U.S. patent application number 17/494545 was filed with the patent office on 2022-02-17 for antibodies.
The applicant listed for this patent is GENMAB A/S. Invention is credited to Sieto BOSGRA, Esther C. W. BREIJ, Frederikke L. EGEROD, Patrick ENGELBERTS, Louise KOOPMAN, Rik RADEMAKER, David SATIJN, Edward N. VAN DEN BRINK, Dennis VERZIJL.
Application Number | 20220048999 17/494545 |
Document ID | / |
Family ID | |
Filed Date | 2022-02-17 |
United States Patent
Application |
20220048999 |
Kind Code |
A1 |
KOOPMAN; Louise ; et
al. |
February 17, 2022 |
ANTIBODIES
Abstract
The present invention relates to antibodies binding to B7H4,
including bispecific antibodies binding to B7H4 and CD3. The
invention further provides pharmaceutical compositions comprising
the antibodies and use of the antibodies for therapeutic and
diagnostic procedures, in particular in cancer therapy.
Inventors: |
KOOPMAN; Louise; (Utrecht,
NL) ; ENGELBERTS; Patrick; (Amersfoort, NL) ;
VERZIJL; Dennis; (Amstelveen, NL) ; VAN DEN BRINK;
Edward N.; (Halfweg, NL) ; RADEMAKER; Rik;
(Utrecht, NL) ; BOSGRA; Sieto; (Amsterdam, NL)
; EGEROD; Frederikke L.; (Copenhagen, DK) ;
SATIJN; David; (Nieuwegein, NL) ; BREIJ; Esther C.
W.; (Driebergen, NL) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GENMAB A/S |
Copenhagen V |
|
DK |
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Appl. No.: |
17/494545 |
Filed: |
October 5, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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17204604 |
Mar 17, 2021 |
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17494545 |
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International
Class: |
C07K 16/28 20060101
C07K016/28; A61P 35/00 20060101 A61P035/00 |
Foreign Application Data
Date |
Code |
Application Number |
Mar 18, 2020 |
EP |
20164059.6 |
Claims
1-78. (canceled)
79. An antibody comprising a first antigen-binding region which
binds to human B7H4, wherein the first antigen-binding region
comprises a variable heavy chain (VH) region comprising: a CDR1
comprising the sequence of SEQ ID NO: 26, a CDR2 comprising the
sequence of SEQ ID NO: 30, and a CDR3 comprising the sequence of
SEQ ID NO: 28 and a variable light chain (VL) region comprising: a
CDR1 comprising the sequence of SEQ ID NO: 34, a CDR2 comprising
the sequence GAS, and a CDR3 comprising the sequence of SEQ ID NO:
35, and said antibody further comprising a second antigen-binding
region which binds to human CD3.
80. An antibody comprising a first antigen-binding region which
binds to human B7H4, wherein the first antigen-binding region
comprises a variable heavy chain (VH) region comprising the
sequence of SEQ ID NO. 29 and a variable light chain (VL) region
comprising the sequence of SEQ ID NO. 33, said antibody further
comprising a second antigen-binding region which binds to human
CD3.
81. The antibody according to claim 79, wherein the second
antigen-binding region binds to human CD3.epsilon..
82. The antibody according to claim 79, wherein the second
antigen-binding region comprises a variable heavy chain (VH) region
comprising: a CDR1 comprising the sequence of SEQ ID NO: 18, a CDR2
comprising the sequence of SEQ ID NO: 19, and a CDR3 comprising the
sequence of SEQ ID NO: 20 or SEQ ID NO: 21, and a variable light
chain (VL) region comprising: a CDR1 comprising the sequence of SEQ
ID NO: 23, a CDR2 comprising the sequence GTN, and a CDR3
comprising the sequence of SEQ ID NO: 24.
83. The antibody according to claim 82, wherein the CDR3 of the VH
region of the second antigen-binding region comprises the sequence
of SEQ ID NO: 21.
84. The antibody according to claim 80, wherein the second
antigen-binding region comprises a heavy chain variable (VH) region
comprising the sequence of SEQ ID NO: 16 or 17 and a light chain
variable (VL) region comprising the sequence of SEQ ID NO: 22.
85. The antibody according to claim 84, wherein the second
antigen-binding region comprises a VH region comprising the
sequence of SEQ ID NO: 17.
86. The antibody according to claim 83, wherein the antibody is a
full-length antibody.
87. The antibody according to claim 85, wherein the antibody is a
full-length antibody.
88. The antibody according to claim 83, wherein the antibody
comprises a human IgG1 heavy chain constant region.
89. The antibody according to claim 85, wherein the antibody
comprises a human IgG1 heavy chain constant region.
90. The antibody according to claim 83, wherein said antibody
comprises a first heavy chain and a second heavy chain, wherein the
amino acid in the position corresponding to K409 in a human IgG1
heavy chain is R in said first heavy chain, and the amino acid in
the position corresponding to F405 in a human IgG1 heavy chain is L
in said second heavy chain, or vice versa, and wherein the amino
acid positions are numbered according to Eu numbering.
91. The antibody according to claim 85, wherein said antibody
comprises a first heavy chain and a second heavy chain, wherein the
amino acid in the position corresponding to K409 in a human IgG1
heavy chain is R in said first heavy chain, and the amino acid in
the position corresponding to F405 in a human IgG1 heavy chain is L
in said second heavy chain, or vice versa, and wherein the amino
acid positions are numbered according to Eu numbering.
92. The antibody according to claim 83, wherein the antibody
comprises a first heavy chain and a second heavy chain, and wherein
in both the first heavy chain and the second heavy chain, the amino
acid residues at the positions corresponding to positions L234,
L235 and D265 in a human IgG1 heavy chain according to Eu numbering
are F, E and A, respectively.
93. The antibody according to claim 85, wherein the antibody
comprises a first heavy chain and a second heavy chain, and wherein
in both the first heavy chain and the second heavy chain, the amino
acid residues at the positions corresponding to positions L234,
L235 and D265 in a human IgG1 heavy chain according to Eu numbering
are F, E and A, respectively.
94. The antibody according to claim 83, wherein said antibody
comprises a human kappa (.kappa.) light chain and/or a human lambda
(k) light chain.
95. The antibody according to claim 85, wherein said antibody
comprises a human kappa (.kappa.) light chain and/or a human lambda
(k) light chain.
96. The antibody according to claim 83, wherein said first antigen
binding region which binds to human B7H4 is comprised in a first
heavy chain and a first light chain, said first heavy chain
comprising said VH region and a human IgG1 heavy chain constant
region and said first light chain comprising said VL region and a
human kappa light chain constant region; and wherein said second
antigen binding region which binds to human CD3 is comprised in a
second heavy chain and a second light chain, said second heavy
chain comprising said VH region and a human IgG1 heavy chain
constant region and said second light chain comprising said VL
region and a human lambda light chain constant region.
97. The antibody according to claim 85, wherein said first antigen
binding region which binds to human B7H4 is comprised in a first
heavy chain and a first light chain, said first heavy chain
comprising said VH region and a human IgG1 heavy chain constant
region and said first light chain comprising said VL region and a
human kappa light chain constant region; and wherein said second
antigen binding region which binds to human CD3 is comprised in a
second heavy chain and a second light chain, said second heavy
chain comprising said VH region and a human IgG1 heavy chain
constant region and said second light chain comprising said VL
region and a human lambda light chain constant region.
98. The antibody according to claim 96, wherein said first IgG1
heavy chain constant region comprises the sequence of SEQ ID NO. 61
and said second IgG1 heavy chain constant region comprises the
sequence of SEQ ID NO. 60, and wherein said kappa light chain
constant region comprises the sequence of SEQ ID NO. 63 and said
lambda light chain constant region comprises the sequence of SEQ ID
NO. 64.
99. The antibody according to claim 97, wherein said first IgG1
heavy chain constant region comprises the sequence of SEQ ID NO. 61
and said second IgG1 heavy chain constant region comprises the
sequence of SEQ ID NO. 60, and wherein said kappa light chain
constant region comprises the sequence of SEQ ID NO. 63 and said
lambda light chain constant region comprises the sequence of SEQ ID
NO. 64.
100. The antibody according to claim 98, wherein at least one of
said IgG1 heavy chain constant regions lacks the C-terminal
lysine.
101. The antibody according to claim 99, wherein at least one of
said IgG1 heavy chain constant regions lacks the C-terminal
lysine.
102. A bispecific antibody comprising a first antigen-binding
region which binds to human B7H4, wherein the first antigen-binding
region comprises a variable heavy chain (VH) region comprising: a
CDR1 comprising the sequence of SEQ ID NO: 26, a CDR2 comprising
the sequence of SEQ ID NO: 30, and a CDR3 comprising the sequence
of SEQ ID NO: 28 and a variable light chain (VL) region comprising:
a CDR1 comprising the sequence of SEQ ID NO: 34, a CDR2 comprising
the sequence GAS, and a CDR3 comprising the sequence of SEQ ID NO:
35, and said bispecific antibody further comprising a second
antigen-binding region which binds to human CD3, wherein the second
antigen-binding region comprises a VH region comprising: a CDR1
comprising the sequence of SEQ ID NO: 18, a CDR2 comprising the
sequence of SEQ ID NO: 19, and a CDR3 comprising the sequence of
SEQ ID NO: 21 and a VL region comprising: a CDR1 comprising the
sequence of SEQ ID NO: 23, a CDR2 comprising the sequence GTN, and
a CDR3 comprising the sequence of SEQ ID NO: 24.
103. A bispecific antibody comprising a first antigen-binding
region which binds to human B7H4, wherein the first antigen-binding
region comprises a variable heavy chain region comprising the
sequence of SEQ ID NO. 29 and a variable light chain region
comprising the sequence of SEQ ID NO. 33, said bispecific antibody
further comprising a second antigen-binding region which binds to
human CD3, wherein the second antigen-binding region comprises a
variable heavy chain region comprising the sequence of SEQ ID NO.
17 and a variable light chain region comprising the sequence of SEQ
ID NO. 22.
104. The bispecific antibody of claim 102, wherein the antibody
further comprises a first heavy chain constant region and a second
heavy chain constant region and a first light chain constant region
and a second light chain constant region, wherein said first heavy
chain constant region comprises the sequence of SEQ ID NO. 61 and
said second heavy chain constant region comprises the sequence of
SEQ ID NO. 60, and wherein said first light chain constant region
comprises the sequence of SEQ ID NO. 63 and said second light chain
constant region comprises the sequence of SEQ ID NO. 64.
105. The bispecific antibody of claim 103, wherein the antibody
further comprises a first heavy chain constant region and a second
heavy chain constant region and a first light chain constant region
and a second light chain constant region, wherein said first heavy
chain constant region comprises the sequence of SEQ ID NO. 61 and
said second heavy chain constant region comprises the sequence of
SEQ ID NO. 60, and wherein said first light chain constant region
comprises the sequence of SEQ ID NO. 63 and said second light chain
constant region comprises the sequence of SEQ ID NO. 64.
106. The bispecific antibody of claim 104, wherein at least one of
the heavy chain constant regions lacks the C-terminal lysine.
107. The bispecific antibody of claim 105, wherein at least one of
the heavy chain constant regions lacks the C-terminal lysine.
108. The bispecific antibody of claim 107, wherein both heavy chain
constant regions lack the C-terminal lysine.
Description
RELATED APPLICATIONS
[0001] This application is a division of U.S. application Ser. No.
17/204,604, filed Mar. 17, 2021, which claims priority to European
Patent Application No. 20164059.6, filed Mar. 18, 2020. The content
of the aforementioned applications are hereby incorporated by
reference.
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 Oct. 5, 2021, is named GMI_204DV_Sequence_Listing.txt and is
78,207 bytes in size.
FIELD OF INVENTION
[0003] The present invention relates to antibodies binding to B7H4,
in particular to bispecific antibodies binding to B7H4 and CD3. The
invention further provides pharmaceutical compositions comprising
the antibodies and use of the antibodies for therapeutic and
diagnostic procedures, in particular in cancer therapy.
INTRODUCTION
[0004] B7H4 (B7-H4, V-set domain containing T cell activation
inhibitor 1 or VTCN1) is a member of the B7 family of proteins,
which family comprises cell-surface protein ligands that bind to
receptors on lymphocytes. The B7 family plays an important role in
the regulation of immune responses. B7H4 negatively regulates T
cell-mediated immune responses by inhibiting T cell activation,
proliferation, cytokine production and cytotoxic activity (Prasad
et al., 2003, Immunity 18: 863-873). B7H4 is a type I transmembrane
protein that includes a short intracellular domain, a hydrophobic
transmembrane domain, and an extracellular domain with an IgV- and
an IgC-like domain with four conserved cysteine residues and seven
sites for N-linked glycosylation. (Sica et al., 2003, Immunity 18:
849-861). To date, no receptor for B7H4 has been identified.
[0005] In normal adult tissue, B7H4 expression is very limited,
whereas B7H4 expression is found on tumor cells in numerous cancer
tissues (Kaur and Janakiram, 2019, ESMO Open 4:e000554). In cancer,
B7H4 expression is correlated with advanced stages of cancer, poor
prognosis, and decreased overall patient survival.
[0006] Hence, targeting of B7H4 has been proposed for the treatment
of cancer (Podojil and Miller, Immunological Reviews, 2017: 276;
40-51). Currently, B7H4 binding antibodies are in development for
cancer therapy. For example, FPA150 is an afucosylated human
antibody that relieves the B7H4-mediated suppression of T cell
activation and exhibits antibody dependent cellular cytotoxicity
(ADCC) activity (Wainberg et al., 2019, Annals of Oncology 30,
Suppl. 5, v 489 (1198P). It is currently in early clinical trials
as a monotherapy or in combination with pembrolizumab in advanced
solid tumors.
[0007] Efforts to target T cells to B7H4 have also been made. A
B7H4/CD3-bispecific single chain antibody, Fab scFv, was made based
on the Fab and single-chain variable fragments (scFv) structure of
a mouse anti-human B7H4 antibody and a mouse anti-human CD3
antibody (Iizuka et al., 2019, Clin Cancer Res 25: 2925-2934).
Smith et al. have described engineered T cells with B7H4-specific
chimeric antigen receptors (CARs) that displayed anti-tumor
activity against B7H4-positive human ovarian tumor xenografts, but
which also showed multi-organ lymphocytic infiltration and lethal
toxicity (Smith et al. 2016, Molecular Therapy, Vol. 24 Iss. 11 pp
1987-99) in mice.
[0008] While there has been some progress made, there is a need for
the development of antibody-based cancer therapy targeting B7H4
that is efficacious and/or safe for human use.
SUMMARY OF INVENTION
[0009] It is an object of the present invention to provide for
antibodies comprising an antigen-binding region capable of binding
to human B7H4 and an antigen-binding region that binds to CD3, such
as human CD3.epsilon. (epsilon). The antigen-binding regions of
such antibodies comprise at least human framework regions, such as
e.g FR1, FR2, FR3 and FR4. Most preferred is that all framework
regions are human. Such antigen-binding regions are humanized
and/or human antigen-binding regions. These antibodies are useful
in the treatment of conditions wherein specific targeting and T
cell mediated killing of B7H4 expressing cells is desired, e.g. in
conditions such as cancer. Preferably, such an antibody is suitable
for human use, e.g. in a medical treatment. Cancers that may be
suitable for treatment are solid tumors. Said B7H4 expression, and
T cell mediated killing, e.g. in cancer cells, may range in
accordance with the invention from relatively low expression of
B7H4, such as in MCF-7 cells, to relatively high expression of
B7H4, such as in SK-BR3 cells, as shown e.g. in example 12. More
preferably such bispecific antibodies have substitutions within the
constant region that renders the Fc region, if present, inert.
[0010] In one embodiment, a bispecific antibody is provided
comprising an antigen-binding region capable of binding to human
B7H4 and an antigen-binding region that is capable of binding to
CD3, such as human CD3.epsilon. (epsilon), wherein the
antigen-binding region capable of binding to human B7H4 comprises a
variable heavy chain (VH) region comprising the CDR1, CDR2 and CDR3
regions of SEQ ID NO. 25, 29 or 31, and a variable light chain
region comprising the CDR1, CDR2 and CDR3 of SEQ ID NO. 33 and
wherein the antigen-binding region that is capable of binding to
CD3 comprises a heavy chain variable region (VH) comprising the
CDR1, CDR2, and CDR3 sequences of 18, 19 and 21 respectively; and,
a light chain variable region (VL) comprising the CDR1, CDR2, and
CDR3 sequences of SEQ ID NO: 23, GTN and 24, respectively.
[0011] In another aspect, nucleic acids, such as DNA or RNA, are
provided encoding antibodies as defined herein, as well as methods
of producing the antibodies, or components thereof, as defined
herein.
[0012] In a further aspect, said antibodies, or nucleic acids, in
accordance with the invention are for use in a medical
treatment.
BRIEF DESCRIPTION OF THE FIGURES
[0013] FIGS. 1A-1D. Determination of B7H4 domain involved in
binding using B7H4-B7H3 chimeric molecules. The B7H4 domain
specificity of the B7H4 antibodies was determined using a panel of
cells transfected to express human B7H4 (FIG. 1A), human B7H4-B7H3
chimeric molecules B7H3-IgV/B7H4-IgC (FIG. 1B) or B7H4-IgV/B7H3-IgC
(FIG. 1C), or human B7H3 (FIG. 1D). Binding was determined by flow
cytometry. I=bsIgG1-huCD3-FEALxB7H4-C4-FEAR;
II=bsIgG1-huCD3-FEALxB7H4-C3-FEAR;
III=bsIgG1-huCD3-FEALxB7H4-C2-FEAR;
IV=bsIgG1-huCD3-FEALxB7H4-C1-FEAR; V=IgG1-B7H3-BRCA84D.
[0014] FIG. 2. Binding of B7H4 antibodies to B7H4, B7H3 or
B7H4-B7H3 chimeric molecules. Binding of
bsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR,
bsIgG1-huCD3-H101G-FEALxB7H4-C2-FEAR,
bsIgG1-huCD3-H101G-FEALxB7H4-C3-FEAR,
bsIgG1-huCD3-H101G-FEALxB7H4-C4-FEAR and
bsIgG1-huCD3-H101G-FEALxB7H4-C5-FEAR to HEK cells transiently
transfected to express human B7H4 or the B7H4-B7H3 chimeric
molecules B7H3-IgV/B7H4-IgC or B7H4-IgV/B7H3-IgC was assessed using
flow cytometry.
[0015] FIGS. 3A-3C. Binding of B7H4 antibodies to B7H4 variants
with alanine mutations in the ECD. Binding was expressed as fold
change compared to a reference antibody. Fold change was defined as
Log 10(Normalized gMFI[ala mutant]/Normalized gMFI[wt]). Residues
where the Fold Change in binding was lower than mean Fold
Change--1.5.times.SD were considered `loss of binding mutants`.
Residues with a positive Fold Change in binding are loss of binding
residues for the reference antibody. Numbers below the x-axis refer
to amino acid positions. (FIG. 3A) Results for C1-N52S, with C2 as
reference antibody. (FIG. 3B) Results for C2, with C1-N52S as
reference antibody. (FIG. 3C) Results for C3, with C2 as reference
antibody.
[0016] FIG. 4. Binding of B7H4 antibody and CD3xB7H4 bispecific
antibody to human and cynomolgus monkey B7H4. Binding of
IgG1-B7H4-C1-N52S-FEAR (I) and
bsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR (II) to HEK-293F cells
transiently transfected with human B7H4 or cynomolgus monkey B7H4
was determined by flow cytometry. Non-transfected HEK-293F cells
(III) were used as negative control; for these binding of
bsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR is shown.
[0017] FIG. 5. Binding of B7H4 antibody and CD3xB7H4 bispecific
antibody to B7H4 from rabbit, rat, mouse, dog and pig. Binding of
IgG1-B7H4-C1-N52S-FEAR (I) and
bsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR (II) to HEK-293F cells
transiently transfected with B7H4 from rabbit, rat, mouse, dog or
pig was determined by flow cytometry. Non-transfected HEK-293F
cells (III) were used as negative control; for these binding of
bsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR is shown.
[0018] FIG. 6. Binding of B7H4 antibodies to HEK-293F cells
transiently transfected with B7H4 from different species. Binding
of IgG1-B7H4-C1-N52S-FEAR (I), IgG1-B7H4-C3-FEAR (II),
IgG1-B7H4-C2-FEAR (III), IgG1-B7H4-C4-FEAR (IV), and
IgG1-B7H4-C5-FEAR (V) to HEK-293F cells transfected with B7H4 from
human, cynomolgus monkey, mouse, rat or pig, or to untransfected
HEK-293F cells, was determined by flow cytometry. IgG1-b12 was used
as non-binding control antibody (not shown).
[0019] FIG. 7. Binding of IgG1-B7H4-C1-N52S-FEAR and
bsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR to MCF-7 and MDA-MB-468
cells. Binding was determined by flow cytometry. I:
IgG1-B7H4-C1-N52S-FEAR. II:
bsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR. IgG1-b12 (III) and
bsIgG1-huCD3-H101G-FEALxb12-FEAR (IV) were used as non-binding
control antibodies.
[0020] FIG. 8. Binding of bsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR
to NIH-OVCAR-3, HCC1954 and HeLa cells. Binding was determined by
flow cytometry. I: bsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR.
BsIgG1-huCD3-H101G-FEALxb12-FEAR (II) was used as non-binding
control antibody.
[0021] FIG. 9. Binding of bsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR
and bsIgG1-huCD3-FEALxB7H4-C1-N52S-FEAR to SK-BR3 and MDA-MB-486
cells. Binding was determined by flow cytometry. I:
bsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR. II:
bsIgG1-huCD3-FEALxB7H4-C1-N52S-FEAR. bsIgG1-huCD3-FEALxb12-FEAR
(III) and bsIgG1-huCD3-H101G-FEALxb12-FEAR (IV) were used as
non-binding control antibodies.
[0022] FIG. 10 Binding of various B7H4 antibodies in homodimer and
bsAb format to MDA-MB-486 and HCC1954 cells. Binding of
IgG1-B7H4-C1-N52S-FEAR (I homodimer), IgG1-B7H4-C2-FEAR (II
homodimer), IgG1-B7H4-C3-FEAR (III homodimer), IgG1-B7H4-C4-FEAR
(IV homodimer), IgG1-B7H4-C5-FEAR (V homodimer),
bsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR (I bsAb),
bsIgG1-huCD3-FEALxB7H4-C2-FEAR [MDA-MB-468] or
bsIgG1-huCD3-H101G-FEALxB7H4-C2-FEAR [HCC1954] (II bsAb),
bsIgG1-huCD3-H101G-FEALxB7H4-C3-FEAR (III bsAb),
bsIgG1-huCD3-H101G-FEALxB7H4-C4-FEAR (IV bsAb), and
bsIgG1-huCD3-H101G-FEALxB7H4-C5-FEAR (V bsAb) was determined by
flow cytometry. bsIgG1-huCD3-H101G-FEALxb12-FEAR (VI bsAb) or
IgG1-b12-K409R (VI homodimer) was used as a non-binding control
antibody.
[0023] FIG. 11. Induction of T cell mediated cytotoxicity of SK-BR3
cells in vitro by CD3xB7H4 bispecific antibodies using purified T
cells as effector cells at varying effector to target ratios (E:T).
bsIgG1-huCD3-FEALxb12-FEAR was used as non-binding control
antibody. I=bsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR;
II=bsIgG1-huCD3-FEALxB7H4-C1-N52S-FEAR;
III=bsIgG1-huCD3-FEALxb12-FEAR.
[0024] FIG. 12. Induction of T cell mediated cytotoxicity in
various tumor cell lines in vitro in the presence of CD3xB7H4
bispecific antibodies with different CD3 arms.
bsIgG1-huCD3-FEALxb12-FEAR was used as non-binding control
antibody. I=bsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR;
II=bsIgG1-huCD3-FEALxB7H4-C1-N52S-FEAR;
III=bsIgG1-huCD3-FEALxb12-FEAR,
IV=bsIgG1-huCD3-H101G-FEALxb12-FEAR.
[0025] FIGS. 13A and 13B. B7H4 expression levels and IC50 of T
cell-mediated tumor cell killing. (FIG. 13A) Quantitative flow
cytometric analysis of B7H4 expression levels on tumor cell lines.
Shown are individual measurements (dots), geometric means (bars)
and standard deviation (error bars). sABC=specific antibody binding
capacity. (FIG. 13B) IC50 of T cell-mediated tumor cell killing in
the presence of bsIgG1-huCD3-FEALxB7H4-C1-N52S-FEAR (I) or
bsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR (II) for the different
tumor cell lines. Each dot represents an experiment performed with
an individual T cell donor (4-6 donors per cell line), horizontal
lines indicate median. Cell lines are ranked according to B7H4
expression level.
[0026] FIGS. 14A and 14B. T cell activation by B7H4 bispecific
antibodies in T cell-tumor cell co-cultures. (FIG. 14A) T cell
activation (% of CD69 on CD8+ cells) in the presence of
bsIgG1-huCD3-FEALxB7H4-C1-N52S-FEAR (I) or
bsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR (II) for various
B7H4-positive tumor cell lines, determined by flow cytometry. (FIG.
14B) EC50 of T cell activation, using T cells derived from 3-5
donors, for each of the target cell lines. Each dot represents an
experiment performed with an individual T cell donor; horizontal
lines indicate geometric mean.
[0027] FIG. 15. IFN.gamma. in the supernatant of T cell-tumor cell
co-cultures at EC50, EC90 and EC99 for
bsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR and
bsIgG1-huCD3-FEALxB7H4-C1-N52S-FEAR using T cells from 3-4 donors,
determined by a multiplex U-plex assay. Shown are individual
measurements (dots), geometric means (horizontal lines) and
standard deviation (error bars). I:
bsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR. II:
bsIgG1-huCD3-FEALxB7H4-C1-N52S-FEAR.
[0028] FIGS. 16A and 16B. IL-6 and MCP-1 levels in the plasma of
cynomolgus monkeys treated with single dose IV infusion of
bsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR (FIG. 16A) or
bsIgG1-huCD3-FEALxB7H4-C1-N52S-FEAR (FIG. 16B).
[0029] FIG. 17. Mean plasma concentration-time profiles following a
single IV infusion of bsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR or
bsIgG1-huCD3-FEALxB7H4-C1-N52S-FEAR. I:
bsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR. II:
bsIgG1-huCD3-FEALxB7H4-C1-N52S-FEAR.
[0030] FIG. 18. B7H4 mRNA expression levels in a range of primary
solid tumors. B7H4 mRNA levels were extracted from the Omicsoft
TCGA database and visualized using Oncoland software. Indications
are ranked according to median of the B7H4 mRNA expression.
THYM=thymoma, UVM=uveal melanoma, PCPG=pheochromocytoma and
paraganglioma, ACC=adrenocortical carcinoma, MESO=mesothelioma,
SKCM=skin cutaneous melanoma, READ=rectum adenocarcinoma,
COAD=colon adenocarcinoma, GMB=glioblastoma multiforme,
SARC=sarcoma, LIHC=liver hepatocellular carcinoma, LGG=brain lower
grade glioma, KIRC=kidney renal clear cell carcinoma,
TGCT=testicular germ cell tumors, KICH=kidney chromophobe,
STAD=stomach adenocarcinoma, THCA=thyroid carcinoma, HNSC=head and
neck squamous cell carcinoma, PRAD=prostate adenocarcinoma,
LUAD=lung adenocarcinoma, ESCA=esophageal carcinoma, CESC=cervical
squamous cell carcinoma and endocervical adenocarcinoma,
KIRP=kidney renal papillary cell carcinoma, UCS=uterine
carcinosarcoma, BLCA=bladder urothelial carcinoma, PAAD=pancreatic
adenocarcinoma, LUSC=lung squamous cell carcinoma, BRCA=breast
invasive carcinoma, UCEC=uterine corpus endometrial carcinoma,
OV=ovarian serous cystadenocarcinoma and
CHOL=cholangiocarcinoma.
TABLE-US-00001 [0031] TABLE 1 Amino acid and nucleic acid sequence
SEQ ID NO: Reference Domain Sequence 1 Human B7H4 ORF
MASLGQILFWSIISIIIILAGAIALIIGFGISGRHSITVTTVAS
AGNIGEDGILSCTFEPDIKLSDIVIQWLKEGVLGLVHEFK
EGKDELSEQDEMFRGRTAVFADQVIVGNASLRLKNVQ
LTDAGTYKCYIITSKGKGNANLEYKTGAFSMPEVNVDY
NASSETLRCEAPRWFPQPTVVWASQVDQGANFSEVS
NTSFELNSENVTMKVVSVLYNVTINNTYSCMIENDIAK
ATGDIKVTESEIKRRSHLQLLNSKASLCVSSFFAISWALLP LSPYLMLK 2 Macaca ORF
MASLGQILFWSIISIIFILAGAIALIIGFGISGRHSITVTTVA fascicularis
SAGNIGEDGILSCTFEPDIKLSDIVIQWLKEGVIGLVHEF B7H4
KEGKDELSEQDEMFRGRTAVFADQVIVGNASLRLKNV transcript 1
QLTDAGTYKCYIITSKGKGNANLEYKTGAFSMPEVNVD
YNASSETLRCEAPRWFPQPTVVWASQVDQGANFSEV
SNTSFELNSENVTMKVVSVLYNVTINNTYSCMIENDIAK
ATGDIKVTESEIKRRSHLQLLNSKASLCVSSFLAISWALLP LAPYLMLK 3 Canis ORF
MASPGQNIFWSIISVIIILAGAIALIIGFGISGRHSITVTTLT familiaris
SAGNIGEDGILSCTFEPDIKLSDIVIQWLKEGVMGLVHE B7H4
FKEGKDDLSDQDEMFRGRTAVFADQVIGGNASLRLKN
VQLTDAGTYKCYIITSKGKGNANLEYKTGAFSIPEVNVD
YNASSENLRCEAPRWFPQPTVVWASQADQGANFSEV
FNTSFELNSENVTMKVVSVLYNVTINNTYSCMIENDIAK
ATGDIKVTDSEIKRRSHLQLLNSKASLGVSSFFAISWVLL PLSSYLMLK 4 Oryctolagus
ORF MASLGQIIFWSIISIIIILAGAIALIIGFGISGRHSITVTTLTS cuniculus
AGNIGEDGILSCTFEPDIRLSDIVIQWLKEGVVGLVHEFK B7H4
EGKDDLSDQDEMFRGRTAVFTDQVIVGNASLRLKNVQ
LTDAGTYKCYIITSKGKGNANLEYKTGAFSMPEVNLDY
NASSESLRCEAPRWFPQPTVVWASQVDQGANFSEVS
NTSFELNSENVTMKVVSVLYNVTVNNTYSCMIENDIAK
ATGDIKVTDSEIKRRSSLQLLNSRAAPSVSPRSAVGWLL LPLSSYVMLK 5 Rattus ORF
MASLGQIIFWSIINVIIILAGAIVLIIGFGISGKHFITVTTFT norvegicus
SAGNIGEDGTLSCTFEPDIKLNGIVIQWLKEGIKGLVHEF B7H4
KEGKDDLSQQHEMFRGRTAVFADQVVVGNASLRLKN
VQLTDAGTYTCYIHTSKGKGNANLEYKTGAFSMPEINV
DYNASSESLRCEAPRWFPQPTVAWASQVDQGANFSE
VSNTSFELNSENVTMKVVSVLYNVTINNTYSCMIENDIA
KATGDIKVTDSEVKRRSQLELLNSGPSPCVSSVSAAGW ALLSLSCCLMLR 6 Mus ORF
MASLGQIIFWSIINIIIILAGAIALIIGFGISGKHFITVTTFTS musculus
AGNIGEDGTLSCTFEPDIKLNGIVIQWLKEGIKGLVHEFK B7H4
EGKDDLSQQHEMFRGRTAVFADQVVVGNASLRLKNV
QLTDAGTYTCYIRTSKGKGNANLEYKTGAFSMPEINVD
YNASSESLRCEAPRWFPQPTVAWASQVDQGANFSEV
SNTSFELNSENVTMKVVSVLYNVTINNTYSCMIENDIAK
ATGDIKVTDSEVKRRSQLQLLNSGPSPCVFSSAFVAGW ALLSLSCCLMLR 7 Sus scrofa
ORF MASLGQVVFWSIISIIIILAGAIAFIIGFGISGRHSITVTTLT B7H4
SAGNIGEDGILSCTFEPDIKLSDIVIQWLKEGVTGLVHEF
KKGKDDLSDQDEMFRGRTAVFADQVIVGNASLRLKNV
QLTDAGTYKCYIITSKGKGNAKLEYKTGAFSIPEVNVDS
NASSESLRCEAPRWFPQPTVVWASQVDQGANFSEVS
NTSFELNPENVTKVVSVLYNVTINTTYSCMIENDIAKA
TGDIRVTDSEIKRQSHLQLLNSKASLCLSSFVAISWVLLP LCPYLMLK 8 Kozak GCCGCCACC
9 B7H3 ORF MLRRRGSPGMGVHVGAALGALWFCLTGALEVQVPED
PVVALVGTDATLCCSFSPEPGFSLAQLNLIWQLTDTKQL
VHSFAEGQDQGSAYANRTALFPDLLAQGNASLRLQRV
RVADEGSFTCFVSIRDFGSAAVSLQVAAPYSKPSMTLEP
NKDLRPGDIVTITCSSYQGYPEAEVFWQDGQGVPLTG
NVTTSQMANEQGLFDVHSILRVVLGANGTYSCLVRNP
VLQQDAHSSVTITPQRSPTGAVEVQVPEDPVVALVGT
DATLRCSFSPEPGFSLAQLNLIWQLTDTKQLVHSFTEGR
DQGSAYANRTALFPDLLAQGNASLRLQRVRVADEGSF
TCFVSIRDFGSAAVSLQVAAPYSKPSMTLEPNKDLRPG
DTVTITCSSYRGYPEAEVFWQDGQGVPLTGNVTTSQM
ANEQGLFDVHSVLRVVLGANGTYSCLVRNPVLQQDAH
GSVTITGQPMTFPPEALWVTVGLSVCLIALLVALAFVC
WRKIKQSCEEENAGAEDQDGEGEGSKTALQPLKHSDS KEDDGQEIA 10 B7H4- ORF
MASLGQILFWSIISIIIILAGAIALIIGFGISGRHSITVTTVAS IgV/B7H3-
AGNIGEDGILSCTFEPDIKLSDIVIQWLKEGVLGLVHEFK IgC
EGKDELSEQDEMFRGRTAVFADQVIVGNASLRLKNVQ
LTDAGTYKCYIITSKGKGNANLEYKTGAPYSKPSMTLEP
NKDLRPGDIVTITCSSYRGYPEAEVFWQDGQGVPLTG
NVTTSQMANEQGLFDVHSVLRVVLGANGTYSCLVRNP
VLQQDAHGSVTITGQPMTFPPEALWVTVGLSVCLIALL
VALAFVCWRKIKQSCEEENAGAEDQDGEGEGSKTALQ PLKHSDSKEDDGQEIA 11 B7H3- ORF
MLRRRGSPGMGVHVGAALGALWFCLTGALEVQVPED IgV/B7H4-
PVVALVGTDATLCCSFSPEPGFSLAQLNLIWQLTDTKQL IgC
VHSFAEGQDQGSAYANRTALFPDLLAQGNASLRLQRV
RVADEGSFTCFVSIRDFGSAAVSLQVAAFSMPEVNVDY
NASSETLRCEAPRWFPQPTVVWASQVDQGANFSEVS
NTSFELNSENVTMKVVSVLYNVTINNTYSCMIENDIAK ATGDIKVTESEI
KRRSHLQLLNSKASLCVSSFFAISWALLP LSPYLMLK 12 B7H4ECD- Mature
LIIGFGISGRHSITVTTVASAGNIGEDGILSCTFEPDIKLSD FcHisC protein
IVIQWLKEGVLGLVHEFKEGKDELSEQDEMFRGRTAVF
ADQVIVGNASLRLKNVQLTDAGTYKCYIITSKGKGNAN
LEYKTGAFSMPEVNVDYNASSETLRCEAPRWFPQPTV
VWASQVDQGANFSEVSNTSFELNSENVTMKVVSVLY
NVTINNTYSCMIENDIAKATGDIKVTESEIKRRSHLQLLN
SKASIEGRMDPKSCDKTHTCPPCPAPEAEGAPSVFLFPP
KPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGV
EVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEY
KCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEM
TKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTAPP
VLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALH NHYTQKSLSLSPGKHHHHHHHHEPEA 13
Mature Mature QDGNEEMGGITQTPYKVSISGTTVILTCPQYPGSEILW Human
CD3.epsilon. protein QHNDKNIGGDEDDKNIGSDEDHLSLKEFSELEQSGYYV
(epsilon) CYPRGSKPEDANFYLYLRARVCENCMEMDVMSVATIV
IVDICITGGLLLLVYYWSKNRKAKAKPVTRGAGAGGRQ
RGQNKERPPPVPNPDYEPIRKGQRDLYSGLNQRRI 14 b12_VH VH
QVQLVQSGAEVKKPGASVKVSCQASGYRFSNFVIHWV
RQAPGQRFEWMGWINPYNGNKEFSAKFQDRVTFTA
DTSANTAYMELRSLRSADTAVYYCARVGPYSWDDSPQ DNYYMDVWGKGTTVIVSS 15 b12_VL
VL EIVLTQSPGTLSLSPGERATFSCRSSHSIRSRRVAWYQH
KPGQAPRLVIHGVSNRASGISDRFSGSGSGTDFTLTITR
VEPEDFALYYCQVYGASSYTFGQGTKLERK 16 VH_huCD3- VH
EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWV H1L1
RQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRD
DSKSSLYLQMNNLKTEDTAMYYCVRHGNFGNSYVSW FAYWGQGTLVTVSS 17 VH_huCD3- VH
EVKLVESGGGLVQPGGSLRLSCAASGFTFNTYAMNWV H1L1-H101G
RQAPGKGLEWVARIRSKYNNYATYYADSVKDRFTISRD
DSKSSLYLQMNNLKTEDTAMYYCVRGGNFGNSYVSW FAYWGQGTLVTVSS 18 VH_huCD3-
VH_CDR1 GFTFNTYA H1L1_CDR1 19 VH_huCD3- VH_CDR2 IRSKYNNYAT
H1L1_CDR2 20 VH_huCD3- VH_CDR3 VRHGNFGNSYVSWFAY H1L1_CDR3 21
VH_huCD3- VH_CDR3 VRGGNFGNSYVSWFAY H1L1- H101G_CDR3 22 VL_huCD3- VL
QAVVTQEPSFSVSPGGTVTLTCRSSTGAVTTSNYANW H1L1
VQQTPGQAFRGLIGGTNKRAPGVPARFSGSLIGDKAAL
TITGAQADDESIYFCALWYSNLWVFGGGTKLTVL 23 VL_huCD3- VL_CDR1 TGAVTTSNY
H1L1_CDR1 VL_huCD3- VL_CDR2 GTN H1L1_CDR2 24 VL_huCD3- VL_CDR3
ALWYSNLWV H1L1_CDR3 25 VH_B7H4-C1 VH
QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWI
RQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISIDTSKN
QFSLKLTSVTAADTAVFYCARGLFNWNFDSWGQGTLV TVSS 26 VH_B7H4- VH_CDR1
GGSFSGYY C1_CDR1 27 VH_B7H4- VH_CDR2 INHSGST C1_CDR2 28 VH_B7H4-
VH_CDR3 ARGLFNWNFDS C1_CDR3 29 VH_B7H4- VH
QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWI C1-N52S
RQPPGKGLEWIGEISHSGSTNYNPSLKSRVTISIDTSKN
QFSLKLTSVTAADTAVFYCARGLFNWNFDSWGQGTLV TVSS 30 VH_B7H4- VH_CDR2
ISHSGST C1-N52S_ CDR2 31 VH_B7H4- VH
QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWI C1-N52Q
RQPPGKGLEWIGEIQHSGSTNYNPSLKSRVTISIDTSKN
QFSLKLTSVTAADTAVFYCARGLFNWNFDSWGQGTLV TVSS 32 VH_B7H4- VH_CDR2
IQHSGST C1- N52Q_CDR2 33 VLB7H4-C1 VL
DIQMTQSPSSLSASVGDRVTITCRASQGIRNDLGWYQ
QKPGKAPKRLIYGASSLQSGVPSRFSGSGSGTEFTLTISS
LQPEDFATYYCLQHNSYPRTFGQGTTVEIK 34 VLB7H4- VL_CDR1 QGIRND C1_CDR1
VLB7H4- VL_CDR2 GAS C1_CDR2 35 VLB7H4- VL_CDR3 LQHNSYPRT C1_CDR3 36
VH_B7H4-C3 VH EVQLVQSGAEVKKPGASVKVSCKASGYTFTNFWIHWV
RQAPGQGLEWIGEIDPSDSYTNYNQKFKGRVTITRDTS
TSTAYLELSSLRSEDTAVYYCAREITTVDYWGQGTLVTV SS 37 VH_B7H4- VH_CDR1
GYTFTNFW C3_CDR1 38 VH_B7H4- VH_CDR2 IDPSDSYT C3_CDR2 39 VH_B7H4-
VH_CDR3 AREITTVDY C3_CDR3 40 VL_B7H4-C3 VL
DIQMTQSPSSLSASVGDRVTITCSATSSISYMHWYQQK
PGKAPKGWIYDTSKLAHGVPSRFSGSGSGTDFTLTISSL
QPEDFATYYCHQRRSYPFTFGQGTKVEIK 41 VLB7H4- VL_CD R1 SSISY C3_CDR1
VLB7H4- VL_CDR2 DTS C3_CDR2 42 VLB7H4- VL_CD R3 HQRRSYPFT C3_CDR3
43 VH_B7H4-C2 VH EVQLVQSGAEVKKPGASVKVSCKASGYTFTSYWIGWV
RQAPGQGLEWIGDIYPGGGYTNYNEKFKGRVTITRDTS
TSTAYLELSSLRSEDTAVYYCARLDGSSYRGAMDSWGQ GTLVTVSS 44 VH_B7H4- VH_CDR1
GYTFTSYW C2_CDR1 45 VH_B7H4- VH_CDR2 IYPGGGYT C2_CDR2
46 VH_B7H4- VH_CDR3 ARLDGSSYRGAMDS C2_CDR3 47 VLB7H4-C2 VL
DIQMTQSPSSLSASVGDRVTITCKASQGFNKYVAWYQ
QKPGKAPKLLIYYTSTLQPGVPSRFSGSGSGRDYTLTISS
LQPEDFATYYCLQYGNLLYAFGQGTKVEIK 48 VLB7H4- VL_CDR1 QGFNKY C2_CDR1
VLB7H4- VL_CDR2 YTS C2_CDR2 49 VLB7H4- VL_CDR3 LQYGNLLYA C2_CDR3 50
VH_B7H4-C4 VH EVQLVESGGGLIQPGGSLRLSCAASGFTVSSNYMNWV
RQAPGKGLEWVSVIYGSGRTYYADSVKGRVTISRDNSK
NTLYLQMNSLRAEDTAVYYCARDTYAMDVWGQGTTV TVSS 51 VH_B7H4- VH_CDR1
GFTVSSNY C4_CDR1 52 VH_B7H4- VH_CDR2 IYGSGRT C4_CDR2 53 VH_B7H4-
VH_CDR3 ARDTYAMDV C4_CDR3 54 VL_B7H4-C4 VL
EIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQ
KPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRL
EPEDFAVYYCQQYGSSPMYTFGQGTKLEIK 55 VLB7H4- VL_CDR1 QSVSSSY C4_CDR1
VLB7H4- VL_CDR2 GAS C4_CDR2 56 VLB7H4- VL_CDR3 QQYGSSPMYT C4_CDR3
57 IgG1-Fc Constant ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV
SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
QTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPE
LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE
VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ
VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNG
QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGK 58
IgG1- Constant ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV Fc_F405L
SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
QTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPE
LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE
VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ
VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNG
QPENNYKTTPPVLDSDGSFLLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGK 59
IgG1-Fc_FEA Constant ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV
SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
QTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPE
FEGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPE
VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ
VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNG
QPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGK 60
IgG1- Constant ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV Fc_FEAL
SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
QTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPE
FEGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPE
VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ
VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNG
QPENNYKTTPPVLDSDGSFLLYSKLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGK 61
IgG1- Constant ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV Fc_FEAR
SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
QTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPE
FEGGPSVFLFPPKPKDTLMISRTPEVTCVVVAVSHEDPE
VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ
VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNG
QPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGK 62
IgG1- Constant ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTV Fc_K409R
SWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGT
QTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPE
LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPE
VKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVL
HQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQ
VYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNG
QPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQQGNV FSCSVMHEALHNHYTQKSLSLSPGK 63
Kappa Constant RIVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKV
QWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA
DYEKHKVYACEVTHQGLSSPVTKSFNRGEC 64 Lambda Constant
GQPKAAPSVTLFPPSSEELQANKATLVCLISDFYPGAVT
VAWKADSSPVKAGVETTTPSKQSNNKYAASSYLSLTPE
QWKSHRSYSCQVTHEGSTVEKTVAPTECS 65 VH_B7H4-05 VH
QLQLQESGPGLVKPSETLSLTCTVSGGSIKSGSYYWGWI
RQPPGKGLEWIGNIYYSGSTYYNPSLRSRVTISVDTSKN
QFSLKLSSVTAADTAVYYCAREGSYPNQFDPWGQGTL VTVSS 66 VH_B7H4- VH_CDR1
GGSIKSGSYY C5_CDR1 67 VH_B7H4- VH_CDR2 IYYSGST C5_CDR2 68 VH_B7H4-
VH_CDR3 AREGSYPNQFDP C5_CDR3 69 VLB7H4-05 VL
EIVMTQSPATLSVSPGERATLSCRASQSVSSNLAWYQQ
KPGQAPRLLIYGASTRATGIPARFSGSGSGTEFTLTISSL
QSEDFAVYYCQQYHSFPFTFGGGTKVEIK 70 VLB7H4- VL_CD R1 QSVSSN C5_CDR1
VLB7H4- VL_CDR2 GAS C5_CDR2 71 VLB7H4- VL_CD R3 QQYHSFPFT
C5_CDR3
[0032] The CDR regions in the table listed above (CDR1, CDR2 and
CDR3, and underlined sequences in VH and VL sequences) have been
annotated according to IMGT (see Lefranc M P. et al., Nucleic Acids
Research, 27, 209-212, 1999] and Brochet X. Nucl. Acids Res. 36,
W503-508 (2008)). The references to K405L and K409R as used in the
table above is in accordance with the Eu-index of numbering
(described in Kabat, E. A. et al., Sequences of proteins of
immunological interest. 5th Edition--US Department of Health and
Human Services, NIH publication No. 91-3242, pp 662,680,689
(1991).
