The present invention is in the field of medicine, particularly in the novel field of
bispecific antibodies directed against Calcitonin Gene Related Peptide (CORP) and
Interleukin-23 (IL-23). The bispecific antibodies of the present invention are expected to
be useful in treating autoimmune diseases including Inflammatory Bowel Disease (IBD),
such as Crohn's Disease (CD) and Ulcerative Colitis (UC), Psoriatic Arthritis (PsA) and
ankylosing spondylitis (AS).
Autoimmune diseases arise from the body's production of an immune response
against its own tissue. Autoimmune diseases are often chronic and can be debilitating
and even life-threatening. IBD, which generically represents a group of disorders such as
CD and UC, is a common chronic relapsing autoimmune disease characterized
pathologically by intestinal inflammation and epithelial injury. Other forms of chronic
autoimmune diseases, such PsA and AS, may affect the axial and I or peripheral skeleton.
Interleukin 23 (IL-23) is a heterodimeric cytokine believed to be important in the
activation of a range of inflammatory cells required for the induction of chronic
inflammation. IL-23, which is believed to be an upstream regulator of IL-6, IL-17, GMCSF
and IL-22 secretion, is composed of a p 19 subunit (IL23p 19) covalently paired to a
p40 subunit (the p40 subunit is also shared with cytokine IL-12). Additionally, IL-23 has
been implicated as playing an important role in memory I pathogenic T -cell inflammatory
response as well as playing a role in the regulation of innate lymphoid cell inflammatory
activity. There is evidence that IL-23 regulation of the cytokines IL-6, IL-17, GM-CSF
and IL-22 is associated with inflammatory diseases including IBD and other autoimmune
diseases.
CORP is a 37 amino acid neuropeptide secreted by the nerves ofthe central and
peripheral nervous systems. CORP is widely distributed in sensory nerves, both in the
peripheral and central nervous system and displays a large number of different biological
activities. For instance, it is a potent vasodilator with microvasculature being sensitive
thereto. When released from trigeminal and other nerve fibers, CORP is thought to
mediate its biological responses by binding to specific cell surface receptors. CORP is
believed to play a role in the modulation and/or transmission of pain signaling and in
neurogenic inflammation. CORP has been reported to play a role in migraines as CORP
2
is released upon stimulation of sensory nerves. The release of CGRP increases vascular
permeability and subsequent plasma protein leakage (plasma protein extravasation) in
tissues innervated by trigeminal nerve fibers upon stimulation of these fibers. In addition,
studies have reported that infusion of CGRP in patients who suffer from migraines has
resulted in migraine-like symptoms.
Current FDA approved treatments for autoimmune diseases such as IBD include
corticosteroids, often used to treat acute inflammation, and bioproducts, many of which
(such as REMICADE®, ENBREL ® and HUMIRA ®) attempt to target and neutralize
TNFa in the body. Another bioproduct approved for treatment ofPsA includes
STELARA ® which attempts to target the shared p40 subunit of cytokines IL-12 and IL-
23. Current treatments have demonstrated efficacy for reducing symptoms and slowing
progression of some autoimmune diseases in a subset of patients. However, a large
percentage of patients are nonresponsive to currently available treatments (for example,
induction of remission occurs in only 30-50% of CD patients treated with TNFa
neutralization, and loss of response to TN Fa neutralization occurs in between 23 and 46%
of patients following 12 months of treatment). Alternative therapies for autoimmune
diseases include antibodies that bind to the p 19 subunit of IL-23, such as those disclosed
in U.S. Patent No. 9,023,358.
While currently approved treatments for autoimmune diseases treat the
inflammatory aspect of the disease, said treatments have proved ineffective in treating
associated pain. Even in patients suffering from IBD (CD and UC) that are responsive to
anti-TNFa therapy, pain remains. It is thought that inflammation associated with
autoimmune diseases drives central sensitization to pain leading to hyperalgesia and
allodynia. The consequence is that pain can be present even after inflammation has
subsided with a high percentage of patients continuing to take pain medication. The
standard therapies for pain in patients suffering from IBD are analgesics including
NSAIDS, COX-2 inhibitors and opiates. At present, patients suffering from IBD are
filling a similar number of analgesic prescriptions both prior to and post the introduction
of biologic therapy. Antibodies that bind to CGRP, such as those described in U.S. Patent
No. 9,073,991, have been suggested as therapeutics for migraine.
