume 1, pp 76-136, 1965, Academic
Press) that is well known in the field of peptide chemistry. The
auristatin/dolastatin drug moieties may be prepared according to
the methods of: U.S. Pat. Nos. 5,635,483; 5,780,588; Pettit et al
(1989) J. Am. Chem. Soc. 111:5463-5465; Pettit et al (1998)
Anti-Cancer Drug Design 13:243-277; Pettit, G. R., et al.
Synthesis, 1996, 719-725; Pettit et al (1996) J. Chem. Soc. Perkin
Trans. 1 5:859-863; and Doronina (2003) Nat Biotechnol
21(7):778-784.
Calicheamicin
[0345] In other embodiments, the ADC comprises an antibody of the
invention conjugated to one or more calicheamicin molecules. For
example, Mylotarg is the first commercial ADC drug and utilizes
calicheamicin .gamma.1 as the payload (see U.S. Pat. No. 4,970,198,
incorporated by reference in its entirety). Additional
calicheamicin derivatives are described in U.S. Pat. Nos.
5,264,586, 5,384,412, 5,550,246, 5,739,116, 5,773,001, 5,767,285
and 5,877,296, all expressly incorporated by reference. The
calicheamicin family of antibiotics are capable of producing
double-stranded DNA breaks at sub-picomolar concentrations. For the
preparation of conjugates of the calicheamicin family, see U.S.
Pat. Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701,
5,770,710, 5,773,001, 5,877,296 (all to American Cyanamid Company).
Structural analogues of calicheamicin which may be used include,
but are not limited to, .gamma.1I, .alpha.2I, .alpha.2I,
N-acetyl-.gamma.1I, PSAG and .theta.I1 (Hinman et al., Cancer
Research 53:3336-3342 (1993), Lode et al., Cancer Research
58:2925-2928 (1998) and the aforementioned U.S. patents to American
Cyanamid). Another anti-tumor drug that the antibody can be
conjugated is QFA which is an antifolate. Both calicheamicin and
QFA have intracellular sites of action and do not readily cross the
plasma membrane. Therefore, cellular uptake of these agents through
antibody mediated internalization greatly enhances their cytotoxic
effects.
Duocarmycins
[0346] CC-1065 (see 4,169,888, incorporated by reference) and
duocarmycins are members of a family of antitumor antibiotics
utilized in ADCs. These antibiotics appear to work through
sequence-selectively alkylating DNA at the N3 of adenine in the
minor groove, which initiates a cascade of events that result in
apoptosis.
[0347] Important members of the duocarmycins include duocarmycin A
(U.S. Pat. No. 4,923,990, incorporated by reference) and
duocarmycin SA (U.S. Pat. No. 5,101,038, incorporated by
reference), and a large number of analogues as described in U.S.
Pat. Nos. 7,517,903, 7,691,962, 5,101,038; 5,641,780; 5,187,186;
5,070,092; 5,070,092; 5,641,780; 5,101,038; 5,084,468, 5,475,092,
5,585,499, 5,846,545, WO2007/089149, WO2009/017394A1, 5,703,080,
6,989,452, 7,087,600, 7,129,261, 7,498,302, and 7,507,420, all of
which are expressly incorporated by reference.
Other Cytotoxic Agents
[0348] Other antitumor agents that can be conjugated to the
antibodies of the invention include BCNU, streptozoicin,
vincristine and 5-fluorouracil, the family of agents known
collectively LL-E33288 complex described in U.S. Pat. Nos.
5,053,394, 5,770,710, as well as esperamicins (U.S. Pat. No.
5,877,296).
[0349] Enzymatically active toxins and fragments thereof which can
be used include diphtheria A chain, nonbinding active fragments of
diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa),
ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin,
Aleurites fordii proteins, dianthin proteins, Phytolaca americana
proteins (PAPI, PAPII, and PAP-S), Momordica charantia inhibitor,
curcin, crotin, Sapaonaria officinalis inhibitor, gelonin,
mitogellin, restrictocin, phenomycin, enomycin and the
tricothecenes. See, for example, WO 93/21232 published Oct. 28,
1993.
[0350] The present invention further contemplates an ADC formed
between an antibody and a compound with nucleolytic activity (e.g.,
a ribonuclease or a DNA endonuclease such as a deoxyribonuclease;
DNase).
[0351] For selective destruction of the tumor, the antibody may
comprise a highly radioactive atom. A variety of radioactive
isotopes are available for the production of radioconjugated
antibodies. Examples include At211, I131, I125, Y90, Re186, Re188,
Sm153, Bi212, P32, Pb212 and radioactive isotopes of Lu.
[0352] The radio- or other labels may be incorporated in the
conjugate in known ways. For example, the peptide may be
biosynthesized or may be synthesized by chemical amino acid
synthesis using suitable amino acid precursors involving, for
example, fluorine-19 in place of hydrogen. Labels such as Tc99m or
I123, Re186, Re188 and In111 can be attached via a cysteine residue
in the peptide. Yttrium-90 can be attached via a lysine residue.
The IODOGEN method (Fraker et al (1978) Biochem. Biophys. Res.
Commun. 80: 49-57 can be used to incorporate Iodine-123.
"Monoclonal Antibodies in Immunoscintigraphy" (Chatal, CRC Press
1989) describes other methods in detail.
[0353] For compositions comprising a plurality of antibodies, the
drug loading is represented by p, the average number of drug
molecules per Antibody. Drug loading may range from 1 to 20 drugs
(D) per Antibody. The average number of drugs per antibody in
preparation of conjugation reactions may be characterized by
conventional means such as mass spectroscopy, ELISA assay, and
HPLC. The quantitative distribution of Antibody-Drug-Conjugates in
terms of p may also be determined.
[0354] In some instances, separation, purification, and
characterization of homogeneous Antibody-Drug-conjugates where p is
a certain value from Antibody-Drug-Conjugates with other drug
loadings may be achieved by means such as reverse phase HPLC or
electrophoresis. In exemplary embodiments, p is 2, 3, 4, 5, 6, 7,
or 8 or a fraction thereof.
[0355] The generation of Antibody-drug conjugate compounds can be
accomplished by any technique known to the skilled artisan.
Briefly, the Antibody-drug conjugate compounds can include a
multispecific antibody as the Antibody unit, a drug, and optionally
a linker that joins the drug and the binding agent.
[0356] A number of different reactions are available for covalent
attachment of drugs and/or linkers to binding agents. This is can
be accomplished by reaction of the amino acid residues of the
binding agent, for example, antibody molecule, including the amine
groups of lysine, the free carboxylic acid groups of glutamic and
aspartic acid, the sulfhydryl groups of cysteine and the various
moieties of the aromatic amino acids. A commonly used non-specific
methods of covalent attachment is the carbodiimide reaction to link
a carboxy (or amino) group of a compound to amino (or carboxy)
groups of the antibody. Additionally, bifunctional agents such as
dialdehydes or imidoesters have been used to link the amino group
of a compound to amino groups of an antibody molecule.