DETAILED DESCRIPTION
Definitions
[0033] The term "antibody" as used herein is intended to refer to
an immunoglobulin molecule, a fragment of an immunoglobulin
molecule, or a derivative of either thereof, which has the ability
to specifically bind to an antigen under typical physiological
and/or tumor-specific conditions with a half-life of significant
periods of time, such as at least about 30 minutes, at least about
45 minutes, at least about one hour, at least about two hours, at
least about four hours, at least about 8 hours, at least about 12
hours, at least about 24 hours or more, at least about 48 hours or
more, at least about 3, 4, 5, 6, 7 or more days, etc., or any other
relevant functionally-defined period (such as a time sufficient to
induce, promote, enhance, and/or modulate a physiological response
associated with antibody binding to the antigen and/or time
sufficient for the antibody to be internalized). An antibody
comprises a binding region (or binding domain which may be used
herein, both having the same meaning) which can interact with an
antigen, a binding region comprising variable regions of both heavy
and light chains of an immunoglobulin molecule, or the like.
Antibodies can comprise constant regions of the antibodies (Abs)
which may mediate the binding of the immunoglobulin to host tissues
or factors, including various cells of the immune system (such as
effector cells) and components of the complement system such as
C1q, the first component in the classical pathway of complement
activation.
[0034] In the context of the present invention, the term "antibody"
includes a monoclonal antibody (mAb), an antibody-like polypeptide,
a chimeric antibody, a humanized antibody, as well as an `antibody
fragment` or a `fragment thereof` retaining the ability to
specifically bind to the antigen (antigen-binding fragment)
provided by any known technique, such as enzymatic cleavage,
peptide synthesis, and recombinant DNA technology. The term
"antibody" includes bispecific antibodies and/or antibodies having
further modifications, e.g. antibody-drug conjugates thereof.
[0035] An antibody as defined according to the invention can
possess any isotype unless the disclosure herein is otherwise
limited.
[0036] It has been shown that the antigen-binding function of an
antibody may be performed by fragments of a full-length antibody.
Examples of binding fragments encompassed within the term
"antibody" include (i) a Fab' or Fab fragment, a monovalent
fragment consisting of the light chain variable domain (VL), heavy
chain variable domain (VH), light chain constant region (CL) and
heavy chain constant region domain 1 (CH1) domains, or a monovalent
antibody as described in WO 2007/059782; (ii) F(ab').sub.2
fragments, bivalent fragments comprising two Fab fragments linked
by a disulfide bridge at the hinge region; (iii) an Fd fragment
consisting essentially of the VH and CH1 domains; (iv) an Fv
fragment consisting essentially of the VL and VH domains of a
single arm of an antibody, (v) a dAb fragment Ward et al., Nature
341, 544-546 (1989), which consists essentially of a VH domain and
is also called domain antibody Holt et al; Trends Biotechnol. 2003
November; 21(11):484-90; (vi) camelid or nanobodies Revets et al;
Expert Opin Biol Ther. 2005 January; 5(1):111-24 and (vii) an
isolated complementarity determining region (CDR). Furthermore,
although the two domains of the Fv fragment, VL and VH, are coded
for by separate genes, they may be joined, using recombinant
methods, by a synthetic linker that enables them to be made as a
single protein chain in which the VL and VH regions pair to form
monovalent molecules (known as single chain antibodies or single
chain Fv (scFv), see for instance Revets et al; Expert Opin Biol
Ther. 2005 January; 5(1):111-24 and Bird et al., Science 242,
423-426 (1988). Such single chain antibodies are encompassed within
the term antibody unless otherwise noted or clearly indicated by
context. Although such fragments are generally included within the
meaning of antibody, they collectively and each independently are
unique features of the present invention, exhibiting different
biological properties and utility. These and other useful antibody
fragments in the context of the present invention are discussed
further herein.
[0037] An antibody can be produced in and collected from different
in vitro or ex vivo expression or production systems, for example
from recombinantly modified host cells, from hybridomas or systems
that use cellular extracts supporting in vitro transcription and/or
translation of nucleic acid sequences encoding the antibody. It is
to be understood that a multitude of different antibodies, the
antibodies being as defined in the context of the present
invention, can be provided by producing each antibody separately in
a production system as mentioned above and thereafter mixing the
antibodies, or by producing several antibodies in the same
production system.
[0038] The term "immunoglobulin heavy chain" or "heavy chain of an
immunoglobulin" as used herein is intended to refer to one of the
heavy chains of an immunoglobulin. A heavy chain is typically
comprised of a heavy chain variable region (abbreviated herein as
VH) and a heavy chain constant region (abbreviated herein as CH)
which defines the isotype of the immunoglobulin. The heavy chain
constant region typically is comprised of three domains, CH1, CH2,
and CH3. The term "immunoglobulin" as used herein is intended to
refer to a class of structurally related glycoproteins consisting
of two pairs of polypeptide chains, one pair of light (L) low
molecular weight chains and one pair of heavy (H) chains, all four
potentially inter-connected by disulfide bonds. The structure of
immunoglobulins has been well characterized (see for instance
Fundamental Immunology Ch. 7 (Paul, W., ed., 2nd ed. Raven Press,
N.Y. (1989)). Within the structure of the immunoglobulin, the two
heavy chains are inter-connected via disulfide bonds in the
so-called "hinge region". Equally to the heavy chains, each light
chain is typically comprised of several regions; a light chain
variable region (abbreviated herein as VL) and a light chain
constant region. The light chain constant region typically is
comprised of one domain, CL. Furthermore, the VH and VL regions may
be further subdivided into regions of hypervariability (or
hypervariable regions which may be hypervariable in sequence and/or
form of structurally defined loops), also termed complementarity
determining regions (CDRs), interspersed with regions that are more
conserved, termed framework regions (FRs). Each VH and VL is
typically composed of three CDRs and four FRs, arranged from
amino-terminus to carboxy-terminus in the following order: FR1,
CDR1, FR2, CDR2, FR3, CDR3, FR4.
[0039] When used herein, the terms "half molecule", "Fab-arm" and
"arm" refer to one heavy chain-light chain pair. When a bispecific
antibody is described to comprise a half-molecule antibody "derived
from" a first antibody, and a half-molecule antibody "derived from"
a second antibody, the term "derived from" indicates that the
bispecific antibody was generated by recombining, by any known
method, said half-molecules from each of said first and second
antibodies into the resulting bispecific antibody. In this context,
"recombining" is not intended to be limited by any particular
method of recombining and thus includes all of the methods for
producing bispecific antibodies described herein below, including
for example recombining by half-molecule exchange, as well as
recombining at nucleic acid level and/or through co-expression of
two half-molecules in the same cells.
[0040] The term "antigen-binding region" or "binding region" as
used herein, refers to a region of an antibody which is capable of
binding to the antigen. The antigen can be any molecule, such as a
polypeptide. Antigens may e.g. be presented on a cell, bacterium,
or virion. The terms "antigen" and "target" may, unless
contradicted by the context, be used interchangeably in the context
of the present invention. The terms "antigen-binding region" and
"antigen-binding site" may, unless contradicted by the context, be
used interchangeably in the context of the present invention.
[0041] The term "blocks binding" or "blocking the binding of an
antibody" or "cross-blocking binding" or "cross-blocks binding"
refers to the situation where one antibody bound to a specific
antigen prevents binding of the second antibody to the same antigen
and vice versa. In the absence of the other antibody, each antibody
has the ability to bind to the antigen as determined by a
significant binding response, whereas one of the antibodies lacks a
binding response when the other antibody is present. The ability of
one antibody to block the binding of another antibody may be
determined by biolayer interferometry in a classical sandwich
epitope binning assay format, for instance as described in Example
5 in the present application and by Abdiche et al. (Abdiche Y N,
Malashock D S, Pinkerton A, Pons J. Exploring blocking assays using
Octet, ProteOn, and Biacore biosensors. Anal Biochem. 2009; 386(2):
172-180). Briefly, in a sandwich epitope binning assay, an antibody
in solution is tested for binding to its specific antigen that is
first captured via an immobilized antibody. In the context of the
present invention, one antibody does not block the binding of a
second antibody if it is capable of binding to the antigen in the
presence the second antibody and vice versa. The terms "blocks
binding" and "blocking the binding of an antibody" and
"cross-blocking binding" and "cross-blocks binding" may, unless
contradicted by the context, be used interchangeably in the context
of the present invention. An antibody that is said to blocks
binding of another antibody, may also be said to compete with the
other antibody for binding to the target.
[0042] The term "K.sub.D" (M), as used herein, refers to the
equilibrium dissociation constant of a particular antibody-antigen
interaction, and is obtained by dividing k.sub.d by k.sub.a.
K.sub.D can also be referred to as "binding affinity".
[0043] The term "k.sub.d" (sec.sup.-1), as used herein, refers to
the dissociation rate constant of a particular antibody-antigen
interaction. Said value is also referred to as the k.sub.off value
or off-rate.
[0044] The term "k.sub.a" (M.sup.-1.times.sec.sup.-1), as used
herein, refers to the association rate constant of a particular
antibody-antigen interaction. Said value is also referred to as the
k.sub.on value or on-rate.
[0045] The term "binding" as used herein refers to the binding of
an antibody to a predetermined antigen or target, typically with a
binding affinity corresponding to a K.sub.D of 1E.sup.-6 M or less,
e.g. 5E.sup.-7 M or less, 1E.sup.-7 M or less, such as 5E.sup.-8 M
or less, such as 1E.sup.-8 M or less, such as 5E.sup.-9 M or less,
or such as 1E.sup.-9 M or less, when determined by biolayer
interferometry using the antibody as the ligand and the antigen as
the analyte and binds to the predetermined antigen with an affinity
corresponding to a K.sub.D that is at least ten-fold lower, such as
at least 100-fold lower, for instance at least 1,000-fold lower,
such as at least 10,000-fold lower, for instance at least
100,000-fold lower than its affinity for binding to a non-specific
antigen (e.g., BSA, casein) other than the predetermined antigen or
a closely-related antigen.
[0046] The term "B7H4" as used herein, refers to a protein entitled
B7H4, which is also referred to as: B7-H4; V-set domain containing
T cell activation inhibitor 1; or VTCN1. B7H4 is a member of the B7
family of proteins, which family comprises cell-surface protein
ligands that bind to receptors on lymphocytes. B7H4 is a type I
transmembrane protein that includes a short intracellular domain, a
hydrophobic transmembrane domain, and an extracellular domain with
an IgV- and an IgC-like domain with four conserved cysteine
residues and seven sites for N-linked glycosylation. (Sica et al.,
2003, Immunity 18: 849-861). B7H4 proteins are known from various
species, such as human (Homo sapiens) B7H4 (Uniprot accession no.
Q7Z7D3), cynomolgus monkey (Macaca fascicularis) B7H4 transcript 1
(Uniprot accession no. A0A2K5U6P5), dog (Canis familiaris) B7H4
(Uniprot accession no. F1P8R9), rabbit (Oryctolagus cuniculus) B7H4
(Uniprot accession no. G1TQE8), rat (Rattus norvegicus) B7H4
(Uniprot accession no. Q501W4), mouse (Mus musculus) B7H4 (Uniprot
accession no. Q7TSP5), and pig (Sus scrofa) B7H4 (Uniprot accession
no. F1SAY4). Natural variants of the listed B7H4 sequences may
exist.
[0047] The term "CD3" as used herein, refers to the human Cluster
of Differentiation 3 protein which is part of the T-cell
co-receptor protein complex and is composed of four distinct
chains. CD3 is found in various species, and thus, the term "CD3"
may not be limited to human CD3, unless contradicted by context. In
mammals, the complex contains a CD3.gamma. (gamma) chain (human
CD3.gamma. chain UniProtKB/Swiss-Prot No P09693, or cynomolgus
monkey CD3.gamma. UniProtKB/Swiss-Prot No Q95LI7), a CD3.delta.
(delta) chain (human CD3.delta. UniProtKB/Swiss-Prot No P04234, or
cynomolgus monkey CD3.delta. UniProtKB/Swiss-Prot No Q95LI8), two
CD3.epsilon. (epsilon) chains (human CD3.epsilon.:
UniProtKB/Swiss-Prot No P07766, of which a sequence herein is
incorporated as SEQ ID NO: 13, in which amino acid residues 1-22
represent a signal peptide and amino acid residues 23-207 represent
the mature CD3.epsilon. polypeptide; cynomolgus monkey CD3.epsilon.
UniProtKB/Swiss-Prot No Q95L15; or rhesus monkey CD3.epsilon.
UniProtKB/Swiss-Prot No G7NCB9), and a CD3.zeta.-chain (zeta) chain
(human CD3.zeta. UniProtKB/Swiss-Prot No P20963, cynomolgus monkey
CD3.zeta. UniProtKB/Swiss-Prot No Q09TKO). These chains associate
with a molecule known as the T cell receptor (TCR) and generate an
activation signal in T lymphocytes. The TCR and CD3 molecules
together comprise the TCR complex.
[0048] The term "antibody binding region" refers to a region of the
antigen, which comprises the epitope to which the antibody binds.
An antibody binding region may be determined by epitope binning
using biolayer interferometry, by alanine scan, or by domain
shuffle assays (using antigen constructs in which regions of the
antigen are exchanged with that of another species and determining
whether the antibody still binds to the antigen or not). The amino
acids within the antibody binding region that are involved in the
interaction with the antibody may be determined by
hydrogen/deuterium exchange mass spectrometry and/or by
crystallography of the antibody bound to its antigen.
[0049] The term "epitope" means an antigenic determinant which is
specifically bound by an antibody. Epitopes usually consist of
surface groupings of molecules such as amino acids, sugar side
chains or a combination thereof and usually have specific three
dimensional structural characteristics, as well as specific charge
characteristics. Conformational and non-conformational epitopes are
distinguished in that the binding to the former but not the latter
is lost in the presence of denaturing solvents. The epitope may
comprise amino acid residues which are directly involved in the
binding, and other amino acid residues, which are not directly
involved in the binding, such as amino acid residues which are
effectively blocked or covered by the antibody when it is bound to
the antigen (in other words, the amino acid residue is within or
closely adjacent to the footprint of the specific antibody).
[0050] The terms "monoclonal antibody", "monoclonal Ab",
"monoclonal antibody composition", "mAb", or the like, as used
herein refer to a preparation of antibody molecules of single
molecular composition and typically displays a single binding
specificity and affinity for a particular epitope. A monoclonal
antibody can be typically made by identical cells that are all
clones of a unique parent cell, such as for example hybridomas,
stable cell lines or the like. Accordingly, the term "human
monoclonal antibody" refers to antibodies displaying a single
binding specificity which have variable and constant regions
derived from human germline immunoglobulin sequences. The human
monoclonal antibodies may be produced by a hybridoma which includes
a B cell obtained from a transgenic or transchromosomal nonhuman
animal, such as a transgenic mouse, having a genome comprising a
human heavy chain transgene and a light chain transgene, fused to
an immortalized cell. Human monoclonal antibodies may be derived
from human B cells or plasma cells. Monoclonal antibodies may also
be produced from recombinantly modified host cells, or systems that
use cellular extracts supporting in vitro transcription and/or
translation of nucleic acid sequences encoding the antibody.
[0051] The term "isotype" as used herein refers to the
immunoglobulin class (for instance IgG1, IgG2, IgG3, IgG4, IgD,
IgA, IgE, or IgM) or any allotypes thereof, such as IgG1m(za) and
IgG1m(f)) that is encoded by heavy chain constant region genes.
Further, each heavy chain isotype can be combined with either a
kappa (.kappa.) or lambda (.lamda.) light chain.
[0052] The term "full-length antibody" when used herein, refers to
an antibody (e.g., a parent or variant antibody) comprising one
pair of a heavy and light chain or two different pairs of heavy and
light chains, each pair containing heavy and light chain constant
and variable domains such as normally found in a heavy chain-light
chain pair of a wild-type antibody of that isotype. In a full
length variant antibody, the heavy and light chain constant and
variable domains may in particular contain amino acid substitutions
that modify and/or improve functional properties of the antibody
when compared to the full length parent or wild-type antibody. A
full-length antibody according to the present invention may be
produced by a method comprising the steps of (i) cloning the CDR
sequences into one or more suitable vectors comprising complete
heavy and light chain sequences, and (ii) expressing the obtained
suitable vectors with the heavy and light chain sequences in
suitable expression systems. It is within the knowledge of the
skilled person to produce a full-length antibody when starting out
from either CDR sequences or full variable region sequences. Thus,
the skilled person knows how to generate a full-length antibody in
accordance with the present invention.
[0053] The term "humanized antibody" as used herein, refers to a
genetically engineered non-human antibody, which contains human
antibody constant domains and non-human variable domains modified
to contain a high level of sequence homology to human variable
domains. This can be achieved by grafting of non-human antibody
complementarity-determining regions (CDRs), which together form the
antigen binding site, onto a homologous human acceptor framework
region (FR) (see i.a. WO92/22653 and EP0629240). In order to fully
reconstitute the binding affinity and specificity of the parental
antibody, substitution of framework residues from the parental
antibody (i.e. the non-human antibody) into the human framework
regions (back-mutations) may be required. Structural homology
modeling may help to identify the amino acid residues in the
framework regions that are important for the binding properties of
the antibody. Thus, a humanized antibody may comprise non-human CDR
sequences, primarily human framework regions optionally comprising
one or more amino acid back-mutations to the non-human amino acid
sequence, and fully human constant regions. Optionally, additional
amino acid modifications, which are not necessarily back-mutations,
may be applied to obtain a humanized antibody with preferred
characteristics, such as particular useful affinity and biochemical
properties, e.g. to include modifications to avoid deamidation,
provide an "inert Fc region", and/or improve manufacturing.
[0054] The term "human antibody", as used herein, is intended to
include antibodies having variable and framework regions derived
from human germline immunoglobulin sequences and a constant domain
derived from a human immunoglobulin constant domain. The human
antibodies of the invention may include amino acid residues not
encoded by human germline immunoglobulin sequences (e.g.,
mutations, insertions or deletions introduced by random or
site-specific mutagenesis in vitro or by somatic mutation in vivo).
A "human antibody" can incorporate VH and VL sequences that have
been generated from human germline immunoglobulin sequences in a
human, in a transgenic animal such as described in the examples
herein, a HIS mouse, or the like. Such VH and VL sequences are
considered human VH and VL sequences, which have been e.g. fused to
constant domains derived from a human immunoglobulin constant
domain.
[0055] Hence, "human antibodies" can be engineered antibodies. A
"human antibody" may have been subjected to further engineering,
e.g. include modifications to avoid deamidation, provide an "inert
Fc region", enable bispecific antibody generation and/or improve
manufacturing. A human antibody may also be produced in non-human
cells, e.g. in CHO cells or the like. However, the term "human
antibody", as used herein, is not intended to include antibodies in
which CDR sequences derived from the germline of another non-human
species, such as a mouse, have been grafted onto human framework
sequences.
[0056] The term "Fc region" as used herein, refers to a region
comprising, in the direction from the N- to C-terminal ends of the
two heavy chains of the antibody, at least a hinge region, a CH2
region and a CH3 region. An Fc region of the antibody may mediate
the binding of the immunoglobulin to host tissues or factors,
including various cells of the immune system (such as effector
cells) and components of the complement system.
[0057] The term "hinge region" as used herein refers to the hinge
region of an immunoglobulin heavy chain. Thus, for example the
hinge region of a human IgG1 antibody corresponds to amino acids
216-230 according to the Eu numbering as set forth in Kabat Kabat,
E. A. et al., Sequences of proteins of immunological interest. 5th
Edition--US Department of Health and Human Services, NIH
publication No. 91-3242, pp 662,680,689 (1991). However, the hinge
region may also be any of the other subtypes as described
herein.
[0058] The term "CH1 region" or "CH1 domain" as used herein refers
to the CH1 region of an immunoglobulin heavy chain. Thus, for
example the CH1 region of a human IgG1 antibody corresponds to
amino acids 118-215 according to the Eu numbering as set forth in
Kabat (ibid). However, the CH1 region may also be any of the other
subtypes as described herein.
[0059] The term "CH2 region" or "CH2 domain" as used herein refers
to the CH2 region of an immunoglobulin heavy chain. Thus, for
example the CH2 region of a human IgG1 antibody corresponds to
amino acids 231-340 according to the Eu numbering as set forth in
Kabat (ibid). However, the CH2 region may also be any of the other
subtypes as described herein.
[0060] The term "CH3 region" or "CH3 domain" as used herein refers
to the CH3 region of an immunoglobulin heavy chain. Thus, for
example the CH3 region of a human IgG1 antibody corresponds to
amino acids 341-447 according to the Eu numbering as set forth in
Kabat (ibid). However, the CH3 region may also be any of the other
subtypes as described herein.
[0061] The term "Fc-mediated effector functions," as used herein,
is intended to refer to functions that are a consequence of binding
a polypeptide or antibody to its target or antigen on a cell
membrane wherein the Fc-mediated effector function is attributable
to the Fc region of the polypeptide or antibody. Examples of
Fc-mediated effector functions include (i) C1q binding, (ii)
complement activation, (iii) complement-dependent cytotoxicity
(CDC), (iv) antibody-dependent cell-mediated cytotoxity (ADCC), (v)
Fc-gamma receptor (FcgR)-binding, (vi) antibody-dependent,
Fc.gamma.R-mediated antigen crosslinking, (vii) antibody-dependent
cellular phagocytosis (ADCP), (viii) complement-dependent cellular
cytotoxicity (CDCC), (ix) complement-enhanced cytotoxicity, (x)
binding to complement receptor of an opsonized antibody mediated by
the antibody, (xi) opsonisation, and (xii) a combination of any of
(i) to (xi).
[0062] The term "inertness", "inert" or "non-activating" as used
herein, refers to an Fc region which is at least not able to bind
any Fc.gamma.R, induce Fc-mediated cross-linking of Fc.gamma.Rs, or
induce Fc.gamma.R-mediated cross-linking of target antigens via two
Fc regions of individual antibodies, or is not able to bind C1q. An
example thereof is FEA substitutions within the constant domain as
described herein. The inertness of an Fc region of an antibody, may
be tested using the antibody in a monospecific or bispecific
format.
[0063] The term "full-length" when used in the context of an
antibody indicates that the antibody is not a fragment, but
contains all of the domains corresponding with the particular
isotype such as normally found for that isotype in nature, e.g. the
VH, CH1, CH2, CH3, hinge, VL and CL domains for an IgG1
antibody.
[0064] The term "monovalent antibody", in the context of the
present invention, refers to an antibody molecule that can interact
with an antigen, with only one antigen-binding domain (e.g. one Fab
arm). In the context of a bispecific antibody, "monovalent antibody
binding" refers to the binding of the bispecific antibody to one
antigen with only one antigen-binding domain (e.g. one Fab
arm).
[0065] The term "monospecific antibody" in the context of the
present invention, refers to an antibody that has binding
specificity to one antigen, one epitope only. The antibody may be a
monospecific, monovalent antibody (i.e. carrying only one
antigen-binding region) or a monospecific, bivalent antibody (e.g.
an antibody with two identical antigen-binding regions).
[0066] The term "bispecific antibody" refers to an antibody having
two antigen-binding domains that bind different epitopes, e.g. two
non-identical pairs of VH and VL regions, two non-identical
Fab-arms or two Fab-arms with non-identical CDR regions. In the
context of this invention, bispecific antibodies have specificity
for at least two different epitopes. Such epitopes may be on the
same or different antigens or targets. If the epitopes are on
different antigens, such antigens may be on the same cell or
different cells, cell types or structures, such as extracellular
matrix or vesicles and soluble protein. A bispecific antibody may
thus be capable of crosslinking multiple antigens, e.g. two
different cells.
[0067] The term "bivalent antibody" refers to an antibody that has
two antigen-binding regions, which bind to two of the same epitopes
on two of the same antigens or binds to two different epitopes on
the same or different antigen(s). Hence, a bivalent antibody may be
a monospecific antibody or a bispecific antibody.
[0068] The term "amino acid" and "amino acid residue" may herein be
used interchangeably, and are not to be understood limiting. Amino
acids are organic compounds containing amine (--NH.sub.2) and
carboxyl (--COOH) functional groups, along with a side chain (R
group) specific to each amino acid. In the context of the present
invention, amino acids may be classified based on structure and
chemical characteristics. Thus, classes of amino acids may be
reflected in one or both of the following tables:
[0069] Main classification based on structure and general chemical
characterization of R group
TABLE-US-00002 TABLE 2 Class Amino acid Acidic Residues D and E
Basic Residues K, R, and H Hydrophilic Uncharged Residues S, T, N,
and Q Aliphatic Uncharged Residues G, A, V, L, and I Non-polar
Uncharged Residues C, M, and P Aromatic Residues F, Y, and W
TABLE-US-00003 TABLE 3 Alternative Physical and Functional
Classifications of Amino Acid Residues Class Amino acid Hydroxyl
group containing residues S and T Aliphatic residues I, L, V, and M
Cycloalkenyl-associated residues F, H, W, and Y Hydrophobic
residues A, C, F, G, H, I, L, M, R, T, V, W, and Y Negatively
charged residues D and E Polar residues C, D, E, H, K, N, Q, R, S,
and T Positively charged residues H, K, and R Small residues A, C,
D, G, N, P, S, T, and V Very small residues A, G, and S Residues
involved in turn formation A, C, D, E, G, H, K, N, Q, R, S, P, and
T Flexible residues Q, T, K, S, G, P, D, E, and R
[0070] Substitution of one amino acid for another may be classified
as a conservative or non-conservative substitution. In the context
of the invention, a "conservative substitution" is a substitution
of one amino acid with another amino acid having similar structural
and/or chemical characteristics, such substitution of one amino
acid residue for another amino acid residue of the same class as
defined in any of the two tables above: for example, leucine may be
substituted with isoleucine as they are both aliphatic, branched
hydrophobes. Similarly, aspartic acid may be substituted with
glutamic acid since they are both small, negatively charged
residues.
[0071] In the context of the present invention, a substitution in
an antibody is indicated as:
[0072] Original amino acid--position--substituted amino acid;
[0073] Referring to the well-recognized nomenclature for amino
acids, the three letter code, or one letter code, is used,
including the codes "Xaa" or "X" to indicate any amino acid
residue. Thus, Xaa or X may typically represent any of the 20
naturally occurring amino acids. The term "naturally occurring" as
used herein refers to any one of the following amino acid residues;
glycine, alanine, valine, leucine, isoleucine, serine, threonine,
lysine, arginine, histidine, aspartic acid, asparagine, glutamic
acid, glutamine, proline, tryptophan, phenylalanine, tyrosine,
methionine, and cysteine.
[0074] Accordingly, the notation "K409R" or "Lys409Arg" means, that
the antibody comprises a substitution of Lysine with Arginine in
amino acid position 409. Substitution of an amino acid at a given
position to any other amino acid is referred to as: Original amino
acid--position; or e.g. "K409". For a modification where the
original amino acid(s) and/or substituted amino acid(s) may
comprise more than one, but not all amino acid(s), the more than
one amino acid may be separated by "," or "/". E.g. the
substitution of Lysine with Arginine, Alanine, or Phenylalanine in
position 409 is: "Lys409Arg,Ala,Phe" or "Lys409Arg/Ala/Phe" or
"K409R,A,F" or "K409R/A/F" or "K409 to R, A, or F". Such
designation may be used interchangeably in the context of the
invention but have the same meaning and purpose.
[0075] Furthermore, the term "a substitution" embraces a
substitution into any one or the other nineteen natural amino
acids, or into other amino acids, such as non-natural amino acids.
For example, a substitution of amino acid K in position 409
includes each of the following substitutions: 409A, 409C, 409D,
409E, 409F, 409G, 409H, 409I, 409L, 409M, 409N, 409O, 409R, 409S,
409T, 409V, 409W, 409P, and 409Y. This is, by the way, equivalent
to the designation 409X, wherein the X designates any amino acid
other than the original amino acid. These substitutions may also be
designated K409A, K409C, etc. or K409A,C, etc. or K409A/C/etc. The
same applies by analogy to each and every position mentioned
herein, to specifically include herein any one of such
substitutions.
[0076] The antibody according to the invention may also comprise a
deletion of an amino acid residue. Such deletion may be denoted
"del", and includes, e.g., writing as K409del. Thus, in case of
such embodiments, the Lysine in position 409 has been deleted from
the amino acid sequence.
[0077] The term "host cell", as used herein, is intended to refer
to a cell into which a nucleic acid such as an expression vector
has been introduced. It should be understood that such terms are
intended to refer not only to the particular subject cell, but may
also include the progeny of such a cell. Because certain
modifications may occur in succeeding generations due to either
mutation or environmental influences, such progeny may not, in
fact, be identical to the parent cell, but are still included
within the scope of the term "host cell" as used herein.
Recombinant host cells include, for example, transfectomas, such as
CHO cells, HEK-293 cells, Expi293F cells, PER.C6 cells, NS0 cells,
and lymphocytic cells, and prokaryotic cells such as E. coli and
other eukaryotic hosts such as plant cells and fungi.
[0078] The term "transfectoma", as used herein, includes
recombinant eukaryotic host cells expressing the antibody or a
target antigen, such as CHO cells, PER.C6 cells, NS0 cells, HEK-293
cells, Expi293F cells, plant cells, or fungi, including yeast
cells.
[0079] For purposes of the present invention, sequence identity
between two amino acid sequences is determined over the length of
the referenced sequence using the Needleman-Wunsch algorithm
(Needleman and Wunsch, 1970, J. Mol. Biol. 48: 443-453) as
implemented in the Needle program of the EMBOSS package (EMBOSS:
The European Molecular Biology Open Software Suite, Rice et al.,
2000, Trends Genet. 16: 276-277), preferably version 5.0.0 or
later. The parameters used are gap open penalty of 10, gap
extension penalty of 0.5, and the EBLOSUM62 (EMBOSS version of
BLOSUM62) substitution matrix. The output of Needle labeled
"longest identity" (obtained using the -nobrief option) is used as
the percent identity and is calculated as follows:
(Identical Residues.times.100)/(Length of Alignment-Total Number of
Gaps in Alignment).
[0080] The retention of similar residues may also or alternatively
be measured by a similarity score, as determined by use of a BLAST
program (e.g., BLAST 2.2.8 available through the NCBI using
standard settings BLOSUM62, Open Gap=11 and Extended Gap=1).
Suitable variants typically exhibit at least about 45%, such as at
least about 55%, at least about 65%, at least about 75%, at least
about 85%, at least about 90%, at least about 95%, or more (e.g.,
about 99%) similarity to the parent or referenced sequence.
[0081] The term "internalized" or "internalization" as used herein,
refers to a biological process in which molecules such as the
antibody according to the present invention, are engulfed by the
cell membrane and drawn into the interior of the cell.