3
One approach to such alternative therapies may include the co·administration of
two different bioproducts (e.g., antibodies) treating different aspects ofthe autoimmune
disease (e.g. pathology of the disease and associated pain). Co·administration requires
either injections of two separate products or a single injection of a co· formulation of two
different antibodies. While two injections permit flexibility of dose amounts and timing,
it is inconvenient to patients both for compliance and pain. Moreover, while a co·
formulation might provide some flexibility of dose amounts, it is often quite challenging
or impossible to find formulation conditions having acceptable viscosity (at relatively
high concentration) and that promote chemical and physical stability of both antibodies
due to different molecular characteristics of the two antibodies. Additionally, co·
administration and co·formulation involve the additive costs of two different drug
therapies which can increase patient and I or payer costs.
Thus, there remains a need for alternative therapies for treatment of autoimmune
diseases that have both disease modification and pain management properties and
preferably such alternative therapies comprise a bispecific antibody.
The present invention provides a bispecific antibody comprising an
immunoglobulin G antibody (lgG) and two single chain variable fragments (scFv).
More specifically, the present invention provides a bispecific antibody comprising
an lgG and two scFv wherein,
(a) said lgG comprises two heavy chains (HC) and two light chains (LC), each
HC comprises a heavy chain variable region (HCVRl) comprising heavy
chain CORs (HCOR) 1·3 and each light chain comprises a light chain
variable region (LCVR1) comprising light chain CORs (LCDR) 1·3,
wherein the amino acid sequence ofHCDR1 is SEQ ID NO: 10, the amino
acid sequence of HCDR2 is SEQ 10 NO: 11, the amino acid sequence of
HCOR3 is SEQ ID NO: 12, the amino acid sequence ofLCOR1 is SEQ 10
NO: 16, the amino acid sequence of LCDR2 is SEQ ID NO: 17, and the
amino acid sequence of LCOR3 is SEQ 10 NO: 18; and
(b) each scFv comprises a heavy chain variable region (HCVR2) and a light
chain variable region (LCVR2), the HCVR2 comprising HCORs 4·6, and
4
the LCVR2 comprising LCDRs 4-6, wherein the amino acid sequence of
HCDR4 is SEQ ID NO: 13, the amino acid sequence ofHCDR5 is SEQ
ID NO: 14, the amino acid sequence ofHCDR6 is SEQ ID NO: 15, the
amino acid sequence of LCDR4 is SEQ ID NO: 19, the amino acid
sequence of LCDR5 is SEQ ID NO: 20, and the amino acid sequence of
LCDR6 is SEQ ID NO: 21 or SEQ ID NO: 22,
wherein each scFv is linked at theN-terminus ofHCVR2 of each scFv to said IgG
antibody at the C-terminus of each IgG HC via a polypeptide linker (L 1 ),
and wherein the HCVR2 of each scFv is linked at the C-terminus of the HCVR2
to the LCVR2 of the same scFv at theN-terminus ofthe LCVR2 ofthe same scFv via a
second polypeptide linker (L2).
The bispecific antibody of the present invention binds to CGRP and the p 19
subunit of IL-23.
Preferably, the amino acid sequence ofLCDR6 is SEQ ID NO: 21.
Further preferably, the amino acid sequence of LCDR6 is SEQ ID NO: 22.
In a further embodiment of the bispecific antibody ofthe present invention, the
amino acid sequence ofHCVR1 of each HC is SEQ ID NO: 5, the amino acid sequence
ofLCVR1 of each LC is SEQ ID NO: 7, the amino acid sequence ofHCVR2 of each
scFv is SEQ ID NO: 6 and the amino acid sequence of LCVR2 of each scFv is SEQ ID
NO: 8 or SEQ ID NO: 9.
Preferably, the amino acid sequence of LCVR2 of each scFv is SEQ ID NO: 8.
Further preferably, the amino acid sequence ofLCVR2 of each scFv is SEQ ID
N0:9.
In a still further embodiment of the bispecific antibody of the present invention,
the amino acid sequence of each HC is SEQ ID NO: 4, the amino acid sequence of each
LC is SEQ ID NO: 3, the amino acid sequence of HCVR2 of each scFv is SEQ ID NO: 6
and the amino acid sequence ofLCVR2 ofeachscFv is SEQ ID NO: 8 or SEQ ID NO: 9.
Preferably, the amino acid sequence of LCVR2 of each scFv is SEQ ID NO: 8.