[0357] Also available for attachment of drugs to binding agents is
the Schiff base reaction. This method involves the periodate
oxidation of a drug that contains glycol or hydroxy groups, thus
forming an aldehyde which is then reacted with the binding agent.
Attachment occurs via formation of a Schiff base with amino groups
of the binding agent. Isothiocyanates can also be used as coupling
agents for covalently attaching drugs to binding agents. Other
techniques are known to the skilled artisan and within the scope of
the present invention.
[0358] In some embodiments, an intermediate, which is the precursor
of the linker, is reacted with the drug under appropriate
conditions. In other embodiments, reactive groups are used on the
drug and/or the intermediate. The product of the reaction between
the drug and the intermediate, or the derivatized drug, is
subsequently reacted with an multispecific antibody of the
invention under appropriate conditions.
[0359] It will be understood that chemical modifications may also
be made to the desired compound in order to make reactions of that
compound more convenient for purposes of preparing conjugates of
the invention. For example a functional group e.g. amine, hydroxyl,
or sulfhydryl, may be appended to the drug at a position which has
minimal or an acceptable effect on the activity or other properties
of the drug
ADC Linker Units
[0360] Typically, the antibody-drug conjugate compounds comprise a
Linker unit between the drug unit and the antibody unit. In some
embodiments, the linker is cleavable under intracellular or
extracellular conditions, such that cleavage of the linker releases
the drug unit from the antibody in the appropriate environment. For
example, solid tumors that secrete certain proteases may serve as
the target of the cleavable linker; in other embodiments, it is the
intracellular proteases that are utilized. In yet other
embodiments, the linker unit is not cleavable and the drug is
released, for example, by antibody degradation in lysosomes.
[0361] In some embodiments, the linker is cleavable by a cleaving
agent that is present in the intracellular environment (for
example, within a lysosome or endosome or caveolea). The linker can
be, for example, a peptidyl linker that is cleaved by an
intracellular peptidase or protease enzyme, including, but not
limited to, a lysosomal or endosomal protease. In some embodiments,
the peptidyl linker is at least two amino acids long or at least
three amino acids long or more.
[0362] Cleaving agents can include, without limitation, cathepsins
B and D and plasmin, all of which are known to hydrolyze dipeptide
drug derivatives resulting in the release of active drug inside
target cells (see, e.g., Dubowchik and Walker, 1999, Pharm.
Therapeutics 83:67-123). Peptidyl linkers that are cleavable by
enzymes that are present in CD38-expressing cells. For example, a
peptidyl linker that is cleavable by the thiol-dependent protease
cathepsin-B, which is highly expressed in cancerous tissue, can be
used (e.g., a Phe-Leu or a Gly-Phe-Leu-Gly linker (SEQ ID NO:
287)). Other examples of such linkers are described, e.g., in U.S.
Pat. No. 6,214,345, incorporated herein by reference in its
entirety and for all purposes.
[0363] In some embodiments, the peptidyl linker cleavable by an
intracellular protease is a Val-Cit linker or a Phe-Lys linker
(see, e.g., U.S. Pat. No. 6,214,345, which describes the synthesis
of doxorubicin with the val-cit linker).
[0364] In other embodiments, the cleavable linker is pH-sensitive,
that is, sensitive to hydrolysis at certain pH values. Typically,
the pH-sensitive linker hydrolyzable under acidic conditions. For
example, an acid-labile linker that is hydrolyzable in the lysosome
(for example, a hydrazone, semicarbazone, thiosemicarbazone,
cis-aconitic amide, orthoester, acetal, ketal, or the like) may be
used. (See, e.g., U.S. Pat. Nos. 5,122,368; 5,824,805; 5,622,929;
Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123; Neville
et al., 1989, Biol. Chem. 264:14653-14661.) Such linkers are
relatively stable under neutral pH conditions, such as those in the
blood, but are unstable at below pH 5.5 or 5.0, the approximate pH
of the lysosome. In certain embodiments, the hydrolyzable linker is
a thioether linker (such as, e.g., a thioether attached to the
therapeutic agent via an acylhydrazone bond (see, e.g., U.S. Pat.
No. 5,622,929).
[0365] In yet other embodiments, the linker is cleavable under
reducing conditions (for example, a disulfide linker). A variety of
disulfide linkers are known in the art, including, for example,
those that can be formed using SATA
(N-succinimidyl-5-acetylthioacetate), SPDP
(N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB
(N-succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT
(N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene)-
-, SPDB and SMPT. (See, e.g., Thorpe et al., 1987, Cancer Res.
47:5924-5931; Wawrzynczak et al., In Immunoconjugates: Antibody
Conjugates in Radioimagery and Therapy of Cancer (C. W. Vogel ed.,
Oxford U. Press, 1987. See also U.S. Pat. No. 4,880,935.)
[0366] In other embodiments, the linker is a malonate linker
(Johnson et al., 1995, Anticancer Res. 15:1387-93), a
maleimidobenzoyl linker (Lau et al., 1995, Bioorg-Med-Chem. 3(10):
1299-1304), or a 3'-N-amide analog (Lau et al., 1995,
Bioorg-Med-Chem. 3(10): 1305-12).
[0367] In yet other embodiments, the linker unit is not cleavable
and the drug is released by antibody degradation. (See U.S.
Publication No. 2005/0238649 incorporated by reference herein in
its entirety and for all purposes).
[0368] In many embodiments, the linker is self-immolative. As used
herein, the term "self-immolative Spacer" refers to a bifunctional
chemical moiety that is capable of covalently linking together two
spaced chemical moieties into a stable tripartite molecule. It will
spontaneously separate from the second chemical moiety if its bond
to the first moiety is cleaved. See for example, WO 2007059404A2,
WO06110476A2, WO05112919A2, WO2010/062171, WO09/017394,
WO07/089149, WO 07/018431, WO04/043493 and WO02/083180, which are
directed to drug-cleavable substrate conjugates where the drug and
cleavable substrate are optionally linked through a self-immolative
linker and which are all expressly incorporated by reference.
[0369] Often the linker is not substantially sensitive to the
extracellular environment. As used herein, "not substantially
sensitive to the extracellular environment," in the context of a
linker, means that no more than about 20%, 15%, 10%, 5%, 3%, or no
more than about 1% of the linkers, in a sample of antibody-drug
conjugate compound, are cleaved when the antibody-drug conjugate
compound presents in an extracellular environment (for example, in
plasma).
[0370] Whether a linker is not substantially sensitive to the
extracellular environment can be determined, for example, by
incubating with plasma the antibody-drug conjugate compound for a
predetermined time period (for example, 2, 4, 8, 16, or 24 hours)
and then quantitating the amount of free drug present in the
plasma.
[0371] In other, non-mutually exclusive embodiments, the linker
promotes cellular internalization. In certain embodiments, the
linker promotes cellular internalization when conjugated to the
therapeutic agent (that is, in the milieu of the linker-therapeutic
agent moiety of the antibody-drug conjugate compound as described
herein). In yet other embodiments, the linker promotes cellular
internalization when conjugated to both the auristatin compound and
the multispecific antibodies of the invention.