Internalization may also be referred to as "endocytosis".
Bispecific Antibodies Targeting CD3xB7H4
[0082] In a first aspect of the invention, an antibody is provided
comprising an antigen-binding region capable of binding to human
B7H4 and an antigen-binding region capable of binding to human CD3,
wherein said antigen-binding regions comprise heavy and light chain
variable regions, wherein said antigen-binding regions are human
variable regions and/or humanized variable regions. For example,
one antigen-binding region may comprise human heavy and light chain
variable regions, and the other antigen-binding region may comprise
humanized heavy and light chain variable regions. Or, both
antigen-binding region may comprise human heavy and light chain
variable regions, or both antigen-binding regions may comprise
humanized heavy and light chain variable regions. Hence,
accordingly, an antibody is provided comprising an antigen-binding
region capable of binding to human B7H4 and an antigen-binding
region capable of binding to human CD3, wherein said
antigen-binding regions comprise heavy and light chain variable
regions, wherein said heavy and light chain variable regions
comprise human framework regions. An antibody in accordance with
the invention as described herein comprising an antigen-binding
region capable of binding to human B7H4 and an antigen-binding
region capable of binding to human CD3, may also be referred to
herein e.g. as a B7H4xCD3 antibody.
[0083] Such antibodies are preferably bispecific antibodies. Such
an antibody as described above are in a further embodiment capable
of binding cancer cells and T-cells, such as e.g. described in the
examples. Cancer cells that may be selected are cancer cells that
express human B7H4 and/or are cancer cells that are of a solid
tumor. Such an antibody preferably is capable of inducing T-cell
mediated cell killing of the cancer cells.
[0084] Capable of binding is understood to comprise, as shown in
the examples, that in a binding assay, an antibody binds to its
target, as shown by e.g. typical binding curves such as shown in
FIGS. 3 and 4 herein, or by determining binding affinity, using
e.g. biolayer interferometry, as shown in examples 3 and 4. An
antigen-binding region not capable of binding to a specified target
has e.g. an undetectable binding affinity to its target, e.g.
having a response of <0.05 nm at the highest concentration used
in a typical biolayer interferometry assay such as shown in example
3. In any case, the skilled person is well aware how to determine
whether or not an antigen-binding region is capable of binding to
its target.
Bispecific Formats
[0085] The present invention provides bispecific CD3xB7H4
antibodies which efficiently promote T cell-mediated killing of
B7H4-expressing tumor cells. Depending on the desired functional
properties for a particular use, particular antigen-binding regions
can be selected from the set of antibodies or antigen-binding
regions provided by the present invention. Many different formats
and uses of bispecific antibodies are known in the art, and were
reviewed by Kontermann; Drug Discov Today, 2015 July; 20(7):838-47
and; MAbs, 2012 March-April; 4(2):182-97. A bispecific antibody
according to the present invention may not be limited to any
particular bispecific format or method of producing it.
[0086] Examples of bispecific antibody molecules which may be used
in the present invention comprise (i) a single antibody that has
two arms comprising different antigen-binding regions; (ii) a
single chain antibody that has specificity to two different
epitopes, e.g., via two scFvs linked in tandem by an extra peptide
linker; (iii) a dual-variable-domain antibody (DVD-Ig), where each
light chain and heavy chain contains two variable domains in tandem
through a short peptide linkage (Wu et al., Generation and
Characterization of a Dual Variable Domain Immunoglobulin
(DVD-Ig.TM.) Molecule, In: Antibody Engineering, Springer Berlin
Heidelberg (2010)); (iv) a chemically-linked bispecific (Fab')2
fragment; (v) a Tandab, which is a fusion of two single chain
diabodies resulting in a tetravalent bispecific antibody that has
two binding sites for each of the target antigens; (vi) a
flexibody, which is a combination of scFvs with a diabody resulting
in a multivalent molecule; (vii) a so-called "dock and lock"
molecule, based on the "dimerization and docking domain" in Protein
Kinase A, which, when applied to Fabs, can yield a trivalent
bispecific binding protein consisting of two identical Fab
fragments linked to a different Fab fragment; (viii) a so-called
Scorpion molecule, comprising, e.g., two scFvs fused to both
termini of a human Fab-arm; and (ix) a diabody.
[0087] In one embodiment, the bispecific antibody of the present
invention is a diabody, a cross-body, or a bispecific antibody
obtained via a controlled Fab-arm exchange (such as described in
WO2011131746 (Genmab)).
[0088] Examples of different classes of bispecific antibodies
include but are not limited to (i) IgG-like molecules with
complementary CH3 domains to force heterodimerization; (ii)
recombinant IgG-like dual targeting molecules, wherein the two
sides of the molecule each contain the Fab fragment or part of the
Fab fragment of at least two different antibodies; (iii) IgG fusion
molecules, wherein full length IgG antibodies are fused to extra
Fab fragment or parts of Fab fragment; (iv) Fc fusion molecules,
wherein single chain Fv molecules or stabilized diabodies are fused
to heavy-chain constant-domains, Fc-regions or parts thereof; (v)
Fab fusion molecules, wherein different Fab-fragments are fused
together, fused to heavy-chain constant-domains, Fc-regions or
parts thereof; and (vi) ScFv- and diabody-based and heavy chain
antibodies (e.g., domain antibodies, nanobodies) wherein different
single chain Fv molecules or different diabodies or different
heavy-chain antibodies (e.g. domain antibodies, nanobodies) are
fused to each other or to another protein or carrier molecule fused
to heavy-chain constant-domains, Fc-regions or parts thereof.
[0089] Examples of IgG-like molecules with complementary CH3 domain
molecules include but are not limited to the Triomab/Quadroma
molecules (Trion Pharma/Fresenius Biotech; Roche, WO2011069104),
the so-called Knobs-into-Holes molecules (Genentech, WO9850431),
CrossMAbs (Roche, WO2011117329) and the electrostatically-matched
molecules (Amgen, EP1870459 and WO2009089004; Chugai,
US201000155133; Oncomed, WO2010129304), the LUZ-Y molecules
(Genentech, Wranik et al. J. Biol. Chem. 2012, 287(52): 43331-9,
doi: 10.1074/jbc.M112.397869. Epub 2012 Nov. 1), DIG-body and
PIG-body molecules (Pharmabcine, WO2010134666, WO2014081202), the
Strand Exchange Engineered Domain body (SEEDbody) molecules (EMD
Serono, WO2007110205), the Biclonics molecules (Merus,
WO2013157953), Fc.DELTA.Adp molecules (Regeneron, WO201015792),
bispecific IgG1 and IgG2 molecules (Pfizer/Rinat, WO11143545),
Azymetric scaffold molecules (Zymeworks/Merck, WO2012058768),
mAb-Fv molecules (Xencor, WO2011028952), bivalent bispecific
antibodies (WO2009080254) and the DuoBody.RTM. molecules (Genmab
A/S, WO2011131746).
[0090] Examples of recombinant IgG-like dual targeting molecules
include but are not limited to Dual Targeting (DT)-Ig molecules
(WO2009058383), Two-in-one Antibody (Genentech; Bostrom, et al
2009. Science 323, 1610-1614.), Cross-linked Mabs (Karmanos Cancer
Center), mAb2 (F-Star, WO2008003116), Zybody molecules (Zyngenia;
LaFleur et al. MAbs. 2013 March-April; 5(2):208-18), approaches
with common light chain (Crucell/Merus, U.S. Pat. No. 7,262,028),
.kappa..lamda.Bodies (NovImmune, WO2012023053) and CovX-body
(CovX/Pfizer; Doppalapudi, V. R., et al 2007. Bioorg. Med. Chem.
Lett. 17, 501-506.).
[0091] Examples of IgG fusion molecules include but are not limited
to Dual Variable Domain (DVD)-Ig molecules (Abbott, U.S. Pat. No.
7,612,181), Dual domain double head antibodies (Unilever; Sanofi
Aventis, WO20100226923), IgG-like Bispecific molecules (ImClone/Eli
Lilly, Lewis et al. Nat Biotechnol. 2014 February; 32(2):191-8),
Ts2Ab (MedImmune/AZ; Dimasi et al. J Mol Biol. 2009 Oct. 30;
393(3):672-92) and BsAb molecules (Zymogenetics, WO2010111625),
HERCULES molecules (Biogen Idec, U.S. Ser. No. 00/795,1918), scFv
fusion molecules (Novartis), scFv fusion molecules (Changzhou Adam
Biotech Inc, CN 102250246) and TvAb molecules (Roche, WO2012025525,
WO2012025530).
[0092] Examples of Fc fusion molecules include but are not limited
to ScFv/Fc Fusions (Pearce et al., Biochem Mol Biol Int. 1997
September; 42(6):1179-88), SCORPION molecules (Emergent
BioSolutions/Trubion, Blankenship J W, et al. AACR 100 th Annual
meeting 2009 (Abstract #5465); Zymogenetics/BMS, WO2010111625),
Dual Affinity Retargeting Technology (Fc-DART) molecules
(MacroGenics, WO2008157379, WO2010080538) and Dual(ScFv)2-Fab
molecules (National Research Center for Antibody
Medicine--China).
[0093] Examples of Fab fusion bispecific antibodies include but are
not limited to F(ab)2 molecules (Medarex/AMGEN; Deo et al J
Immunol. 1998 Feb. 15; 160(4):1677-86.), Dual-Action or Bis-Fab
molecules (Genentech, Bostrom, et al 2009. Science 323,
1610-1614.), Dock-and-Lock (DNL) molecules (ImmunoMedics,
WO2003074569, WO2005004809), Bivalent Bispecific molecules
(Biotecnol, Schoonjans, J Immunol. 2000 Dec. 15; 165(12):7050-7.)
and Fab-Fv molecules (UCB-Celltech, WO 2009040562 A1).
[0094] Examples of ScFv-, diabody-based and domain antibodies
include but are not limited to Bispecific T Cell Engager (BiTE)
molecules (Micromet, WO2005061547), Tandem Diabody molecules
(TandAb) (Affimed) Le Gall et al., Protein Eng Des Sel. 2004 April;
17(4):357-66.), Dual Affinity Retargeting Technology (DART)
molecules (MacroGenics, WO2008157379, WO2010080538), Single-chain
Diabody molecules (Lawrence, FEBS Lett. 1998 Apr. 3;
425(3):479-84), TCR-like Antibodies (AIT, ReceptorLogics), Human
Serum Albumin ScFv Fusion (Merrimack, WO2010059315) and COMBODY
molecules (Epigen Biotech, Zhu et al. Immunol Cell Biol. 2010
August; 88(6):667-75.), dual targeting nanobodies (Ablynx, Hmila et
al., FASEB J. 2010) and dual targeting heavy chain only domain
antibodies.
[0095] The bispecific antibody of the invention can be of any
isotype. Exemplary isotypes include but are not limited to either
of the human IgG1, IgG2, IgG3, and IgG4 isotypes. Preferably,
bispecific antibodies may be selected to be of the human IgG1
isotype, as shown in the examples. Either of the human light chain
constant regions, kappa or lambda, may be used. In one embodiment,
both heavy chains of an antibody of the present invention are of
the IgG1 isotype, for instance an IgG1,.kappa.. In one embodiment,
the two heavy chains of a bispecific antibody are of the IgG1 and
IgG4 isotypes, respectively. Preferably, bispecific antibodies may
be selected to be of the human IgG1 isotype, as shown in the
examples. Optionally, and preferably, the heavy chain and Fc
sequences thereof of the selected isotype, may be modified in the
hinge and/or CH3 region as described herein to enable the
generation of bispecific antibodies and introduce inertness.
[0096] In one aspect, the bispecific antibody of the invention
comprises an Fc-region comprising a first heavy chain with a first
Fc sequence comprising a first CH3 region, and a second heavy chain
with a second Fc sequence comprising a second CH3 region, wherein
the sequences of the first and second CH3 regions are different and
are such that the heterodimeric interaction between said first and
second CH3 regions is stronger than each of the homodimeric
interactions of said first and second CH3 regions. More details on
these interactions and how they can be achieved are provided in
WO2011131746 and WO2013060867 (Genmab), which are hereby
incorporated by reference.
[0097] As described further herein, a stable bispecific CD3xB7H4
antibody can be obtained at high yield on the basis of one B7H4
antibody and one CD3 antibody, each composed of two identical heavy
chains and two identical light chains, each antibody containing
only a few, fairly conservative, (asymmetrical) mutations in the
CH3 regions. Asymmetrical mutations mean that the sequences of said
first and second CH3 regions contain one or more amino acid
substitutions at non-identical positions.
Antigen-Binding Region Capable of Binding CD3
[0098] As said, the invention provides an antibody according to the
invention comprising an antigen-binding region capable of binding
to human B7H4 and an antigen-binding region capable of binding to
human CD3. Furthermore, the invention provides an antibody
according to the invention comprising an antigen-binding region
capable of binding to human B7H4 and an antigen-binding region
capable of binding to human CD3, wherein the antigen-binding region
capable of binding CD3, is capable of binding human CD3.epsilon.
(epsilon), such as human CD3.epsilon. (epsilon) as specified in SEQ
ID NO: 13. Such antigen-binding region is capable of binding human
CD3.epsilon. (epsilon), as presented on a T cell, such as a primary
human T cell.
[0099] Said antibody according to the invention may be an antibody
comprising an antigen-binding region capable of binding to human
B7H4 and an antigen-binding region capable of binding to human CD3,
wherein the antigen-binding region that binds to CD3 comprises
[0100] a heavy chain variable region (VH) comprising the CDR1,
CDR2, and CDR3 regions of SEQ ID NO: 16 or of SEQ ID NO. 17, and,
optionally, [0101] a light chain variable region (VL) comprising
the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 22.
[0102] CDR1, CDR2 and CDR3 regions can be identified from variable
heavy and light chain regions using methods known in the art. The
CDR regions from said variable heavy and light chain regions can be
annotated according to IMGT (see Lefranc M P. et al., Nucleic Acids
Research, 27, 209-212, 1999] and Brochet X. Nucl. Acids Res. 36,
W503-508 (2008)). Hence, also disclosed are antibodies comprising
an antigen-binding region capable of binding to human B7H4 and an
antigen-binding region capable of binding to human CD3, wherein the
antigen-binding region that binds to CD3 comprises [0103] a heavy
chain variable region (VH) comprising the CDR1, CDR2, and CDR3
sequences of SEQ ID NOs.: 18, 19 and 20 or 18, 19 and 21
respectively; and, optionally [0104] a light chain variable region
(VL) comprising the CDR1, CDR2, and CDR3 sequences of SEQ ID NO:
23, GTN and 24, respectively.
[0105] Further disclosed are antibodies comprising an
antigen-binding region capable of binding to human B7H4 and an
antigen-binding region capable of binding to human CD3, wherein the
antigen-binding region that binds to CD3 comprises [0106] a heavy
chain variable region (VH) comprising the sequence of SEQ ID NO:
16, or a sequence having at least 90%, at least 95%, at least 97%,
or at least 99% amino acid sequence identity to the sequence of SEQ
ID NO: 16; and; optionally [0107] a light chain variable region
(VL) comprising the sequence of SEQ ID NO: 22 or a sequence having
at least 90%, at least 95%, at least 97%, or at least 99% amino
acid sequence identity to the sequence of SEQ ID NO: 22.
[0108] Such antigen-binding regions that are capable of binding
human CD3 have been described i.a. in WO2015001085, and
WO2017009442. Further antigen-binding regions that are capable of
binding human CD3 are disclosed and described in WO2015001085 and
WO2017009442, which can be further contemplated and serve as the
basis for generating antibodies in accordance with the current
invention, which are incorporated by reference herein.
[0109] The said antibody in accordance with the invention, may bind
with an equilibrium dissociation constant K.sub.D between the
antigen-binding region that binds to human CD3, and human CD3 is
within the range of 1-1000 nM.
[0110] The said antibody in accordance with the invention, may bind
with a equilibrium dissociation constant K.sub.D between the
antigen-binding region that binds to human CD3, and human CD3 is
within the range of 1-100 nM, such as within the range of 5-100 nM,
within the range of 10-100 nM, within the range of 1-80 nM, within
the range of 1-60 nM within the range of 1-40 nM, within the range
of 1-20 nM, within the range of 5-80 nM, within the range of 5-60
nM, within the range of 5-40 nM, within the range of 5-20 nM,
within the range of 10-80 nM, within the range of 10-60 nM, within
the range of 10-40 nM, or such as within the range of 10-20 nM. An
exemplary and suitable antigen-binding region comprises a heavy
chain variable region (VH) of SEQ ID NO: 16 and a light chain
variable region (VL) regions of SEQ ID NO: 22. Such variable
regions have been described i.a. in WO2015001085.
[0111] In another aspect of the invention, said antibody has a
lower binding affinity for human CD3.epsilon. than an antibody
having an antigen-binding region comprising a VH sequence as set
forth in SEQ ID NO: 16, and a VL sequence as set forth in SEQ ID
NO: 22, preferably wherein said affinity is at least 5-fold lower,
such as at least 10-fold lower, e.g. at least 20-fold lower, at
least 30 fold lower, at least 40 fold lower, at least 45 fold lower
or such as at least 50-fold lower.
[0112] In another aspect of the invention, said antibody may bind
with an equilibrium dissociation constant K.sub.D between the
antigen-binding region that binds to human CD3, and human CD3
antigen-binding which is within the range of 200-1000 nM, such as
within the range of 300-1000 nM, within the range of 400-1000 nM,
within the range of 500-1000 nM, within the range of 300-900 nM
within the range of 400-900 nM, within the range of 400-700 nM,
within the range of 500-900 nM, within the range of 500-800 nM,
within the range of 500-700 nM, within the range of 600-1000 nM,
within the range of 600-900 nM, within the range of 600-800 nM, or
such as within the range of 600-700 nM. An exemplary and suitable
antigen-binding region comprises a heavy chain variable region (VH)
of SEQ ID NO: 16 or of SEQ ID NO. 17, and, a light chain variable
region (VL) regions of SEQ ID NO: 22. Such variable regions have
been described i.a. in WO2017009442.
[0113] Said binding affinity can be determined by biolayer
interferometry, optionally as set forth in Example 4 herein. Hence,
the antibody according to the invention having a binding affinity
to human CD3 as defined herein, may have the binding affinity
determined using biolayer interferometry comprising the steps of:
[0114] I) immobilizing the antibody at an amount of 1 .mu.g/mL for
600 seconds on an anti-human IgG Fc Capture biosensor; [0115] II)
determining association over a time period of 1000 seconds and
dissociation over a time period of 2000 seconds of human
recombinant soluble CD3.epsilon. (CD3E27-GSKa) (mature protein of
SEQ ID NO: 13) using a 3-fold dilution series ranging from 1.40 nM
to 1000 nM; and [0116] III) referencing the data to a buffer
control (0 nM).
[0117] Furthermore, said binding affinity may be determined using
an antibody such as a monospecific, bivalent antibody, such as an
antibody which is a full length IgG1.
[0118] Hence, in a further embodiment, the antibody according to
the invention is an antibody, wherein [0119] the antigen-binding
region that binds to CD3 comprises a heavy chain variable (VH)
region, as defined herein, comprising a CDR1 sequence, a CDR2
sequence and a CDR3 sequence, when compared to a heavy chain
variable (VH) region comprising the sequence set forth in SEQ ID
NO: 16 has an amino acid substitution being at a position selected
from the group consisting of: T31, N57, H101, G105, S110 and Y114,
the positions being numbered according to the sequence of SEQ ID
NO: 16; and [0120] the wild type light chain variable (VL) region
comprises the CDR1, CDR2 and CDR3 sequences set forth in SEQ ID NO:
23, GTN and SEQ ID NO: 24, respectively.
[0121] In particular, the antibody according to the invention is an
antibody, wherein the antigen-binding region that binds to CD3
comprises in the heavy chain variable (VH) region as defined herein
comprises a substitution selected from the group consisting of:
T31M, T31P, N57E, H101G, H101N, G105P, S110A, S110G, Y114M, Y114R,
Y114V.
[0122] Furthermore, the antibody according to the invention is an
antibody wherein the antigen-binding region that binds to CD3
comprises a heavy chain variable region as defined herein having at
the amino acid position 31 an M or P, or at the amino acid position
57 an E, or at the amino acid position 101 a G or N, or at the
amino acid 105 a P, or at the amino acid position 110 and A or G,
or at the amino acid position 114 an M, R or V, said positions
corresponding with the amino acid position numbering of the heavy
chain variable (VH) region having the sequence set forth in SEQ ID
NO: 16.
[0123] Still further, the antibody according to the invention is an
antibody wherein the CDR1, CDR2 and CDR3 of the heavy chain
variable (VH) region of the antigen-binding region that binds to
CD3 as defined herein comprises, in total, at the most 1, 2, 3, 4
or 5 amino acid substitutions, when compared with the CDR1, CDR2
and CDR3 of the sequences of SEQ ID NO: 16, said amino acid
substitutions comprising preferably amino acid substitutions as
defined above.
Antigen-Binding Region Capable of Binding B7H4
[0124] In particular, the invention provides an antibody according
to the invention comprising an antigen-binding region capable of
binding to human B7H4 and an antigen-binding region capable of
binding to human CD3, wherein said human B7H4 is human B7H4 of SEQ
ID NO. 1. Preferably, said antibody in accordance with the
invention comprises an antigen-binding region capable of binding to
human CD3.epsilon. (epsilon) as specified in SEQ ID NO: 13, and an
antigen-binding region capable of binding human B7H4 of SEQ ID NO.
1.
[0125] In particular, the antibody according to the invention is an
antibody wherein said antigen-binding region capable of binding to
human B7H4 is capable of binding to the extracellular domain of
human B7H4.
[0126] Preferably, said B7H4 is expressed on a cell, more
preferably a human cell.
[0127] In a further embodiment, the antibody according to the
invention is an antibody wherein said antigen-binding region
capable of binding to human B7H4 is capable of binding to the
IgC-like constant region of human B7H4. In another further
embodiment, the antibody according to the invention is an antibody
wherein said antigen-binding region capable of binding to human
B7H4 is capable of binding to B7H3-IgV/B7H4-IgC. B7H3-IgV/B7H4-IgC
represents a fusion between human B7H3 and B7H4, wherein the B7H3
IgV-like domain is fused with the B7H4 IgC-like domain,
corresponding with SEQ ID NO. 11. Said B7H3-IgV/B7H4-IgC being
expressed by a cell such as described in the example 7 herein. In
still another further embodiment, the antibody according to the
invention is an antibody wherein said antigen-binding region
capable of binding to human B7H4 is not capable of binding to
B7H4-IgV/B7H3-IgC. B7H4-IgV/B7H3-IgC represents a fusion between
human B7H3 and B7H4, wherein the B7H4 IgV-like domain is fused with
the B7H3 IgC-like domain, corresponding with SEQ ID NO. 10. Said
B7H4-IgV/B7H3-IgC being expressed by a cell such as described in
the example 7 herein.
[0128] Suitable antigen-binding regions capable of binding to human
B7H4, that are contemplated according to the invention as described
herein comprise: [0129] a) a variable heavy chain (VH) region
comprising the CDR1, CDR2 and CDR3 regions of SEQ ID NO. 25: and a
variable light chain region comprising the CDR1, CDR2 and CDR3
regions respectively of SEQ ID NO. 33; [0130] b) a variable heavy
chain (VH) region comprising the CDR1, CDR2 and CDR3 regions of SEQ
ID NO. 29: and a variable light chain region comprising the CDR1,
CDR2 and CDR3 regions respectively of SEQ ID NO. 33; [0131] c) a
variable heavy chain (VH) region comprising the CDR1, CDR2 and CDR3
regions of SEQ ID NO. 36: and a variable light chain region
comprising the CDR1, CDR2 and CDR3 regions respectively of SEQ ID
NO. 40; [0132] d) a variable heavy chain (VH) region comprising the
CDR1, CDR2 and CDR3 regions of SEQ ID NO. 43: and a variable light
chain region comprising the CDR1, CDR2 and CDR3 regions
respectively of SEQ ID NO. 47; [0133] e) a variable heavy chain
(VH) region comprising the CDR1, CDR2 and CDR3 regions of SEQ ID
NO. 50: and a variable light chain region comprising the CDR1, CDR2
and CDR3 regions respectively of SEQ ID NO. 54; [0134] f) a
variable heavy chain (VH) region comprising the CDR1, CDR2 and CDR3
regions of SEQ ID NO. 31: and a variable light chain region
comprising the CDR1, CDR2 and CDR3 regions respectively of SEQ ID
NO. 33; or [0135] g) a variable heavy chain (VH) region comprising
the CDR1, CDR2 and CDR3 regions of SEQ ID NO. 65: and a variable
light chain region comprising the CDR1, CDR2 and CDR3 regions
respectively of SEQ ID NO. 69.
[0136] CDR1, CDR2 and CDR3 regions can be identified from variable
heavy and light chain regions using methods known in the art. The
CDR regions from said variable heavy and light chain regions can be
annotated according to IMGT (see Lefranc M P. et al., Nucleic Acids
Research, 27, 209-212, 1999] and Brochet X. Nucl. Acids Res. 36,
W503-508 (2008)). Hence, suitable antigen-binding regions capable
of binding to human B7H4, that are contemplated according to the
invention as described herein comprise: [0137] a) a variable heavy
chain (VH) region comprising the CDR1, CDR2 and CDR3 regions
respectively of SEQ ID NOs.: 26, 27 and 28, and a variable light
chain region comprising the CDR1, CDR2 and CDR3 respectively of SEQ
ID NO. 34, GAS and SEQ ID NO. 35; [0138] b) a variable heavy chain
(VH) region comprising the CDR1, CDR2 and CDR3 regions respectively
of SEQ ID NOs.: 26, 30 and 28, and a variable light chain region
comprising the CDR1, CDR2 and CDR3 respectively of SEQ ID NO. 34,
GAS and SEQ ID NO. 35; [0139] c) a variable heavy chain (VH) region
comprising the CDR1, CDR2 and CDR3 regions respectively of SEQ ID
NOs.: 37, 38 and 39, and a variable light chain region comprising
the CDR1, CDR2 and CDR3 respectively of SEQ ID NO. 41, DTS and SEQ
ID NO. 42; [0140] d) a variable heavy chain (VH) region comprising
the CDR1, CDR2 and CDR3 regions respectively of SEQ ID NOs.: 44, 45
and 46, and a variable light chain region comprising the CDR1, CDR2
and CDR3 respectively of SEQ ID NO. 48, YTS and SEQ ID NO. 49;
[0141] e) a variable heavy chain (VH) region comprising the CDR1,
CDR2 and CDR3 regions respectively of SEQ ID NOs.: 51, 52 and 53,
and a variable light chain region comprising the CDR1, CDR2 and
CDR3 respectively of SEQ ID NO. 55, GAS and SEQ ID NO. 56; [0142]
f) a variable heavy chain (VH) region comprising the CDR1, CDR2 and
CDR3 regions respectively of SEQ ID NOs.: 26, 32 and 28, and a
variable light chain region comprising the CDR1, CDR2 and CDR3
respectively of SEQ ID NO. 34, GAS and SEQ ID NO. 35; or [0143] g)
a variable heavy chain (VH) region comprising the CDR1, CDR2 and
CDR3 regions respectively of SEQ ID NOs.: 66, 67 and 68, and a
variable light chain region comprising the CDR1, CDR2 and CDR3
respectively of SEQ ID NO. 70, GAS and SEQ ID NO. 71.
[0144] Still further suitable antigen-binding regions capable of
binding to human B7H4, that are contemplated according to the
invention as described herein comprise: [0145] a) a variable heavy
chain (VH) region of SEQ ID NO. 25: and a variable light chain
region of SEQ ID NO. 33; [0146] b) a variable heavy chain (VH)
region of SEQ ID NO. 29: and a variable light chain region of SEQ
ID NO. 33; [0147] c) a variable heavy chain (VH) region of SEQ ID
NO. 36: and a variable light chain region of SEQ ID NO. 40; [0148]
d) a variable heavy chain (VH) region of SEQ ID NO. 43: and a
variable light chain region of SEQ ID NO. 47; [0149] e) a variable
heavy chain (VH) region of SEQ ID NO. 50: and a variable light
chain region of SEQ ID NO. 54; [0150] f) a variable heavy chain
(VH) region of SEQ ID NO. 31: and a variable light chain region of
SEQ ID NO. 33; or [0151] g) a variable heavy chain (VH) region of
SEQ ID NO. 65: and a variable light chain region of SEQ ID NO.
69.
[0152] Optionally, said antigen-binding regions that binds to B7H4
comprise heavy and light chain variable regions (VH) having at
least 90%, at least 95%, at least 97%, or at least 99% amino acid
sequence identity with: [0153] a) a variable heavy chain (VH)
region of SEQ ID NO. 25: and a variable light chain region of SEQ
ID NO. 33; [0154] b) a variable heavy chain (VH) region of SEQ ID
NO. 29: and a variable light chain region of SEQ ID NO. 33; [0155]
c) a variable heavy chain (VH) region of SEQ ID NO. 36: and a
variable light chain region of SEQ ID NO. 40; [0156] d) a variable
heavy chain (VH) region of SEQ ID NO. 43: and a variable light
chain region of SEQ ID NO. 47; [0157] e) a variable heavy chain
(VH) region of SEQ ID NO. 50: and a variable light chain region of
SEQ ID NO. 54; [0158] f) a variable heavy chain (VH) region of SEQ
ID NO. 31: and a variable light chain region of SEQ ID NO. 33; or
[0159] g) a variable heavy chain (VH) region of SEQ ID NO. 65: and
a variable light chain region of SEQ ID NO. 69.
[0160] The antibody according to the invention may have an
antigen-binding region capable of binding to B7H4 having a binding
affinity to human B7H4 that corresponds to a K.sub.D value of 5E-7
M or less, such as 1E-7 M or less, such as with a binding affinity
corresponding to a K.sub.D value which is within the range of 5E-7
to 2E-10 M, such as within the range of 2E-7 to 1E-10 M or 1E-7 to
5E-9 M.
[0161] Said binding affinity can be determined by biolayer
interferometry, optionally as set forth in Example 3 herein. Hence,
the antibody according to the invention having a binding affinity
to human B7H4 as defined herein, may have the binding affinity
determined using biolayer interferometry comprising the steps of:
[0162] I) immobilizing the antibody at an amount of 1 .mu.g/mL for
600 seconds on an anti-human IgG Fc Capture biosensor; [0163] II)
determining association over a time period of 300 seconds and
dissociation over a time period of 1000 seconds of human
recombinant His tagged B7H4 protein (Sino Biological cat no
10738-H08H; a protein expressed from a construct of DNA sequence
encoding the human VTCN1(Uniprot accession no. Q7Z7D3)
(Phe29-Ala258) with a C-terminal polyhistidine tag) using a 2-fold
dilution series ranging from 1.56 nM to 100 nM; and [0164] III)
referencing the data to a buffer control (0 nM).
[0165] Furthermore, said binding affinity may be determined using
an antibody such as a monospecific, bivalent antibody, such as an
antibody which is a full length IgG1.
[0166] In a further embodiment, an antibody in accordance with the
invention is provided, comprising an antigen region capable of
binding to human B7H4, wherein said antigen-binding region is
capable of crossblocking: [0167] an antibody comprising a variable
heavy chain (VH) region of SEQ ID NO. 29 and a variable light chain
region of SEQ ID NO. 33; and [0168] an antibody comprising a
variable heavy chain (VH) region of SEQ ID NO. 36: and a variable
light chain region of SEQ ID NO. 40; and [0169] wherein said
antigen-binding region is not capable of crossblocking [0170] an
antibody comprising a variable heavy chain (VH) region of SEQ ID
NO. 43: and a variable light chain region of SEQ ID NO. 47; [0171]
an antibody comprising a variable heavy chain (VH) region of SEQ ID
NO. 50: and a variable light chain region of SEQ ID NO. 54; and
[0172] an antibody comprising a variable heavy chain (VH) region of
SEQ ID NO. 65 and a variable light chain region of SEQ ID NO.
69.
[0173] In still another further embodiment, said antibody in
accordance with the invention, comprises an antigen region capable
of binding to human B7H4, said antigen-binding region capable of
crossblocking [0174] an antibody comprising a variable heavy chain
(VH) region of SEQ ID NO. 43: and a variable light chain region of
SEQ ID NO. 47; [0175] an antibody comprising a variable heavy chain
(VH) region of SEQ ID NO. 50: and a variable light chain region of
SEQ ID NO. 54, and [0176] an antibody comprising a variable heavy
chain (VH) region of SEQ ID NO. 65 and a variable light chain
region of SEQ ID NO. 69; [0177] and wherein said antigen-binding
region is not capable of crossblocking an antibody comprising
[0178] an antibody comprising a variable heavy chain (VH) region of
SEQ ID NO. 29 and a variable light chain region of SEQ ID NO. 33;
and [0179] an antibody comprising a variable heavy chain (VH)
region of SEQ ID NO. 36: and a variable light chain region of SEQ
ID NO. 40.
[0180] In particular, "cross-blocking", or the ability of an
antibody according to the invention to block binding of another
antibody to B7H4, is defined as the ability of a first antibody
bound to B7H4 to block binding of a second antibody to the B7H4
bound to the first antibody. Crossblocking can be determined using
an assay as described in example 5. Such crossblocking can also be
determined e.g. in a procedure comprising the steps of: [0181] i)
providing a set of samples, each sample comprising an antibody
which binds to B7H4; [0182] ii) immobilizing a first antibody from
the set of samples at an amount of 20 .mu.g/mL for 600 seconds on
Amine Reactive 2.sup.nd Generation biosensor (AR2G); [0183] iii)
loading the ARG2 biosensor with immobilized antibody with human
B7H4 (100 nM of human recombinant His tagged B7H4 protein (Sino
Biological cat no 10738-H08H; a protein expressed from a construct
of DNA sequence encoding the human VTCN1(Uniprot accession no.
Q7Z7D3) (Phe29-Ala258) with a C-terminal polyhistidine tag); [0184]
iv) determining the association of a second antibody from the set
of samples at an amount of 10 .mu.g/mL for 300 seconds.
[0185] When the second antibody is not capable of association, the
first antibody is considered to cross-block the second antibody.
The skilled person will be familiar with suitable technologies for
determining the ability of an antibody to crossblock the binding of
another antibody to its target, the present application discloses
procedures suitable for determining blocking of binding and
displacement. In a further embodiment, crossblocking as described
herein is determined as described in Example 5.
[0186] In a further embodiment, the antibody in accordance with the
invention, having an antigen-binding region capable of binding to
human B7H4 complying with a crossblocking feature as described
above, wherein said an antigen-binding region capable of binding to
human B7H4 is capable of binding to B7H3-IgV/B7H4-IgC (SEQ ID NO.
11), and optionally is not capable of binding to B7H4-IgV/B7H3-IgC
(SEQ ID NO. 10).
CD3 and B7H4 Antigen-Binding Region Combinations
[0187] The present disclosure further provides an antibody
according to the invention comprising an antigen-binding region
capable of binding to human B7H4 and an antigen-binding region
capable of binding to human CD3, wherein the antigen-binding region
that binds to CD3 comprises [0188] a heavy chain variable region
(VH) comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 16,
and, a light chain variable region (VL) comprising the CDR1, CDR2,
and CDR3 regions of SEQ ID NO: 22, and wherein the antigen-binding
region capable of binding to B7H4 comprises: [0189] a) a variable
heavy chain (VH) region comprising the CDR1, CDR2 and CDR3 regions
of SEQ ID NO. 25: and a variable light chain region comprising the
CDR1, CDR2 and CDR3 regions respectively of SEQ ID NO. 33; [0190]
b) a variable heavy chain (VH) region comprising the CDR1, CDR2 and
CDR3 regions of SEQ ID NO. 29: and a variable light chain region
comprising the CDR1, CDR2 and CDR3 regions respectively of SEQ ID
NO. 33; [0191] c) a variable heavy chain (VH) region comprising the
CDR1, CDR2 and CDR3 regions of SEQ ID NO. 36: and a variable light
chain region comprising the CDR1, CDR2 and CDR3 regions
respectively of SEQ ID NO. 40; [0192] d) a variable heavy chain
(VH) region comprising the CDR1, CDR2 and CDR3 regions of SEQ ID
NO. 43: and a variable light chain region comprising the CDR1, CDR2
and CDR3 regions respectively of SEQ ID NO. 47; [0193] e) a
variable heavy chain (VH) region comprising the CDR1, CDR2 and CDR3
regions of SEQ ID NO. 50: and a variable light chain region
comprising the CDR1, CDR2 and CDR3 regions respectively of SEQ ID
NO. 54; [0194] f) a variable heavy chain (VH) region comprising the
CDR1, CDR2 and CDR3 regions of SEQ ID NO. 31: and a variable light
chain region comprising the CDR1, CDR2 and CDR3 regions
respectively of SEQ ID NO. 33; or [0195] g) a variable heavy chain
(VH) region comprising the CDR1, CDR2 and CDR3 regions of SEQ ID
NO. 65: and a variable light chain region comprising the CDR1, CDR2
and CDR3 regions respectively of SEQ ID NO. 69.
[0196] The present disclosure further provides an antibody
according to the invention may be an antibody comprising an
antigen-binding region capable of binding to human B7H4 and an
antigen-binding region capable of binding to human CD3, wherein the
antigen-binding region that binds to CD3 comprises [0197] a heavy
chain variable region (VH) comprising the CDR1, CDR2, and CDR3
regions of SEQ ID NO. 17, and, a light chain variable region (VL)
comprising the CDR1, CDR2, and CDR3 regions of SEQ ID NO: 22, and
wherein the antigen-binding region capable of binding to B7H4
comprises: [0198] a) a variable heavy chain (VH) region comprising
the CDR1, CDR2 and CDR3 regions of SEQ ID NO. 25: and a variable
light chain region comprising the CDR1, CDR2 and CDR3 regions
respectively of SEQ ID NO. 33; [0199] b) a variable heavy chain
(VH) region comprising the CDR1, CDR2 and CDR3 regions of SEQ ID
NO. 29: and a variable light chain region comprising the CDR1, CDR2
and CDR3 regions respectively of SEQ ID NO. 33; [0200] c) a
variable heavy chain (VH) region comprising the CDR1, CDR2 and CDR3
regions of SEQ ID NO. 36: and a variable light chain region
comprising the CDR1, CDR2 and CDR3 regions respectively of SEQ ID
NO. 40; [0201] d) a variable heavy chain (VH) region comprising the
CDR1, CDR2 and CDR3 regions of SEQ ID NO. 43: and a variable light
chain region comprising the CDR1, CDR2 and CDR3 regions
respectively of SEQ ID NO. 47; [0202] e) a variable heavy chain
(VH) region comprising the CDR1, CDR2 and CDR3 regions of SEQ ID
NO. 50: and a variable light chain region comprising the CDR1, CDR2
and CDR3 regions respectively of SEQ ID NO. 54; [0203] f) a
variable heavy chain (VH) region comprising the CDR1, CDR2 and CDR3
regions of SEQ ID NO. 31: and a variable light chain region
comprising the CDR1, CDR2 and CDR3 regions respectively of SEQ ID
NO. 33; or [0204] g) a variable heavy chain (VH) region comprising
the CDR1, CDR2 and CDR3 regions of SEQ ID NO. 65: and a variable
light chain region comprising the CDR1, CDR2 and CDR3 regions
respectively of SEQ ID NO. 69.