Further preferably, the amino acid sequence of LCVR2 of each scFv is SEQ ID
N0:9.
5
In a preferred aspect of the above embodiments of the present invention, the
amino acid sequence of L1 is SEQ ID NO: 23 and the amino acid sequence of L2 is SEQ
IDNO: 24.
In a preferred embodiment of the bispecific antibody of the present invention, the
amino acid sequence of each HC is SEQ ID NO: 4, the amino acid sequence of each LC
is SEQ ID NO: 3, the amino acid sequence ofHCVR2 of each scFv is SEQ ID NO: 6, the
amino acid sequence of LCVR2 of each scFv is SEQ ID NO: 8, the amino acid sequence
ofLl is SEQ ID NO: 23 and the amino acid sequence ofL2 is SEQ ID NO: 24.
In a further preferred embodiment of the bispecific antibody of the present
invention, the amino acid sequence of each HC is SEQ ID NO: 4, the amino acid
sequence of each LC is SEQ ID NO: 3, the amino acid sequence of HCVR2 of each scFv
is SEQ ID NO: 6, the amino acid sequence of LCVR2 of each scFv is SEQ IO NO: 9, the
amino acid sequence ofLl is SEQ 10 NO: 23 and the amino acid sequence ofL2 is SEQ
ID NO: 24.
Significant problems associated with chemical and physical stability were
addressed when building a bispecific antibody of the present invention. Many changes
were required in the starting bispecific antibody to sufficiently overcome a myriad of
issues that can be associated with bispecific antibodies, such as expressing a physically
stable molecule, stabilizing the VHNL interface of the single chain fragment variable
region (scFv), increasing thermal and salt-dependent stability, decreasing aggregation,
increasing solubility at high concentrations, and/or rebalancing the electrostatic
distribution in the binding surfaces of the bispecific antibody, all while maintaining
binding affinity for both targeted antigens; CORP and the p19 subunit ofiL-23.
Bispecific antibodies of the present invention are thermally stable and physically
stable. Moreover, bispecific antibodies of the present invention may also exhibit low
aggregation. Furthermore, bispecific antibodies of the present invention may also
neutralize human CORP and human IL23p19 (the pl9 subunit ofiL-23), as well as
simultaneously binding both ligands. The presently claimed antibodies may also avoid
the challenges of finding formulation conditions that must satisfy the different molecular
characteristics of two different, separate antibodies.
6
The IgG part of a first bispecific antibody of the present invention comprises two
identical heavy chains (IgG HC)(SEQ ID NO: 4).
Each IgG HC is attached at its C-terminus via a first polypeptide linker (L1)(SEQ
ID NO: 23) to an identical scFv portion that specifically binds to the p 19 subunit ofiL-
23.
Each heavy chain scFv portion (HCVR2)(SEQ ID NO: 6) is attached at its Cterminus
via a second polypeptide linker (L2)(SEQ ID NO: 24) to a scFv light chain
(LCVR2)(SEQ ID NO: 8).
The complete linear amino acid sequence of each identical heavy chain part of the
first bispecific antibody of the invention, starting from theN-terminal residue of the IgG4
HC and ending at the C-terminal residue ofthe scFv LC is provided in SEQ ID NO: 1.
Similarly, the complete amino acid sequence of each identical LC of the first
bispecific antibody ofthe invention, starting from theN-terminal residue of the variable
domain and ending at the C-terminal residue ofthe LC kappa constant region is provided
in SEQ ID NO: 3.