[0372] A variety of exemplary linkers that can be used with the
present compositions and methods are described in WO 2004-010957,
U.S. Publication No. 2006/0074008, U.S. Publication No.
20050238649, and U.S. Publication No. 2006/0024317 (each of which
is incorporated by reference herein in its entirety and for all
purposes).
Drug Loading
[0373] Drug loading is represented by p and is the average number
of Drug moieties per antibody in a molecule. Drug loading ("p") may
be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,
19, 20 or more moieties (D) per antibody, although frequently the
average number is a fraction or a decimal. Generally, drug loading
of from 1 to 4 is frequently useful, and from 1 to 2 is also
useful. ADCs of the invention include collections of antibodies
conjugated with a range of drug moieties, from 1 to 20. The average
number of drug moieties per antibody in preparations of ADC from
conjugation reactions may be characterized by conventional means
such as mass spectroscopy and, ELISA assay.
[0374] The quantitative distribution of ADC in terms of p may also
be determined. In some instances, separation, purification, and
characterization of homogeneous ADC where p is a certain value from
ADC with other drug loadings may be achieved by means such as
electrophoresis.
[0375] For some antibody-drug conjugates, p may be limited by the
number of attachment sites on the antibody. For example, where the
attachment is a cysteine thiol, as in the exemplary embodiments
above, an antibody may have only one or several cysteine thiol
groups, or may have only one or several sufficiently reactive thiol
groups through which a linker may be attached. In certain
embodiments, higher drug loading, e.g. p>5, may cause
aggregation, insolubility, toxicity, or loss of cellular
permeability of certain antibody-drug conjugates. In certain
embodiments, the drug loading for an ADC of the invention ranges
from 1 to about 8; from about 2 to about 6; from about 3 to about
5; from about 3 to about 4; from about 3.1 to about 3.9; from about
3.2 to about 3.8; from about 3.2 to about 3.7; from about 3.2 to
about 3.6; from about 3.3 to about 3.8; or from about 3.3 to about
3.7. Indeed, it has been shown that for certain ADCs, the optimal
ratio of drug moieties per antibody may be less than 8, and may be
about 2 to about 5. See US 2005-0238649 A1 (herein incorporated by
reference in its entirety).
[0376] In certain embodiments, fewer than the theoretical maximum
of drug moieties are conjugated to an antibody during a conjugation
reaction. An antibody may contain, for example, lysine residues
that do not react with the drug-linker intermediate or linker
reagent, as discussed below. Generally, antibodies do not contain
many free and reactive cysteine thiol groups which may be linked to
a drug moiety; indeed most cysteine thiol residues in antibodies
exist as disulfide bridges. In certain embodiments, an antibody may
be reduced with a reducing agent such as dithiothreitol (DTT) or
tricarbonylethylphosphine (TCEP), under partial or total reducing
conditions, to generate reactive cysteine thiol groups. In certain
embodiments, an antibody is subjected to denaturing conditions to
reveal reactive nucleophilic groups such as lysine or cysteine.
[0377] The loading (drug/antibody ratio) of an ADC may be
controlled in different ways, e.g., by: (i) limiting the molar
excess of drug-linker intermediate or linker reagent relative to
antibody, (ii) limiting the conjugation reaction time or
temperature, (iii) partial or limiting reductive conditions for
cysteine thiol modification, (iv) engineering by recombinant
techniques the amino acid sequence of the antibody such that the
number and position of cysteine residues is modified for control of
the number and/or position of linker-drug attachments (such as
thioMab or thioFab prepared as disclosed herein and in
WO2006/034488 (herein incorporated by reference in its
entirety)).
[0378] It is to be understood that where more than one nucleophilic
group reacts with a drug-linker intermediate or linker reagent
followed by drug moiety reagent, then the resulting product is a
mixture of ADC compounds with a distribution of one or more drug
moieties attached to an antibody. The average number of drugs per
antibody may be calculated from the mixture by a dual ELISA
antibody assay, which is specific for antibody and specific for the
drug. Individual ADC molecules may be identified in the mixture by
mass spectroscopy and separated by HPLC, e.g. hydrophobic
interaction chromatography.
[0379] In some embodiments, a homogeneous ADC with a single loading
value may be isolated from the conjugation mixture by
electrophoresis or chromatography.
Methods of Determining Cytotoxic Effect of ADCs
[0380] Methods of determining whether a Drug or Antibody-Drug
conjugate exerts a cytostatic and/or cytotoxic effect on a cell are
known. Generally, the cytotoxic or cytostatic activity of an
Antibody Drug conjugate can be measured by: exposing mammalian
cells expressing a target protein of the Antibody Drug conjugate in
a cell culture medium; culturing the cells for a period from about
6 hours to about 5 days; and measuring cell viability. Cell-based
in vitro assays can be used to measure viability (proliferation),
cytotoxicity, and induction of apoptosis (caspase activation) of
the Antibody Drug conjugate.
[0381] For determining whether an Antibody Drug conjugate exerts a
cytostatic effect, a thymidine incorporation assay may be used. For
example, cancer cells expressing a target antigen at a density of
5,000 cells/well of a 96-well plated can be cultured for a 72-hour
period and exposed to 0.5 .mu.Ci of 3H-thymidine during the final 8
hours of the 72-hour period. The incorporation of 3H-thymidine into
cells of the culture is measured in the presence and absence of the
Antibody Drug conjugate.
[0382] For determining cytotoxicity, necrosis or apoptosis
(programmed cell death) can be measured. Necrosis is typically
accompanied by increased permeability of the plasma membrane;
swelling of the cell, and rupture of the plasma membrane. Apoptosis
is typically characterized by membrane blebbing, condensation of
cytoplasm, and the activation of endogenous endonucleases.
Determination of any of these effects on cancer cells indicates
that an Antibody Drug conjugate is useful in the treatment of
cancers.
[0383] Cell viability can be measured by determining in a cell the
uptake of a dye such as neutral red, trypan blue, or ALAMAR.TM.
blue (see, e.g., Page et al., 1993, Intl. J. Oncology 3:473-476).
In such an assay, the cells are incubated in media containing the
dye, the cells are washed, and the remaining dye, reflecting
cellular uptake of the dye, is measured spectrophotometrically. The
protein-binding dye sulforhodamine B (SRB) can also be used to
measure cytotoxicity (Skehan et al., 1990, J. Natl. Cancer Inst.
82:1107-12).
[0384] Alternatively, a tetrazolium salt, such as MTT, is used in a
quantitative colorimetric assay for mammalian cell survival and
proliferation by detecting living, but not dead, cells (see, e.g.,
Mosmann, 1983, J. Immunol. Methods 65:55-63).