[0205] Also, the present disclosure further provides an antibody
comprising an antigen-binding region capable of binding to human
B7H4 and an antigen-binding region capable of binding to human CD3,
wherein the antigen-binding region capable of binding to CD3
comprises: [0206] a heavy chain variable region (VH) comprising the
CDR1, CDR2, and CDR3 sequences of SEQ ID NOs.: 18, 19 and 20
respectively; and, a light chain variable region (VL) comprising
the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 23, GTN and 24,
respectively; and wherein the antigen-binding region capable of
binding to B7H4 comprises: [0207] a) a variable heavy chain (VH)
region comprising the CDR1, CDR2 and CDR3 regions respectively of
SEQ ID NOs.: 26, 27 and 28, and a variable light chain region
comprising the CDR1, CDR2 and CDR3 respectively of SEQ ID NO. 34,
GAS and SEQ ID NO. 35; [0208] b) a variable heavy chain (VH) region
comprising the CDR1, CDR2 and CDR3 regions respectively of SEQ ID
NOs.: 26, 30 and 28, and a variable light chain region comprising
the CDR1, CDR2 and CDR3 respectively of SEQ ID NO. 34, GAS and SEQ
ID NO. 35; [0209] c) a variable heavy chain (VH) region comprising
the CDR1, CDR2 and CDR3 regions respectively of SEQ ID NOs.: 37, 38
and 39, and a variable light chain region comprising the CDR1, CDR2
and CDR3 respectively of SEQ ID NO. 41, DTS and SEQ ID NO. 42;
[0210] d) a variable heavy chain (VH) region comprising the CDR1,
CDR2 and CDR3 regions respectively of SEQ ID NOs.: 44, 45 and 46,
and a variable light chain region comprising the CDR1, CDR2 and
CDR3 respectively of SEQ ID NO. 48, YTS and SEQ ID NO. 49; [0211]
e) a variable heavy chain (VH) region comprising the CDR1, CDR2 and
CDR3 regions respectively of SEQ ID NOs.: 51, 52 and 53, and a
variable light chain region comprising the CDR1, CDR2 and CDR3
respectively of SEQ ID NO. 55, GAS and SEQ ID NO. 56; [0212] f) a
variable heavy chain (VH) region comprising the CDR1, CDR2 and CDR3
regions respectively of SEQ ID NOs.: 26, 32 and 28, and a variable
light chain region comprising the CDR1, CDR2 and CDR3 respectively
of SEQ ID NO. 34, GAS and SEQ ID NO. 35; or [0213] g) a variable
heavy chain (VH) region comprising the CDR1, CDR2 and CDR3 regions
respectively of SEQ ID NOs.: 66, 67 and 68, and a variable light
chain region comprising the CDR1, CDR2 and CDR3 respectively of SEQ
ID NO. 70, GAS and SEQ ID NO. 71.
[0214] The present disclosure further provides an antibody
comprising an antigen-binding region capable of binding to human
B7H4 and an antigen-binding region capable of binding to human CD3,
wherein the antigen-binding region capable of binding to CD3
comprises: [0215] a heavy chain variable region (VH) comprising the
CDR1, CDR2, and CDR3 sequences of SEQ ID NOs.: 18, 19 and 21
respectively; and, a light chain variable region (VL) comprising
the CDR1, CDR2, and CDR3 sequences of SEQ ID NO: 23, GTN and 24,
respectively; and wherein the antigen-binding region capable of
binding to B7H4 comprises: [0216] a) a variable heavy chain (VH)
region comprising the CDR1, CDR2 and CDR3 regions respectively of
SEQ ID NOs.: 26, 27 and 28, and a variable light chain region
comprising the CDR1, CDR2 and CDR3 respectively of SEQ ID NO. 34,
GAS and SEQ ID NO. 35; [0217] b) a variable heavy chain (VH) region
comprising the CDR1, CDR2 and CDR3 regions respectively of SEQ ID
NOs.: 26, 30 and 28, and a variable light chain region comprising
the CDR1, CDR2 and CDR3 respectively of SEQ ID NO. 34, GAS and SEQ
ID NO. 35; [0218] c) a variable heavy chain (VH) region comprising
the CDR1, CDR2 and CDR3 regions respectively of SEQ ID NOs.: 37, 38
and 39, and a variable light chain region comprising the CDR1, CDR2
and CDR3 respectively of SEQ ID NO. 41, DTS and SEQ ID NO. 42;
[0219] d) a variable heavy chain (VH) region comprising the CDR1,
CDR2 and CDR3 regions respectively of SEQ ID NOs.: 44, 45 and 46,
and a variable light chain region comprising the CDR1, CDR2 and
CDR3 respectively of SEQ ID NO. 48, YTS and SEQ ID NO. 49; [0220]
e) a variable heavy chain (VH) region comprising the CDR1, CDR2 and
CDR3 regions respectively of SEQ ID NOs.: 51, 52 and 53, and a
variable light chain region comprising the CDR1, CDR2 and CDR3
respectively of SEQ ID NO. 55, GAS and SEQ ID NO. 56; [0221] f) a
variable heavy chain (VH) region comprising the CDR1, CDR2 and CDR3
regions respectively of SEQ ID NOs.: 26, 32 and 28, and a variable
light chain region comprising the CDR1, CDR2 and CDR3 respectively
of SEQ ID NO. 34, GAS and SEQ ID NO. 35; or [0222] g) a variable
heavy chain (VH) region comprising the CDR1, CDR2 and CDR3 regions
respectively of SEQ ID NOs.: 66, 67 and 68, and a variable light
chain region comprising the CDR1, CDR2 and CDR3 respectively of SEQ
ID NO. 70, GAS and SEQ ID NO. 71.
[0223] Further disclosed are antibodies comprising an
antigen-binding region capable of binding to human B7H4 and an
antigen-binding region capable of binding to human CD3, wherein the
antigen-binding region that binds to CD3 comprises: [0224] a heavy
chain variable region (VH) comprising the sequence of SEQ ID NO: 16
and, a light chain variable region (VL) comprising the sequence of
SEQ ID NO: 22; and wherein the antigen-binding region capable of
binding to B7H4 comprises an antigen-binding regions that bind to
B7H4 comprise heavy and light chain variable regions (VH) having:
[0225] a) a variable heavy chain (VH) region of SEQ ID NO. 25: and
a variable light chain region of SEQ ID NO. 33; [0226] b) a
variable heavy chain (VH) region of SEQ ID NO. 29: and a variable
light chain region of SEQ ID NO. 33; [0227] c) a variable heavy
chain (VH) region of SEQ ID NO. 36: and a variable light chain
region of SEQ ID NO. 40; [0228] d) a variable heavy chain (VH)
region of SEQ ID NO. 43: and a variable light chain region of SEQ
ID NO. 47; [0229] e) a variable heavy chain (VH) region of SEQ ID
NO. 50: and a variable light chain region of SEQ ID NO. 54; [0230]
f) a variable heavy chain (VH) region of SEQ ID NO. 31: and a
variable light chain region of SEQ ID NO. 33; or [0231] g) a
variable heavy chain (VH) region of SEQ ID NO. 65: and a variable
light chain region of SEQ ID NO. 69.
[0232] Still further disclosed are antibodies comprising an
antigen-binding region capable of binding to human B7H4 and an
antigen-binding region capable of binding to human CD3, wherein the
antigen-binding region that binds to CD3 comprises: [0233] a heavy
chain variable region (VH) comprising the sequence of SEQ ID NO: 17
and, a light chain variable region (VL) comprising the sequence of
SEQ ID NO: 22; and wherein the antigen-binding region capable of
binding to B7H4 comprises antigen-binding heavy and light chain
variable regions (VH) having: [0234] a) a variable heavy chain (VH)
region of SEQ ID NO. 25: and a variable light chain region of SEQ
ID NO. 33; [0235] b) a variable heavy chain (VH) region of SEQ ID
NO. 29: and a variable light chain region of SEQ ID NO. 33; [0236]
c) a variable heavy chain (VH) region of SEQ ID NO. 36: and a
variable light chain region of SEQ ID NO. 40; [0237] d) a variable
heavy chain (VH) region of SEQ ID NO. 43: and a variable light
chain region of SEQ ID NO. 47; [0238] e) a variable heavy chain
(VH) region of SEQ ID NO. 50: and a variable light chain region of
SEQ ID NO. 54; [0239] f) a variable heavy chain (VH) region of SEQ
ID NO. 31: and a variable light chain region of SEQ ID NO. 33; or
[0240] g) a variable heavy chain (VH) region of SEQ ID NO. 65: and
a variable light chain region of SEQ ID NO. 69.
[0241] In a further embodiment, in such a bispecific antibody, said
antigen binding region capable of binding to human B7H4 is
comprised in an heavy chain and a light chain, said heavy chain
comprising said VH region and an IgG1 heavy chain constant region
and said light chain comprising said VL region and a kappa light
chain constant region; and wherein said antigen binding region
capable of binding to human CD3 is comprised in a heavy chain and a
light chain, said heavy chain comprising said VH region and an IgG1
heavy chain constant region and said light chain comprising said VL
region and a lambda light chain constant region. More preferably,
in such a bispecific antibody, one IgG1 heavy chain constant region
is as defined in SEQ ID NO. 60 and the other is as defined in SEQ
ID NO. 61, and wherein said kappa light chain constant region is as
defined in SEQ ID NO. 63 and said lambda light chain constant
region is as defined in SEQ ID NO. 64. It is understood that
optionally, of said IgG1 heavy chain constant regions as defined in
SEQ ID NO. 60 and 61, the terminal lysines can be deleted.
[0242] As will be well-known to the skilled person, each
antigen-binding region of an antibody generally comprise a heavy
chain variable region (VH) and a light chain variable region (VL),
and each of the variable regions comprises three CDR sequences,
CDR1, CDR2 and CDR3, respectively, and may comprise four framework
sequences, FR1, FR2, FR3 and FR4, respectively. Each
antigen-binding region of an antibody may generally comprise a
heavy chain variable region (VH) and a light chain variable region
(VL), and each of the variable regions comprises three CDR
sequences, CDR1, CDR2 and CDR3, respectively, and may comprise four
human framework sequences, FR1, FR2, FR3 and FR4, respectively.
This structure is preferably also found in the antibodies according
to the present invention. Furthermore, the antibodies according to
the invention may comprise two heavy chain constant regions (CH),
and two light chain constant regions (CL). Examples of constant
regions are provided i.a. in SEQ ID NOs. 57-64.
[0243] In particular embodiments, the antibody according to the
invention comprises a first and a second heavy chain, such as a
first and second heavy chain each comprising at least a hinge
region, a CH2 and CH3 region. Stable, heterodimeric antibodies can
be obtained at high yield for instance by so-called Fab-arm
exchange as provided in WO 2008/119353 and WO 2011/131746, on the
basis of two homodimeric starting proteins containing only a few,
asymmetrical mutations in the CH3 regions. Hence, in some
embodiments of the invention, the antibody comprises a first heavy
chain wherein at least one of the amino acids at the positions
corresponding to positions selected from the group consisting of
T366, L368, K370, D399, F405, Y407 and K409 in a human IgG1 heavy
chain has been substituted, and a second heavy chain wherein at
least one of the amino acids in the positions corresponding to a
position selected from the group consisting of T366, L368, K370,
D399, F405, Y407, and K409 in a human IgG1 heavy chain has been
substituted, wherein said substitutions of said first and said
second heavy chains are not in the same positions, and wherein the
amino acid positions are numbered according to Eu numbering. For
example, constant domains having such a substitution are provided
i.a. in SEQ ID NO. 58 and 62, which can be compared with SEQ ID NO.
57, which does not have such a substitution.
[0244] The term "amino acid corresponding to positions" as used
herein refers to an amino acid position number in a human IgG1
heavy chain. Corresponding amino acid positions in other
immunoglobulins may be found by alignment with human IgG1. Unless
otherwise stated or contradicted by context, the amino acids of the
constant region sequences are herein numbered according to the
EU-index of numbering (described in Kabat, E. A. et al., 1991,
Sequences of proteins of immunological interest. 5th Edition--US
Department of Health and Human Services, NIH publication No.
91-3242, pp 662, 680, 689). Thus, an amino acid or segment in one
sequence that "corresponds to" an amino acid or segment in another
sequence is one that aligns with the other amino acid or segment
using a standard sequence alignment program such as ALIGN, ClustalW
or similar, typically at default settings and has at least 50%, at
least 80%, at least 90%, or at least 95% identity to a human IgG1
heavy chain. It is considered well-known in the art how to align a
sequence or segment in a sequence and thereby determine the
corresponding position in a sequence to an amino acid position
according to the present invention.
[0245] In particular embodiments, the invention provides an
antibody, wherein the amino acid in the position corresponding to
K409 in a human IgG1 heavy chain is R in said first heavy chain,
and the amino acid in the position corresponding to F405 in a human
IgG1 heavy chain is L in said second heavy chain, or vice
versa.
[0246] In some embodiments, the antibody according to the present
invention comprises, in addition to the antigen-binding regions,
comprises an Fc region with Fc sequences of the two heavy chains.
The first and second Fc sequence may each be of any isotype,
including any human isotype, such as an IgG1, IgG2, IgG3, IgG4,
IgE, IgD, IgM, or IgA isotype or a mixed isotype. Preferably, the
Fc region is a human IgG1, IgG2, IgG3, IgG4 isotype or a mixed
isotype, such as a human IgG1 isotype. In some embodiments, it is
preferred that the antibody according to the invention is a
full-length antibody, most preferably it is of the IgG1 type.
[0247] Antibodies according to the present invention may comprise
modifications in the Fc region to render the antibody an inert, or
non-activating, antibody. Hence, in the antibodies disclosed
herein, one or both heavy chains may be modified so that the
antibody induces Fc-mediated effector function to a lesser extent
relative to an antibody which is identical, except for comprising
non-modified first and second heavy chains. The Fc-mediated
effector function may be measured by determining Fc-mediated CD69
expression on T cells (i.e. CD69 expression as a result of CD3
antibody-mediated, Fc.gamma. receptor-dependent CD3 crosslinking),
by binding to Fc.gamma. receptors, by binding to C1q, or by
induction of Fc-mediated cross-linking of Fc.gamma.Rs. In
particular, the heavy chain constant sequences may be modified so
that the Fc-mediated CD69 expression is reduced by at least 50%, at
least 60%, at least 70%, at least 80%, at least 90%, at least 99%
or 100% when compared to a wild-type (unmodified) antibody, wherein
said Fc-mediated CD69 expression is determined in a PBMC-based
functional assay, e.g. as described in Example 3 of WO2015001085.
Modifications of the heavy and light chain constant sequences may
also result in reduced binding of C1q to said antibody. As compared
to an unmodified antibody the reduction may be by at least 70%, at
least 80%, at least 90%, at least 95%, at least 97%, or 100% and
the C1q binding may be determined by ELISA. Further, the Fc region
which may be modified so that said antibody mediates reduced
Fc-mediated T-cell proliferation compared to an unmodified antibody
by at least 50%, at least 60%, at least 70%, at least 80%, at least
90%, at least 99% or 100%, wherein said T-cell proliferation is
measured in a PBMC-based functional assay.
[0248] A wide range of different non-activating antibody formats
have been developed in which amino acid substitutions, and
combinations thereof, have been introduced in the constant heavy
chain region of an IgG1 isotype antibody to eliminate Fc-mediated
effector functions (e.g. Chiu et al., Antibodies 2019 December;
8(4): 55; Liu et al., Antibodies, 2020 Nov. 17; 9(4):64;
29(10):457-66; Shields et al., J Biol Chem., 2001 Mar. 2;
276(9):6591-604).
[0249] Examples of amino acid positions that may be modified, e.g.
in an IgG1 isotype antibody, include positions L234 and L235.
Hence, the antibody according to the invention may comprises a
first and a second heavy chain, and wherein in both the first and
the second heavy chain, the amino acid residues at the positions
corresponding to positions L234 and L235 in a human IgG1 heavy
chain according to Eu numbering are F and E, respectively. It is
understood that in addition to modifications of amino acid
positions L234 and L235, further positions may be modified.
[0250] In addition, a D265A amino acid substitution can decrease
binding to all Fc.gamma. receptors and prevent ADCC (Shields et
al., 2001, J. Biol. Chem. (276):6591-604). Therefore, the antibody
according to the invention may comprise a first and a second heavy
chain, wherein in both the first and the second heavy chain, the
amino acid residue at the position corresponding to position D265
in a human IgG1 heavy chain according to Eu numbering is A. Further
embodiments of the invention provide antibodies wherein, in at
least one, such as in both, of said first and second heavy chains
the amino acids in the positions corresponding to positions L234,
L235, and D265 in a human IgG1 heavy chain, are F, E, and A,
respectively. In the present application antibodies, which have the
combination of three amino acid substitutions L234F, L235E and
D265A and in addition the K409R or the F405L mutation disclosed
herein above may be termed with the suffix "FEAR" or "FEAL",
respectively.
[0251] An amino acid sequence of a wild type IgG1 heavy chain
constant region is identified herein as SEQ ID NO: 57. Consistent
with the embodiments disclosed above, the antibody of the invention
may comprise an IgG1 heavy chain constant region carrying the F405L
substitution and may have the amino acid sequence set forth in SEQ
ID NO: 58 and/or an IgG1 heavy chain constant region carrying the
K409R substitution and may have the amino acid sequence set forth
in SEQ ID NO: 62.
[0252] An amino acid sequence of an IgG1 heavy chain constant
region carrying the L234F, L235E and D265A substitutions is
identified herein as SEQ ID NO: 59. An amino acid sequence of an
IgG1 heavy chain constant region carrying the L234F, L235E, D265A
and F405L substitutions is identified herein as SEQ ID NO: 60. An
amino acid sequence of an IgG1 heavy chain constant region carrying
the L234F, L235E, D265A and K409R substitutions is identified
herein as SEQ ID NO: 61.
[0253] The constant region sequences listed in SEQ ID NOs. 57-62
list a terminal lysine (K), such sequences were used in the example
section herein. The origin of this lysine is a naturally occurring
sequence found in humans from which these Fc regions are derived.
During cell culture production of recombinant antibodies, this
terminal lysine can be cleaved off by proteolysis by endogenous
carboxypeptidase(s), resulting in a constant region having the same
sequence but lacking the C-terminal lysine. For manufacturing
purposes of antibodies, the DNA encoding this terminal lysine can
be omitted from the sequence such that antibodies are produced
without the lysine. Antibodies produced from nucleic acid sequences
that either do, or do not encode a terminal lysine are
substantially identical in sequence and in function since the
degree of processing of the terminal lysine is typically high when
e.g. using antibodies produced in CHO-based production systems
(Dick, L. W. et al. Biotechnol. Bioeng. 2008; 100: 1132-1143).
Hence, it is understood that antibodies in accordance with the
invention can be generated without encoding or having a terminal
lysine such as listed herein. For manufacturing purposes,
antibodies can thus be generated without having a terminal
lysine.
[0254] The present invention further provides an antibody, wherein
[0255] a) the antigen-binding region capable of binding to B7H4 is
human, and [0256] b) the antigen-binding region capable of binding
to CD3, is humanized.
[0257] Also, the invention provides an antibody, wherein [0258] a)
the antigen-binding region capable of binding to B7H4 is human,
and/or the antigen-binding region capable of binding to CD3, is
humanized.
[0259] In some embodiments of the invention, the antibody comprises
a kappa (.kappa.) light chain. The sequence of particular
embodiments of the invention concerning bispecific antibodies, the
kappa light chain comprises the CDR1, -2 and -3 sequences of a B7H4
antibody light chain as disclosed above.
[0260] In further embodiments of the invention, the antibody
according to any one of the preceding claims, wherein said antibody
comprises a lambda (.lamda.) light chain. In particular embodiments
of the invention concerning bispecific antibodies, the lambda light
chain comprises the CDR1, -2 and -3 sequences of a CD3 antibody
light chain as disclosed above, in particular a the CDR1, -2 and -3
sequences of a CD3 antibody having reduced affinity for CD3 as
disclosed above. The amino acid sequence of a kappa light chain
constant region is included herein as SEQ ID NO: 63 and the amino
acid sequence of a lambda light chain constant region is included
herein as SEQ ID NO: 64.
[0261] In particular embodiments, the antibody comprises a lambda
(.lamda.) light chain and a kappa (.kappa.) light chain; e.g. an
antibody with a heavy chain and a lambda light chain which comprise
the binding region capable of binding to CD3, and a heavy chain and
a kappa light chain which comprise the binding region capable of
binding to B7H4.
[0262] Hence, in a further embodiment, in a bispecific antibody as
defined herein, said antigen binding region capable of binding to
human B7H4 is comprised in a heavy chain and a light chain, said
heavy chain comprising said VH region and an IgG1 heavy chain
constant region and said light chain comprising said VL region and
a kappa light chain constant region; and said antigen binding
region capable of binding to human CD3 is comprised in a heavy
chain and a light chain, said heavy chain comprising said VH region
and an IgG1 heavy chain constant region and said light chain
comprising said VL region and a lambda light chain constant region.
More preferably, in said bispecific antibody, one IgG1 heavy chain
constant region is as defined in SEQ ID NO. 60 and the other is as
defined in SEQ ID NO. 61, and said kappa light chain constant
region is as defined in SEQ ID NO. 63 and said lambda light chain
constant region is as defined in SEQ ID NO. 64. It is understood
that said IgG1 heavy chain constant regions as defined in SEQ ID
NO. 60 and 61 may have their terminal lysines deleted.
Binding, Cytotoxicity and T-Cell Activation
[0263] Antibodies, such as bispecific antibodies, as described
herein that can bind to human CD3 and human B7H4 can advantageously
target T cells to human B7H4 expressing cancer cells, thereby
inducing T-cell mediated killing of said cancer cells. By having
reduced or inert Fc-functionality in such antibodies, as shown in
the example section, safe, effective and sufficient antibody can be
administered to human patients, while being efficacious against a
wide range of cancers varying in B7H4 expression levels.
[0264] As said, preferably, the antibody in accordance with the
invention is devoid of, or has reduced Fc-mediated effector
function, and furthermore, the antibody: [0265] a) is capable of
binding to B7H4-expressing human tumor cells as described in
Example 9 and 10 herein, [0266] b) is capable of mediating
concentration-dependent cytotoxicity of B7H4-expressing human tumor
cells when using e.g. purified PBMCs or T cells as effector cells
when assayed as described in Example 11 and 12 herein, [0267] c) is
capable of mediating concentration-dependent cytotoxicity of one or
more human B7H4-expressing tumor cell lines selected from the group
consisting of MCF-7, MDA-MB-468, SK-BR3, NIH-OVCAR-3, HCC1954, and
NCI-H1650, when using e.g. purified PBMCs or T cells as effector
cells when assayed as described in Example 11 and 12 herein, [0268]
d) is capable of activating T cells in vitro in the presence of
B7H4-expressing human tumor cells; e.g. when assayed as described
in Example 13 herein, [0269] e) is capable of activating T-cells in
vitro in the presence of one or more B7H4-expressing human tumor
cell lines selected from the group consisting of MCF-7, MDA-MB-468,
SK-BR3, NIH-OVCAR-3, HCC1954, and NCI-H1650; e.g. when assayed as
described in Example 13 herein, [0270] f) is capable of inducing
cytotoxicity of B7H4-expressing human tumor cells; e.g. when
assayed as described in Example 11 and 12 herein, and/or [0271] g)
is capable of inducing T cell mediated cytotoxicity in one or more
B7H4-expressing human tumor cell lines selected from the group
consisting of MCF-7, MDA-MB-468, SK-BR3, NIH-OVCAR-3, HCC1954, and
NCI-H1650; e.g. when assayed as described in Example 11 and 12
herein.
[0272] Furthermore, the antibody in accordance with the invention
may be devoid of, or has reduced Fc-mediated effector function,
and, furthermore capable of inducing T-cell mediated cytotoxicity
antibody, wherein cytotoxicity is assessed in an in vitro IC50
assay comprising: [0273] i) providing isolated peripheral blood
mononuclear cells (PBMCs), or purified T-cells, from healthy human
donor buffy coats; [0274] ii) providing B7H4-expressing tumor
cells, such as a human B7H4-expressing tumor cell line selected
from the group consisting of MCF-7, MDA-MB-468, SK-BR3,
NIH-OVCAR-3, HCC1954, and NCI-H1650; [0275] iii) combining said
PBMCs or said purified T-cells with a plurality of samples of said
B7H4-expressing tumor cells, wherein the ratio of the number of
T-cells from said PBMCs, or said purified T-cells, to the selected
tumor cell is 8:1; [0276] iv) providing said antibody in a dilution
series to said samples, ranging e.g. from 0.0128 ng/mL to 10,000
ng/mL for a selected human B7H4 expressing tumor cell; and [0277]
v) incubating the samples obtained in step iv), e.g. for 72 hours
at 37.degree. C.; and subsequently; [0278] vi) assessing the
viability of the B7H4-expressing tumor cells; [0279] vii)
determining the percentage of viable cells for each dilution
sample; and [0280] viii) determining the IC50.
[0281] Instead of isolated peripheral blood mononuclear cells
(PBMCs), purified T-cells may also be provided in step i).
[0282] Accordingly, the antibody may have an IC50 in the range of
0.001-2 microgram/ml, wherein the IC50 is determined in an in vitro
cytotoxicity assay comprising the steps of: [0283] i) providing
isolated peripheral blood mononuclear cells (PBMCs) from healthy
human donor buffy coats; [0284] ii) providing B7H4-expressing tumor
cells, such as a human B7H4-expressing tumor cell line selected
from the group consisting of MCF-7, MDA-MB-468, SK-BR3,
NIH-OVCAR-3, and HCC1954; [0285] iii) combining said PBMCs with a
plurality of samples of said B7H4-expressing tumor cells, wherein
the ratio of the number of T-cells from said PBMCs to the selected
tumor cell is 8:1; [0286] iv) providing said antibody in a dilution
series to said samples, ranging e.g. from 0.0128 ng/mL to 10,000
ng/mL for a selected human B7H4 expressing tumor cell; and [0287]
v) incubating the samples obtained in step iv), e.g. for 72 hours
at 37.degree. C.; and subsequently, [0288] vi) assessing the
viability of the B7H4-expressing tumor cells; [0289] vii)
determining the percentage of viable cells for each dilution
sample; and [0290] viii) determining the IC50.
[0291] Accordingly, the antibody may have an IC50 in the range of
0.001-5 microgram/ml, wherein the IC50 is determined in an in vitro
cytotoxicity assay comprising the steps of: [0292] i) providing
isolated peripheral blood mononuclear cells (PBMCs), or purified
T-cells, from healthy human donor buffy coats; [0293] ii) providing
B7H4-expressing tumor cells, such as a human B7H4-expressing tumor
cell line selected from the group consisting of MCF-7, MDA-MB-468,
SK-BR3, NIH-OVCAR-3, HCC1954, and NCI-H1650; [0294] iii) combining
said PBMCs or said purified T-cells with a plurality of samples of
said B7H4-expressing tumor cells, wherein the ratio of the number
of T-cells from said PBMCs, or said purified T-cells, to the
selected tumor cell is 8:1; [0295] iv) providing said antibody in a
dilution series to said samples, ranging e.g. from 0.0128 ng/mL to
10,000 ng/mL for a selected human B7H4 expressing tumor cell; and
[0296] v) incubating the samples obtained in step iv), e.g. for 72
hours at 37.degree. C.; and subsequently, [0297] vi) assessing the
viability of the B7H4-expressing tumor cells; [0298] vii)
determining the percentage of viable cells for each dilution
sample; and [0299] viii) determining the IC50.
[0300] In one embodiment, the antibody in accordance with the
invention may have an IC50 in the range of 0.001-5 microgram/ml. In
one embodiment, the antibody in accordance with the invention may
have an IC50 in the range of 0.001-2 microgram/ml. In another
embodiment, the antibody in accordance with the invention may have
an IC50 is in the range of 0.001-0.03 microgram/ml. In still a
further embodiment, the IC50 may be in the range of 0.05-2
microgram/ml. In yet another further embodiment, the IC50 may be in
the range of 0.05-5 microgram/ml. Said IC50 may be determined using
a method such as described in Example 12.
[0301] In a further embodiment, the ability of the antibody in
accordance with the invention to mediate T cell activation is
determined in an in vitro assay comprising the steps of: [0302] i)
providing isolated peripheral blood mononuclear cells (PBMCs) from
healthy human donor buffy coats; [0303] ii) providing
B7H4-expressing tumor cells; [0304] iii) combining PBMCs and
B7H4-expressing tumor cells in a plurality of samples, wherein the
ratio of the number of PBMCs to tumor cells is 8:1; [0305] iv)
providing said antibody in a dilution series to said samples,
ranging e.g. from 0.0128 ng/mL to 10,000 ng/mL; [0306] v)
incubating the samples, e.g. for 72 hours at 37.degree. C.; and
[0307] vi) subsequently detecting cytokines.
[0308] Exemplary cytokines that can be e.g. detected are e.g.
IFN-.gamma., such as e.g. described in example 13. Preferably
B7H4-expressing tumor cells are human B7H4-expressing tumors, such
as primary tumors, or tumor cell lines selected from the group
consisting of MCF-7, MDA-MB-468, SK-BR3, NIH-OVCAR-3, and
HCC1954.
B7H4 Antibodies
[0309] In another embodiment, an antibody is provided comprising an
antigen-binding region capable of binding to human B7H4, wherein
said antigen-binding region capable of binding to human B7H4
comprises: [0310] a) a variable heavy chain (VH) region comprising
the CDR1, CDR2 and CDR3 regions of SEQ ID NO. 25: and a variable
light chain region comprising the CDR1, CDR2 and CDR3 regions
respectively of SEQ ID NO. 33; [0311] b) a variable heavy chain
(VH) region comprising the CDR1, CDR2 and CDR3 regions of SEQ ID
NO. 29: and a variable light chain region comprising the CDR1, CDR2
and CDR3 regions respectively of SEQ ID NO. 33; [0312] c) a
variable heavy chain (VH) region comprising the CDR1, CDR2 and CDR3
regions of SEQ ID NO. 31: and a variable light chain region
comprising the CDR1, CDR2 and CDR3 regions respectively of SEQ ID
NO. 33; [0313] d) a variable heavy chain (VH) region comprising the
CDR1, CDR2 and CDR3 regions respectively of SEQ ID NOs.: 26, 27 and
28, and a variable light chain region comprising the CDR1, CDR2 and
CDR3 respectively of SEQ ID NO. 34, GAS and SEQ ID NO. 35; [0314]
e) a variable heavy chain (VH) region comprising the CDR1, CDR2 and
CDR3 regions respectively of SEQ ID NOs.: 26, 30 and 28, and a
variable light chain region comprising the CDR1, CDR2 and CDR3
respectively of SEQ ID NO. 34, GAS and SEQ ID NO. 35; [0315] f) a
variable heavy chain (VH) region comprising the CDR1, CDR2 and CDR3
regions respectively of SEQ ID NOs.: 26, 32 and 28, and a variable
light chain region comprising the CDR1, CDR2 and CDR3 respectively
of SEQ ID NO. 34, GAS and SEQ ID NO. 35; [0316] g) a variable heavy
chain (VH) region of SEQ ID NO. 25: and a variable light chain
region of SEQ ID NO. 33; [0317] h) a variable heavy chain (VH)
region of SEQ ID NO. 29: and a variable light chain region of SEQ
ID NO. 33; [0318] i) a variable heavy chain (VH) region of SEQ ID
NO. 31: and a variable light chain region of SEQ ID NO. 33; or
[0319] j) having a heavy (VH) and light (VH) chain variable regions
having at least 90%, at least 95%, at least 97%, or at least 99%
amino acid sequence identity with the respective variable heavy
chain (VH) region of SEQ ID NO. 25 and the variable light chain
region of SEQ ID NO. 33.
[0320] Such antibodies do not necessarily comprise an
antigen-binding region that binds to CD3. Such antibodies may be
useful, e.g. in kits and assays for detecting B7H4. Such antibodies
may also be useful in the treatment of cancer. Hence, such an
antibody may be monospecific antibody binding to B7H4. Such an
antibody may be a bivalent antibody.
[0321] Preferably, such an antibody is an antibody comprising a
heavy chain constant region which is a human IgG1 constant region.
For example, a heavy chain constant region such as listed in SEQ ID
NO. 57-62. A preferred light chain constant region is a kappa light
chain, such as listed in SEQ ID NO. 63.
[0322] In one embodiment, the antibody provided herein may bind to
an epitope or antibody binding region on human B7H4 comprising one
or more of the amino acid residues S151, V157, D158, Y159, E164,
L166, W173, P175, P177, V179, W181, F199, M208, V210, T222, Y223,
V240, E242 and I245; the numbering of each amino acid residue
referring to its position in SEQ ID NO: 1. In a further embodiment,
the antibody provided herein may bind to an epitope or antibody
binding region on human B7H4 comprising one or more of the amino
acid residues V157, D158, Y159, E164, L166; the numbering of each
amino acid residue referring to its position in SEQ ID NO: 1.
[0323] In another embodiment, the antibody provided herein may bind
to an epitope or antibody binding region on human B7H4 comprising
the amino acid residues S151, V157, D158, Y159, E164, L166, W173,
P175, P177, V179, W181, F199, M208, V210, T222, Y223, V240, E242
and I245; the numbering of each amino acid residue referring to its
position in SEQ ID NO: 1. In a further embodiment, the antibody
provided herein may bind to an epitope or antibody binding region
on human B7H4 comprising the amino acid residues V157, D158, Y159,
E164, L166; the numbering of each amino acid residue referring to
its position in SEQ ID NO: 1.
[0324] Based on the results provided in Example 7 herein it is
hypothesized, without any wish to be bound by theory, that any one
or more of these amino acid residues (i.e. S151, V157, D158, Y159,
E164, L166, W173, P175, P177, V179, W181, F199, M208, V210, T222,
Y223, V240, E242 and I245) is/are directly involved in binding of
the antibody, such as by way of non-covalent interactions; e.g with
amino acid residues within the CDR sequences of the antibody.
[0325] The amino acid residues comprised by said epitope or
antibody binding region and optionally the one or more additional
amino acid residues which are indirectly involved in binding may be
identified by alanine scanning of human B7H4 having the amino acid
sequence set forth in SEQ ID NO: 1 or the extracellular domain
sequence of SEQ ID NO: 1. The alanine scanning may in particular be
performed as set forth or essentially as set forth in Example 7
herein.
[0326] Further, the alanine scanning may be performed by a
procedure comprising the steps of: [0327] i) Expressing mutant
human B7H4 polypeptides in which amino acid residues in the
extracellular domain of human B7H4, except cysteines and alanines,
are individually substituted with alanine, and corresponding wild
type B7H4 polypeptides individually in human embryonic kidney
cells, e.g. HEK 293 cells, such that for each mutant or wild type
B7H4 a sample comprising 40-60.000 cells, such as 50.000 cells is
provided, [0328] ii) Incubating the cells in each sample with 20
.mu.L of said antibody, wherein said antibody consists of a single
heavy chain and a single light chain, which antibody is labelled,
e.g. with a suitable label for flow cytometry analysis such as an
mNeogreen label, and incubated for an hour at room temperature, and
subsequently washing with FACS buffer (e.g. phosphate-buffered
saline [PBS; Lonza, cat. no. BE17-517]+0.1% [w/v] BSA [Roche, cat.
no. 10735086001]+0.02% [w/v] sodium azide [Na N3; EMELCA
Bioscience, cat. no. 41920044-3]) and resuspending the cells in
each sample in 30 .mu.L FACS buffer, [0329] iii) Determining, for
each sample, the average amount of antibody bound per cell as the
geometric mean of the fluorescence intensity (gMFI) for the viable,
single cell population in said sample and normalizing the data for
each test antibody against the binding intensity of a non-cross
blocking B7H4-specific reference antibody using the equation:
[0329] Normalized .times. .times. gMFI aa .times. .times. position
= L .times. o .times. g 1 .times. 0 .function. ( gMFI Test .times.
.times. Ab gMFI Control .times. .times. Ab ) ##EQU00001## [0330]
wherein `aa position` refers to the position that was mutated into
an alanine, [0331] wherein the fold-change or Z-score is calculated
to express loss or gain of binding of the antibody, according to
the calculation:
[0331] Fold .times. .times. Change = Lo .times. g 1 .times. 0
.function. ( Normalized .times. .times. gMFI ala .times. .times.
mutant Normalized .times. .times. gMFI w .times. t ) ##EQU00002##
[0332] wherein amino acid positions for which, upon replacing the
amino acid with alanine, there is no loss or gain of binding by a
particular antibody will give as result `0`, and gain of binding
will result in `>0` and loss of binding will result in `<0`,
and wherein, only B7H4 amino acid residues where the Fold Change in
binding was lower than the mean Fold Change--1.5.times.SD, where SD
is the standard deviation of calculated fold changes from four
independent experiments for a particular test antibody, were
considered `loss of binding mutants`, and, wherein, in case the
gMFI of the reference antibody for a particular B7H4 mutant was
lower than the mean gMFI--2.5.times.SD of the mean gMFI Control Ab,
data were excluded from analysis.
[0333] Furthermore, such an antibody may also be a bispecific
antibody comprising in addition to an antigen-binding region
capable of binding to B7H4 another antigen-binding region. Such
another antigen-binding region may be an antigen-binding region
capable of binding to human CD3. Such antigen-binding region
capable of binding to human CD3 may be antigen-binding regions
capable of binding to CD3 as described and disclosed herein.
[0334] In a further embodiment, in such a bispecific antibody, said
antigen binding region capable of binding to human B7H4 is
comprised in an heavy chain and a light chain, said heavy chain
comprising said VH region and an IgG1 heavy chain constant region
and said light chain comprising said VL region and a kappa light
chain constant region; and wherein said antigen binding region
capable of binding to human CD3 is comprised in a heavy chain and a
light chain, said heavy chain comprising said VH region and an IgG1
heavy chain constant region and said light chain comprising said VL
region and a lambda light chain constant region. More preferably,
in such a bispecific antibody, one IgG1 heavy chain constant region
is as defined in SEQ ID NO. 60 and the other is as defined in SEQ
ID NO. 61, and wherein said kappa light chain constant region is as
defined in SEQ ID NO. 63 and said lambda light chain constant
region is as defined in SEQ ID NO. 64. It is understood that
optionally, of said IgG1 heavy chain constant regions as defined in
SEQ ID NO. 60 and 61, the terminal lysines can be deleted.