The relationship of the various regions and linkers of an exemplified first
bispecific antibody of the present invention is as follows (numbering of amino acids
applies linear numbering; assignment of amino acids to variable domains is based on the
International Immunogenetics Information System® available at www.imgt.org;
assignment of amino acids to CDR domains is based on the well-known Kabat (Kabat et
al., Ann. NY Acad. Sci. 190:382-93 (1971); Kabat et al., Sequences of Proteins of
Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services,
NIH Publication No. 91-3242 (1991)) and North (North et al., A New Clustering of
Antibody CDR Loop Conformations, Journal of Molecular Biology, 406:228-256 (2011))
numbering conventions as reflected in Tables 1(a)-(c)):
7
Table 1(a): Amino acid regions ofBispecific Antibody 1- IgG HC-L1-scFv HCVR2-
L2-scFv LCVR2
SEQID NO: 1
Re2ion Positions
FRHl-1 (SEQ 1-22
ID NO: 25) QVQLVQSGAEVKKPGSSVKVSC
HCDR1 (SEQ 23-35
ID NO: 10) KASGYTFGNYWMQ
FRHl-2 (SEQ 36-49
ID NO: 26) WVRQAPGQGLEWMG
HCVR1CGRP HCDR2 (SEQ 50-66
(SEQ ID NO: 5) IDNO: 11) AIYEGTGKTVYIQKF AD
FRH1-3 (SEQ 67-96
ID NO: 27) RVTITADKSTSTAYMELSSLRSEDTAVYYC
HCDR3 (SEQ 97-108
ID NO: 12) ARLSDYVSGFGY
FRH1-4 (SEQ 109-119
ID NO: 28) WGQGTTVTVSS
120-444
ASTKGPSVFPLAPCSRSTSESTAALGCLVKDY
FPEPVTVSWNSGALTSGVHTFPAVLQSSGLYS
LSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDK
RVESKYGPPCPPCPAPEAAGGPSVFLFPPKPK
Constant
CH (SEQ ID DTLMISRTPEVTCVVVDVSQEDPEVQFNWYV
NO: 41) DGVEVHNAKTKPREEQFNSTYRVVSVLTVLH
QDWLNGKEYKCKVSNKGLPSSIEKTISKAKG
QPREPQVYTLPPSQEEMTKNQVSL TCL VKGF
YPSDIA VEWESNGQPENNYKTTPPVLDSDGSF
FL YSRLTVDKSRWQEGNVFSCSVMHEALHNH
YTQKSLSLSL
Linker
Ll (SEQ ID 445-459
NO: 23) GGGGSGGGGSGGGGS
FRH2-1 (SEQ 460-481
ID NO: 29) QVQLVQSGAEVKKPGSSVKVSC
HCDR4 (SEQ 482-494
ID NO: 13) KASGYPFTRYVMH
FRH2-2 (SEQ 495-508
HCVR2 IL-23
ID NO: 30) WVRQAPGQCLEWMG
(SEQ ID NO: 6)
HCDR5 (SEQ 509-525
ID NO: 14) YINPYNDGVNYNEKFKG
FRH2-3 (SEQ 526-555
ID NO: 31) RVTIT ADESTSTA YMELSSLRSEDT A VYYC
HCDR6 (SEQ 556-563
ID NO: 15) ARNWDTGL
FRH2-4 (SEQ 564-574
8
SEQ ID NO: 1
ID NO: 32) WGQGTTVTVSS
Linker
L2 (SEQ ID 575-594
NO: 24) GGGGSGGGGSGGGGSGGGGS
FRL2-1 (SEQ 595-617
ID NO: 33) DIQMTQSPSSLSASVGDRVTITC
LCDR4 (SEQ 618-628
ID NO: 19) KASDHIGKFLT
FRL2-2 (SEQ 629-642
ID NO: 34) WYQQKPGKAPKLLI
LCVR2 IL-23 LCDR5 (SEQ 643-650
(SEQ ID NO: 8) ID NO: 20) YGATSKLT
FRL2-3 (SEQ 651-682
ID NO: 35) GVPSRFSGSGSGTDFTL TISSLQPEDF A TYYC
LCDR6 (SEQ 683-691
ID NO: 21) QQYWSTPFT
FRL2-4 (SEQ 692-701
ID NO: 36) FGCGTKVEIK
Table l(b): Amino acid regions ofBispecific Antibodyl- lgG LC
SEQ ID NO: 3
Regions Positions
FRL1-1 (SEQ 1-23
ID NO: 37) DIQMTQSPSSLSASVGDRVTITC
LCDR1 (SEQ 24-34
ID NO: 16) RASKDISKYLN
FRL1-2 (SEQ 35-48
LCVR1 CGRP
ID NO: 38) WYQQKPGKAPKLLI
LCDR2 (SEQ 49-56
(SEQ ID NO: 7) IDNO: 17) YYTSGYHS
FRLl-3 (SEQ 57-88
ID NO: 39) GVPSRFSGSGSGTDFTL TISSLQPEDF ATYYC
LCDR3 (SEQ 89-97
ID NO: 18) QQGDALPPT
FRL1-4 (SEQ 98-107
ID NO: 40) FGGGTKVEIK
108-214
CL (SEQ ID
RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNF
Constant YPREAKVQWKVDNALQSGNSQESVTEQDSK
NO: 42)
DSTYSLSSTLTLSKADYEKHKVY ACEVTHQG
LSSPVTKSFNRGEC
9
According to a second exemplified bispecific antibody of the present invention,
LCDR6 incorporates an engineered single amino acid change that substitutes threonine
(T) for glutamine (Q) at position 684 (Q684T) in SEQ ID NO: 1 such that the LCDR6 of
the second exemplified bispecific antibody of the present invention has the following
sequence QTYWSTPFT (SEQ ID NO: 22). No additional changes were made and,
consequently, all remaining amino acid sequences ofthe second exemplified bispecific
antibody of the present invention are identical to those of the first exemplified bispecific
antibody described above.