[0385] Apoptosis can be quantitated by measuring, for example, DNA
fragmentation. Commercial photometric methods for the quantitative
in vitro determination of DNA fragmentation are available. Examples
of such assays, including TUNEL (which detects incorporation of
labeled nucleotides in fragmented DNA) and ELISA-based assays, are
described in Biochemica, 1999, no. 2, pp. 34-37 (Roche Molecular
Biochemicals).
[0386] Apoptosis can also be determined by measuring morphological
changes in a cell. For example, as with necrosis, loss of plasma
membrane integrity can be determined by measuring uptake of certain
dyes (e.g., a fluorescent dye such as, for example, acridine orange
or ethidium bromide). A method for measuring apoptotic cell number
has been described by Duke and Cohen, Current Protocols in
Immunology (Coligan et al. eds., 1992, pp. 3.17.1-3.17.16). Cells
also can be labeled with a DNA dye (e.g., acridine orange, ethidium
bromide, or propidium iodide) and the cells observed for chromatin
condensation and margination along the inner nuclear membrane.
Other morphological changes that can be measured to determine
apoptosis include, e.g., cytoplasmic condensation, increased
membrane blebbing, and cellular shrinkage.
[0387] The presence of apoptotic cells can be measured in both the
attached and "floating" compartments of the cultures. For example,
both compartments can be collected by removing the supernatant,
trypsinizing the attached cells, combining the preparations
following a centrifugation wash step (e.g., 10 minutes at 2000
rpm), and detecting apoptosis (e.g., by measuring DNA
fragmentation). (See, e.g., Piazza et al., 1995, Cancer Research
55:3110-16).
[0388] In vivo, the effect of a therapeutic composition of the
multispecific antibody of the invention can be evaluated in a
suitable animal model. For example, xenogenic cancer models can be
used, wherein cancer explants or passaged xenograft tissues are
introduced into immune compromised animals, such as nude or SCID
mice (Klein et al., 1997, Nature Medicine 3: 402-408). Efficacy can
be measured using assays that measure inhibition of tumor
formation, tumor regression or metastasis, and the like.
[0389] The therapeutic compositions used in the practice of the
foregoing methods can be formulated into pharmaceutical
compositions comprising a carrier suitable for the desired delivery
method. Suitable carriers include any material that when combined
with the therapeutic composition retains the anti-tumor function of
the therapeutic composition and is generally non-reactive with the
patient's immune system. Examples include, but are not limited to,
any of a number of standard pharmaceutical carriers such as sterile
phosphate buffered saline solutions, bacteriostatic water, and the
like (see, generally, Remington's Pharmaceutical Sciences 16th
Edition, A. Osal., Ed., 1980).
Antibody Compositions for In Vivo Administration
[0390] Formulations of the antibodies used in accordance with the
present invention are prepared for storage by mixing an antibody
having the desired degree of purity with optional pharmaceutically
acceptable carriers, excipients or stabilizers (Remington's
Pharmaceutical Sciences 16th edition, Osol, A. Ed. [1980]), in the
form of lyophilized formulations or aqueous solutions. Acceptable
carriers, excipients, or stabilizers are nontoxic to recipients at
the dosages and concentrations employed, and include buffers such
as phosphate, citrate, and other organic acids; antioxidants
including ascorbic acid and methionine; preservatives (such as
octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;
benzalkonium chloride, benzethonium chloride; phenol, butyl or
benzyl alcohol; alkyl parabens such as methyl or propyl paraben;
catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low
molecular weight (less than about 10 residues) polypeptides;
proteins, such as serum albumin, gelatin, or immunoglobulins;
hydrophilic polymers such as polyvinylpyrrolidone; amino acids such
as glycine, glutamine, asparagine, histidine, arginine, or lysine;
monosaccharides, disaccharides, and other carbohydrates including
glucose, mannose, or dextrins; chelating agents such as EDTA;
sugars such as sucrose, mannitol, trehalose or sorbitol;
salt-forming counter-ions such as sodium; metal complexes (e.g.
Zn-protein complexes); and/or non-ionic surfactants such as
TWEEN.TM., PLURONICS.TM. or polyethylene glycol (PEG).
[0391] The formulation herein may also contain more than one active
compound as necessary for the particular indication being treated,
preferably those with complementary activities that do not
adversely affect each other. For example, it may be desirable to
provide antibodies with other specificities. Alternatively, or in
addition, the composition may comprise a cytotoxic agent, cytokine,
growth inhibitory agent and/or small molecule antagonist. Such
molecules are suitably present in combination in amounts that are
effective for the purpose intended.
[0392] The active ingredients may also be entrapped in
microcapsules prepared, for example, by coacervation techniques or
by interfacial polymerization, for example, hydroxymethylcellulose
or gelatin-microcapsules and poly-(methylmethacylate)
microcapsules, respectively, in colloidal drug delivery systems
(for example, liposomes, albumin microspheres, microemulsions,
nano-particles and nanocapsules) or in macroemulsions. Such
techniques are disclosed in Remington's Pharmaceutical Sciences
16th edition, Osol, A. Ed. (1980).
[0393] The formulations to be used for in vivo administration
should be sterile, or nearly so. This is readily accomplished by
filtration through sterile filtration membranes.
[0394] Sustained-release preparations may be prepared. Suitable
examples of sustained-release preparations include semipermeable
matrices of solid hydrophobic polymers containing the antibody,
which matrices are in the form of shaped articles, e.g. films, or
microcapsules. Examples of sustained-release matrices include
polyesters, hydrogels (for example,
poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),
polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic
acid and .gamma. ethyl-L-glutamate, non-degradable ethylene-vinyl
acetate, degradable lactic acid-glycolic acid copolymers such as
the LUPRON DEPOT.TM. (injectable microspheres composed of lactic
acid-glycolic acid copolymer and leuprolide acetate), and
poly-D-(-)-3-hydroxybutyric acid. While polymers such as
ethylene-vinyl acetate and lactic acid-glycolic acid enable release
of molecules for over 100 days, certain hydrogels release proteins
for shorter time periods.
[0395] When encapsulated antibodies remain in the body for a long
time, they may denature or aggregate as a result of exposure to
moisture at 37.degree. C., resulting in a loss of biological
activity and possible changes in immunogenicity. Rational
strategies can be devised for stabilization depending on the
mechanism involved. For example, if the aggregation mechanism is
discovered to be intermolecular S--S bond formation through
thio-disulfide interchange, stabilization may be achieved by
modifying sulfhydryl residues, lyophilizing from acidic solutions,
controlling moisture content, using appropriate additives, and
developing specific polymer matrix compositions.
Administrative Modalities
[0396] The antibodies and chemotherapeutic agents of the invention
are administered to a subject, in accord with known methods, such
as intravenous administration as a bolus or by continuous infusion
over a period of time, by intramuscular, intraperitoneal,
intracerobrospinal, subcutaneous, intra-articular, intrasynovial,
intrathecal, oral, topical, or inhalation routes. Intravenous or
subcutaneous administration of the antibody is preferred.