[0335] A highly preferred bispecific antibody in accordance with
the invention is as described and used in the example section, and
is referred to as BsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR.
[0336] Hence, In a preferred embodiment, a bispecific antibody
capable of binding human CD3 and human B7H4 is provided comprising:
[0337] a first heavy chain and a first light chain which comprise
the binding region capable of binding to human CD3, wherein said
first heavy chain comprises a heavy chain variable region as
defined by SEQ ID NO: 17 and a human IgG1 heavy chain constant
region as defined herein, and wherein said first light chain
comprises a light chain variable region as defined by SEQ ID NO: 22
and a human lambda light chain constant region; and [0338] a second
heavy chain and a second light chain which comprise the binding
region capable of binding to human B7H4, wherein said second heavy
chain comprises a heavy chain variable region as defined by SEQ ID
NO: 29 and a human IgG1 heavy chain constant region as defined
herein, and wherein said second light chain comprises a light chain
variable region as defined by SEQ ID NO: 33 and a human kappa light
chain constant region.
[0339] It is understood that the human IgG1 heavy chain constant
regions as defined herein may encompass substitutions as defined
herein (e.g. FEAR/FEAL), or the like. It is also understood that
the human IgG1 heavy chain constant region may have its terminal
lysine (K) deleted.
[0340] In a further preferred embodiment, a bispecific antibody
capable of binding human CD3 and human B7H4 is provided comprising:
[0341] a first heavy chain and a first light chain which comprise
the binding region capable of binding to human CD3, wherein said
first heavy chain comprises a heavy chain variable region as
defined by SEQ ID NO: 17 and a heavy chain constant region as
defined by SEQ ID NO: 60, and wherein said first light chain
comprises a light chain variable region as defined by SEQ ID NO: 22
and a light chain constant region as defined by SEQ ID NO: 64; and
[0342] a second heavy chain and a second light chain which comprise
the binding region capable of binding to human B7H4, wherein said
second heavy chain comprises a heavy chain variable region as
defined by SEQ ID NO: 29 and a heavy chain constant region as
defined by SEQ ID NO: 61, and wherein said second light chain
comprises a light chain variable region as defined by SEQ ID NO: 33
and a light chain constant region as defined by SEQ ID NO: 63.
[0343] Likewise, it is understood that the human IgG1 heavy chain
constant region may have its terminal lysine (K) deleted.
[0344] In yet another further preferred embodiment, a bispecific
antibody capable of binding human CD3 and human B7H4 is provided
comprising: [0345] a first heavy chain and a first light chain
which comprise the binding region capable of binding to human CD3,
wherein said first heavy chain consists of a heavy chain variable
region as defined by SEQ ID NO: 17 and a heavy chain constant
region as defined by SEQ ID NO: 60, and wherein said first light
chain consists of a light chain variable region as defined by SEQ
ID NO: 22 and a light chain constant region as defined by SEQ ID
NO: 64; and [0346] a second heavy chain and a second light chain
which comprise the binding region capable of binding to human B7H4,
wherein said second heavy chain consists of a heavy chain variable
region as defined by SEQ ID NO: 29 and a heavy chain constant
region as defined by SEQ ID NO: 61, and wherein said second light
chain consists of a light chain variable region as defined by SEQ
ID NO: 33 and a light chain constant region as defined by SEQ ID
NO: 63.
[0347] In another further preferred embodiment, a bispecific
antibody capable of binding human CD3 and human B7H4 is provided
comprising: [0348] a first heavy chain and a first light chain
which comprise the binding region capable of binding to human CD3,
wherein said first heavy chain consists of a heavy chain variable
region as defined by SEQ ID NO: 17 and a heavy chain constant
region as defined by SEQ ID NO: 60 with the terminal lysine (K)
deleted, and wherein said first light chain consists of a light
chain variable region as defined by SEQ ID NO: 22 and a light chain
constant region as defined by SEQ ID NO: 64; and a second heavy
chain and a second light chain which comprise the binding region
capable of binding to human B7H4, wherein said second heavy chain
consists of a heavy chain variable region as defined by SEQ ID NO:
29 and a heavy chain constant region as defined by SEQ ID NO: 61
with the terminal lysine (K) deleted, and wherein said second light
chain consists of a light chain variable region as defined by SEQ
ID NO: 33 and a light chain constant region as defined by SEQ ID
NO: 63.
Methods of Preparing Bispecific Antibodies
[0349] Traditional methods such as the hybrid hybridoma and
chemical conjugation methods (Marvin and Zhu (2005) Acta Pharmacol
Sin 26:649) can be used in the preparation of the bispecific
antibodies of the invention. Co-expression in a host cell of two
antibodies, consisting of different heavy and light chains, leads
to a mixture of possible antibody products in addition to the
desired bispecific antibody, which can then be isolated by, e.g.,
affinity chromatography or similar methods.
[0350] Strategies favoring the formation of a functional
bispecific, product, upon co-expression of different antibody
constructs can also be used, e.g., the method described by
Lindhofer et al. (1995 J Immunol 155:219). Fusion of rat and mouse
hydridomas producing different antibodies leads to a limited number
of heterodimeric proteins because of preferential
species-restricted heavy/light chain pairing. Another strategy to
promote formation of heterodimers over homodimers is a
"knob-into-hole" strategy in which a protuberance is introduced on
a first heavy-chain polypeptide and a corresponding cavity in a
second heavy-chain polypeptide, such that the protuberance can be
positioned in the cavity at the interface of these two heavy chains
so as to promote heterodimer formation and hinder homodimer
formation. "Protuberances" are constructed by replacing small
amino-acid side-chains from the interface of the first polypeptide
with larger side chains. Compensatory "cavities" of identical or
similar size to the protuberances are created in the interface of
the second polypeptide by replacing large amino-acid side-chains
with smaller ones (U.S. Pat. No. 5,731,168). EP1870459 (Chugai) and
WO2009089004 (Amgen) describe other strategies for favoring
heterodimer formation upon co-expression of different antibody
domains in a host cell. In these methods, one or more residues that
make up the CH3-CH3 interface in both CH3 domains are replaced with
a charged amino acid such that homodimer formation is
electrostatically unfavorable and heterodimerization is
electrostatically favorable. WO2007110205 (Merck) describe yet
another strategy, wherein differences between IgA and IgG CH3
domains are exploited to promote heterodimerization.
[0351] Another in vitro method for producing bispecific antibodies
has been described in WO2008119353 (Genmab), wherein a bispecific
antibody is formed by "Fab-arm" or "half-molecule" exchange
(swapping of a heavy chain and attached light chain) between two
monospecific IgG4- or IgG4-like antibodies upon incubation under
reducing conditions. The resulting product is a bispecific antibody
having two Fab arms which may comprise different sequences.
[0352] A preferred method for preparing the bispecific CD3xB7H4
antibodies of the present invention includes methods described in
WO2011131746 and WO13060867 (Genmab) comprising the following
steps:
[0353] a) providing a first antibody comprising an Fc region, said
Fc region comprising a first CH3 region;
[0354] b) providing a second antibody comprising a second Fc
region, said Fc region comprising a second CH3 region, wherein the
first antibody is a CD3 antibody and the second antibody is a B7H4
antibody, or vice versa;
[0355] wherein the sequences of said first and second CH3 regions
are different and are such that the heterodimeric interaction
between said first and second CH3 regions is stronger than each of
the homodimeric interactions of said first and second CH3
regions;
[0356] c) incubating said first antibody together with said second
antibody under reducing conditions; and
[0357] d) obtaining said bispecific CD3xB7H4 antibody.
[0358] In one embodiment, the said first antibody together with
said second antibody are incubated under reducing conditions
sufficient to allow the cysteines in the hinge region to undergo
disulfide-bond isomerization, wherein the heterodimeric interaction
between said first and second antibodies in the resulting
heterodimeric antibody is such that no Fab-arm exchange occurs at
0.5 mM GSH after 24 hours at 37.degree. C.
[0359] Without being limited to theory, in step c), the heavy-chain
disulfide bonds in the hinge regions of the parent antibodies are
reduced and the resulting cysteines are then able to form inter
heavy-chain disulfide bond with cysteine residues of another parent
antibody molecule (originally with a different specificity). In one
embodiment of this method, the reducing conditions in step c)
comprise the addition of a reducing agent, e.g. a reducing agent
selected from the group consisting of: 2-mercaptoethylamine
(2-MEA), dithiothreitol (DTT), dithioerythritol (DTE), glutathione,
tris(2-carboxyethyl)phosphine (TCEP), L-cysteine and
beta-mercapto-ethanol, preferably a reducing agent selected from
the group consisting of: 2-mercaptoethylamine, dithiothreitol and
tris(2-carboxyethyl)phosphine. In a further embodiment, step c)
comprises restoring the conditions to become non-reducing or less
reducing, for example by removal of a reducing agent, e.g. by
desalting.
[0360] For this method any of the CD3 and B7H4 antibodies described
herein may be used. In a particular embodiment the CD3 and B7H4
antibodies, respectively, may be chosen so as to obtain a
bispecific CD3xB7H4 antibody as described herein.
[0361] In one embodiment of this method, said first and/or second
antibodies are full-length antibodies.
[0362] The Fc regions of the first and second antibodies may be of
any isotype, including, but not limited to, IgG1, IgG2, IgG3 or
IgG4. In one embodiment of this method, the Fc regions of both said
first and said second antibodies are of the IgG1 isotype. In
another embodiment, one of the Fc regions of said antibodies is of
the IgG1 isotype and the other of the IgG4 isotype. In the latter
embodiment, the resulting bispecific antibody comprises an Fc
region of an IgG1 and an Fc region of IgG4 and may thus have
interesting intermediate properties with respect to activation of
effector functions.
[0363] In a further embodiment, one of the antibody starting
proteins has been engineered to not bind Protein A, thus allowing
to separate the heterodimeric protein from said homodimeric
starting protein by passing the product over a protein A
column.
[0364] As described above, the sequences of the first and second
CH3 regions of the homodimeric starting antibodies are different
and are such that the heterodimeric interaction between said first
and second CH3 regions is stronger than each of the homodimeric
interactions of said first and second CH3 regions. More details on
these interactions and how they can be achieved are provided in
WO2011131746 and WO2013060867 (Genmab), which are hereby
incorporated by reference in their entirety.
[0365] In particular, a stable bispecific CD3xB7H4 antibody can be
obtained at high yield using the above method of the invention on
the basis of two homodimeric starting antibodies which bind CD3 and
B7H4, respectively, and contain only a few, fairly conservative,
asymmetrical mutations in the CH3 regions. Asymmetrical mutations
mean that the sequences of said first and second CH3 regions
contain amino acid substitutions at non-identical positions.
[0366] The bispecific antibodies of the invention may also be
obtained by co-expression of constructs encoding the first and
second polypeptides in a single cell.
[0367] Thus, in a further aspect, the invention relates to a method
for producing a bispecific antibody, said method comprising the
following steps:
[0368] a) providing a first nucleic-acid construct encoding a first
polypeptide comprising a first Fc region and a first
antigen-binding region of a first antibody heavy chain, said first
Fc region comprising a first CH3 region,
[0369] b) providing a second nucleic-acid construct encoding a
second polypeptide comprising a second Fc region and a second
antigen-binding region of a second antibody heavy chain, said
second Fc region comprising a second CH3 region,
[0370] wherein the sequences of said first and second CH3 regions
are different and are such that the heterodimeric interaction
between said first and second CH3 regions is stronger than each of
the homodimeric interactions of said first and second CH3 regions,
and wherein said first homodimeric protein has an amino acid other
than Lys, Leu or Met at position 409 and said second homodimeric
protein has an amino-acid substitution at a position selected from
the group consisting of: 366, 368, 370, 399, 405 and 407,
[0371] optionally wherein said first and second nucleic acid
constructs encode light chain sequences of said first and second
antibodies
[0372] c) co-expressing said first and second nucleic-acid
constructs in a host cell, and
[0373] d) obtaining said heterodimeric protein from the cell
culture.
[0374] Thus, the present invention also relates to a recombinant
eukaryotic or prokaryotic host cell which produces a bispecific
antibody of the present invention.
[0375] Suitable expression vectors, including promoters, enhancers,
etc., and suitable host cells for the production of antibodies are
well-known in the art. Examples of host cells include yeast,
bacterial and mammalian cells, such as CHO or HEK cells.
[0376] In embodiment, a method for producing an antibody capable of
binding to both B7H4 and CD3 in accordance with the invention is
provided, comprising the steps of: [0377] a) providing an antibody
capable of binding to B7H4, said antibody comprising an
antigen-binding region capable of binding to B7H4 as defined
herein; [0378] b) providing an antibody capable of binding to CD3,
said antibody comprising an antigen-binding region capable of
binding to CD3 as defined herein; [0379] c) incubating said
antibody capable of binding to B7H4 together with said antibody
capable of binding to CD3 under reducing conditions sufficient to
allow cysteines in the hinge region to undergo disulfide-bond
isomerization; and [0380] d) obtaining said antibody capable of
binding to B7H4 and CD3.
[0381] In such methods, the steps of providing an antibody capable
of binding to B7H4 and/or CD3, may comprise the steps of [0382]
providing cells containing expression vectors for producing said
antibody or said antibodies; and [0383] allowing the cells to
produce said antibody or said antibodies and subsequently, [0384]
obtaining said antibody or said antibodies, thereby providing said
antibody or said antibodies
[0385] The invention furthermore provides for [0386] a) a nucleic
acid sequence encoding a heavy chain sequence of an antigen-binding
region capable of binding to B7H4 as defined herein, and/or [0387]
b) a nucleic acid sequence encoding the corresponding light chain
sequence of the antigen-binding region capable of binding to
B7H4.
[0388] Furthermore, the invention provides for one or more nucleic
acids comprising: [0389] a) a nucleic acid sequence encoding a
heavy chain sequence of an antigen-binding region capable of
binding to B7H4 as defined herein, [0390] b) a nucleic acid
sequence encoding the corresponding light chain sequence of said
antigen-binding region capable of binding to B7H4, [0391] c) a
nucleic acid sequence encoding a heavy chain sequence of an
antigen-binding region capable of binding to CD3 as defined herein,
and [0392] d) a nucleic acid sequence encoding the corresponding
light chain sequence of said antigen-binding region capable of
binding to CD3.
[0393] The nucleic acid, or one or more nucleic acids, as defined
herein can be RNA or DNA. The nucleic acid, or one or more nucleic
acids, as defined herein may be for use in expression in mammalian
cells. Hence, furthermore the invention provides for a cell or
cells, comprising a nucleic acid, or comprising one or more nucleic
acids, as defined herein.
[0394] The nucleic acid in the context of the present invention may
be an expression vector, which may be any suitable vector,
including chromosomal, non-chromosomal, and synthetic nucleic acid
vectors (a nucleic acid sequence comprising a suitable set of
expression control elements). Examples of such vectors include
derivatives of SV40, bacterial plasmids, phage DNA, baculovirus,
yeast plasmids, vectors derived from combinations of plasmids and
phage DNA, and viral nucleic acid (RNA or DNA) vectors. In one
embodiment, a B7H4 or a CD3 antibody-encoding nucleic acid is
comprised in a naked DNA or RNA vector, including, for example, a
linear expression element (as described in for instance Sykes and
Johnston, Nat Biotech 17, 355 59 (1997)), a compacted nucleic acid
vector (as described in for instance U.S. Pat. No. 6,077,835 and/or
WO 00/70087), a plasmid vector such as pBR322, pUC 19/18, or pUC
118/119, a "midge" minimally-sized nucleic acid vector (as
described in for instance Schakowski et al., Mol Ther 3, 793 800
(2001)), or as a precipitated nucleic acid vector construct, such
as a CaPO4-precipitated construct (as described in for instance
WO200046147, Benvenisty and Reshef, PNAS USA 83, 9551 55 (1986),
Wigler et al., Cell 14, 725 (1978), and Coraro and Pearson, Somatic
Cell Genetics 7, 603 (1981)). Such nucleic acid vectors and the
usage thereof are well known in the art (see for instance U.S. Pat.
Nos. 5,589,466 and 5,973,972).
[0395] In one embodiment, the vector is suitable for expression of
the B7H4 antibody and/or the CD3 antibody in a bacterial cell.
Examples of such vectors include expression vectors such as
BlueScript (Stratagene), pIN vectors (Van Heeke & Schuster, J
Biol Chem 264, 5503 5509 (1989), pET vectors (Novagen, Madison
Wis.) and the like).
[0396] An expression vector may also or alternatively be a vector
suitable for expression in a yeast system. Any vector suitable for
expression in a yeast system may be employed. Suitable vectors
include, for example, vectors comprising constitutive or inducible
promoters such as alpha factor, alcohol oxidase and PGH (reviewed
in: F. Ausubel et al., ed. Current Protocols in Molecular Biology,
Greene Publishing and Wiley InterScience New York (1987), and Grant
et al., Methods in Enzymol 153, 516 544 (1987)).
[0397] A nucleic acid and/or expression vector may also comprises a
nucleic acid sequence encoding a secretion/localization sequence,
which can target a polypeptide, such as a nascent polypeptide
chain, to the periplasmic space or into cell culture media. Such
sequences are known in the art, and include secretion leader or
signal peptides. The nucleic acid and/or expression vector may
comprise any suitable elements facilitating expression, i.e.
transcription and/or translation of the nucleic acid such that the
components of the (bispecific) antibodies are expressed. The
nucleic acid and/or vector be associated with any suitable
promoter, enhancer, and other expression-facilitating elements.
Examples of such elements include strong expression promoters (e.
g., human CMV IE promoter/enhancer as well as RSV, SV40, SL3 3,
MMTV, and HIV LTR promoters), effective poly (A) termination
sequences, an origin of replication for plasmid product in E. coli,
an antibiotic resistance gene as selectable marker, and/or a
convenient cloning site (e.g., a polylinker). Nucleic acids may
also comprise an inducible promoter as opposed to a constitutive
promoter such as CMV IE.
[0398] In one embodiment, the B7H4 and/or CD3 antibody-encoding
expression vector may be positioned in and/or delivered to a cell.
Hence, in a further aspect, the invention relates to a host cell
comprising the nucleic acid or vector as defined herein. The cell
may be of human origin, such as a human embryonic kidney (HEK)
cell, such as a HEK/Expi cell, or can be of rodent origin, such as
a Chinese hamster ovary cell, such as a CHO/N50 cell.
Compositions and (Medical) Uses
[0399] Furthermore, the invention provides for a composition
comprising an antibody as defined herein. Preferably, such a
composition is a pharmaceutical composition, i.e. the antibody is
comprised in a pharmaceutically acceptable carrier. The
pharmaceutical composition of the present invention may contain a
bispecific antibody of the present invention targeting both B7H4
and CD3. The pharmaceutical composition may also comprise an
antibody targeting B7H4. The pharmaceutical composition may also
comprise a combination of antibodies, including an antibody
targeting B7H4 and/or a bispecific antibody in accordance with the
present invention.
[0400] A pharmaceutical composition may be formulated in accordance
with conventional techniques such as those disclosed in Remington:
The Science and Practice of Pharmacy, 19th Edition, Gennaro, Ed.,
Mack Publishing Co., Easton, Pa., 1995. A pharmaceutical
composition of the present invention may e.g. include diluents,
fillers, salts, buffers, detergents (e. g., a nonionic detergent,
such as Tween-20 or Tween-80), stabilizers (e. g., sugars or
protein-free amino acids), preservatives, tissue fixatives,
solubilizers, and/or other materials suitable for inclusion in a
pharmaceutical composition.
[0401] The antibody, composition, or pharmaceutical composition in
accordance with the invention is preferably for use as a
medicament. The antibody, composition, or pharmaceutical
composition in accordance with the invention is preferably for use
in the treatment of disease. Bispecific antibodies of the invention
may be used for a number of purposes. In particular, the bispecific
antibodies of the invention may be used for the treatment of
various forms of cancer, including metastatic cancer and refractory
cancer. Preferably, the cancer may be of the solid tumor type.
[0402] In particular, the bispecific antibodies according to the
invention may be useful in therapeutic settings in which specific
targeting and T cell-mediated killing of cells that express B7H4 is
desired.
[0403] In one embodiment, the present invention provides a method
for treating a cancer in a subject, which method comprises
administration of a therapeutically effective amount of a
bispecific B7H4xCD3 antibody of the present invention. In a further
embodiment, the present invention provides a method for treating a
disorder involving cells expressing B7H4, in a subject, which
method comprises administration of a therapeutically effective
amount of a bispecific antibody of the present invention.
[0404] In another embodiment, the present invention provides a
method for treating a cancer in a subject, which method comprises
administration of a therapeutically effective amount of an antibody
capable of binding to human B7H4 of the present invention. In a
further embodiment, the present invention provides a method for
treating a disorder involving cells expressing B7H4, in a subject,
which method comprises administration of a therapeutically
effective amount of a monospecific antibody of the present
invention that is capable of binding to human B7H4.
[0405] As said, suitable diseases that can be contemplated in
methods and uses in accordance with the invention are cancer. Said
cancer most preferably is characterized by expression of B7H4.
Expression of B7H4 in a cancer can easily be determined using
methods known in the art, such as PCR, immunostaining, or FACS
analysis, i.e. detecting expression of B7H4 transcript and/or
protein. The antibodies as described herein that are capable of
binding to human B7H4 may be used e.g. in immunostaining and/or
FACS analysis or the like.
[0406] Cancers that can express B7H4 include Breast cancer,
Uterine/endometrial cancer, Uterine carcinosarcoma cancer, Ovarian
cancer, Cervical cancer, Non-small cell lung cancer (squamous cell
carcinoma and adenocarcinoma), Head and neck squamous cell
carcinoma, Bladder cancer, esophageal cancer, cholangiocarcinoma,
Pancreatic cancer, Stomach cancer, Renal cancer and Prostate
cancer.
[0407] Cancers that can express B7H4 include cancers such as
cancers of the stomach, cholangiocarcinoma, bladder cancer, non
small cell lung cancer (in particular squamous NSCLC), pancreatic
cancer, cervical cancer, head and neck cancer, breast cancer
(including triple negative breast cancer), ovarian cancer and
uterine cancer. Types of cancers that may be preferred are cancers
selected from uterine carcinosarcoma (UCS), bladder urothelial
carcinoma (BLCA), pancreatic adenocarcinoma (PAAD), lung squamous
cell carcinoma (LUSC), breast invasive carcinoma (BRCA), uterine
corpus endometrial carcinoma (UCEC), ovarian serous
cystadenocarcinoma (OV) and cholangiocarcinoma (CHOL).
[0408] In a further embodiment, a patient being diagnosed with
cancer may be subjected to an assessment of B7H4 expression in the
cancer cells, and when B7H4 is detected, which may be in the range
from low to high, such a patient may be selected for treatment with
an antibody in accordance with the invention. Patients diagnosed
with having cancer of the stomach, cholangiocarcinoma, bladder
cancer, non small cell lung cancer (in particular squamous NSCLC),
pancreatic cancer, cervical cancer, head and neck cancer, breast
cancer (including triple negative breast cancer), ovarian cancer or
uterine cancer, may be subjected to such test. In a further
embodiment, a patient being diagnosed with having uterine
carcinosarcoma (UCS), bladder urothelial carcinoma (BLCA),
pancreatic adenocarcinoma (PAAD), lung squamous cell carcinoma
(LUSC), breast invasive carcinoma (BRCA), uterine corpus
endometrial carcinoma (UCEC), ovarian serous cystadenocarcinoma
(OV) or cholangiocarcinoma (CHOL), may be subjected to such test.
However, it may not necessarily be a requirement to include such an
assessment in selecting a patient for treatment.
Kits
[0409] The invention further provides a kit-of-parts comprising an
antibody as disclosed above, such as a kit for use as a companion
diagnostic/for identifying within a population of patients, those
patients which have a propensity to respond to treatment with an
antibody as defined herein above or an immunoconjugate or
antibody-drug conjugate (ADC) as defined herein above, or for
predicting efficacy or anti-tumor activity of said antibody or
immunoconjugate or ADC when used in treatment of a patient, the kit
comprising an antibody as defined above; and instructions for use
of said kit.
[0410] A kit-of-parts, such as a kit for use as a companion
diagnostic/for identifying within a population of patients those
patients which have a propensity to respond to treatment with an
antibody as defined in any one of claims 1 to 55, comprising an
antibody as defined in any one of claims 1 to 55; and instructions
for use of said kit.
[0411] Hence, in one aspect, the invention relates to a diagnostic
composition comprising a bispecific CD3xB7H4 antibody as defined
herein, or a B7H4 antibody as defined herein, and to its use.
[0412] In another aspect, the invention relates to a kit for
detecting cross-linking between CD3- and B7H4 expressing cells, in
a sample derived from a patient, comprising
[0413] i) a bispecific antibody according to any one of the
embodiments as disclosed herein; and
[0414] ii) instructions for use of said kit.
[0415] In one embodiment, the present invention provides a kit for
diagnosis of cancer comprising a container comprising a bispecific
CD3xB7H4 antibody, and one or more reagents for detecting
cross-linking of B7H4 expressing cells and CD3 expressing cells.
Reagents may include, for example, fluorescent tags, enzymatic
tags, or other detectable tags. The reagents may also include
secondary or tertiary antibodies or reagents for enzymatic
reactions, wherein the enzymatic reactions produce a product that
may be visualized.
[0416] In a further aspect, the invention relates to a method for
detecting whether cross-linking between CD3- and B7H4-expressing
cells occurs in a sample derived from a patient, upon
administration of a bispecific antibody according to any one of the
embodiments as disclosed herein, comprising the steps of:
[0417] (i) contacting the sample with a bispecific antibody
according to any one of the embodiments as disclosed herein under
conditions that allow for formation of a complex between said
bispecific antibody and the CD3-expressing cells and the
B7H4-expressing cells; and
[0418] (ii) analyzing whether a complex has been formed.
[0419] The present invention is further illustrated by the
following examples, which should not be construed as limiting the
scope of the invention.
Example 1--Generation of B7H4 Antibodies and Screenings
Materials
Expression of B7H4 Constructs
[0420] Constructs encoding various full length B7H4 variants were
generated: human (Homo sapiens) B7H4 (Uniprot accession no.
Q7Z7D3), cynomolgus monkey (Macaca fascicularis) B7H4 transcript 1
(Uniprot accession no. A0A2K5U6P5), dog (Canis familiaris) B7H4
(Uniprot accession no. F1P8R9), Rabbit (Oryctolagus cuniculus) B7H4
(Uniprot accession no. G1TQE8), rat (Rattus norvegicus) B7H4
(Uniprot accession no. Q501W4), mouse (Mus musculus) B7H4 (Uniprot
accession no. Q7TSP5), and pig (Sus scrofa) B7H4 (Uniprot accession
no. F1SAY4) (see Table 1).
[0421] In addition, a construct for the extracellular domain (ECD
of human B7H4 (aa 25-259 from Uniprot accession no. Q7Z7D3) fused
to human IgG1 Fc domain with a C-terminal His tag and C tag
(B7H4ECD-FcHisC) (SEQ ID NO: 12) was generated. In SEQ ID NO: 1,
amino acid residues 1-24 are a signal peptide; hence the mature
B7H4ECD-FcHisC protein corresponds to amino acid residues 25-259 of
SEQ ID NO: 1.
[0422] Constructs contained suitable restriction sites for cloning
and an optimal Kozak (GCCGCCACC) sequence (Kozak, M., Gene 1999;
234(2):187-208). The full length and ECD of B7H4 constructs were
cloned in pSB, a mammalian expression vector containing Sleeping
Beauty inverted terminal repeats flanking an expression cassette
consisting of a CMV promoter and HSV-TK polyA signal.
Generation of HEK-293F Cell Lines Transiently Expressing Full
Length B7H4 Variants
[0423] Freestyle.TM. 293-F (a HEK-293 subclone adapted to
suspension growth and chemically defined Freestyle medium
[HEK-293F]) cells were obtained from Invitrogen (cat. no. R790-07)
and transfected with the constructs described supra, using
293fectin (Invitrogen, cat. no. 12347-019) according to the
manufacturer's instructions.
Purification of His-Tagged B7H4
[0424] B7H4ECD-FcHisC was expressed using the Expi293F expression
platform (Thermo Fisher Scientific, Waltham, Mass., USA, cat. no.
A14527) essentially as described by the manufacturer.
[0425] The His-tag enables purification with immobilized metal
affinity chromatography Ni-NTA. The His-tagged protein binds
strongly to the column material, while other proteins present in
the culture supernatant do not bind or bind weakly compared to the
His-tagged proteins and elute in the flow-through. The column was
washed in order to remove weakly bound proteins. The strongly bound
His-tagged proteins were then eluted with a buffer containing
imidazole, which competes with the binding of His to Ni.sup.2+. The
eluent was removed by buffer exchange on a desalting column.
Immunization
[0426] OmniRat.RTM. animals (transgenic rats expressing a
diversified repertoire of antibodies with fully human idiotypes;
Ligand Pharmaceuticals Inc., San Diego, USA) were immunized by
subcutaneous injections in the hocks of both hind legs (twice
weekly for 7 weeks) with 50 .mu.g B7H4ECD-FcHisC in PBS mixed with
an equal volume of adjuvant (Sigma adjuvant system (Sigma-Aldrich,
St. Louis, Mo., USA, cat. no. S6322) or CFA, Complete Freund
Adjuvant (1.sup.st injection) and IFA, Incomplete Freund Adjuvant
(Sigma-Aldrich, St. Louis, Mo., USA, cat. no. F5881/F5506)
(subsequent injections), followed by a final boost s.c. injection
of antigen in PBS without adjuvant.
Antibody Generation
[0427] Lymph node cells from immunized animals were fused to mouse
myeloma SP2.0 cells according to standard procedures 3 days after
the final boost. RNA from hybridomas producing B7H4 specific
antibody was extracted and 5'-RACE-complementary DNA (cDNA) was
prepared from 100 ng total RNA, using the SMART RACE cDNA
Amplification kit (Clontech), according to the manufacturer's
instructions. VH and VL coding regions were amplified by PCR and
cloned directly, in frame, in the p33G1f, p33Kappa and p33Lambda
expression vectors (pcDNA3.3 based vectors with codon optimized
human IgG1m(f), Kappa and Lambda constant domains respectively), by
ligation independent cloning (Aslanidis, C. and P. J. de Jong,
Nucleic Acids Res 1990; 18(20): 6069-74). The variable domains from
these expression vectors were sequenced and CDRs were annotated
according to IMGT definitions (Lefranc M P. et al., Nucleic Acids
Research, 27, 209-212, 1999 and Brochet X. Nucl. Acids Res. 36,
W503-508 (2008)). Clones with a correct Open Reading Frame (ORF)
were expressed and tested for binding to the antigen. After antigen
specific screening assay was performed, the sequences of variable
regions of heavy and light chain were gene synthesized and cloned
into an expression vector including a human IgG1 heavy chain
containing the following amino acid mutations: L234F, L235E, D265A
and K409R (FEAR) wherein the amino acid position number is
according to Eu numbering (correspond to SEQ ID NO 60), and into
expression vectors including human kappa or lambda light chain. For
some of the antibodies, a variant with point mutation in the
variable domains was generated to remove a cysteine residue, which
potentially could generate undesired disulphide bridge formation,
or to replace an Asparagine to Serine or germline residue to remove
a potential N-linked glycosylation site. For example, from the C1
heavy and light chain variable region sequences, a variant with an
N52S substitution was made corresponding with a substitution in
CDR2 (see TABLE 1, SEQ ID NOs. 25 and 29), and a further variant
can have an N52Q substitution (SEQ ID NO. 31).
Antigen Specific Screening Assay
[0428] The presence of B7H4 antibodies in sera of immunized
animals, or hybridoma and transfectoma culture supernatant was
determined in a homogeneous binding assay. Samples were analyzed
for binding of antibodies to HEK-293F cells transiently transfected
with the constructs made to express full length B7H4 variants
expressing human B7H4, cynomolgus monkey B7H4 or murine B7H4, or
HEK-293F wild-type cells (negative control). Samples were added to
the cells to allow antibody binding to B7H4. Subsequently, antibody
binding was detected using an appropriate fluorescent conjugate
(AffiniPure Goat Anti-Rat IgG (H+L) Alexa Fluor.RTM. 647; Jackson
ImmunoResearch, cat no. 112-605-143; AffiniPure Goat Anti-Human IgG
Fc gamma-Alexa Fluor.RTM. 647; Jackson ImmunoResearch, cat no.
109-605-098). Cells (2.5.times.10.sup.5 cells/ml) were mixed with
goat anti-human AffiniPure Goat Anti-Human IgG Fc gamma-Alexa
Fluor.RTM. 647 (0.2 .mu.g/ml; Jackson ImmunoResearch Laboratories,
109-605-098) or AffiniPure Goat Anti-Rat IgG (H+L) Alexa Fluor.RTM.
647 (0.2 .mu.g/ml; Jackson ImmunoResearch, 112-605-143) depending
on the backbone of the antibody. Serial dilutions of test and
control antibodies (range 0.003 to 3 .mu.g/mL in 2-fold dilution
steps) were prepared and 2 .mu.l antibody dilution was added to 5
.mu.l of the cell/conjugate mixture in 1536 well plates (Greiner,
cat. no. 789866). Plates were incubated at room temperature for 9
hours, and after which fluorescence intensity was determined using
an ImageXpress Velos Laser Scanning Cytometer (Molecular Devices,
LLC, Sunnyvale, Calif., USA) and total fluorescence was used as
read-out. Samples were stated positive when counts were higher than
50 and counts x fluorescence was at least three times higher than
the negative control.
Results from B7H4 Antibody Panel Generation
[0429] From 176 out of 193 hybridomas produced, heavy and light
chain variable region sequences were successfully obtained. Of 351
heavy chain/light chain combinations tested, 98 showed binding in
antigen screenings assays using human B7H4-transfected HEK-293F
cells as described above. 35 antibodies were selected: 26 with
original sequences and 9 variants with point mutations introduced
in the variable domains. Antibodies were produced as monovalent
binding antibodies (as CD3 bispecifics) and bivalent binding
antibodies (as IgG1 molecules), and tested for binding to tumor
cells as described below. Of the antibodies from the panel
generated, only antibody B7H4-C1 and its variant B7H4-C1-N52S, of
which the corresponding VH and VL antibody variable domain encoding
sequences are listed in TABLE 1, provided for antibodies that bound
to tumor cells as described below.
Further B7H4 Antibodies
[0430] In the examples, further antibodies specific for B7H4 were
used containing the variable domains previously described in
WO2014159835 (referenced therein as SEQ ID NOs 38 and 35),
corresponding herein to B7H4-C2, relevant sequences of the variable
domains are listed herein in TABLE 1 and include SEQ ID NO. 43 and
47; WO2014159835 (referenced therein as SEQ ID NO 56 and 55),
corresponding herein to B7H4-C3, relevant sequences of the variable
domains are listed herein in TABLE 1 and include SEQ ID NO. 36 and
40; WO2009073533 (referenced therein as SEQ ID No 2 and 7),
corresponding herein to B7H4-C4 and relevant sequences of the
variable domains are listed herein in TABLE 1 and include SEQ ID
NO. 50 and 54; and US20190085080A1 corresponding herein to B7H4-05
and relevant sequences of the variable domains are listed herein in
TABLE 1 and include SEQ ID NO. 65 and 69. The corresponding VH and
VL antibody variable domain encoding sequences were synthesized and
cloned into pcDNA3.3 based vectors with codon optimized human
IgG1m(f) and Kappa or Lambda constant domains, or variants thereof,
to produce monospecific and bispecific antibodies. When reference
is made to antibody IgG1-B7H4-CX-FEAL, this represents an antibody
having the B7H4-CX variable regions, being of the IgG1 isotype, and
having amino acid substitutions L234F, L235E, D265A and F409R in
the constant region of the heavy chain.
IgG1-b12 Antibody
[0431] The antibody b12, an HIV-1 gp120 specific antibody (Barbas,
C F. J Mol Biol. 1993 Apr. 5; 230(3):812-23) was used in some
examples as a negative control IgG1, or as the non-binding control
Fab-arm of a control bispecific. The codon optimized antibody
encoding sequences for this control antibody were synthesized and
cloned into pcDNA3.3 based vectors with codon optimized human
IgG1m(f) and Kappa constant domains, or variants thereof. The
sequence of the variable heavy chain (VH) region and the sequence
of the variable light chain (VL) region are included herein as SEQ
ID NOs.: 14 and 15, respectively.
Example 2--Humanized CD3 Antibodies for the Generation of CD3xB7H4
Bispecific Antibodies
[0432] The generation of humanized antibody IgG1-huCD3-H1L1 (of
which the variable heavy and light chain region sequences are
listed herein in SEQ ID NO: 16 and 22) is described in Example 1 of
WO2015/001085. IgG1-huCD3-H1L1 is referred to herein as
`IgG1-huCD3`. Antibody IgG1-huCD3-H1L1-FEAL is a variant hereof
with three amino acid substitutions in the Fc domain (L234F, L235E,
D265A), in addition to an amino acid substitution that allows the
generation of bispecific antibodies through controlled Fab-arm
exchange (F405L), as described herein below. It has been shown that
such mutations did not have effect on target binding of the
antibodies in which they are introduced (see e.g. US 2015/0337049
and Engelberts et al., 2020, EBioMedicine 52: 102625).
[0433] The generation of humanized antibody IgG1-huCD3-H1L1-H101G
(of which the variable heavy chain and light chain region sequences
are listed as SEQ ID NO: 17 and 22 herein) is described in Example
2 of WO2017/009442. IgG1-huCD3-H1L1-H101G will be referred to as
`IgG1-huCD3-H101G`. This variant comprises a substitution H101G in
the variable heavy chain region sequence (compare SEQ ID NO. 16 and
17), and has the same light chain as IgG1-huCD3-H1L1. Antibody
IgG1-huCD3-H101G-FEAL is a variant hereof with amino acid
substitutions L234F, L235E, D265A and F405L.