The complete linear amino acid sequence of each identical heavy chain part of the
second bispecific antibody ofthe invention, which comprises LCDR6 having SEQ ID
NO: 22, starting from theN-terminal residue of the lgG4 HC and ending at the C-terminal
residue of the scFv LC is provided in SEQ ID NO: 2.
Similarly, the complete amino acid sequence of each identical LC of the second
bispecific antibody ofthe invention, starting from theN-terminal residue of the LC
variable domain and ending at the C-terminal residue of the LC constant region is
provided in SEQ ID NO: 3.
The present invention further provides a bispecific antibody wherein each of the
HCs form an inter-chain disulfide bond with each of the LCs; wherein each of the HCs
forms an inter-chain disulfide bond with the other HC; and wherein each of the scFvs
forms an intra-chain disulfide bond between HCVR2 and LCVR2. According to the
exemplified bispecific antibody ofthe present invention presented in Tables 1(a) and (b),
an inter-chain disulfide bond of each ofthe HCs and each of the LCs forms between
cysteine residue 133 (of SEQ ID NO: 1 and SEQ ID NO: 2) ofthe HC, and cysteine
residue 214 (of SEQ ID NO: 3) ofthe LC; at least two inter-chain disulfide bonds form
between the two HCs, the first inter-chain disulfide bond forming between cysteine
residue 225 (of SEQ ID NO: 1 or SEQ ID NO: 2) ofthe HC and cysteine residue 225 (of
SEQ ID NO: 1 or SEQ ID NO: 2) of the other HC, the second inter-chain disulfide bond
forming between cysteine residue 228 (of SEQ ID NO: 1 or SEQ ID NO: 2) ofthe HC
and cysteine residue 228 (of SEQ ID NO: 1 or SEQ ID NO: 2) of the other HC; and an
10
intra-chain disulfide bond of the scFv is formed between cysteine residue 503 (of SEQ ID
NO: 1 or SEQ ID NO: 2) ofthe HCVR2 and cysteine residue 694 (of SEQ ID NO: I or
SEQ ID NO: 2) ofthe LCVR2.
According to some embodiments of the present invention, a bispecific antibody
comprising glycosylation of the HC is provided. According to the exemplified bispecific
antibody ofthe present invention presented in Tables l(a) and (b), glycosylation ofthe
HC occurs at the asparagine residue 296 of SEQ ID NO: 1 or SEQ ID NO: 2.
Given the amino acid sequences provided herein, one of ordinary skill in the art
could use this knowledge to design a DNA molecule to encode and express any bispecific
antibody, or fragment thereof, described hereinabove. The present invention thus
encompasses all DNA sequences encoding a bispecific antibody or fragment thereof
according to the invention.
In particular, the present invention provides a DNA molecule comprising a
polynucleotide sequence encoding a polypeptide chain comprising a HC, a scFv, a first
polypeptide linker L 1 and a second polypeptide linker L2 of the bispecific antibody of
present invention.
According to an embodiment of the present invention, the amino acid sequence of
the encoded polypeptide chain is SEQ ID NO: 1.
According to an alternative embodiment ofthe present invention, the amino acid
sequence ofthe encoded polypeptide is SEQ ID NO: 2.
The present invention also provides an expression vector comprising a
polynucleotide sequence encoding the polypeptide of SEQ ID NO: 1 and a polynucleotide
sequence encoding the polypeptide of SEQ ID NO: 3.
The present invention also provides an expression vector comprising a
polynucleotide sequence encoding the polypeptide of SEQ ID NO: 2 and a polynucleotide
sequence encoding the polypeptide of SEQ ID NO: 3.