Treatment Modalities
[0397] In the methods of the invention, therapy is used to provide
a positive therapeutic response with respect to a disease or
condition. By "positive therapeutic response" is intended an
improvement in the disease or condition, and/or an improvement in
the symptoms associated with the disease or condition. For example,
a positive therapeutic response would refer to one or more of the
following improvements in the disease: (1) a reduction in the
number of neoplastic cells; (2) an increase in neoplastic cell
death; (3) inhibition of neoplastic cell survival; (5) inhibition
(i.e., slowing to some extent, preferably halting) of tumor growth;
(6) an increased patient survival rate; and (7) some relief from
one or more symptoms associated with the disease or condition.
[0398] Positive therapeutic responses in any given disease or
condition can be determined by standardized response criteria
specific to that disease or condition. Tumor response can be
assessed for changes in tumor morphology (i.e., overall tumor
burden, tumor size, and the like) using screening techniques such
as magnetic resonance imaging (MRI) scan, x-radiographic imaging,
computed tomographic (CT) scan, bone scan imaging, endoscopy, and
tumor biopsy sampling including bone marrow aspiration (BMA) and
counting of tumor cells in the circulation.
[0399] In addition to these positive therapeutic responses, the
subject undergoing therapy may experience the beneficial effect of
an improvement in the symptoms associated with the disease.
[0400] Thus for B cell tumors, the subject may experience a
decrease in the so-called B symptoms, i.e., night sweats, fever,
weight loss, and/or urticaria. For pre-malignant conditions,
therapy with an multispecific therapeutic agent may block and/or
prolong the time before development of a related malignant
condition, for example, development of multiple myeloma in subjects
suffering from monoclonal gammopathy of undetermined significance
(MGUS).
[0401] An improvement in the disease may be characterized as a
complete response. By "complete response" is intended an absence of
clinically detectable disease with normalization of any previously
abnormal radiographic studies, bone marrow, and cerebrospinal fluid
(CSF) or abnormal monoclonal protein in the case of myeloma.
[0402] Such a response may persist for at least 4 to 8 weeks, or
sometimes 6 to 8 weeks, following treatment according to the
methods of the invention. Alternatively, an improvement in the
disease may be categorized as being a partial response. By "partial
response" is intended at least about a 50% decrease in all
measurable tumor burden (i.e., the number of malignant cells
present in the subject, or the measured bulk of tumor masses or the
quantity of abnormal monoclonal protein) in the absence of new
lesions, which may persist for 4 to 8 weeks, or 6 to 8 weeks.
[0403] Treatment according to the present invention includes a
"therapeutically effective amount" of the medicaments used. A
"therapeutically effective amount" refers to an amount effective,
at dosages and for periods of time necessary, to achieve a desired
therapeutic result.
[0404] A therapeutically effective amount may vary according to
factors such as the disease state, age, sex, and weight of the
individual, and the ability of the medicaments to elicit a desired
response in the individual. A therapeutically effective amount is
also one in which any toxic or detrimental effects of the antibody
or antibody portion are outweighed by the therapeutically
beneficial effects.
[0405] A "therapeutically effective amount" for tumor therapy may
also be measured by its ability to stabilize the progression of
disease. The ability of a compound to inhibit cancer may be
evaluated in an animal model system predictive of efficacy in human
tumors.
[0406] Alternatively, this property of a composition may be
evaluated by examining the ability of the compound to inhibit cell
growth or to induce apoptosis by in vitro assays known to the
skilled practitioner. A therapeutically effective amount of a
therapeutic compound may decrease tumor size, or otherwise
ameliorate symptoms in a subject. One of ordinary skill in the art
would be able to determine such amounts based on such factors as
the subject's size, the severity of the subject's symptoms, and the
particular composition or route of administration selected.
[0407] Dosage regimens are adjusted to provide the optimum desired
response (e.g., a therapeutic response). For example, a single
bolus may be administered, several divided doses may be
administered over time or the dose may be proportionally reduced or
increased as indicated by the exigencies of the therapeutic
situation. Parenteral compositions may be formulated in dosage unit
form for ease of administration and uniformity of dosage. Dosage
unit form as used herein refers to physically discrete units suited
as unitary dosages for the subjects to be treated; each unit
contains a predetermined quantity of active compound calculated to
produce the desired therapeutic effect in association with the
required pharmaceutical carrier.
[0408] The specification for the dosage unit forms of the present
invention are dictated by and directly dependent on (a) the unique
characteristics of the active compound and the particular
therapeutic effect to be achieved, and (b) the limitations inherent
in the art of compounding such an active compound for the treatment
of sensitivity in individuals.
[0409] The efficient dosages and the dosage regimens for the
multispecific antibodies used in the present invention depend on
the disease or condition to be treated and may be determined by the
persons skilled in the art.
[0410] An exemplary, non-limiting range for a therapeutically
effective amount of an multispecific antibody used in the present
invention is about 0.1-100 mg/kg, such as about 0.1-50 mg/kg, for
example about 0.1-20 mg/kg, such as about 0.1-10 mg/kg, for
instance about 0.5, about such as 0.3, about 1, or about 3 mg/kg.
In another embodiment, he antibody is administered in a dose of 1
mg/kg or more, such as a dose of from 1 to 20 mg/kg, e.g. a dose of
from 5 to 20 mg/kg, e.g. a dose of 8 mg/kg.
[0411] A medical professional having ordinary skill in the art may
readily determine and prescribe the effective amount of the
pharmaceutical composition required. For example, a physician or a
veterinarian could start doses of the medicament employed in the
pharmaceutical composition at levels lower than that required in
order to achieve the desired therapeutic effect and gradually
increase the dosage until the desired effect is achieved.
[0412] In one embodiment, the multispecific antibody is
administered by infusion in a weekly dosage of from 10 to 500 mg/kg
such as of from 200 to 400 mg/kg Such administration may be
repeated, e.g., 1 to 8 times, such as 3 to 5 times. The
administration may be performed by continuous infusion over a
period of from 2 to 24 hours, such as of from 2 to 12 hours.
[0413] In one embodiment, the multispecific antibody is
administered by slow continuous infusion over a long period, such
as more than 24 hours, if required to reduce side effects including
toxicity.
[0414] In one embodiment the multispecific antibody is administered
in a weekly dosage of from 250 mg to 2000 mg, such as for example
300 mg, 500 mg, 700 mg, 1000 mg, 1500 mg or 2000 mg, for up to 8
times, such as from 4 to 6 times. The administration may be
performed by continuous infusion over a period of from 2 to 24
hours, such as of from 2 to 12 hours. Such regimen may be repeated
one or more times as necessary, for example, after 6 months or 12
months. The dosage may be determined or adjusted by measuring the
amount of compound of the present invention in the blood upon
administration by for instance taking out a biological sample and
using anti-idiotypic antibodies which target the antigen binding
region of the multispecific antibody.
[0415] In a further embodiment, the multispecific antibody is
administered once weekly for 2 to 12 weeks, such as for 3 to 10
weeks, such as for 4 to 8 weeks.