Example 3--B7H4 Binding Affinity Determination Using Biolayer
Interferometry
[0434] Target binding affinity of B7H4 antibodies was determined by
label-free biolayer interferometry (BLI) on an Octet HTX instrument
(ForteBio). Experiments were carried out while shaking at 1,000 RPM
at 30.degree. C. Initially, the affinity of IgG1-B7H4-C1-N52S-FEAR,
IgG1-B7H4-C2-FEAR, IgG1-B7H4-C3-FEAR, and IgG1-B7H4-C4-FEAR for
human and mouse B7H4 was determined using BLI. Anti-Human IgG Fc
Capture (AHC) biosensors (ForteBio, cat. no. 18-5060) were
pre-conditioned by exposure to 10 mM glycine (Sigma-Aldrich, cat.
no. 15527) buffer pH 1.7 for 5 s, followed by neutralization in
Sample Diluent (ForteBio, cat. no. 18-1048) for 5 s; both steps
were repeated 2 times. Next, AHC sensors were loaded with antibody
(1 .mu.g/mL in Sample diluent) for 600 s. After a baseline
measurement in Sample Diluent (100 s), the association (300 s) and
dissociation (1,000 s) of human B7H4 (Sino Biological, cat. no.
10738-H08H-100) or mouse B7H4 (R&D Systems, cat. no.
2154-B7-050) was determined using a concentration range of 1.56-100
nM (0.04-2.68 .mu.g/mL) and 5.9-375 nM (0.16-10 .mu.g/mL) for human
and mouse B7H4 respectively, with two-fold dilution steps in Sample
Diluent. The theoretical molecular mass of human B7H4 and mouse
B7H4 (as ECD-His tagged molecules) based on their amino acid
sequences (26.8 kDa and 26.6 kDa respectively) were used for
calculations. For each antibody a reference sensor was used, which
was incubated with Sample Diluent instead of antigen. AHC sensors
were regenerated by exposure to 10 mM glycine buffer pH 1.7 for 5
s, followed by neutralization in Sample Diluent for 5 s; both steps
were repeated twice. Subsequently sensors were loaded again with
antibody for the next cycle of kinetics measurements.
[0435] Data were acquired using Data Acquisition Software
v9.0.0.49d (ForteBio) and analyzed with Data Analysis Software
v9.0.0.12 (ForteBio). Data traces were corrected per antibody by
subtraction of the reference sensor. The Y-axis was aligned to the
last 10 s of the baseline, Interstep Correction alignment to
dissociation and Savitzky-Golay filtering were applied. Data traces
with a response <0.05 nm were excluded from analysis. The data
was fitted with the 1:1 Global Full fit model using a window of
interest for the association and dissociation times set at 300 s
and 200 s respectively.
[0436] In a second experiment, the affinity of
IgG1-B7H4-C1-N52S-FEAR, IgG1-B7H4-C2-FEAR, IgG1-B7H4-C3-FEAR,
IgG1-B7H4-C4-FEAR, and IgG1-B7H4-C5-FEAR for human and mouse B7H4
was determined using BLI. The experiment was performed as described
above, with some small exceptions. The preconditioning steps were
repeated 5 times. The association (200 s) and dissociation (1,000
s) of human or mouse B7H4 were determined using a concentration
range of 0.78-800 nM with two-fold dilution steps in Sample
Diluent. Data were acquired using Data Acquisition Software
v12.0.1.8 (ForteBio) and analyzed with Data Analysis Software
v12.0.1.2 (ForteBio). The data was fitted with the 1:1 Global Full
Fit model using a window of interest for the association time of
200 s and a window of interest for the dissociation time of 200 s,
except for IgG1-B7H4-C2-FEAR for which a 1,000 s dissociation time
was used. The dissociation time was chosen based upon R.sup.2
value, visual inspection of the curve and at least 5% signal decay
during the dissociation step. Data traces generated with antigen
concentrations higher than 100 nM were excluded from analysis for
antibodies with an affinity below 50 nM.
[0437] In addition, the affinity of for cynomolgus monkey B7H4 was
determined by BLI. In a first experiment, the affinity of
bsIgG1-huCD3-FEALxB7H4-C1-FEAR,
bsIgG1-huCD3-FEALxB7H4-C1-N52S-FEAR,
bsIgG1-huCD3-FEALxB7H4-C2-FEAR, bsIgG1-huCD3-FEALxB7H4-C3-FEAR, and
bsIgG1-huCD3-H101G-FEALxB7H4-C4-FEAR for cynomolgus monkey B7H4 was
determined. Amine Reactive 2.sup.nd Generation (AR2G) biosensors
(ForteBio, cat. no. 18-5092) were activated by reaction with 20 mM
EDC (N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride)
(ForteBio, cat. no. 18-1033) and 10 mM s-NHS
(N-hydroxysulfosuccinimide sodium salt) (ForteBio, cat. no.
18-1067) for 300 s. The activated sensors were loaded with 10
.mu.g/mL recombinant hIgG1 Fc-tagged cynomolgus monkey B7H4
(Creative BioMart, cat. no. VTCN1-1517R) in 10 mM Sodium Acetate pH
4.0 (ForteBio, cat. no. 18-1068) for 600 s and quenched with 1 M
ethanolamine pH 8.5 (ForteBio, cat. no. 18-1071) for 300 s. After a
baseline measurement in Sample Diluent (300 s; ForteBio, cat. no.
18-1048), the association (100 s) and dissociation (1,000 s) of
functionally monovalent B7H4 binding by CD3xB7H4 bispecific
antibodies (as indicated in Table 8) was determined using a
concentration range of 0.23-15 .mu.g/mL (1.56-100 nM) with two-fold
dilution steps in Sample Diluent. A molecular mass of 150 kDa of
the antibodies was used for calculations. For each antibody a
reference sensor was used, which was incubated with Sample Diluent
instead of antibody.
[0438] Data were acquired using Data Acquisition Software
v9.0.0.49d (ForteBio) and analyzed with Data Analysis Software
v9.0.0.12 (ForteBio). Data traces were corrected per antibody by
subtraction of the reference sensor. The Y-axis was aligned to the
last 10 s of the baseline, Interstep Correction alignment to
dissociation and Savitzky-Golay filtering were applied. Data traces
with a response <0.05 nm were excluded from analysis. The data
was fitted with the 1:1 Global Full fit model using a window of
interest for the association and dissociation times set at 100 s
and 200 s respectively.
[0439] In a second experiment to determine the affinity of the B7H4
antibodies for cynomolgus monkey B7H4, the affinity of
bsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR and
bsIgG1-huCD3-H101G-FEALxB7H4-C5-FEAR was determined. The experiment
was performed as described above, with some small exceptions. After
a baseline measurement in Sample Diluent of 600 s, the association
(200 s) and dissociation (1,000 s) of functionally monovalent B7H4
binding by CD3xB7H4 bispecific antibodies (as indicated in Table 9)
was determined using a concentration range of approximately 0.1-116
.mu.g/mL (0.78-800 nM) with two-fold dilution steps in Sample
Diluent. The specific molecular mass of each antibody
(approximately 145 kDa) was used for calculations. Data were
acquired using Data Acquisition Software v12 (ForteBio) and
analyzed with Data Analysis Software v12 (ForteBio). Data traces
with a response <0.03 nm were excluded from analysis. The data
was fitted with the 1:1 Global Full fit model using a window of
interest for the association time and dissociation time of 200 s.
The dissociation time was chosen based upon R.sup.2 value, visual
inspection of the curve and at least 5% signal decay during the
dissociation step. Data traces generated with antibody
concentrations higher than 200 nM were excluded from analysis for
antibodies with an affinity below 50 nM. All results were
determined with an R.sup.2 of at least 0.98.
[0440] "K.sub.D" (M) refers to the equilibrium dissociation
constant of the antibody-antigen interaction, and is obtained by
dividing k.sub.d by k.sub.a. "k.sub.d" (sec.sup.-1) refers to the
dissociation rate constant of the antibody-antigen interaction.
This is sometimes also referred to as the k.sub.off value or
off-rate. "k.sub.a" (M.sup.-1.times.sec.sup.-1) refers to the
association rate constant of the antibody-antigen interaction. This
is sometimes also referred to as the k.sub.on value or on-rate.
[0441] Tables 4 and 5 show the results of the first and the second
experiment in which the association rate constant k.sub.a (1/Ms),
dissociation rate constant k.sub.d (1/s) and equilibrium
dissociation constant K.sub.D (M) of the indicated antibodies for
human B7H4 were determined by biolayer interferometry.
TABLE-US-00004 TABLE 4 Binding affinities of antibodies to human
B7H4 extracellular domain as determined by label-free biolayer
interferometry. On-rate Off-rate Antibody k.sub.a (1/Ms) k.sub.d
(1/s) K.sub.D (M) IgG1-B7H4-C1-FEAR ND ND ND IgG1-B7H4-C1-N52S-FEAR
9.4E+04 5.4E-03 5.7E-08 IgG1-B7H4-C2-FEAR 5.2E+04 8.8E-04 1.7E-08
IgG1-B7H4-C3-FEAR 9.9E+04 4.1E-03 4.2E-08 IgG1-B7H4-C4-FEAR 1.5E+05
1.6E-03 1.1E-08 ND = not determined.
TABLE-US-00005 TABLE 5 Binding affinities of antibodies to human
B7H4 extracellular domain as determined by label-free biolayer
interferometry. On-rate Off-rate Antibody k.sub.a (1/Ms) k.sub.d
(1/s) K.sub.D (M) IgG1-B7H4-C1-N52S-FEAR.sup.1 8.4E+04 4.7E-03
5.7E-08 IgG1-B7H4-C2-FEAR 5.9E+04 1.7E-04 3.0E-09 IgG1-B7H4-C3-FEAR
8.1E+04 4.4E-03 5.4E-08 IgG1-B7H4-C4-FEAR 2.2E+05 1.7E-03 7.9E-09
IgG1-B7H4-C5-FEAR 2.5E+05 2.5E-03 9.9E-09 .sup.1Shown are the
averaged results of n = 3 experiments.
[0442] Tables 6 and 7 show the results of two experiments in which
the k.sub.a (1/Ms), k.sub.d (1/s), and K.sub.D (M) of the indicated
antibodies for mouse B7H4 were determined by biolayer
interferometry.
TABLE-US-00006 TABLE 6 Binding affinities of antibodies to mouse
B7H4 extracellular domain as determined by label-free biolayer
interferometry. On-rate Off-rate Antibody k.sub.a (1/Ms) k.sub.d
(1/s) K.sub.D (M) IgG1-B7H4-C1-FEAR ND ND ND IgG1-B7H4-C1-N52S-FEAR
-- -- -- IgG1-B7H4-C2-FEAR 3.3E+04 7.7E-04 2.4E-08
IgG1-B7H4-C3-FEAR 5.1E+04 2.0E-02 3.9E-07 IgG1-B7H4-C4-FEAR 8.4E+04
1.4E-03 1.6E-08 ND = not determined; -- = no binding (response
<0.05 nm at the highest concentration used).
TABLE-US-00007 TABLE 7 Binding affinities of antibodies to mouse
B7H4 extracellular domain as determined by label-free biolayer
interferometry. On-rate Off-rate Antibody k.sub.a (1/Ms) k.sub.d
(1/s) K.sub.D (M) IgG1-B7H4-C1-N52S-FEAR -- -- -- IgG1-B7H4-C2-FEAR
6.3E+04 1.3E-04 2.1E-09 IgG1-B7H4-C3-FEAR 5.9E+04 1.8E-02 3.0E-07
IgG1-B7H4-C4-FEAR 1.4E+05 1.4E-03 9.7E-09 IgG1-B7H4-C5-FEAR 1.7E+05
2.4E-03 1.4E-08 -- = no binding (response <0.05 nm at the
highest concentration used).
[0443] Tables 8 and 9 show the results of two experiments in which
the k.sub.a (1/Ms), k.sub.d (1/s), and K.sub.D (M) of the indicated
antibodies for cynomolgus monkey B7H4 were determined by biolayer
interferometry.
TABLE-US-00008 TABLE 8 Binding affinities of functionally
monovalent antibodies to cynomolgus monkey B7H4extracellular domain
as determined by label-free biolayer interferometry. On-rate
Off-rate Antibody k.sub.a (1/Ms) k.sub.d (1/s) K.sub.D (M)
bsIgG1-huCD3-FEALxB7H4- 2.7E+05 1.4E-03 5.1E-09 C1-FEAR
bsIgG1-huCD3-FEALxB7H4- 1.4E+05 3.0E-03 2.1E-08 C1-N52S-FEAR
bsIgG1-huCD3-FEALxB7H4- 1.3E+05 4.1E-04 3.1E-09 C2-FEAR
bsIgG1-huCD3-FEALxB7H4- 2.8E+05 4.1E-03 1.5E-08 C3-FEAR
bsIgG1-huCD3-H101G- 3.5E+05 1.5E-03 4.2E-09 FEALxB7H4-C4-FEAR
TABLE-US-00009 TABLE 9 Binding affinities of functionally
monovalent antibodies to cynomolgus monkey B7H4 extracellular
domain as determined by label-free biolayer interferometry. On-rate
Off-rate Antibody R.sup.2 k.sub.a (1/Ms) k.sub.d (1/s) K.sub.D (M)
bsIgG1-huCD3-H101G- 0.99 1.2E+05 2.7E-03 2.5E-08
FEALxB7H4-C1-N52S-FEAR.sup.a bsIgG1-huCD3-H101G- 0.97 4.2E+05
2.5E-03 6.0E-09 FEALxB7H4-C5-FEAR.sup.b .sup.aShown are the
averaged results of n = 3 experiments. .sup.bDid not meet a
stringent quality control R.sup.2 threshold of 0.98.
Example 4--CD3 Binding Affinity Determination Using Biolayer
Interferometry
[0444] Binding affinities of IgG1-huCD3-FEAL and
IgG1-huCD3-H101G-FEAL were determined as described in Example 7 of
WO2017/009442.
[0445] In short, binding affinities of selected CD3 antibodies in
an IgG1-huCD3-FEAL format for recombinant soluble CD3.epsilon.
(CD3E27-GSKa) (mature protein of SEQ ID NO: 13) were determined
using biolayer interferometry on a ForteBio Octet HTX (ForteBio).
Anti-human Fc capture biosensors (ForteBio, cat. no. 18-5060) were
loaded for 600 s with hIgG (1 .mu.g/mL). After a baseline
measurement (200 s), the association (1000 s) and dissociation
(2000 s) of CD3E27-GSKa was determined, using a CD3E27-GSKa
concentration range of 27.11 .mu.g/mL-0.04 .mu.g/mL (1000 nM-1.4
nM) with three-fold dilution steps (sample diluent, ForteBio, cat.
no. 18-5028). For calculations, the theoretical molecular mass of
CD3E27-GSKa based on the amino acid sequence was used, i.e. 27.11
kDa. Experiments were carried out while shaking at 1000 rpm and at
30.degree. C. Each antibody was tested in at least two independent
experiments. Data was analyzed with ForteBio Data Analysis Software
v8.1, using the 1:1 model and a global full fit with 1000 s
association time and 100 s dissociation time. Data traces were
corrected by subtraction of a reference curve (antibody on
biosensor, measurement with sample diluent only), the Y-axis was
aligned to the last 10 s of the baseline, and interstep correction
as well as Savitzky-Golay filtering was applied. Data traces with a
response <0.05 nm were excluded from analysis.
[0446] Table 10 shows the association rate constant k.sub.a (1/Ms),
dissociation rate constant k.sub.d (1/s) and equilibrium
dissociation constant K.sub.D (M) for recombinant CD3.epsilon.
determined by biolayer interferometry. IgG1-huCD3-FEAL showed a
relatively high (K.sub.D: 15 nM) binding affinity to recombinant
CD3.epsilon. compared to IgG1-huCD3-H101G-FEAL (K.sub.D: 683
nM).
TABLE-US-00010 TABLE 10 Binding affinities of monospecific,
bivalent CD3 antibodies to recombinant CD3E as determined by
label-free biolayer interferometry On-rate Off-rate Antibody
k.sub.a (1/Ms) k.sub.d (1/s) K.sub.D (M) IgG1-huCD3-FEAL 2.7E+05
4.0E-03 15 IgG1-huCD3-H101G-FEAL 3.0E+04 2.0E-02 683
Example 5--Cross-Block of B7H4 Antibodies Determined by Biolayer
Interferometry
[0447] Antibody cross-block analysis (epitope binning) in classical
Sandwich format was performed by BLI on an Octet HTX instrument
(ForteBio). A first cross-block experiment with
IgG1-B7H4-C1-N52S-FEAR, IgG1-B7H4-C2-FEAR, IgG1-B7H4-C3-FEAR, and
IgG1-B7H4-C4-FEAR was carried out while shaking at 1,000 RPM and at
30.degree. C.
[0448] Amine Reactive 2.sup.nd Generation (AR2G) biosensors
(ForteBio, cat. no. 18-5092) were activated for 300 s with a
solution of 20 mM EDC
(N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride)
(Sigma-Aldrich, cat. no. 03449) and 10 mM s-NHS
(N-Hydroxysulfosuccinimide sodium salt) (Sigma-Aldrich, cat. no.
56485). The activated AR2G sensors were loaded with 20 .mu.g/mL
first antibody in 10 mM Sodium Acetate pH 6.0 (ForteBio, cat. no.
18-1070) for 600 s and quenched with 1 M ethanolamine pH 8.5
(ForteBio cat. no. 18-1071) for 300 s. After a baseline measurement
in Sample Diluent (50 s; ForteBio, cat. no. 18-1048), the AR2G
biosensors containing immobilized antibodies were loaded for 300 s
with human B7H4 (100 nM or 2.68 .mu.g/mL diluted in Sample Diluent;
Sino Biological, cat. no 10738-H08H). The theoretical molecular
mass of human B7H4 based on the amino acid sequence (26.8 kDa) was
used for calculations. The association (300 s) of a second antibody
(10 .mu.g/mL in Sample Diluent) was determined. Sensors were
regenerated by exposure to 10 mM glycine (Riedel-de Haen, cat. no.
15527) buffer pH 2.5 for 5 s, followed by neutralization in Sample
Diluent for 5 s; both steps were repeated twice. Subsequently the
sensors containing immobilized first antibody were used again,
starting with the baseline step.
[0449] Data were acquired using Data Acquisition Software
v9.0.0.49d (ForteBio) and analyzed with Data Analysis HT Software
v10.0.17 (ForteBio). Data traces were corrected by subtraction of a
reference curve (Sample Diluent instead of second antibody) in
order to correct for the dissociation of B7H4 from the immobilized
first antibody. The Y-axis was aligned to the start of the
association step and Savitzky-Golay filtering was applied. The
corrected association responses of the second antibodies were
plotted in a matrix format. In general, responses >0.05 nm were
considered non-cross-blocking antibodies, while responses <0.05
nm were considered to be blocking antibody pairs.
[0450] The cross-block experiment was repeated to also include
IgG1-B7H4-C5-FEAR and was performed as described above, with minor
adaptations. The experiment was carried out while shaking at 1,000
RPM and at 22.degree. C. Data were acquired using Data Acquisition
Software v12.0.1.8 (ForteBio) and analyzed with Data Analysis HT
Software v12.0.1.55 (ForteBio). In general, responses >0.1 nm
were considered non-cross-blocking antibodies, while responses
<0.1 nm were considered to be blocking antibody pairs.
[0451] Initial cross-block experiments were performed for
antibodies IgG1-B7H4-C1-N52S-FEAR, IgG1-B7H4-C3-FEAR,
IgG1-B7H4-C4-FEAR and IgG1-B7H4-C2-FEAR. The results are summarized
in Table 11. A second set of cross-block experiments was performed
to also include IgG1-B7H4-C5-FEAR. These results are summarized in
Table 12. The first column shows the immobilized antibodies; the
first row shows the antibodies in solution (referred to as `the
second antibodies` above). Corrected association responses of the
antibodies in solution are shown. Cross-block of antibodies is
indicated by bold font, and non-blocking antibody combinations are
unmarked (transparent background), showing that
IgG1-B7H4-C1-N52S-FEAR, IgG1-B7H4-C3-FEAR, and IgG1-B7H4-C5-FEAR
are cross-blocking with each other and not with IgG1-B7H4-C4-FEAR
and IgG1-B7H4-C2-FEAR, and vice versa.
TABLE-US-00011 TABLE 11 First antibody cross-block experiment using
biolayer interferometry. The first column shows the immobilized
antibodies and the first row shows the antibodies in solution.
Corrected association responses of the antibodies in solution are
shown. Cross-block of antibodies is indicated by bold font,
non-blocking antibody combinations are unmarked (transparent
background). IgG1- IgG1- IgG1- IgG1- B7H4- B7H4- B7H4- B7H4-
C1-N52S- C3- C4- C2- Antibody cross-block FEAR FEAR FEAR FEAR
IgG1-B7H4-C1-N52S-FEAR -0.01 0.00 0.80 0.56 IgG1-B7H4-C3-FEAR -0.02
-0.01 0.97 0.53 IgG1-B7H4-C4-FEAR 0.82 0.56 -0.02 -0.01
IgG1-B7H4-C2-FEAR 0.74 0.54 0.00 0.00
TABLE-US-00012 TABLE 12 Second antibody cross-block experiment
using biolayer interferometry. The first column shows the
immobilized antibodies and the first row shows the antibodies in
solution. Corrected association responses of the antibodies in
solution are shown. Cross-block of antibodies is indicated by bold
font, non-blocking antibody combinations are unmarked (transparent
background). IgG1- B7H4- IgG1- IgG1- IgG1- IgG1- C1- B7H4- B7H4-
B7H4- B7H4- N52S- C3- C2- C4- C5- Antibody cross-block FEAR FEAR
FEAR FEAR FEAR IgG1-B7H4-C1-N525- 0 0.01 0.38 0.42 0.43 FEAR
IgG1-B7H4-C3-FEAR 0.01 0 0.44 0.57 0.61 IgG1-B7H4-C2-FEAR 0.28 0.25
0.01 0.01 0.02 IgG1-B7H4-C4-FEAR 0.5 0.39 0 -0.01 0
IgG1-B7H4-C5-FEAR 0.67 0.57 -0.03 -0.03 -0.01
Example 6--Generation of Bispecific Antibodies by 2-MEA-Induced
Fab-Arm Exchange
[0452] Bispecific antibodies were generated in vitro using the
DuoBody.RTM. platform technology, i.e. 2-MEA-induced Fab-arm
exchange as described in WO2011147986, WO2011131746 and
WO2013060867 (Genmab) and Labrijn et al. (Labrijn et al., PNAS
2013, 110: 5145-50; Gramer et al., MAbs 2013, 5: 962-973). To
enable the production of bispecific antibodies by this method, IgG1
molecules carrying specific point mutations in the CH3 domain were
generated: in one parental IgG1 antibody the F405L mutation (i.e.
the CD3 antibodies in this application), in the other parental IgG1
antibody the K409R mutation (i.e. the B7H4 or control, HIV-1
gp120-specific, antibodies in this application). In addition to
these mutations, the parental IgG1 antibodies included
substitutions L234F, L235E, D265A (FEA).
[0453] To generate bispecific antibodies, the two parental
antibodies were mixed in equal mass amounts in PBS buffer
(Phosphate Buffered Saline; 8.7 mM HPO.sub.4.sup.2-, 1.8 mM
H.sub.2PO.sub.4.sup.-, 163.9 mM Na.sup.+, 140.3 mM Cl.sup.-, pH
7.4). 2-mercaptoethylamine-HCl (2-MEA) was added to a final
concentration of 75 mM and the reaction mixture was incubated at
31.degree. C. for 5 h. The 2-MEA was removed by dialysis into PBS
buffer using 10 kDa molecular-weight cutoff Slide-A-Lyzer carriages
(Thermo Fisher Scientific) according to the manufacturer's protocol
in order to allow re-oxidation of the inter-chain disulfide bonds
and formation of intact bispecific antibodies.
[0454] The following antibodies were used in the examples:
B7H4 Antibodies
[0455] IgG1-B7H4-C1-FEAR (having the VH and VL sequences set forth
in SEQ ID NO: 25 and SEQ ID NO: 33).
[0456] IgG1-B7H4-C1-N52S-FEAR (having the VH and VL sequences set
forth in SEQ ID NO: 29 and SEQ ID NO: 33).
[0457] IgG1-B7H4-C2-FEAR having the VH and VL sequences set forth
in SEQ ID NO: 43 and SEQ ID NO: 47).
[0458] IgG1-B7H4-C3-FEAR having the VH and VL sequences set forth
in SEQ ID NO: 36 and SEQ ID NO: 40).
[0459] IgG1-B7H4-C4-FEAR having the VH and VL sequences set forth
in SEQ ID NO: 50 and SEQ ID NO: 54).
[0460] IgG1-B7H4-C5-FEAR having the VH and VL sequences set forth
in SEQ ID NO: 65 and SEQ ID NO: 69).
[0461] The annotation IgG1 indicates that full length antibodies of
the IgG1 isotype were made, and the FEAR annotation indicates that
the heavy chain constant regions contains amino acid substitutions
L234F, L235E, D265A and K409R and the light chain constant regions
were of the kappa type (SEQ ID NO. 61 and 63, respectively).
CD3 Antibodies
[0462] IgG1-huCD3-FEAL (having the VH and VL sequences set forth in
SEQ ID NO: 16 and SEQ ID NO: 22).
[0463] IgG1-huCD3-H101G-FEAL (having the VH and VL sequences set
forth in SEQ ID NO: 17 and SEQ ID NO: 22).
[0464] The annotation IgG1 indicates that full length antibodies of
the IgG1 isotype were made, and the FEAL annotation indicates that
the heavy chain constant regions contains amino acid substitutions
L234F, L235E, D265A and F405L and the light chain constant regions
were of the lambda type (SEQ ID NO. 60 and 64, respectively).
Control Antibodies
[0465] IgG1-b12-K409R (having the VH and VL sequences set forth in
SEQ ID NO: 14 and SEQ ID NO: 15).
[0466] The annotation IgG1 indicates that full length antibodies of
the IgG1 isotype were made, and the K409R annotation indicates that
the heavy chain constant regions contains amino acid substitution
K409R and the light chain constant regions were of the kappa type
(SEQ ID NO. 62 and 63, respectively). Bispecific antibodies
[0467] The CD3 and B7H4 antibodies described above were combined to
generate a bispecific antibody, having one antigen-binding region
capable of binding human CD3 and the other antigen-binding region
capable of binding B7H4, providing a bispecific antibody of the
isotype IgG1, which is annotated as bsIgG1.
TABLE-US-00013 bsIgG1-huCD3-FEALxB7H4-C1-FEAR
bsIgG1-huCD3-FEALxB7H4-C1-N52S-FEAR
bsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR
bsIgG1-huCD3-FEALxB7H4-C2-FEAR bsIgG1-huCD3-FEALxB7H4-C3-FEAR
bsIgG1-huCD3-FEALxB7H4-C4-FEAR bsIgG1-huCD3-H101G-FEALxB7H4-C2-FEAR
bsIgG1-huCD3-H101G-FEALxB7H4-C3-FEAR
bsIgG1-huCD3-H101G-FEALxB7H4-C4-FEAR
bsIgG1-huCD3-H101G-FEALxB7H4-05-FEAR bsIgG1-huCD3-FEALxb12-FEAR
(for the b12 arm having the VH and VL sequences set forth in SEQ ID
NO: 14 and SEQ ID NO: 15) bsIgG1-huCD3-H101G-FEALxb12-FEAR
Example 7--Determining the B7H4 Domain and Functional Epitope
Involved in Binding Using B7H4-B7H3 Chimeric Molecules and a B7H4
Alanine Scanning Library
Domain Mapping Using B7H4-B7H3 Chimeric Molecules Using End-Point
Analysis
[0468] The B7H4 domain specificity of the B7H4 antibodies was
determined using a panel of cells transfected to express human
B7H4, human B7H3 (a structurally comparable protein with sufficient
amino acid sequence difference in the extracellular domain) or two
different human B7H4-B7H3 chimeric molecules. Expression constructs
were prepared encoding human B7H4, human B7H3 (Uniprot accession
no. Q5ZPR3-1; SEQ ID NO: 9), or a chimeric molecule containing the
IgV domain of B7H3 and the IgC domain of B7H4 (B7H3-IgV/B7H4-IgC;
SEQ ID NO: 11), or a chimeric molecule containing the IgV domain of
B7H4 and the IgC domain of B7H3 (B7H4-IgV/B7H3-IgC; SEQ ID NO: 10).
HEK cells were transiently transfected to express these
constructs.
[0469] Cells (3.times.10.sup.4 cell/well) were incubated in
polystyrene 96-well round-bottom plates (Greiner bio-one, cat. no.
650101) with serial dilutions of antibodies (range 0.0046 to 10
.mu.g/mL in 3-fold dilution steps) in 50 .mu.L PBS/0.1% BSA/0.02%
azide (FACS buffer) at 4.degree. C. for 30 min. After washing twice
in FACS buffer, cells were incubated with secondary antibody at
4.degree. C. for 30 min. As a secondary antibody, R-Phycoerythrin
(PE)-conjugated goat-anti-human IgG F(ab').sub.2 (1:500 in staining
buffer; Jackson ImmunoResearch Laboratories, Inc., West Grove, Pa.,
cat. no. 109-116-098) was used. Next, cells were washed twice in
FACS buffer, re-suspended in 20 .mu.L FACS buffer and analyzed on
an iQue Screener (Intellicyt Corporation, USA). The binding of
bsIgG1-huCD3-FEALxB7H4-C1-N52S-FEAR,
bsIgG1-huCD3-FEALxB7H4-C4-FEAR, bsIgG1-huCD3-FEALxB7H4-C3-FEAR and
bsIgG1-huCD3-FEALxB7H4-C2-FEAR at 10 .mu.g/mL was determined as %
mean fluorescence intensity (MFI) of the binding 10 .mu.g/mL of:
[0470] IgG1-B7H3-BRCA84D (a B7H3-specific IgG1 antibody, generated
as described above with CDR sequences as described for antibody
BRCA84D in WO2011109400) to B7H3 expressing cells, [0471]
bsIgG1-huCD3-FEALxB7H4-C4-FEAR to B7H3-IgV/B7H4-IgC expressing
cells, [0472] bsIgG1-huCD3-FEALxB7H4-C2-FEAR to B7H4-IgV/B7H3-IgC
expressing cells, [0473] and bsIgG1-huCD3-FEALxB7H4-C3-FEAR to B7H4
expressing cells.
[0474] FIG. 1 shows that the IgC domain of B7H4 is involved in
binding of bsIgG1-huCD3-FEALxB7H4-C1-N52S-FEAR and
bsIgG1-huCD3-FEALxB7H4-C4-FEAR, both the IgC and IgV domain of B7H4
are involved in binding of bsIgG1-huCD3-FEALxB7H4-C3-FEAR, and at
least the IgV domain of B7H4 is involved in binding of
bsIgG1-huCD3-FEALxB7H4-C2-FEAR. For the C2 antibody from which the
variable domains were used to created
bsIgG1-huCD3-FEALxB7H4-C2-FEAR, it has been described that it binds
to the IgV domain; the data in FIG. 1 indicates that the IgC domain
is also involved in binding (WO2014159835 and Leong et al 2015,
Mol. Pharmaceutics 12, 1717-1729).
Domain Mapping Using B7H4-B7H3 Chimeric Molecules Using Analysis of
Full Dose-Response Curves
[0475] Further experiments were conducted to study the B7H4 domain
specificity of the B7H4 antibodies in more detail, by analysis of
full dose-response curves. In these experiments, the domain
specificity of bsIgG1-huCD3-H101G-FEALxB7H4-C5-FEAR was also
determined. Binding of serial dilutions (0.014 to 30 .mu.g/mL in
3-fold dilution steps) of
bsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR,
bsIgG1-huCD3-H101G-FEALxB7H4-C2-FEAR,
bsIgG1-huCD-H101G-FEALxB7H4-C3-FEAR,
bsIgG1-huCD3-H101G-FEALxB7H4-C4-FEAR and
bsIgG1-huCD3-H101G-FEALxB7H4-C5-FEAR to HEK cells transiently
transfected to express human B7H4 or the B7H4-B7H3 chimeric
molecules B7H3-IgV/B7H4-IgC or B7H4-IgV/B7H3-IgC was determined as
described above. FIG. 2 shows the dose-response curves, showing
that the IgC domain of B7H4 is involved in binding of
bsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR, in line with the
findings of the alanine scanning library experiments. Furthermore,
the IgV domain is involved in the binding of
bsIgG1-huCD3-H101G-FEALxB7H4-C2-FEAR,
bsIgG1-huCD3-H101G-FEALxB7H4-C4-FEAR and
bsIgG1-huCD3-H101G-FEALxB7H4-C5-FEAR, whereas both the IgC and IgV
domain appear involved in the binding of
bsIgG1-huCD3-H101GFEALxB7H4-C3-FEAR.
Determination of the Contribution of B7H4 Amino Acid Residues to
Binding of B7H4 Antibodies Using a B7H4 Alanine Scanning
Library
Library Design
[0476] A human B7H4 (Uniprot Q7Z7D3-1) single residue alanine
library was synthesized (GeneArt) in which all amino acid residues
in the extracellular domain of human B7H4 were individually mutated
to alanines except for positions containing alanines or cysteines.
Cysteines were not mutated to minimize the chance of structural
disruption of the antigen. The library was cloned in the pMAC
expression vector containing a CMV/TK-polyA expression cassette, an
Amp resistance gene and a pBR322 replication origin.
Library Production and Screening
[0477] The antibodies C1-N52S, C2 and C3 were generated as
recombinant monovalent antibodies as described in WO2007059782 with
a mNeonGreen tag. The wild type B7H4 and alanine mutants were
expressed individually in FreeStyle HEK293 cells according to the
manufacturer's instructions (Thermo Scientific). One day post
transfection the cells were harvested. Approximately 50,000 cells
were incubated with 20 .mu.L mNeoGreen labeled antibody of
interest. Cells were incubated for 1 hour at room temperature.
Subsequently, 150 .mu.L FACS buffer was added and cells were washed
twice with FACS buffer. Cells were resuspended in 30 .mu.L fresh
FACS buffer and analyzed by flow cytometry using an iQue Screener
(Intellicyt Corporation, USA).
[0478] The entire experiment was performed 2 times in
duplicate.
Data Analysis
[0479] For every sample, the average antibody binding per cell was
determined as the geometric mean of the fluorescence intensity
(gMFI) for the ungated cell population. The gMFI is influenced by
the affinity of the antibody for the B7H4 mutant and the expression
level of the B7H4 mutant per cell. Since specific alanine mutations
can impact the surface expression level of the mutant B7H4, and to
correct for expression differences for each B7H4 mutant in general,
data were normalized against the binding intensity of a non-cross
blocking B7H4 specific reference antibody, using the following
equation:
Normalized .times. .times. gMFI aa .times. .times. position = gMFI
Test .times. .times. Ab gMFI Reference .times. .times. Ab
##EQU00003##
in which C2 was used as reference antibody for C1-N52S and C3, and
C1-N52S was used as reference antibody for C2, and in which `aa
position` refers to either a particular ala mutant of B7H4 or wild
type (wt) B7H4.
[0480] To express loss or gain of binding of the antibodies on a
linear Fold Change scale, the following calculation was used:
Fold .times. .times. Change = Lo .times. g 1 .times. 0 .function. (
Normalized .times. .times. gMFI ala .times. .times. mutant
Normalized .times. .times. gMFI w .times. t ) ##EQU00004##
[0481] Gain of binding in most cases will be caused by loss of
binding of the reference antibody to specific ala mutants.
[0482] Upon these calculations, amino acid positions for which,
upon replacing the amino acid with alanine, there is no loss or
gain of binding by a particular antibody will give as result `0`,
gain of binding will result in `>0` and loss of binding will
result in `<0`. To correct for sample variation, only B7H4 amino
acid residues where the Fold Change in binding was lower than the
mean Fold Change--1.5.times.SD, where SD is the standard deviation
of calculated fold changes from four independent experiments for a
particular test antibody, were considered `loss of binding
mutants`.
[0483] In case the gMFI of the reference antibody for a particular
B7H4 mutant was lower than the mean gMFI-2.5.times.SD of the mean
gMFI.sub.Control Ab, data were excluded from analysis (as for those
B7H4 mutants it was assumed expression levels were not
sufficient).
[0484] FIG. 3 shows the Fold Change in binding of the B7H4
antibodies to B7H4 variants with ala mutations in the ECD, with the
amino acid residues where the Fold Change in binding was lower than
the mean Fold Change--1.5.times.SD annotated. The Fold Change is
indicated in FIG. 3 as Z-score. The results indicate that: [0485]
binding of antibody C1-N52S is at least dependent on aa S151, V157,
D158, Y159, E164, L166, W173, P175, P177, V179, W181, F199, M208,
V210, T222, Y223, V240, E242 and I245, which are in the IgC domain
of human B7H4, [0486] binding of antibody C2 is at least dependent
on aa R98, G99, R116, K118, N119 and D124, which are in the IgV of
human B7H4, and [0487] binding of antibody C3 is at least dependent
on aa N156, E164, V217 and R248, which are in the IgC domain of
human B7H4, and [0488] antibodies C1-N52S, C2 and C3 recognize
distinct functional epitopes on B7H4.
Example 8--Binding of B7H4 Monospecific and CD3xB7H4 Bispecific
Antibodies to B7H4 from Various Species
[0489] First, binding of bispecific CD3xB7H4 antibodies and
monospecific B7H4 antibodies to HEK-293F cells transiently
transfected with human B7H4 or with cynomolgus monkey (Macaca
fascicularis) B7H4 was analyzed by flow cytometry. Non-transfected
HEK-293F cells were used as negative control; these cells were
(also) confirmed not to express CD3.
[0490] Cells (3.times.10.sup.4 cells/well) were incubated in
polystyrene 96-well round-bottom plates (Greiner bio-one, cat. no.
650180) with serial dilutions of antibodies (ranging from 0.000458
to 30 .mu.g/mL in 4-fold dilution steps) in 100 .mu.L PBS/0.1%
BSA/0.02% azide (staining buffer) at 4.degree. C. for 30 min.
Experiments were performed in technical duplicate. After washing
twice in staining buffer, cells were incubated in 50 .mu.L
secondary antibody at 4.degree. C. for 30 min. As a secondary
antibody, R-Phycoerythrin (PE)-conjugated goat-anti-human IgG
F(ab').sub.2 (1:500 in FACS buffer; Jackson ImmunoResearch
Laboratories, Inc., West Grove, Pa., cat. no. 109-116-098), was
used. Cells were washed twice in staining buffer, re-suspended in
30 .mu.L FACS buffer containing Topro-3 (1:10,000 dilution) and
analyzed on an iQue Screener (Intellicyt Corporation, USA). Binding
curves were analyzed using non-linear regression (sigmoidal
dose-response with variable slope) using Graph Pad Prism V7.02
software (GraphPad Software, San Diego, Calif., USA).
[0491] FIG. 4 shows that both IgG1-B7H4-C1-N52S-FEAR and
bsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR bound to cells expressing
human B7H4 or cynomolgus monkey B7H4.
[0492] Next, binding to HEK-293F cells transiently transfected with
B7H4 from dog, rabbit, rat, mouse or pig was determined as
described above. FIG. 5 shows that IgG1-B7H4-C1-N52S-FEAR and
bsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR bound to B7H4 from dog,
rabbit, rat and mouse to varying degrees; for each the apparent
affinity (EC50) of the bsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR
was lower than that of IgG1-B7H4-C1-N52S-FEAR.
bsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR was not able to bind to
pig B7H4, while IgG1-B7H4-C1-N52S-FEAR bound weakly and only at the
highest antibody concentrations tested.
[0493] The EC50s for binding to human and cynomolgus monkey B7H4 of
bsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR and
IgG1-B7H4-C1-N52S-FEAR were in a similar range.
[0494] Similar studies were performed to compare binding of
IgG1-B7H4-C1-052S-FEAR, IgG1-B7H4-C3-FEAR, IgG1-B7H4-C4-FEAR,
IgG1-B7H4-C2-FEAR and IgG1-B7H4-C5-FEAR to B7H4 from different
species (human, cynomolgus, mouse, rat, rabbit, dog, and pig). FIG.
6 shows that binding to HEK cells transfected with human and
cynomolgus B7H4 was similar for the tested antibodies. Similar
results were obtained with cells expressing rabbit and dog B7H4.
However, binding to mouse B7H4 of IgG1-B7H4-C1-N52S-FEAR appeared
lower relative to the binding of IgG1-B7H4-C3-FEAR,
IgG1-B7H4-C4-FEAR, IgG1-B7H4-C2-FEAR, and IgG1-B7H4-C5-FEAR, which
is in conformity with the results in example 3. Also, binding of
IgG1-B7H4-C1-N52S-FEAR and IgG1-B7H4-C3-FEAR to rat B7H4 appeared
lower relative to IgG1-B7H4-C4-FEAR, IgG1-B7H4-C2-FEAR, and
IgG1-B7H4-C5-FEAR. Furthermore, while IgG1-B7H4-C4-FEAR,
IgG1-B7H4-C2-FEAR and IgG1-B7H4-C5-FEAR bound to pig B7H4, binding
of IgG1-B7H4-C1-052S-FEAR was very weak and only apparent at the
highest antibody concentration tested. Binding of IgG1-B7H4-C3-FEAR
to pig B7H4 was undetectable.
Example 9--Binding of B7H4 Monospecific and CD3xB7H4 Bispecific
Antibodies to B7H4-Expressing Human Tumor Cell Lines
[0495] Binding of IgG1-B7H4-C1-N52S-FEAR and/or
bsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR and/or
bsIgG1-huCD3-FEALxB7H4-C1-N52S-FEAR to the B7H4-expressing human
tumor cell lines MCF-7 (breast adenocarcinoma; ATCC, cat. No.
HTB-22), MDA-MB-468 (breast adenocarcinoma; ATCC, cat. no. HTB-132)
and SK-BR3 (breast adenocarcinoma; ATCC, cat. No. HTB-30), and of
bsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR to B7H4-expressing human
tumor cell lines NIH-OVCAR-3 (ovarian adenocarcinoma; ATCC, cat.
no. HTB-161) or HCC1954 (breast ductal carcinoma; ATCC, cat. no.
CRL-2338) was determined. Furthermore, binding of
IgG1-B7H4-C1-N52S-FEAR, bsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR,
IgG1-B7H4-C2-FEAR, bsIgG1-huCD3-FEALxB7H4-C2-FEAR or
bsIgG1-huCD3-H101G-FEALxB7H4-C2-FEAR, IgG1-B7H4-C3-FEAR,
bsIgG1-huCD3-H101G-FEALxB7H4-C3-FEAR, IgG1-B7H4-C4-FEAR,
bsIgG1-huCD3-H101G-FEALxB7H4-C4-FEAR, IgG1-B7H4-C5-FEAR, and/or
bsIgG1-huCD3-H101G-FEALxB7H4-C5-FEAR to MDA-MB-468 and HCC1954
cells was determined. Solid tumor cell lines typically do not
express CD3. As negative control, tumor cell line HeLa that showed
no detectable B7H4 expression (cervix adenocarcinoma; ATCC, cat.
no. CCL-2) was used. Binding was analyzed by flow cytometry as
described above.
[0496] FIG. 7 shows that IgG1-B7H4-C1-N52S-FEAR and
bsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR showed comparable
dose-dependent binding to MCF-7 and MDA-MB-468 cells, with
comparable maximum binding levels.
[0497] FIG. 8 shows dose-dependent binding of
bsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR to NIH-OVCAR-3 and
HCC1954 cells, and lack of detectable binding to a non-B7H4
expressing cell line, HeLa.
[0498] Binding of bsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR and
bsIgG1-huCD3-FEALxB7H4-C1-N52S-FEAR to B7H4-expressing tumor cells
was compared using MDA-MB-486 and SK-BR3 cells. FIG. 9 shows that
bsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR and
bsIgG1-huCD3-FEALxB7H4-C1-N52S-FEAR showed comparable
dose-dependent binding to these cells, with comparable maximum
binding levels.
[0499] FIG. 10 shows dose-dependent binding of the C1-N52S, C2, C3,
C4, and C5 B7H4 antibodies in homodimer or bispecific antibody
format to MDA-MB-468 and HCC1954 cells. The antibodies based on C4
and C5 showed most efficient binding, the antibodies based on
C1-N52S and C2 showed intermediate binding efficiency, and the
antibodies based on C3 showed the lowest binding efficiency.
Maximum binding was comparable between the antibodies based on
C1-N52S, C2, C4 and C5, but lower for the antibodies based on
C3.
Example 10--Binding of B7H4 Antibody to Primary Tumor Cells
[0500] Primary tumor cells from an ovarian cancer patient were
obtained from Discovery Life Sciences (Huntsville, Ala., USA;
patient ID 110045042). Binding of IgG1-B7H4-C1-N52S-FEAR to tumor
cells was assessed by flow cytometry: cells were seeded at
2.times.10.sup.4 cells/well in polystyrene 96-well round-bottom
plates (Greiner bio-one, cat. no. 650180), centrifuged and
incubated with 50 .mu.l Fixable Viability Stain FVS-BV510 (BD
Biosciences, cat. no. 564406), 1:1000 diluted in PBS, at 4.degree.
C. for 30 min. After washing in staining buffer, cells were
incubated with FITC-labeled IgG1-B7H4-C1-N52S-FEAR and a panel of
CD3 (EF450 labeled; eBioscience, cat. no. 48-0037-42), CD45 (BV786
labeled; Biolegend, cat. no. 304048), CD14 (PE-Cy7 labeled; BD
Biosciences, cat. no. 557742), CD86 (PerCP-Cy5.5 labeled;
Biolegend, cat. no. 305420), CD163 (APC-Cy7 labeled; Biolegend,
cat. no. 333622) and EpCAM (AF700 labeled; R&D systems, cat.
no. FAB9601N) specific antibodies, at 4.degree. C. for 30 min.
After washing cells were resuspended in staining buffer and
analyzed using a FACS Fortessa (BD Biosciences). Single cells were
gated based on scatter FSC/SSC and live cells were identified by
exclusion of FVS-BV510 positive cells. Tumor cells were identified
as EpCAM positive cells.
[0501] Flow cytometric analysis showed that IgG1-B7H4-N52S-FEAR
bound EpCAM-positive live tumor cells but not to monocytes or T
cells within a dissociated tumor cell suspension of an ovarian
cancer sample.
Example 11--Induction of T Cell Mediated Cytotoxicity In Vitro by
CD3xB7H4 Bispecific Antibodies, Using Purified T Cells as Effector
Cells at Varying Effector to Target Ratios
[0502] To determine the efficiency of the T cell-mediated tumor
cell kill in presence of bispecific antibodies
bsIgG1-huCD3-FEALxB7H4-C1-N52S-FEAR and
bsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR, an in vitro cytotoxicity
assay was performed using B7H4-positive tumor cell lines as target
cells and purified T cells as effector cells, with varying effector
to target cell (E:T) ratios.
[0503] T cells were obtained from healthy human donor buffy coats
(Sanquin, Amsterdam, The Netherlands) and isolated using the
RosetteSep.TM. human T cell enrichment cocktail (Stemcell
Technologies, France, cat. no. 15061) according to the
manufacturer's instructions. SK-BR3 cells (16,000 cells/well) were
seeded into flat bottom 96-well plates (Greiner-bio-one, The
Netherlands, cat. no. 655180) and left to adhere for 4 hours at
37.degree. C. T cells were added to tumor cells at an effector to
target (E:T) ratio of 2:1, 4:1 or 8:1. Serial dilutions of
bsIgG1-huCD3-FEALxB7H4-C1-N52S-FEAR or
bsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR were added (final
concentration ranging from 10,000 to 0.0128 ng/mL; 5-fold
dilutions) and plates were incubated for 72 hours at 37.degree. C.
Plates were washed 3 times with PBS, and cells were incubated with
150 .mu.l/well of 10% alamarBlue.RTM. solution (Invitrogen, cat.
no. DAL1100) for 4 hours at 37.degree. C. As a positive control for
cytotoxicity, cells were incubated with 16 .mu.g/mL phenylarsine
oxide (PAO; Sigma-Aldrich, cat. no. P3075; dissolved in
dimethylsulfoxide [DMSO; Sigma-Adrich, cat. no. D2438]). AlamarBlue
fluorescence, as a measure of metabolic activity of the tumor cell
cultures and thus of viable tumor cells, was measured at 615 nm
(OD615) on an EnVision plate reader (PerkinElmer). The absorbance
of PAO-treated tumor cell samples was set as 0% viability and the
absorbance of untreated tumor cell samples was set as 100%
viability. The `percentage viable cells` was calculated as
follows:
% viable cells=([absorbance sample-absorbance PAO-treated target
cells]/[absorbance untreated target cells-absorbance PAO-treated
target cells]).times.100.
[0504] Dose-response curves and IC50 values were generated using
non-linear regression analysis (sigmoidal dose-response with
variable slope) using Graph Pad Prism V7.02 software (GraphPad
Software, San Diego, Calif., USA).
[0505] FIG. 11 shows that T cell mediated cytotoxicity was observed
at all E:T ratio's, with maximal tumor cell killing (less than 10%
viable tumor cells) observed at an E:T ratio of 8:1.
Example 12--Induction of Cytotoxicity In Vitro in Various Tumor
Cell Lines by CD3xB7H4 Bispecific Antibodies and Correlation with
B7H4 Expression Level
[0506] The T cell-mediated kill of bispecific antibodies
bsIgG1-huCD3-FEALxB7H4-C1-N52S-FEAR and
bsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR of various B7H4
expressing tumor cell lines was determined in an in vitro
cytotoxicity assay as described above, using an E:T ratio of 8:1.
The following cell lines were used: MCF-7, MDA-MB-486, SK-BR3,
NIH-OVCAR-3, HCC1954, and NCI-H1650. From each incubation, 150
.mu.L supernatants containing T cells was transferred to U-bottom
96 Well culture plates (CellStar, cat. no. 650180) prior to washing
and alamarBlue incubation (to determine T cell activation and
cytokine release, as described below)
[0507] For these tumor cell lines, the expression of B7H4 was
quantified by quantitative flow cytometry (Human IgG calibrator,
BioCytex) according to the manufacturer's instructions, using
bsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR to detect B7H4.
[0508] FIG. 12 shows both bsIgG1-huCD3-FEALxB7H4-C1-N52S-FEAR and
bsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR induced dose-dependent T
cell mediated cytotoxicity in MCF-7, MDA-MB-486, SK-BR3,
NIH-OVCAR-3 and HCC1954 cells in vitro. While maximum cytotoxic
activity (<10% viable tumor cells) was achieved for both bsAb
variants, this occurred at lower concentrations for
bsIgG1-huCD3-FEALxB7H4-C1-N52S-FEAR in comparison with
bsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR (Table 13).
[0509] No significant relation between tumor cell lysis and the
level of B7H4 expression (FIG. 13A) was observed for either
bsIgG1-huCD3-FEALxB7H4-C1-N52S-FEAR or
bsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR (FIG. 13B). FIG. 13B
shows the IC50 of T cell-mediated kill, using T cells derived from
4-6 donors, in the presence of bsIgG1-huCD3-FEALxB7H4-C1-N52S-FEAR
or bsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR for each cell line,
with the cell lines arranged from lowest to highest level of B7H4
expression. This means that T cell mediated killing can occur over
a wide range of B7H4 expression levels.
[0510] Table 13 summarizes results across a panel of 5 cell lines
and 4 donors.
TABLE-US-00014 TABLE 13 Induction of cytotoxicity in vitro in
various tumor cell lines by CD3x67H4 bispecific antibodies. IC50
range (4 donors each cell line) (.mu.g/ml) CD3- H101GXB7H4 CD3x67H4
cell line lowest highest lowest highest MCF7 0.55 1.29 0.012 0.025
OVCAR3 0.09 1.629 0.003 0.012 NCI-H16650 1.67 5.07 N.D. N.D.
MDA-MB-468 0.08 0.16 0.001 0.004 HCC1954 0.06 0.22 0.001 0.008
SK-BR3 0.09 0.22 0.002 0.016
[0511] bsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR also induced
dose-dependent T-cell mediated cytotoxicity of the tested NCI-H1650
NSCLC cell line.
Example 13--Induction of T Cell Activation and Cytokine Production
In Vitro by CD3xB7H4 Bispecific Antibodies in the Presence of
B7H4-Positive Tumor Cells
[0512] The U-bottom 96 well culture plates containing the
supernatants collected during the in vitro T cell-mediated
cytotoxicity experiments described in example 12 were centrifuged
(300.times.g) for 3 min at 4.degree. C., after which 75 .mu.L of
supernatant was transferred to a new plate for cytokine production
measurement, and T cells were kept to assess T cell activation
(described below). Cytokine production was analyzed by a multiplex
U-plex assay (MeSo Scale Discovery, USA, cat. no. K15049K)
according to manufacturer's instructions.
[0513] T cells were stained for T cell markers CD3 (1:200;
eBioscience, clone OKT3, conjugated to eFluor450), CD4 (1:50;
eBioscience, clone OKT4, conjugated to APC-eFluor780), CD8 (1:100;
Biolegend, clone RPA-T8, conjugated to AF700) and T cell activation
markers CD69 (1:50; BD Biosciences, clone AB2439, conjugated to
APC), CD25 (1:50; eBioscience, clone BC96, conjugated to PE-Cy7)
and CD279/PD1 (1:50; Biolegend, clone EH12.2H7, conjugated to
BV605). Single stained samples with Ultracomp beads (5 .mu.L;
Invitrogen, cat. no. 01-2222-42) were included and used for
compensation adjustments of the flow cytometer. After 30 min of
incubation at 4.degree. C., plates were washed three times with
PBS/0.1% BSA/0.02% azide (staining buffer). Cells were resuspended
in 120 .mu.L staining buffer and analyzed using a FACS Fortessa (BD
Biosciences). Data were processed using FlowJo (BD
Biosciences).
[0514] Dose-response curves, EC50, EC90 and EC99 values were
calculated using non-linear regression analysis (sigmoidal
dose-response with variable slope) using GraphPad Prism V7.02
software (GraphPad Software, San Diego, Calif., USA).
[0515] FIG. 14A shows T cell activation in the presence of
bsIgG1-huCD3-FEALxB7H4-C1-N52S-FEAR or
bsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR for the B7H4-positive
tumor cell lines, as defined by the expression of activation
markers CD69 on CD8+ T cells (determined by flow cytometry). FIG.
14B shows the EC50 of T cell activation, using T cells derived from
3-4 donors, for each of the tumor cell lines.
[0516] Overall, a subset (approximately 20-50% at the highest
antibody concentration) of CD8+ T cells became activated in the
presence of either bsIgG1-huCD3-FEALxB7H4-C1-N52S-FEAR or
bsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR. T cell activation
induced by bsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR generally
occurred at higher concentrations than that induced by
bsIgG1-huCD3-FEALxB7H4-C1-N52S-FEAR (FIG. 14A). The EC50 of T cell
activation for both bispecific antibodies was variable between
target cell line used and between donors (FIG. 14B).
[0517] Production of cytokines was assessed in supernatants of the
tumor cell-T cell cultures by Mesoscale Discovery U-plex multiplex
ELISA. Of the 10 cytokines analyzed across the cell line panel,
using T cells from 4 donors, significant increases in cytokine
levels were primarily observed for IFN-gamma and IL-8 (>2000
pg/ml). IL-4, IL-6 and IL-13 were modulated at much lower levels
(<500 pg/ml), while IL-1beta, IL-2, IL-10, IL-12p70, and
TNFalpha levels were generally below 50 pg/ml. Because IFN-gamma
changes were robustly and consistently detected and IFN-gamma is
one of the core cytokines elevated in serum of patients with
cytokine release syndrome, the data for this cytokine is
represented.
[0518] FIG. 15 shows the levels of IFN-gamma in the supernatant of
T cell-tumor cell co-cultures at antibody concentrations that
induced T cell mediated cytotoxicity in 50%, 90% and 99% of tumor
cells (EC50, EC90, EC99, resp) in the presence of
bsIgG1-huCD3-FEALxB7H4-C1-N52S-FEAR and
bsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR, using T cells from at
least 3 donors analyzed per cell line. Cytokine production levels
varied per donor and per target tumor cell line. Nevertheless, at
antibody concentrations that induced the same level (%) of tumor
cell killing, in general lower cytokine production levels were seen
after exposure of T cell-tumor cell co-cultures to
bsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR compared to that after
exposure to bsIgG1-huCD3-FEALxB7H4-C1-N52S-FEAR. Thus, at the same
level of tumor cell killing, incubation with
bsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR resulted in lower
cytokine production than bsIgG1-huCD3-FEALxB7H4-C1-N52S-FEAR.
Example 14--Non-Clinical Safety Studies of CD3xB7H4 Bispecific
Antibodies in Cynomolgus Monkeys
[0519] The non-clinical safety profile of
bsIgG1-huCD3-FEALxB7H4-C1-N52S-FEAR and
bsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR was evaluated in
non-human primates (cynomolgus monkeys, Macaca fascicularis,
originating from Mauritius) at Citoxlab, France. Cynomolgus monkeys
were considered the only relevant species for non-clinical safety
studies based on the species-specificity of the CD3 arms of
bsIgG1-huCD3-FEALxB7H4-C1-N52S-FEAR and
bsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR, and furthermore due to
similar binding of the B7H4 arm to human and cynomolgus B7H4 and
further pharmacological findings. These studies were conducted in
compliance with animal health regulations (Council Directive No.
2010/63/EU of 22 Sep. 2010 and French decret No. 2013-118 of 1 Feb.
2013 on the protection of animals used for scientific
purposes).
[0520] The aim of the studies were to determine the potential
toxicity and toxicokinetics of the CD3xB7H4 bispecific antibodies.
Here only the results of the toxicokinetics and the determination
of cytokine levels in plasma are described.
[0521] In two separate studies, the animals were treated with a
single dose of 0.1, 1, 3 or 10 mg/kg
bsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR or
bsIgG1-huCD3-FEALxB7H4-C1-N52S-FEAR (one female animal per dose) by
intravenous (IV) infusion. The day of infusion was indicated as Day
1 in the study. Blood samples were obtained twice before dosing and
0.5h, 2h 4h, 12h, 24h and 48h after dosing for evaluation of the
toxicokinetic profile and plasma cytokine levels, and additionally
168, 336 and 504 hours after dosing for toxicokinetics.
Cytokine Levels
[0522] Plasma samples were analyzed for cytokine levels
(IL-1.beta., IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, TNF, IL-12p70,
IL-15 and CCL2/MCP1) using Luminex xMAP technology.
[0523] BsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR administration to
cynomolgus monkey produced only minor changes in plasma cytokine
levels, which were considered unrelated to test compound, whereas
administration of bsIgG1-huCD3-FEALxB7H4-C1-N52S-FEAR resulted in
dose-dependent increase of IL-6 and MCP-1 levels, as shown in FIG.
16.
[0524] The lower cytokine levels produced after treatment with
bispecific BsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR, as compared
with BsIgG1-huCD3-FEALxB7H4-C1-N52S-FEAR antibody, may offer an
advantage in a clinical setting.
Toxicokinetics
[0525] Plasma concentrations of CD3xB7H4 bispecifics were
determined using a generic IgG PK ECLIA method. Toxicokinetic
parameters were estimated using Certara Phoenix WinNonlin
pharmacokinetic software version 8.1 using a non-compartmental
approach consistent with the intravenous infusion injection route
of administration. FIG. 17 shows that the toxicokinetic profiles of
both CD3xB7H4 bispecific antibodies were highly comparable up to 7
days post-dose, with both showing dose-related plasma exposure.
[0526] A pharmacokinetic modeling exercise was undertaken to assess
whether the projected clinical dose range required by the
BsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR variant with lower CD3
affinity would be unsustainably high. A PK model was used that was
informed by observations in cynomolgus monkey. The clinical dose
range was derived that is expected to give rise to one-week average
plasma exposure equal to the EC50 to EC90 for T cell mediated cell
kill as observed in vitro. The resulting dose range was considered
feasible and this aspect gave no reason a priori to favor one type
of bispecific antibody over the other
(BsIgG1-huCD3-H101G-FEALxB7H4-C1-N52S-FEAR vs. the
BsIgG1-huCD3-FEALxB7H4-C1-N52S-FEAR).
Example 15--B7H4 Expression in Various Human Cancer Indications
[0527] B7H4 mRNA levels were extracted from the Omicsoft TCGA
database and visualized using Oncoland software (Qiagen, USA).
[0528] FIG. 18 shows the B7H4 mRNA expression levels in a range of
primary solid tumors, ranked according to median of the expression.
mRNA expression was found in a wide range of cancer indication and
varied within each indication, with highest median expression found
in uterine carcinosarcoma (UCS), bladder urothelial carcinoma
(BLCA), pancreatic adenocarcinoma (PAAD), lung squamous cell
carcinoma (LUSC), breast invasive carcinoma (BRCA), uterine corpus
endometrial carcinoma (UCEC), ovarian serous cystadenocarcinoma
(OV) and cholangiocarcinoma (CHOL).
[0529] Protein expression of B7H4 in colon, lung (small cell lung
cancer, SCLC and non-small cell lung cancer, NSCLC), stomach,
pancreatic, bladder, cervical, head and neck, breast (including
triple-negative breast cancer, TNBC), ovarian, esophageal, kidney,
prostate and uterine cancer and cholangiocarcinoma, was analyzed by
immunohistochemistry (IHC) on tissue microarrays (TMA; all
purchased from BioMax). Prior to staining, freshly cut TMA sections
(5 .mu.m) were deparaffinized and incubated with Target Retrieval
Solution pH9 (DAKO, S2367; 30 min at 97.degree. C., 60 min cool
down). B7H4 IHC was performed using a commercial rabbit anti-human
B7-H4 monoclonal antibody (clone D1M8I, #14572, Cell Signaling
Technologies) at optimal dilution (1:25; final concentration 2.6
.mu.g/mL) for 30 min (RT) on a LabVision autostainer platform.
Subsequently, sections were incubated with anti-rabbit IgG polymer
(Envision.TM. FLEX+ rabbit (DAKO, S2022), washed and incubated with
DAKO Liquid DAB+ Substrate chromogen system (DAKO, K3468).
Hematoxylin (DAKO, S3301) was used to detect nucleated cells.
Cytokeratin (to determine the tumor region of interest, ROI) IHC
was performed with mouse anti-cytokeratin antibody mix (clones
AE1/AE3) on Ventana Benchmark using OptiView detection. Cytokeratin
was visualized with DAB and nuclei counterstained with hematoxylin
using default Ventana reagents. Stained TMA sections were digitized
at 20.times. magnification on a AxioScan (Zeiss). Initially, manual
scoring was performed to determine the average B7H4 staining
intensity (negative-low-medium-high) and the percentage of tumor
cores with >10% B7H4-positive tumor cells.
[0530] Subsequently, automated scoring was performed. The tumor ROI
was defined using cytokeratin mask on TMA sections adjacent to
those stained for B7H4. B7H4 staining intensity in the tumor ROI
was quantified (negative, weak (1), moderate (2) or string (3) and
the percentage of B7H4 percentage positive tumor cells (range
0-100%) was determined using HALO image analysis software. For each
indication, the percentage of tumor cores with >10%
B7H4-positive tumor cells was determined.
[0531] Table 14 shows B7H4 protein expression determined by IHC
analysis of BioMax TMAs. No to very low B7H4 expression was seen in
colon, prostate, kidney, and small cell lung cancer samples. In
samples from the other indications the B7H4 expression varied, with
increasing B7H4 expression found in stomach cancer, pancreatic
cancer, cholangiocarcinoma, oesophageal cancer, bladder cancer,
non-small cell lung cancer (in particular squamous NSCLC), cervical
cancer, head and neck cancer, breast cancer (triple negative breast
cancer [TNBC] and non-TNBC), ovarian cancer, and uterine
cancer.
TABLE-US-00015 TABLE 14 B7H4 protein expression determined by IHC
analysis of BioMax TMAs. Automated Manual scoring scoring >10%
>10% B7H4 B7H4 positive positive (any (1+ and intensity, above,
by Indication (BioMax by visual Staining digital image TMA)
assessment) intensity analysis) Colon cancer (n = 64) 0% Negative
Lung SCLC 1% Negative-Low cancer (n = 60) NSCLC AC 17% Low ND (n =
82) SQCC 48% Medium ND (n = 95) Stomach cancer 17% Low (n = 90)
Pancreatic cancer 25% Low ND (n = 60) Cholangiocarcinoma 31% Low
16% (n = 98) Bladder cancer 43% Low-Medium 25% (n = 60) Cervical
cancer 52% Low-Medium 27% (n = 60) Head and Neck 47% Low-Medium 23%
cancer (n = 92) Breast all 78% Medium-High 72% cancer (n = 232)
TNBC 89% Medium-High ND (n = 35) Ovarian cancer 82% Medium-High 68%
(n = 74) Uterine cancer 82% Medium-High 75% (n = 73) Esophageal
cancer 36% Low ND (n = 53) Kidney cancer 9% Negative ND (n = 83)
Prostate cancer 1% Negative ND (n = 57) B7H4 protein expression
determined by IHC analysis of BioMax TMAs. ND = not determined.
Sequence CWU 1
1
711282PRTHomo Sapiens 1Met Ala Ser Leu Gly Gln Ile Leu Phe Trp Ser
Ile Ile Ser Ile Ile1 5 10 15Ile Ile Leu Ala Gly Ala Ile Ala Leu Ile
Ile Gly Phe Gly Ile Ser 20 25 30Gly Arg His Ser Ile Thr Val Thr Thr
Val Ala Ser Ala Gly Asn Ile 35 40 45Gly Glu Asp Gly Ile Leu Ser Cys
Thr Phe Glu Pro Asp Ile Lys Leu 50 55 60Ser Asp Ile Val Ile Gln Trp
Leu Lys Glu Gly Val Leu Gly Leu Val65 70 75 80His Glu Phe Lys Glu
Gly Lys Asp Glu Leu Ser Glu Gln Asp Glu Met 85 90 95Phe Arg Gly Arg
Thr Ala Val Phe Ala Asp Gln Val Ile Val Gly Asn 100 105 110Ala Ser
Leu Arg Leu Lys Asn Val Gln Leu Thr Asp Ala Gly Thr Tyr 115 120
125Lys Cys Tyr Ile Ile Thr Ser Lys Gly Lys Gly Asn Ala Asn Leu Glu
130 135 140Tyr Lys Thr Gly Ala Phe Ser Met Pro Glu Val Asn Val Asp
Tyr Asn145 150 155 160Ala Ser Ser Glu Thr Leu Arg Cys Glu Ala Pro
Arg Trp Phe Pro Gln 165 170 175Pro Thr Val Val Trp Ala Ser Gln Val
Asp Gln Gly Ala Asn Phe Ser 180 185 190Glu Val Ser Asn Thr Ser Phe
Glu Leu Asn Ser Glu Asn Val Thr Met 195 200 205Lys Val Val Ser Val
Leu Tyr Asn Val Thr Ile Asn Asn Thr Tyr Ser 210 215 220Cys Met Ile
Glu Asn Asp Ile Ala Lys Ala Thr Gly Asp Ile Lys Val225 230 235
240Thr Glu Ser Glu Ile Lys Arg Arg Ser His Leu Gln Leu Leu Asn Ser
245 250 255Lys Ala Ser Leu Cys Val Ser Ser Phe Phe Ala Ile Ser Trp
Ala Leu 260 265 270Leu Pro Leu Ser Pro Tyr Leu Met Leu Lys 275
2802282PRTMacaca fascicularis 2Met Ala Ser Leu Gly Gln Ile Leu Phe
Trp Ser Ile Ile Ser Ile Ile1 5 10 15Phe Ile Leu Ala Gly Ala Ile Ala
Leu Ile Ile Gly Phe Gly Ile Ser 20 25 30Gly Arg His Ser Ile Thr Val
Thr Thr Val Ala Ser Ala Gly Asn Ile 35 40 45Gly Glu Asp Gly Ile Leu
Ser Cys Thr Phe Glu Pro Asp Ile Lys Leu 50 55 60Ser Asp Ile Val Ile
Gln Trp Leu Lys Glu Gly Val Ile Gly Leu Val65 70 75 80His Glu Phe
Lys Glu Gly Lys Asp Glu Leu Ser Glu Gln Asp Glu Met 85 90 95Phe Arg
Gly Arg Thr Ala Val Phe Ala Asp Gln Val Ile Val Gly Asn 100 105
110Ala Ser Leu Arg Leu Lys Asn Val Gln Leu Thr Asp Ala Gly Thr Tyr
115 120 125Lys Cys Tyr Ile Ile Thr Ser Lys Gly Lys Gly Asn Ala Asn
Leu Glu 130 135 140Tyr Lys Thr Gly Ala Phe Ser Met Pro Glu Val Asn
Val Asp Tyr Asn145 150 155 160Ala Ser Ser Glu Thr Leu Arg Cys Glu
Ala Pro Arg Trp Phe Pro Gln 165 170 175Pro Thr Val Val Trp Ala Ser
Gln Val Asp Gln Gly Ala Asn Phe Ser 180 185 190Glu Val Ser Asn Thr
Ser Phe Glu Leu Asn Ser Glu Asn Val Thr Met 195 200 205Lys Val Val
Ser Val Leu Tyr Asn Val Thr Ile Asn Asn Thr Tyr Ser 210 215 220Cys
Met Ile Glu Asn Asp Ile Ala Lys Ala Thr Gly Asp Ile Lys Val225 230
235 240Thr Glu Ser Glu Ile Lys Arg Arg Ser His Leu Gln Leu Leu Asn
Ser 245 250 255Lys Ala Ser Leu Cys Val Ser Ser Phe Leu Ala Ile Ser
Trp Ala Leu 260 265 270Leu Pro Leu Ala Pro Tyr Leu Met Leu Lys 275
2803282PRTCanis familiaris 3Met Ala Ser Pro Gly Gln Asn Ile Phe Trp
Ser Ile Ile Ser Val Ile1 5 10 15Ile Ile Leu Ala Gly Ala Ile Ala Leu
Ile Ile Gly Phe Gly Ile Ser 20 25 30Gly Arg His Ser Ile Thr Val Thr
Thr Leu Thr Ser Ala Gly Asn Ile 35 40 45Gly Glu Asp Gly Ile Leu Ser
Cys Thr Phe Glu Pro Asp Ile Lys Leu 50 55 60Ser Asp Ile Val Ile Gln
Trp Leu Lys Glu Gly Val Met Gly Leu Val65 70 75 80His Glu Phe Lys
Glu Gly Lys Asp Asp Leu Ser Asp Gln Asp Glu Met 85 90 95Phe Arg Gly
Arg Thr Ala Val Phe Ala Asp Gln Val Ile Gly Gly Asn 100 105 110Ala
Ser Leu Arg Leu Lys Asn Val Gln Leu Thr Asp Ala Gly Thr Tyr 115 120
125Lys Cys Tyr Ile Ile Thr Ser Lys Gly Lys Gly Asn Ala Asn Leu Glu
130 135 140Tyr Lys Thr Gly Ala Phe Ser Ile Pro Glu Val Asn Val Asp
Tyr Asn145 150 155 160Ala Ser Ser Glu Asn Leu Arg Cys Glu Ala Pro
Arg Trp Phe Pro Gln 165 170 175Pro Thr Val Val Trp Ala Ser Gln Ala
Asp Gln Gly Ala Asn Phe Ser 180 185 190Glu Val Phe Asn Thr Ser Phe
Glu Leu Asn Ser Glu Asn Val Thr Met 195 200 205Lys Val Val Ser Val
Leu Tyr Asn Val Thr Ile Asn Asn Thr Tyr Ser 210 215 220Cys Met Ile
Glu Asn Asp Ile Ala Lys Ala Thr Gly Asp Ile Lys Val225 230 235
240Thr Asp Ser Glu Ile Lys Arg Arg Ser His Leu Gln Leu Leu Asn Ser
245 250 255Lys Ala Ser Leu Gly Val Ser Ser Phe Phe Ala Ile Ser Trp
Val Leu 260 265 270Leu Pro Leu Ser Ser Tyr Leu Met Leu Lys 275
2804282PRTOryctolagus cuniculus 4Met Ala Ser Leu Gly Gln Ile Ile
Phe Trp Ser Ile Ile Ser Ile Ile1 5 10 15Ile Ile Leu Ala Gly Ala Ile
Ala Leu Ile Ile Gly Phe Gly Ile Ser 20 25 30Gly Arg His Ser Ile Thr
Val Thr Thr Leu Thr Ser Ala Gly Asn Ile 35 40 45Gly Glu Asp Gly Ile
Leu Ser Cys Thr Phe Glu Pro Asp Ile Arg Leu 50 55 60Ser Asp Ile Val
Ile Gln Trp Leu Lys Glu Gly Val Val Gly Leu Val65 70 75 80His Glu
Phe Lys Glu Gly Lys Asp Asp Leu Ser Asp Gln Asp Glu Met 85 90 95Phe
Arg Gly Arg Thr Ala Val Phe Thr Asp Gln Val Ile Val Gly Asn 100 105
110Ala Ser Leu Arg Leu Lys Asn Val Gln Leu Thr Asp Ala Gly Thr Tyr
115 120 125Lys Cys Tyr Ile Ile Thr Ser Lys Gly Lys Gly Asn Ala Asn
Leu Glu 130 135 140Tyr Lys Thr Gly Ala Phe Ser Met Pro Glu Val Asn
Leu Asp Tyr Asn145 150 155 160Ala Ser Ser Glu Ser Leu Arg Cys Glu
Ala Pro Arg Trp Phe Pro Gln 165 170 175Pro Thr Val Val Trp Ala Ser
Gln Val Asp Gln Gly Ala Asn Phe Ser 180 185 190Glu Val Ser Asn Thr
Ser Phe Glu Leu Asn Ser Glu Asn Val Thr Met 195 200 205Lys Val Val
Ser Val Leu Tyr Asn Val Thr Val Asn Asn Thr Tyr Ser 210 215 220Cys
Met Ile Glu Asn Asp Ile Ala Lys Ala Thr Gly Asp Ile Lys Val225 230
235 240Thr Asp Ser Glu Ile Lys Arg Arg Ser Ser Leu Gln Leu Leu Asn
Ser 245 250 255Arg Ala Ala Pro Ser Val Ser Pro Arg Ser Ala Val Gly
Trp Leu Leu 260 265 270Leu Pro Leu Ser Ser Tyr Val Met Leu Lys 275
2805282PRTRattus norvegicus 5Met Ala Ser Leu Gly Gln Ile Ile Phe
Trp Ser Ile Ile Asn Val Ile1 5 10 15Ile Ile Leu Ala Gly Ala Ile Val
Leu Ile Ile Gly Phe Gly Ile Ser 20 25 30Gly Lys His Phe Ile Thr Val
Thr Thr Phe Thr Ser Ala Gly Asn Ile 35 40 45Gly Glu Asp Gly Thr Leu
Ser Cys Thr Phe Glu Pro Asp Ile Lys Leu 50 55 60Asn Gly Ile Val Ile
Gln Trp Leu Lys Glu Gly Ile Lys Gly Leu Val65 70 75 80His Glu Phe
Lys Glu Gly Lys Asp Asp Leu Ser Gln Gln His Glu Met 85 90 95Phe Arg
Gly Arg Thr Ala Val Phe Ala Asp Gln Val Val Val Gly Asn 100 105
110Ala Ser Leu Arg Leu Lys Asn Val Gln Leu Thr Asp Ala Gly Thr Tyr
115 120 125Thr Cys Tyr Ile His Thr Ser Lys Gly Lys Gly Asn Ala Asn
Leu Glu 130 135 140Tyr Lys Thr Gly Ala Phe Ser Met Pro Glu Ile Asn
Val Asp Tyr Asn145 150 155 160Ala Ser Ser Glu Ser Leu Arg Cys Glu
Ala Pro Arg Trp Phe Pro Gln 165 170 175Pro Thr Val Ala Trp Ala Ser
Gln Val Asp Gln Gly Ala Asn Phe Ser 180 185 190Glu Val Ser Asn Thr
Ser Phe Glu Leu Asn Ser Glu Asn Val Thr Met 195 200 205Lys Val Val
Ser Val Leu Tyr Asn Val Thr Ile Asn Asn Thr Tyr Ser 210 215 220Cys
Met Ile Glu Asn Asp Ile Ala Lys Ala Thr Gly Asp Ile Lys Val225 230
235 240Thr Asp Ser Glu Val Lys Arg Arg Ser Gln Leu Glu Leu Leu Asn
Ser 245 250 255Gly Pro Ser Pro Cys Val Ser Ser Val Ser Ala Ala Gly
Trp Ala Leu 260 265 270Leu Ser Leu Ser Cys Cys Leu Met Leu Arg 275
2806283PRTMus musculus 6Met Ala Ser Leu Gly Gln Ile Ile Phe Trp Ser
Ile Ile Asn Ile Ile1 5 10 15Ile Ile Leu Ala Gly Ala Ile Ala Leu Ile
Ile Gly Phe Gly Ile Ser 20 25 30Gly Lys His Phe Ile Thr Val Thr Thr
Phe Thr Ser Ala Gly Asn Ile 35 40 45Gly Glu Asp Gly Thr Leu Ser Cys
Thr Phe Glu Pro Asp Ile Lys Leu 50 55 60Asn Gly Ile Val Ile Gln Trp
Leu Lys Glu Gly Ile Lys Gly Leu Val65 70 75 80His Glu Phe Lys Glu
Gly Lys Asp Asp Leu Ser Gln Gln His Glu Met 85 90 95Phe Arg Gly Arg
Thr Ala Val Phe Ala Asp Gln Val Val Val Gly Asn 100 105 110Ala Ser
Leu Arg Leu Lys Asn Val Gln Leu Thr Asp Ala Gly Thr Tyr 115 120
125Thr Cys Tyr Ile Arg Thr Ser Lys Gly Lys Gly Asn Ala Asn Leu Glu