[0416] In one embodiment, the multispecific antibody is
administered by maintenance therapy, such as, e.g., once a week for
a period of 6 months or more.
[0417] In one embodiment, the multispecific antibody is
administered by a regimen including one infusion of an
multispecific antibody followed by an infusion of an multispecific
antibody conjugated to a radioisotope. The regimen may be repeated,
e.g., 7 to 9 days later.
[0418] As non-limiting examples, treatment according to the present
invention may be provided as a daily dosage of an antibody in an
amount of about 0.1-100 mg/kg, such as 0.5, 0.9, 1.0, 1.1, 1.5, 2,
3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90
or 100 mg/kg, per day, on at least one of day 1, 2, 3, 4, 5, 6, 7,
8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,
25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40,
or alternatively, at least one of week 1, 2, 3, 4, 5, 6, 7, 8, 9,
10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 after initiation of
treatment, or any combination thereof, using single or divided
doses of every 24, 12, 8, 6, 4, or 2 hours, or any combination
thereof.
[0419] In some embodiments the multispecific antibody molecule
thereof is used in combination with one or more additional
therapeutic agents, e.g. a chemotherapeutic agent. Non-limiting
examples of DNA damaging chemotherapeutic agents include
topoisomerase I inhibitors (e.g., irinotecan, topotecan,
camptothecin and analogs or metabolites thereof, and doxorubicin);
topoisomerase II inhibitors (e.g., etoposide, teniposide, and
daunorubicin); alkylating agents (e.g., melphalan, chlorambucil,
busulfan, thiotepa, ifosfamide, carmustine, lomustine, semustine,
streptozocin, decarbazine, methotrexate, mitomycin C, and
cyclophosphamide); DNA intercalators (e.g., cisplatin, oxaliplatin,
and carboplatin); DNA intercalators and free radical generators
such as bleomycin; and nucleoside mimetics (e.g., 5-fluorouracil,
capecitibine, gemcitabine, fludarabine, cytarabine, mercaptopurine,
thioguanine, pentostatin, and hydroxyurea).
[0420] Chemotherapeutic agents that disrupt cell replication
include: paclitaxel, docetaxel, and related analogs; vincristine,
vinblastin, and related analogs; thalidomide, lenalidomide, and
related analogs (e.g., CC-5013 and CC-4047); protein tyrosine
kinase inhibitors (e.g., imatinib mesylate and gefitinib);
proteasome inhibitors (e.g., bortezomib); NF-KB inhibitors,
including inhibitors of IKB kinase; antibodies which bind to
proteins overexpressed in cancers and thereby downregulate cell
replication (e.g., trastuzumab, rituximab, cetuximab, and
bevacizumab); and other inhibitors of proteins or enzymes known to
be upregulated, overexpressed or activated in cancers, the
inhibition of which downregulates cell replication.
[0421] In some embodiments, the antibodies of the invention can be
used prior to, concurrent with, or after treatment with
Velcade.RTM. (bortezomib).
[0422] All cited references are herein expressly incorporated by
reference in their entirety.
[0423] Whereas particular embodiments of the invention have been
described above for purposes of illustration, it will be
appreciated by those skilled in the art that numerous variations of
the details may be made without departing from the invention as
described in the appended claims.
EXAMPLES
[0424] Examples are provided below are for illustrative purposes
only. These examples are not meant to constrain any embodiment
disclosed herein to any particular application or theory of
operation.
Example 1. Constructing Anti-CD4.times.Anti-CD25 Bispecific
Antibodies
[0425] A concept for suppressing Treg cells with
anti-CD4.times.anti-CD25 bispecifics while not affecting other T
cell types is shown schematically in FIG. 1.
[0426] The ability of various anti-CD25 heavy chains to pair with
anti-CD4 light chains and anti-CD4 heavy chains to pair with
anti-CD25 light chains in order to create a "common light-chain"
anti-CD4.times.CD25 bispecific antibody was evaluated. Desired gene
segments were synthesized by Blue Heron Biotechnologies (Bothell,
Wash.) from synthetic oligonucleotides and PCR products by
automated gene synthesis. Antibody constructs in the pTT5 vector
were expressed in 293E cells and purified by standard Protein A,
followed by IEX chromatography in order to isolate the desired
heterodimeric bispecific. Biacore was used to examine binding of
the various pairs to both CD4 and CD25 and the results tabulated
(FIG. 2). 100 nM of each variant was immobilized on a Protein A
chip for 1 min, followed by flowing antigen (CD4 or CD25) at 100 nM
for 2 min dissociation. As can be seen from the data, the HuMax-TAC
anti-CD25 heavy chain has the unique ability to pair with the
anti-CD4 lights chains of OKT4A and zanolimumab, with the
HuMax-TAC/OKT4A pair showing the strongest binding.
[0427] "Common light-chain" anti-CD4.times.CD25 bispecific
antibodies were constructed by co-transfecting (in 293E cells) DNA
encoding the heavy chain of anti-CD4 antibody OKT4A_H0L0 with the
heavy chain of anti-CD25 antibody HuMAX-Tac and the light chain of
anti-CD4 antibody OKTH0L0. These bispecific antibodies express well
and have biophysical properties equivalent to normal monovalent IgG
antibodies. Utilizing a heterodimeric Fc format, dual scFv-Fc
anti-CD4.times.CD25 bispecific antibodies were also constructed and
expressed. A third format, with a normal Fab-Fc on one side and
scFv-Fc on the other side was also constructed. Control "one-armed"
antibodies were also constructed to evaluate the effects of
monovalent antigen binding (e.g. Anti-CD4.times.empty-Fc or
Anti-CD25 by empty-Fc). For all three formats, variants with
different Fc regions were produced: IgG1, high ADCC (S239D/I332E),
and Fc knockout (G236R/L328R or PVA_/S267K). Bispecific formats are
shown schematically in FIG. 3. These bispecific antibody variants
were evaluated for the ability to simultaneously bind both CD4 and
CD25 on Biacore. 100 nM of each variant was bound to a CD25
surface, followed by flowing of 100 nM of CD4 over the chip
surface. An example of the data is shown in FIG. 4.
[0428] Although CD4 and CD25 antigens were initially targeted for
suppressing Tregs, other combinations of Treg markers may also be
used in accordance with the methods described herein, including
combinations listed in FIG. 32. Anti-CTLA4.times.Anti-CD25,
Anti-PD-1.times.Anti-CD25, and Anti-CCR4.times.Anti-CD25 bispecific
antibodies were also constructed. Any of the formats shown in FIG.
3 can be made to bind to any combinations of the targets listed in
FIG. 32.
Example 2. Suppression of Regulatory T Cells with
Anti-CD4.times.Anti-CD25 Bispecific Antibodies
[0429] Treg cells were generated in vitro using the following
method. CD4.sup.+ enriched T cells (isolated using the EasySep.TM.
Human CD4.sup.+ T Cell Enrichment Kit from Stemcell Technologies)
from PBMC were incubated with anti-CD3/anti-CD28 beads (20 .mu.l
beads in 100 .mu.l volume, or 4:1 beads to cell ratio using
Dynabeads.RTM. Regulatory CD4.sup.+CD25.sup.+ T Cell Kit) with 500
U/mL of IL2 in the presence of 0.1 .mu.g/ml rapamycin for a week.
Cells were replaced with new culture with anti-CD3 (OKT3,
eBiosciences) plate bound at 0.5 .mu.g/mL and soluble 0.5 .mu.g/mL
anti-CD28 (clone 28.2, eBiosciences) with 100 U/mL of IL2 and 0.1
.mu.g/mL rapamycin.
[0430] Proliferation of Treg cells was assayed using CFSE cell
proliferation assay or Alamar Blue cell viability assays in the
presence of bispecific or control antibodies with 15 U/mL IL2.
Results are shown in FIG. 5, FIG. 7, FIG. 12, FIG. 13, and FIG. 15.
Anti-CD4.times.Anti-CD25 bispecifics 11209 and 12143 (IgG1 and FcKO
Fc, respectively) were able to suppress proliferation of Treg cells
more strongly compared to anti-CD25 (6368) antibody alone, and no
effect was seen with anti-CD4 mAb (10966) alone. These results
demonstrate the increased suppression of Treg cells with avid
targeting using anti-CD4.times.anti-CD25 bispecific antibodies. The
Fv of OKT4A was also humanized (OKT4A_H1L1) using the method of
Lazar et al., 2007, Molecular Immunology, 44:1986-1998, hereby
incorporated by reference in its entirety for all purposes and in
particular for all teachings related to OKT4A, and this Fv was
tested (FIG. 12 and FIG. 15), along with bispecifics containing the
alternative anti-CD4 Fv Ibalizumab (FIG. 13). The epitopes of OKT4A
and Ibalizumab differ, with binding of OKT4A to CD4 expected to
block MHC II binding to CD4 whereas the epitope of Ibalizumab is
away from the MHC II binding site on CD4 and its binding is not
expected to be blocking. The precursor murine Fv of Ibalizumab
(5A8) was also humanized to generate 5A8_H1L1. Bispecifics with the
anti-CD4 Fv 5A8_H1L1 were also generated.
Example 3. Effect of Altering Antigen Binding Affinity of
Anti-CD4.times.Anti-CD25 Bispecific Antibodies
[0431] Variant bispecific antibodies and one-armed antibody
controls were constructed in which the CD25 binding affinity was
altered. The Anti-TAC_H1.8L1 Fv (in 13531 and 13532) has 6-fold
increased affinity for CD25. Conversely, the Anti-TAC_H1L1.12 Fv
(in 13533 and 13534) has 17-fold lower CD25 affinity. These
variants were assessed in cell proliferation assays (FIG. 15). A
clear correlation between CD25 affinity and potency can be seen.
13531 with increased CD25 affinity has the strongest inhibition of
cell proliferation, while lower affinity resulted in a reduced
effect on cell proliferation. A similar pattern is also expected if
CD4 affinity was altered. However, increasing the affinity for CD4
may result in even greater potency on Tregs due to its lower
expression level compared to CD25. This can be shown by lower
binding of anti-CD4 mAbs on Tregs compared to anti-CD25 mAbs (shown
in FIG. 8).
Example 4. Direct Binding of Anti-CD4.times.Anti-CD25 Bispecific
Antibodies to Tregs and Naive CD4+ T Cells
[0432] Binding of Anti-CD4.times.anti-CD25 bispecifics and control
antibodies was measured to Tregs, naive CD4+ T cells, and activated
CD4+ and CD8+ T cells. 200k Tregs were plated with antibodies at 4
.mu.g/mL (4.times. serial dilutions, 8 total dilutions). Cells and
Abs were incubated at 45 min on ice and then washed and stained
with secondary Ab anti-human F(ab)'2 Fcgamma specific PE labeled at
1 .mu.g/mL. Cells were washed and fixed with 1% PFA overnight and
data acquired on a FACS Canto II. Results are shown in FIGS. 8-11.
Bispecifics and anti-CD25 mAbs bound more strongly to Tregs
compared to anti-CD4 mAbs, indicating that there may be a higher
density of CD25 on Tregs compared to CD4. A clear avidity effect
was seen with the bispecifics. Direct binding to purified naive
human CD4+ T cells, activated CD4+, and activated CD8+ T cells was
also assessed in a similar manner. Results for these binding assays
are shown in FIG. 14 and FIGS. 16-22.
Example 5. Effect of Anti-CD4.times.Anti-CD25 Bispecific Antibodies
on Cell Proliferation of CD4+CD25+(Helper T Cells) and CD8+CD25
(Cytotoxic T Cells)
[0433] For suppression of Tregs, it is desirable to suppress Treg
cells and have little or no impact on other T cell types. To assess
the impact of Anti-CD4.times.Anti-CD25 bispecific antibodies on
other T cell types, CFSE labeled PBMC were incubated with 12.5
ng/mL anti-CD3 and 15 U/mL IL2 for 4 days in the presence of
bispecific or control antibodies. Results are shown in FIG. 6 and
FIG. 7. In this format, a clear dependence on Fc.gamma.R binding
ability is seen. 11209 - Anti-CD4.times.Anti-CD25 IgG1 causes
suppression of T-helper cells, while 12143 -
Anti-CD4.times.Anti-CD25 FcKO has a much reduced level of
suppression. Anti-CD25 (6368) antibody alone is also able to cause
suppression of this T cell type, while anti-CD4 mAb (10966) alone
shows limited activity (both are IgG1 Fc).
[0434] For cytotoxic T cells (CD8+CD25+), suppression was only seen
with 11209 - Anti-CD4.times.CD25 IgG1 and Anti-CD25 (6368). No
suppression was seen with 12143 - Anti-CD4.times.Anti-CD25 FcKO or
anti-CD4 mAb (10966). Again, a clear dependence on Fc.gamma.R
binding ability of the bispecifics is seen.
Example 6. Constructing Bispecific Anti-CD4.times.IL2
Fc-Fusions
[0435] The concept of inducing Treg cells with anti-CD4.times.IL2
Fc-fusions while not affecting other T cell types is shown
schematically in FIG. 24.
[0436] Anti-CD4.times.IL2 Fc-fusions were designed and constructed
from the sequences of human IL2 and the anti-CD4 antibody OKT4A
(FIG. 25). Constructs in the pTT5 vector were expressed in 293E
cells and purified using Protein A and IEX chromatography to
isolate the desired heterodimeric Fc-fusion. SEC and SDS-PAGE
analysis of the Protein A purified material as well as the final
IEX purified material are shown in FIG. 26. Anti-CD4.times.IL2
Fc-fusions were homogeneous and obtained in high purity. All
Fc-fusions were expressed with a Fc.gamma.R knocked out binding Fc
region (IgG1 G236R/L328R or IgG1 PVA_/S267K). Anti-CD4.times.IL2
Fc-fusions using the Anti-CD4 mAbs Ibalizumab and 5A8_H1L1 were
also constructed, as were Anti-CCR4.times.IL2,
Anti-CTLA4.times.IL2, and Anti-PD1.times.IL2 antibody Fc-fusions.