130 135 140Tyr Lys Thr Gly Ala Phe Ser Met Pro Glu Ile Asn Val Asp
Tyr Asn145 150 155 160Ala Ser Ser Glu Ser Leu Arg Cys Glu Ala Pro
Arg Trp Phe Pro Gln 165 170 175Pro Thr Val Ala Trp Ala Ser Gln Val
Asp Gln Gly Ala Asn Phe Ser 180 185 190Glu Val Ser Asn Thr Ser Phe
Glu Leu Asn Ser Glu Asn Val Thr Met 195 200 205Lys Val Val Ser Val
Leu Tyr Asn Val Thr Ile Asn Asn Thr Tyr Ser 210 215 220Cys Met Ile
Glu Asn Asp Ile Ala Lys Ala Thr Gly Asp Ile Lys Val225 230 235
240Thr Asp Ser Glu Val Lys Arg Arg Ser Gln Leu Gln Leu Leu Asn Ser
245 250 255Gly Pro Ser Pro Cys Val Phe Ser Ser Ala Phe Val Ala Gly
Trp Ala 260 265 270Leu Leu Ser Leu Ser Cys Cys Leu Met Leu Arg 275
2807282PRTSus scrofa 7Met Ala Ser Leu Gly Gln Val Val Phe Trp Ser
Ile Ile Ser Ile Ile1 5 10 15Ile Ile Leu Ala Gly Ala Ile Ala Phe Ile
Ile Gly Phe Gly Ile Ser 20 25 30Gly Arg His Ser Ile Thr Val Thr Thr
Leu Thr Ser Ala Gly Asn Ile 35 40 45Gly Glu Asp Gly Ile Leu Ser Cys
Thr Phe Glu Pro Asp Ile Lys Leu 50 55 60Ser Asp Ile Val Ile Gln Trp
Leu Lys Glu Gly Val Thr Gly Leu Val65 70 75 80His Glu Phe Lys Lys
Gly Lys Asp Asp Leu Ser Asp Gln Asp Glu Met 85 90 95Phe Arg Gly Arg
Thr Ala Val Phe Ala Asp Gln Val Ile Val Gly Asn 100 105 110Ala Ser
Leu Arg Leu Lys Asn Val Gln Leu Thr Asp Ala Gly Thr Tyr 115 120
125Lys Cys Tyr Ile Ile Thr Ser Lys Gly Lys Gly Asn Ala Lys Leu Glu
130 135 140Tyr Lys Thr Gly Ala Phe Ser Ile Pro Glu Val Asn Val Asp
Ser Asn145 150 155 160Ala Ser Ser Glu Ser Leu Arg Cys Glu Ala Pro
Arg Trp Phe Pro Gln 165 170 175Pro Thr Val Val Trp Ala Ser Gln Val
Asp Gln Gly Ala Asn Phe Ser 180 185 190Glu Val Ser Asn Thr Ser Phe
Glu Leu Asn Pro Glu Asn Val Thr Met 195 200 205Lys Val Val Ser Val
Leu Tyr Asn Val Thr Ile Asn Thr Thr Tyr Ser 210 215 220Cys Met Ile
Glu Asn Asp Ile Ala Lys Ala Thr Gly Asp Ile Arg Val225 230 235
240Thr Asp Ser Glu Ile Lys Arg Gln Ser His Leu Gln Leu Leu Asn Ser
245 250 255Lys Ala Ser Leu Cys Leu Ser Ser Phe Val Ala Ile Ser Trp
Val Leu 260 265 270Leu Pro Leu Cys Pro Tyr Leu Met Leu Lys 275
28089PRTArtificial sequenceNucleic acid motif for translation 8Gly
Cys Cys Gly Cys Cys Ala Cys Cys1 59534PRTHomo Sapiens 9Met Leu Arg
Arg Arg Gly Ser Pro Gly Met Gly Val His Val Gly Ala1 5 10 15Ala Leu
Gly Ala Leu Trp Phe Cys Leu Thr Gly Ala Leu Glu Val Gln 20 25 30Val
Pro Glu Asp Pro Val Val Ala Leu Val Gly Thr Asp Ala Thr Leu 35 40
45Cys Cys Ser Phe Ser Pro Glu Pro Gly Phe Ser Leu Ala Gln Leu Asn
50 55 60Leu Ile Trp Gln Leu Thr Asp Thr Lys Gln Leu Val His Ser Phe
Ala65 70 75 80Glu Gly Gln Asp Gln Gly Ser Ala Tyr Ala Asn Arg Thr
Ala Leu Phe 85 90 95Pro Asp Leu Leu Ala Gln Gly Asn Ala Ser Leu Arg
Leu Gln Arg Val 100 105 110Arg Val Ala Asp Glu Gly Ser Phe Thr Cys
Phe Val Ser Ile Arg Asp 115 120 125Phe Gly Ser Ala Ala Val Ser Leu
Gln Val Ala Ala Pro Tyr Ser Lys 130 135 140Pro Ser Met Thr Leu Glu
Pro Asn Lys Asp Leu Arg Pro Gly Asp Thr145 150 155 160Val Thr Ile
Thr Cys Ser Ser Tyr Gln Gly Tyr Pro Glu Ala Glu Val 165 170 175Phe
Trp Gln Asp Gly Gln Gly Val Pro Leu Thr Gly Asn Val Thr Thr 180 185
190Ser Gln Met Ala Asn Glu Gln Gly Leu Phe Asp Val His Ser Ile Leu
195 200 205Arg Val Val Leu Gly Ala Asn Gly Thr Tyr Ser Cys Leu Val
Arg Asn 210 215 220Pro Val Leu Gln Gln Asp Ala His Ser Ser Val Thr
Ile Thr Pro Gln225 230 235 240Arg Ser Pro Thr Gly Ala Val Glu Val
Gln Val Pro Glu Asp Pro Val 245 250 255Val Ala Leu Val Gly Thr Asp
Ala Thr Leu Arg Cys Ser Phe Ser Pro 260 265 270Glu Pro Gly Phe Ser
Leu Ala Gln Leu Asn Leu Ile Trp Gln Leu Thr 275 280 285Asp Thr Lys
Gln Leu Val His Ser Phe Thr Glu Gly Arg Asp Gln Gly 290 295 300Ser
Ala Tyr Ala Asn Arg Thr Ala Leu Phe Pro Asp Leu Leu Ala Gln305 310
315 320Gly Asn Ala Ser Leu Arg Leu Gln Arg Val Arg Val Ala Asp Glu
Gly 325 330 335Ser Phe Thr Cys Phe Val Ser Ile Arg Asp Phe Gly Ser
Ala Ala Val 340 345 350Ser Leu Gln Val Ala Ala Pro Tyr Ser Lys Pro
Ser Met Thr Leu Glu 355 360 365Pro Asn Lys Asp Leu Arg Pro Gly Asp
Thr Val Thr Ile Thr Cys Ser 370 375 380Ser Tyr Arg Gly Tyr Pro Glu
Ala Glu Val Phe Trp Gln Asp Gly Gln385 390 395 400Gly Val Pro Leu
Thr Gly Asn Val Thr Thr Ser Gln Met Ala Asn Glu 405 410 415Gln Gly
Leu Phe Asp Val His Ser Val Leu Arg Val Val Leu Gly Ala 420 425
430Asn Gly Thr Tyr Ser Cys Leu Val Arg Asn Pro Val Leu Gln Gln Asp
435 440 445Ala His Gly Ser Val Thr Ile Thr Gly Gln Pro Met Thr Phe
Pro Pro 450 455 460Glu Ala Leu Trp Val
Thr Val Gly Leu Ser Val Cys Leu Ile Ala Leu465 470 475 480Leu Val
Ala Leu Ala Phe Val Cys Trp Arg Lys Ile Lys Gln Ser Cys 485 490
495Glu Glu Glu Asn Ala Gly Ala Glu Asp Gln Asp Gly Glu Gly Glu Gly
500 505 510Ser Lys Thr Ala Leu Gln Pro Leu Lys His Ser Asp Ser Lys
Glu Asp 515 520 525Asp Gly Gln Glu Ile Ala 53010325PRTArtificial
sequenceDomain swap 10Met Ala Ser Leu Gly Gln Ile Leu Phe Trp Ser
Ile Ile Ser Ile Ile1 5 10 15Ile Ile Leu Ala Gly Ala Ile Ala Leu Ile
Ile Gly Phe Gly Ile Ser 20 25 30Gly Arg His Ser Ile Thr Val Thr Thr
Val Ala Ser Ala Gly Asn Ile 35 40 45Gly Glu Asp Gly Ile Leu Ser Cys
Thr Phe Glu Pro Asp Ile Lys Leu 50 55 60Ser Asp Ile Val Ile Gln Trp
Leu Lys Glu Gly Val Leu Gly Leu Val65 70 75 80His Glu Phe Lys Glu
Gly Lys Asp Glu Leu Ser Glu Gln Asp Glu Met 85 90 95Phe Arg Gly Arg
Thr Ala Val Phe Ala Asp Gln Val Ile Val Gly Asn 100 105 110Ala Ser
Leu Arg Leu Lys Asn Val Gln Leu Thr Asp Ala Gly Thr Tyr 115 120
125Lys Cys Tyr Ile Ile Thr Ser Lys Gly Lys Gly Asn Ala Asn Leu Glu
130 135 140Tyr Lys Thr Gly Ala Pro Tyr Ser Lys Pro Ser Met Thr Leu
Glu Pro145 150 155 160Asn Lys Asp Leu Arg Pro Gly Asp Thr Val Thr
Ile Thr Cys Ser Ser 165 170 175Tyr Arg Gly Tyr Pro Glu Ala Glu Val
Phe Trp Gln Asp Gly Gln Gly 180 185 190Val Pro Leu Thr Gly Asn Val
Thr Thr Ser Gln Met Ala Asn Glu Gln 195 200 205Gly Leu Phe Asp Val
His Ser Val Leu Arg Val Val Leu Gly Ala Asn 210 215 220Gly Thr Tyr
Ser Cys Leu Val Arg Asn Pro Val Leu Gln Gln Asp Ala225 230 235
240His Gly Ser Val Thr Ile Thr Gly Gln Pro Met Thr Phe Pro Pro Glu
245 250 255Ala Leu Trp Val Thr Val Gly Leu Ser Val Cys Leu Ile Ala
Leu Leu 260 265 270Val Ala Leu Ala Phe Val Cys Trp Arg Lys Ile Lys
Gln Ser Cys Glu 275 280 285Glu Glu Asn Ala Gly Ala Glu Asp Gln Asp
Gly Glu Gly Glu Gly Ser 290 295 300Lys Thr Ala Leu Gln Pro Leu Lys
His Ser Asp Ser Lys Glu Asp Asp305 310 315 320Gly Gln Glu Ile Ala
32511273PRTArtificial sequenceDomain swap 11Met Leu Arg Arg Arg Gly
Ser Pro Gly Met Gly Val His Val Gly Ala1 5 10 15Ala Leu Gly Ala Leu
Trp Phe Cys Leu Thr Gly Ala Leu Glu Val Gln 20 25 30Val Pro Glu Asp
Pro Val Val Ala Leu Val Gly Thr Asp Ala Thr Leu 35 40 45Cys Cys Ser
Phe Ser Pro Glu Pro Gly Phe Ser Leu Ala Gln Leu Asn 50 55 60Leu Ile
Trp Gln Leu Thr Asp Thr Lys Gln Leu Val His Ser Phe Ala65 70 75
80Glu Gly Gln Asp Gln Gly Ser Ala Tyr Ala Asn Arg Thr Ala Leu Phe
85 90 95Pro Asp Leu Leu Ala Gln Gly Asn Ala Ser Leu Arg Leu Gln Arg
Val 100 105 110Arg Val Ala Asp Glu Gly Ser Phe Thr Cys Phe Val Ser
Ile Arg Asp 115 120 125Phe Gly Ser Ala Ala Val Ser Leu Gln Val Ala
Ala Phe Ser Met Pro 130 135 140Glu Val Asn Val Asp Tyr Asn Ala Ser
Ser Glu Thr Leu Arg Cys Glu145 150 155 160Ala Pro Arg Trp Phe Pro
Gln Pro Thr Val Val Trp Ala Ser Gln Val 165 170 175Asp Gln Gly Ala
Asn Phe Ser Glu Val Ser Asn Thr Ser Phe Glu Leu 180 185 190Asn Ser
Glu Asn Val Thr Met Lys Val Val Ser Val Leu Tyr Asn Val 195 200
205Thr Ile Asn Asn Thr Tyr Ser Cys Met Ile Glu Asn Asp Ile Ala Lys
210 215 220Ala Thr Gly Asp Ile Lys Val Thr Glu Ser Glu Ile Lys Arg
Arg Ser225 230 235 240His Leu Gln Leu Leu Asn Ser Lys Ala Ser Leu
Cys Val Ser Ser Phe 245 250 255Phe Ala Ile Ser Trp Ala Leu Leu Pro
Leu Ser Pro Tyr Leu Met Leu 260 265 270Lys12484PRTArtificial
sequenceTagged protein 12Leu Ile Ile Gly Phe Gly Ile Ser Gly Arg
His Ser Ile Thr Val Thr1 5 10 15Thr Val Ala Ser Ala Gly Asn Ile Gly
Glu Asp Gly Ile Leu Ser Cys 20 25 30Thr Phe Glu Pro Asp Ile Lys Leu
Ser Asp Ile Val Ile Gln Trp Leu 35 40 45Lys Glu Gly Val Leu Gly Leu
Val His Glu Phe Lys Glu Gly Lys Asp 50 55 60Glu Leu Ser Glu Gln Asp
Glu Met Phe Arg Gly Arg Thr Ala Val Phe65 70 75 80Ala Asp Gln Val
Ile Val Gly Asn Ala Ser Leu Arg Leu Lys Asn Val 85 90 95Gln Leu Thr
Asp Ala Gly Thr Tyr Lys Cys Tyr Ile Ile Thr Ser Lys 100 105 110Gly
Lys Gly Asn Ala Asn Leu Glu Tyr Lys Thr Gly Ala Phe Ser Met 115 120
125Pro Glu Val Asn Val Asp Tyr Asn Ala Ser Ser Glu Thr Leu Arg Cys
130 135 140Glu Ala Pro Arg Trp Phe Pro Gln Pro Thr Val Val Trp Ala
Ser Gln145 150 155 160Val Asp Gln Gly Ala Asn Phe Ser Glu Val Ser
Asn Thr Ser Phe Glu 165 170 175Leu Asn Ser Glu Asn Val Thr Met Lys
Val Val Ser Val Leu Tyr Asn 180 185 190Val Thr Ile Asn Asn Thr Tyr
Ser Cys Met Ile Glu Asn Asp Ile Ala 195 200 205Lys Ala Thr Gly Asp
Ile Lys Val Thr Glu Ser Glu Ile Lys Arg Arg 210 215 220Ser His Leu
Gln Leu Leu Asn Ser Lys Ala Ser Ile Glu Gly Arg Met225 230 235
240Asp Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala
245 250 255Pro Glu Ala Glu Gly Ala Pro Ser Val Phe Leu Phe Pro Pro
Lys Pro 260 265 270Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys Val Val 275 280 285Val Asp Val Ser His Glu Asp Pro Glu Val
Lys Phe Asn Trp Tyr Val 290 295 300Asp Gly Val Glu Val His Asn Ala
Lys Thr Lys Pro Arg Glu Glu Gln305 310 315 320Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu His Gln 325 330 335Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala 340 345 350Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro 355 360
365Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr
370 375 380Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr
Pro Ser385 390 395 400Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn Asn Tyr 405 410 415Lys Thr Ala Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe Leu Tyr 420 425 430Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn Val Phe 435 440 445Ser Cys Ser Val Met
His Glu Ala Leu His Asn His Tyr Thr Gln Lys 450 455 460Ser Leu Ser
Leu Ser Pro Gly Lys His His His His His His His His465 470 475
480Glu Pro Glu Ala13186PRTHomo Sapiens 13Gln Asp Gly Asn Glu Glu
Met Gly Gly Ile Thr Gln Thr Pro Tyr Lys1 5 10 15Val Ser Ile Ser Gly
Thr Thr Val Ile Leu Thr Cys Pro Gln Tyr Pro 20 25 30Gly Ser Glu Ile
Leu Trp Gln His Asn Asp Lys Asn Ile Gly Gly Asp 35 40 45Glu Asp Asp
Lys Asn Ile Gly Ser Asp Glu Asp His Leu Ser Leu Lys 50 55 60Glu Phe
Ser Glu Leu Glu Gln Ser Gly Tyr Tyr Val Cys Tyr Pro Arg65 70 75
80Gly Ser Lys Pro Glu Asp Ala Asn Phe Tyr Leu Tyr Leu Arg Ala Arg
85 90 95Val Cys Glu Asn Cys Met Glu Met Asp Val Met Ser Val Ala Thr
Ile 100 105 110Val Ile Val Asp Ile Cys Ile Thr Gly Gly Leu Leu Leu
Leu Val Tyr 115 120 125Tyr Trp Ser Lys Asn Arg Lys Ala Lys Ala Lys
Pro Val Thr Arg Gly 130 135 140Ala Gly Ala Gly Gly Arg Gln Arg Gly
Gln Asn Lys Glu Arg Pro Pro145 150 155 160Pro Val Pro Asn Pro Asp
Tyr Glu Pro Ile Arg Lys Gly Gln Arg Asp 165 170 175Leu Tyr Ser Gly
Leu Asn Gln Arg Arg Ile 180 18514127PRTArtificial sequencebinding
domain sequence 14Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys
Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Gln Ala Ser Gly Tyr
Arg Phe Ser Asn Phe 20 25 30Val Ile His Trp Val Arg Gln Ala Pro Gly
Gln Arg Phe Glu Trp Met 35 40 45Gly Trp Ile Asn Pro Tyr Asn Gly Asn
Lys Glu Phe Ser Ala Lys Phe 50 55 60Gln Asp Arg Val Thr Phe Thr Ala
Asp Thr Ser Ala Asn Thr Ala Tyr65 70 75 80Met Glu Leu Arg Ser Leu
Arg Ser Ala Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Arg Val Gly Pro
Tyr Ser Trp Asp Asp Ser Pro Gln Asp Asn Tyr 100 105 110Tyr Met Asp
Val Trp Gly Lys Gly Thr Thr Val Ile Val Ser Ser 115 120
12515108PRTArtificial sequencebinding domain sequence 15Glu Ile Val
Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly1 5 10 15Glu Arg
Ala Thr Phe Ser Cys Arg Ser Ser His Ser Ile Arg Ser Arg 20 25 30Arg
Val Ala Trp Tyr Gln His Lys Pro Gly Gln Ala Pro Arg Leu Val 35 40
45Ile His Gly Val Ser Asn Arg Ala Ser Gly Ile Ser Asp Arg Phe Ser
50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Thr Arg Val
Glu65 70 75 80Pro Glu Asp Phe Ala Leu Tyr Tyr Cys Gln Val Tyr Gly
Ala Ser Ser 85 90 95Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Arg Lys
100 10516125PRTArtificial sequencebinding domain sequence 16Glu Val
Lys Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25
30Ala Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45Ala Arg Ile Arg Ser Lys Tyr Asn Asn Tyr Ala Thr Tyr Tyr Ala
Asp 50 55 60Ser Val Lys Asp Arg Phe Thr Ile Ser Arg Asp Asp Ser Lys
Ser Ser65 70 75 80Leu Tyr Leu Gln Met Asn Asn Leu Lys Thr Glu Asp
Thr Ala Met Tyr 85 90 95Tyr Cys Val Arg His Gly Asn Phe Gly Asn Ser
Tyr Val Ser Trp Phe 100 105 110Ala Tyr Trp Gly Gln Gly Thr Leu Val
Thr Val Ser Ser 115 120 12517125PRTArtificial sequencebinding
domain sequence 17Glu Val Lys Leu Val Glu Ser Gly Gly Gly Leu Val
Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
Thr Phe Asn Thr Tyr 20 25 30Ala Met Asn Trp Val Arg Gln Ala Pro Gly
Lys Gly Leu Glu Trp Val 35 40 45Ala Arg Ile Arg Ser Lys Tyr Asn Asn
Tyr Ala Thr Tyr Tyr Ala Asp 50 55 60Ser Val Lys Asp Arg Phe Thr Ile
Ser Arg Asp Asp Ser Lys Ser Ser65 70 75 80Leu Tyr Leu Gln Met Asn
Asn Leu Lys Thr Glu Asp Thr Ala Met Tyr 85 90 95Tyr Cys Val Arg Gly
Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe 100 105 110Ala Tyr Trp
Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125188PRTArtificial
sequencebinding domain sequence 18Gly Phe Thr Phe Asn Thr Tyr Ala1
51910PRTArtificial sequencebinding domain sequence 19Ile Arg Ser
Lys Tyr Asn Asn Tyr Ala Thr1 5 102016PRTArtificial sequencebinding
domain sequence 20Val Arg His Gly Asn Phe Gly Asn Ser Tyr Val Ser
Trp Phe Ala Tyr1 5 10 152116PRTArtificial sequencebinding domain
sequence 21Val Arg Gly Gly Asn Phe Gly Asn Ser Tyr Val Ser Trp Phe
Ala Tyr1 5 10 1522109PRTArtificial sequencebinding domain sequence
22Gln Ala Val Val Thr Gln Glu Pro Ser Phe Ser Val Ser Pro Gly Gly1
5 10 15Thr Val Thr Leu Thr Cys Arg Ser Ser Thr Gly Ala Val Thr Thr
Ser 20 25 30Asn Tyr Ala Asn Trp Val Gln Gln Thr Pro Gly Gln Ala Phe
Arg Gly 35 40 45Leu Ile Gly Gly Thr Asn Lys Arg Ala Pro Gly Val Pro
Ala Arg Phe 50 55 60Ser Gly Ser Leu Ile Gly Asp Lys Ala Ala Leu Thr
Ile Thr Gly Ala65 70 75 80Gln Ala Asp Asp Glu Ser Ile Tyr Phe Cys
Ala Leu Trp Tyr Ser Asn 85 90 95Leu Trp Val Phe Gly Gly Gly Thr Lys
Leu Thr Val Leu 100 105239PRTArtificial sequencebinding domain
sequence 23Thr Gly Ala Val Thr Thr Ser Asn Tyr1 5249PRTArtificial
sequencebinding domain sequence 24Ala Leu Trp Tyr Ser Asn Leu Trp
Val1 525117PRTArtificial sequencebinding domain sequence 25Gln Val
Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu1 5 10 15Thr
Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly Tyr 20 25
30Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu Trp Ile
35 40 45Gly Glu Ile Asn His Ser Gly Ser Thr Asn Tyr Asn Pro Ser Leu
Lys 50 55 60Ser Arg Val Thr Ile Ser Ile Asp Thr Ser Lys Asn Gln Phe
Ser Leu65 70 75 80Lys Leu Thr Ser Val Thr Ala Ala Asp Thr Ala Val
Phe Tyr Cys Ala 85 90 95Arg Gly Leu Phe Asn Trp Asn Phe Asp Ser Trp
Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser
115268PRTArtificial sequencebinding domain sequence 26Gly Gly Ser
Phe Ser Gly Tyr Tyr1 5277PRTArtificial sequencebinding domain
sequence 27Ile Asn His Ser Gly Ser Thr1 52811PRTArtificial
sequencebinding domain sequence 28Ala Arg Gly Leu Phe Asn Trp Asn
Phe Asp Ser1 5 1029117PRTArtificial sequencebinding domain sequence
29Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro Ser Glu1
5 10 15Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe Ser Gly
Tyr 20 25 30Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu
Trp Ile 35 40 45Gly Glu Ile Ser His Ser Gly Ser Thr Asn Tyr Asn Pro
Ser Leu Lys 50 55 60Ser Arg Val Thr Ile Ser Ile Asp Thr Ser Lys Asn
Gln Phe Ser Leu65 70 75 80Lys Leu Thr Ser Val Thr Ala Ala Asp Thr
Ala Val Phe Tyr Cys Ala 85 90 95Arg Gly Leu Phe Asn Trp Asn Phe Asp
Ser Trp Gly Gln Gly Thr Leu 100 105 110Val Thr Val Ser Ser
115307PRTArtificial sequencebinding domain sequence 30Ile Ser His
Ser Gly Ser Thr1 531117PRTArtificial sequencebinding domain
sequence 31Gln Val Gln Leu Gln Gln Trp Gly Ala Gly Leu Leu Lys Pro
Ser Glu1 5 10 15Thr Leu Ser Leu Thr Cys Ala Val Tyr Gly Gly Ser Phe
Ser Gly Tyr 20 25 30Tyr Trp Ser Trp Ile Arg Gln Pro Pro Gly Lys Gly
Leu Glu Trp Ile 35 40 45Gly Glu Ile Gln His Ser Gly Ser Thr Asn Tyr
Asn Pro Ser Leu Lys 50 55 60Ser Arg Val Thr Ile Ser Ile Asp Thr Ser
Lys Asn Gln Phe Ser Leu65 70 75
80Lys Leu Thr Ser Val Thr Ala Ala Asp Thr Ala Val Phe Tyr Cys Ala
85 90 95Arg Gly Leu Phe Asn Trp Asn Phe Asp Ser Trp Gly Gln Gly Thr
Leu 100 105 110Val Thr Val Ser Ser 115327PRTArtificial
sequencebinding domain sequence 32Ile Gln His Ser Gly Ser Thr1
533107PRTArtificial sequencebinding domain sequence 33Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg
Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Asp 20 25 30Leu
Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Leu Ile 35 40
45Tyr Gly Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln
Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Ser
Tyr Pro Arg 85 90 95Thr Phe Gly Gln Gly Thr Thr Val Glu Ile Lys 100
105346PRTArtificial sequencebinding domain sequence 34Gln Gly Ile
Arg Asn Asp1 5359PRTArtificial sequencebinding domain sequence
35Leu Gln His Asn Ser Tyr Pro Arg Thr1 536116PRTArtificial
sequencebinding domain sequence 36Glu Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Asn Phe 20 25 30Trp Ile His Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Glu Ile Asp Pro
Ser Asp Ser Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55 60Lys Gly Arg Val
Thr Ile Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80Leu Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Glu Ile Thr Thr Val Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105
110Thr Val Ser Ser 115378PRTArtificial sequencebinding domain
sequence 37Gly Tyr Thr Phe Thr Asn Phe Trp1 5388PRTArtificial
sequencebinding domain sequence 38Ile Asp Pro Ser Asp Ser Tyr Thr1
5399PRTArtificial sequencebinding domain sequence 39Ala Arg Glu Ile
Thr Thr Val Asp Tyr1 540106PRTArtificial sequencebinding domain
sequence 40Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser
Val Gly1 5 10 15Asp Arg Val Thr Ile Thr Cys Ser Ala Thr Ser Ser Ile
Ser Tyr Met 20 25 30His Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Gly Trp Ile Tyr 35 40 45Asp Thr Ser Lys Leu Ala His Gly Val Pro Ser
Arg Phe Ser Gly Ser 50 55 60Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile
Ser Ser Leu Gln Pro Glu65 70 75 80Asp Phe Ala Thr Tyr Tyr Cys His
Gln Arg Arg Ser Tyr Pro Phe Thr 85 90 95Phe Gly Gln Gly Thr Lys Val
Glu Ile Lys 100 105415PRTArtificial sequencebinding domain sequence
41Ser Ser Ile Ser Tyr1 5429PRTArtificial sequencebinding domain
sequence 42His Gln Arg Arg Ser Tyr Pro Phe Thr1 543121PRTArtificial
sequencebinding domain sequence 43Glu Val Gln Leu Val Gln Ser Gly
Ala Glu Val Lys Lys Pro Gly Ala1 5 10 15Ser Val Lys Val Ser Cys Lys
Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30Trp Ile Gly Trp Val Arg
Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45Gly Asp Ile Tyr Pro
Gly Gly Gly Tyr Thr Asn Tyr Asn Glu Lys Phe 50 55 60Lys Gly Arg Val
Thr Ile Thr Arg Asp Thr Ser Thr Ser Thr Ala Tyr65 70 75 80Leu Glu
Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala
Arg Leu Asp Gly Ser Ser Tyr Arg Gly Ala Met Asp Ser Trp Gly 100 105
110Gln Gly Thr Leu Val Thr Val Ser Ser 115 120448PRTArtificial
sequencebinding domain sequence 44Gly Tyr Thr Phe Thr Ser Tyr Trp1
5458PRTArtificial sequencebinding domain sequence 45Ile Tyr Pro Gly
Gly Gly Tyr Thr1 54614PRTArtificial sequencebinding domain sequence
46Ala Arg Leu Asp Gly Ser Ser Tyr Arg Gly Ala Met Asp Ser1 5
1047107PRTArtificial sequencebinding domain sequence 47Asp Ile Gln
Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly1 5 10 15Asp Arg
Val Thr Ile Thr Cys Lys Ala Ser Gln Gly Phe Asn Lys Tyr 20 25 30Val
Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40
45Tyr Tyr Thr Ser Thr Leu Gln Pro Gly Val Pro Ser Arg Phe Ser Gly
50 55 60Ser Gly Ser Gly Arg Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln
Pro65 70 75 80Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Tyr Gly Asn
Leu Leu Tyr 85 90 95Ala Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100
105486PRTArtificial sequencebinding domain sequence 48Gln Gly Phe
Asn Lys Tyr1 5499PRTArtificial sequencebinding domain sequence
49Leu Gln Tyr Gly Asn Leu Leu Tyr Ala1 550115PRTArtificial
sequencebinding domain sequence 50Glu Val Gln Leu Val Glu Ser Gly
Gly Gly Leu Ile Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Val Ser Ser Asn 20 25 30Tyr Met Asn Trp Val Arg
Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45Ser Val Ile Tyr Gly
Ser Gly Arg Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60Gly Arg Val Thr
Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu65 70 75 80Gln Met
Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Arg
Asp Thr Tyr Ala Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr 100 105
110Val Ser Ser 115518PRTArtificial sequencebinding domain sequence
51Gly Phe Thr Val Ser Ser Asn Tyr1 5527PRTArtificial
sequencebinding domain sequence 52Ile Tyr Gly Ser Gly Arg Thr1
5539PRTArtificial sequencebinding domain sequence 53Ala Arg Asp Thr
Tyr Ala Met Asp Val1 554109PRTArtificial sequencebinding domain
sequence 54Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser
Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val
Ser Ser Ser 20 25 30Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala
Pro Arg Leu Leu 35 40 45Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile
Pro Asp Arg Phe Ser 50 55 60Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Arg Leu Glu65 70 75 80Pro Glu Asp Phe Ala Val Tyr Tyr
Cys Gln Gln Tyr Gly Ser Ser Pro 85 90 95Met Tyr Thr Phe Gly Gln Gly
Thr Lys Leu Glu Ile Lys 100 105557PRTArtificial sequencebinding
domain sequence 55Gln Ser Val Ser Ser Ser Tyr1 55610PRTArtificial
sequencebinding domain sequence 56Gln Gln Tyr Gly Ser Ser Pro Met
Tyr Thr1 5 1057330PRTHomo Sapiens 57Ala Ser Thr Lys Gly Pro Ser Val
Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala
Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr
Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile
Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Arg
Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105
110Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro
115 120 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
Thr Cys 130 135 140Val Val Val Asp Val Ser His Glu Asp Pro Glu Val
Lys Phe Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu Val His Asn
Ala Lys Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr Asn Ser Thr Tyr
Arg Val Val Ser Val Leu Thr Val Leu 180 185 190His Gln Asp Trp Leu
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205Lys Ala Leu
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220Gln
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu225 230
235 240Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe
Tyr 245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln
Pro Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser
Asp Gly Ser Phe Phe 275 280 285Leu Tyr Ser Lys Leu Thr Val Asp Lys
Ser Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser Cys Ser Val Met
His Glu Ala Leu His Asn His Tyr Thr305 310 315 320Gln Lys Ser Leu
Ser Leu Ser Pro Gly Lys 325 33058330PRTArtificial sequenceConstant
region 58Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser
Ser Lys1 5 10 15Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val
Lys Asp Tyr 20 25 30Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser 35 40 45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser
Ser Gly Leu Tyr Ser 50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser
Ser Leu Gly Thr Gln Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys
Pro Ser Asn Thr Lys Val Asp Lys 85 90 95Arg Val Glu Pro Lys Ser Cys
Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110Pro Ala Pro Glu Leu
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125Lys Pro Lys
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140Val
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150
155 160Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu 165 170 175Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu 180 185 190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn 195 200 205Lys Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys Ala Lys Gly 210 215 220Gln Pro Arg Glu Pro Gln Val
Tyr Thr Leu Pro Pro Ser Arg Glu Glu225 230 235 240Met Thr Lys Asn
Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255Pro Ser
Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265
270Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Leu
275 280 285Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
Gly Asn 290 295 300Val Phe Ser Cys Ser Val Met His Glu Ala Leu His
Asn His Tyr Thr305 310 315 320Gln Lys Ser Leu Ser Leu Ser Pro Gly
Lys 325 33059330PRTArtificial sequenceConstant region 59Ala Ser Thr
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr
Ser Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe
Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40
45Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser
50 55 60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln
Thr65 70 75 80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys
Val Asp Lys 85 90 95Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr
Cys Pro Pro Cys 100 105 110Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser
Val Phe Leu Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr Leu Met Ile
Ser Arg Thr Pro Glu Val Thr Cys 130 135 140Val Val Val Ala Val Ser
His Glu Asp Pro Glu Val Lys Phe Asn Trp145 150 155 160Tyr Val Asp
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175Glu
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185
190His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn
195 200 205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
Lys Gly 210 215 220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro
Ser Arg Glu Glu225 230 235 240Met Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305 310
315 320Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325
33060330PRTArtificial sequenceConstant region 60Ala Ser Thr Lys Gly
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly
Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu
Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75
80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro
Cys 100 105 110Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys 130 135 140Val Val Val Ala Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200
205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Glu Glu225 230 235 240Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Leu 275 280 285Leu Tyr Ser Lys Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305 310 315
320Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325
33061330PRTArtificial sequenceConstant region 61Ala Ser Thr Lys
Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro
Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly
Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55
60Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65
70 75 80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp
Lys 85 90 95Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro
Pro Cys 100 105 110Pro Ala Pro Glu Phe Glu Gly Gly Pro Ser Val Phe
Leu Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg
Thr Pro Glu Val Thr Cys 130 135 140Val Val Val Ala Val Ser His Glu
Asp Pro Glu Val Lys Phe Asn Trp145 150 155 160Tyr Val Asp Gly Val
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190His
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200
205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Glu Glu225 230 235 240Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285Leu Tyr Ser Arg Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305 310 315
320Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325
33062330PRTArtificial sequenceConstant region 62Ala Ser Thr Lys Gly
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys1 5 10 15Ser Thr Ser Gly
Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30Phe Pro Glu
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45Gly Val
His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60Leu
Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr65 70 75
80Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys
85 90 95Arg Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro
Cys 100 105 110Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu
Phe Pro Pro 115 120 125Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr
Pro Glu Val Thr Cys 130 135 140Val Val Val Asp Val Ser His Glu Asp
Pro Glu Val Lys Phe Asn Trp145 150 155 160Tyr Val Asp Gly Val Glu
Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175Glu Gln Tyr Asn
Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190His Gln
Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200
205Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly
210 215 220Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
Glu Glu225 230 235 240Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu
Val Lys Gly Phe Tyr 245 250 255Pro Ser Asp Ile Ala Val Glu Trp Glu
Ser Asn Gly Gln Pro Glu Asn 260 265 270Asn Tyr Lys Thr Thr Pro Pro
Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285Leu Tyr Ser Arg Leu
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300Val Phe Ser
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr305 310 315
320Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 33063107PRTHomo
Sapiens 63Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser
Asp Glu1 5 10 15Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu
Asn Asn Phe 20 25 30Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp
Asn Ala Leu Gln 35 40 45Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln
Asp Ser Lys Asp Ser 50 55 60Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu
Ser Lys Ala Asp Tyr Glu65 70 75 80Lys His Lys Val Tyr Ala Cys Glu
Val Thr His Gln Gly Leu Ser Ser 85 90 95Pro Val Thr Lys Ser Phe Asn
Arg Gly Glu Cys 100 10564106PRTHomo Sapiens 64Gly Gln Pro Lys Ala
Ala Pro Ser Val Thr Leu Phe Pro Pro Ser Ser1 5 10 15Glu Glu Leu Gln
Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp 20 25 30Phe Tyr Pro
Gly Ala Val Thr Val Ala Trp Lys Ala Asp Ser Ser Pro 35 40 45Val Lys
Ala Gly Val Glu Thr Thr Thr Pro Ser Lys Gln Ser Asn Asn 50 55 60Lys
Tyr Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys65 70 75
80Ser His Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val
85 90 95Glu Lys Thr Val Ala Pro Thr Glu Cys Ser 100
10565120PRTArtificial sequencebinding domain sequence 65Gln Leu Gln
Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu1 5 10 15Thr Leu
Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Lys Ser Gly 20 25 30Ser
Tyr Tyr Trp Gly Trp Ile Arg Gln Pro Pro Gly Lys Gly Leu Glu 35 40
45Trp Ile Gly Asn Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser
50 55 60Leu Arg Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln
Phe65 70 75 80Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala
Val Tyr Tyr 85 90 95Cys Ala Arg Glu Gly Ser Tyr Pro Asn Gln Phe Asp
Pro Trp Gly Gln 100 105 110Gly Thr Leu Val Thr Val Ser Ser 115
1206610PRTArtificial sequencebinding domain sequence 66Gly Gly Ser
Ile Lys Ser Gly Ser Tyr Tyr1 5 10677PRTArtificial sequencebinding
domain sequence 67Ile Tyr Tyr Ser Gly Ser Thr1 56812PRTArtificial
sequencebinding domain sequence 68Ala Arg Glu Gly Ser Tyr Pro Asn
Gln Phe Asp Pro1 5 1069107PRTArtificial sequencebinding domain
sequence 69Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Val Ser
Pro Gly1 5 10 15Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val
Ser Ser Asn 20 25 30Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro
Arg Leu Leu Ile 35 40 45Tyr Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro
Ala Arg Phe Ser Gly 50 55 60Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr
Ile Ser Ser Leu Gln Ser65 70 75 80Glu Asp Phe Ala Val Tyr Tyr Cys
Gln Gln Tyr His Ser Phe Pro Phe 85 90 95Thr Phe Gly Gly Gly Thr Lys
Val Glu Ile Lys 100 105706PRTArtificial sequencebinding domain
sequence 70Gln Ser Val Ser Ser Asn1 5719PRTArtificial
sequencebinding domain sequence 71Gln Gln Tyr His Ser Phe Pro Phe
Thr1 5
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