Bispecific IL2 Fc-fusions with antibodies against any of the Treg
markers listed in Table 1 could also be constructed. Alternatively,
similar variants may possess superior selectivity for Treg agonism
versus other T cell types.
Example 7. Induction of Regulatory T Cells (Tregs) by
Anti-CD4.times.IL2 Fc-Fusions
[0437] Treg cells were generated in vitro using the following
method. CD4.sup.+ enriched T cells (isolated using the EasySep.TM.
Human CD4+ T Cell Enrichment Kit from Stemcell Technologies) from
PBMC were incubated with anti-CD3/anti-CD28 beads (20 .mu.l beads
in 100 .mu.l volume, or 4:1 beads to cell ratio using
Dynabeads.RTM. Regulatory CD4.sup.+CD25.sup.+ T Cell Kit) with 500
U/mL of IL2 in the presence of 0.1 .mu.g/mL rapamycin for a week.
Cells were replaced with new culture with anti-CD3 (OKT3,
eBiosciences) plate bound at 0.5 .mu.g/mL and soluble 0.5 .mu.g/mL
anti-CD28 (clone 28.2, eBiosciences) with 100 U/mL of IL2 and 0.1
.mu.g/mL rapamycin.
[0438] Induction of Treg cells was assayed using the alamar blue
cell viability assay in the presence of anti-CD4.times.IL2
Fc-fusions or control antibodies. Results are shown in FIG. 27.
Increased viability of Treg cells was seen for the
anti-CD4.times.IL2 Fc-fusions as well as IL2-only Fc-fusions.
Anti-CD25 and anti-CD4 control antibodies showed no induction. The
IL2-only Fc fusion (13044) served as a proxy for the reduced level
of induction expected for cytotoxic T cells
(CD8.sup.+CD25.sup.+).
Example 8. Suppression or Induction of Regulatory T Cells Using
Anti-CD4.times.1L2 Fc-Fusions Engineered for Reduced or Increased
IL2-Receptor Signaling
[0439] IL2 are engineered in order to alter the ratio of induction
for Treg cells versus other types of IL2 receptor expressing cells
(i.e. NK cells). For example, a dominant-negative IL2 Fc-fusion is
created by engineering IL2 to have reduced ability to bind to
IL2R.beta., IL2R.gamma., and or IL2R.alpha. in order to ablate IL2
receptor signaling. When coupled with an anti-CD4 antibody (or
other Treg surface marker antibody), this results in an
anti-CD4.times.IL2 Fc-fusion capable of suppressing Treg cells
through targeted binding to CD4 and CD25, but without the ability
to induce Treg proliferation. This Fc-fusion blocks endogenous IL2
from binding to receptor.
[0440] Likewise, more potent Anti-CD4.times.IL2 Fc-fusions inducers
are engineered by increasing the affinity of IL2 for IL2R.alpha..
Exemplary variants of IL2 of use in the present invention are
listed in FIG. 23.
Example 9. Suppression and Induction of Cytotoxic T Cells with
Anti-CD8.times.Anti-CD25 Bispecific Antibodies or
Anti-CD8.times.IL2 Fc-Fusions
[0441] Anti-CD8 antibodies including MCD8, 3B5, SK1, OKT-8, 51.1 or
DK-25 are combined with an anti-CD25 antibody to make a bispecific
antibody for suppression of cytotoxic T cells. Alternatively, in
order to induce cytotoxic T cells, an Fc-fusion consisting of IL2
combined with an anti-CD8 antibody are used. Avidity may also drive
IL-2 activation by binding the low affinity (beta/gamma) IL-2
receptor, circumventing the requirement for CD25, thus also being
effective on non-activated CD8. Methods for suppression and
induction are shown schematically in FIG. 28 and FIG. 29. These
approaches are useful for treating cancer or autoimmune diseases,
respectively.
Example 10. Evaluation of Treg Suppressor and Inducer Variants in a
GVHD Mouse Model
[0442] Variants of the invention are evaluated in a
Graft-versus-Host Disease model conducted in NSG SCID mice such as
those conducted in Mutis et al., Clin Cancer Res (12), 2006. When
NSG SCID mice are injected with human PBMCs they develop an
autoimmune response against the human PBMCs, and this has been
shown to be Treg dependent. NSG SCID mice injected with human PBMCs
and then treated with a Treg suppression bispecific antibody such
as 12143, 12462, 13025, or 13529 will have an exacerbation of
disease and will die more quickly compared to untreated mice.
Conversely, mice can be given a Treg inducing bispecific IL2-Fc
fusion such as 13027 and they have a less severe disease and live
longer than untreated mice.
Example 11. Evaluation of Treg Suppressor Mouse Surrogate Variants
in Syngeneic Mouse Tumor Models
[0443] Mouse surrogate bispecific antibodies and IL2-Fc fusions can
be made and studied in syngeneic mouse tumor models. The Treg
suppressor bispecific Anti-mCD4.times.Anti-mCD25 can be made using
the Anti-mouse CD4 antibody GK1.5 and the Anti-mouse CD25 antibody
PC61. Tumors can be introduced in normal mice and then the mice
treated with surrogate bispecific antibody. Suppression of the
mouse Tregs should allow the mouse cytotoxic T cells to fight the
tumor, resulting in a decreased tumor volume.
Example 12. Evaluation of Treg Inducer Mouse Surrogate Variants in
an EAE Mouse Model
[0444] Mouse surrogate Treg inducer IL2-Fc fusion bispecifics can
be created by using human IL2 with an anti-mouse CD4 antibody such
as GK1.5. Human IL2 is known to bind to the mouse IL2 receptor.
Experimental autoimmune encephalomyelitis (EAE) is a mouse model of
autoimmunity. Mice can be induced for EAE and then treated with
mouse surrogate Anti-mCD4.times.IL2 bispecific Fc-fusions.
Induction of mouse Tregs should result in less severe disease.
[0445] All cited references are herein expressly incorporated by
reference in their entirety.
[0446] Whereas particular embodiments have been described above for
purposes of illustration, it will be appreciated by those skilled
in the art that numerous variations of the details may be made
without departing from the invention as described in the appended
claims.
Sequence CWU 0 SQTB SEQUENCE LISTING The patent application
contains a lengthy "Sequence Listing" section. A copy of the
"Sequence Listing" is available in electronic form from the USPTO
web site
(https://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20200339624A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
0 SQTB SEQUENCE LISTING The patent application contains a lengthy
"Sequence Listing" section. A copy of the "Sequence Listing" is
available in electronic form from the USPTO web site
(https://seqdata.uspto.gov/?pageRequest=docDetail&DocID=US20200339624A1).
An electronic copy of the "Sequence Listing" will also be available
from the USPTO upon request and payment of the fee set forth in 37
CFR 1.19(b)(3).
* * * * *
References