The present invention also provides a recombinant host cell comprising a DNA
molecule comprising a polynucleotide sequence encoding a polypeptide chain comprising
a HC, a scFv, a first polypeptide linker L1 and a second polypeptide linker L2 of the
bispecific antibody of present invention, wherein the amino acid sequence ofthe
11
polypeptide chain is SEQ ID NO: 1 or SEQ ID NO: 2, and a DNA molecule comprising a
polynucleotide sequence encoding a polypeptide chain comprising a LC of the bispecific
antibody the present invention, wherein the amino acid sequence of the LC is SEQ ID
NO: 3, wherein the cell is capable of expressing a bispecific antibody of the present
invention, said bispecific antibody comprising an IgO that binds CORP conjugated to two
scFvs that bind IL23p19.
The present invention also provides a recombinant host cell transformed with a
DNA molecule comprising a polynucleotide sequence encoding a polypeptide chain
comprising a HC, a scFv, a first polypeptide linker L1 and a second polypeptide linker L2
of the bispecific antibody of present invention, wherein the amino acid sequence ofthe
polypeptide chain is SEQ ID NO: 1 or SEQ ID NO: 2, and a DNA molecule comprising a
polynucleotide sequence encoding a polypeptide chain comprising a LC ofthe bispecific
antibody the present invention, wherein the amino acid sequence of the LC is SEQ ID
NO: 3, said bispecific antibody comprising an IgO that binds CORP conjugated to two
scFvs that bind IL23p19.
The present invention also provides a process for producing a bispecific antibody
ofthe present invention, the process comprising cultivating a recombinant host cell of the
present invention under conditions such that the bispecific antibody is expressed, and
recovering the expressed bispecific antibody.
The present invention also provides a bispecific antibody according to the present
invention produced by said process.
Preferably, the recombinant host cells is a mammalian host cell selected from the
group consisting of CHO, NSO, HEK293 and COS cells.
The present invention also provides a method of treating autoimmune diseases
comprising administering to a patient in need thereof an effective amount of a bispecific
antibody of the present invention.
The present invention also provides a method of treating IBD, such as CD and/or
UC, comprising administering to a patient in need thereof an effective amount of a
bispecific antibody ofthe present invention.
12
The present invention also provides a method of treating PsA and/or ankylosing
spondylitis comprising administering to a patient in need thereof an effective amount of a
bispecific antibody of the present invention.
The present invention also provides a bispecific antibody of the present invention
for use in therapy.
The present invention also provides a bispecific antibody of the present invention
for use in the treatment of autoimmune diseases including IBD, such as CD and/or UC.
The present invention also provides a bispecific antibody of the present invention
for use in the treatment of autoimmune diseases including PsA and/or ankylosing
spondylitis.
The present invention also provides a pharmaceutical composition comprising a
bispecific antibody of the present invention and one or more pharmaceutically acceptable
carriers, diluents or excipients.
Another embodiment of the present invention comprises use of a bispecific
antibody of the present invention in the manufacture of a medicament for the treatment of
ulcerative colitis and/or Crohn's disease.
An additional embodiment of the present invention comprises use of a bispecific
antibody of the present invention in the manufacture of a medicament for the treatment of
psoriatic arthritis and/or ankylosing spondylitis.
Definitions
When used herein the term "bispecific antibody" refers to a molecule comprising
an immunoglobulin G antibody (lgG) conjugated to two single chain variable fragments
(scFv). As referred to herein, a bispecific antibody of the present invention comprises an
IgG and two scFv's, wherein each scFv is linked at theN-terminus of HCVR2 of each
scFv to said IgG antibody at the C-terminus of each IgG HC via a polypeptide linker (L 1)
and wherein the HCVR2 of each scFv is linked at the C-terminus of the HCVR2 to the
LCVR2 of the same scFv at theN-terminus of the LCVR2 of the same scFv via a second
polypeptide linker (L2). The IgG and scFvs of a bispecific antibody of the present
invention specifically bind different antigens (CORP and the p 19 subunit ofiL-23,
13
respectively). Notably, the bispecific antibody of the present invention binds to the p 19
subunit ofiL-23 but does not bind to the p40 subunit ofiL-23 that is shared with IL-12.
As referred to herein, the term "single chain variable fragment" (scFv), refers to a
polypeptide chain comprising a heavy chain variable region (HCVR2) and a light chain
variable region (LCVR2) connected via a polypeptide linker (L2). Additionally, as
referred to herein (and as represented in the following schematic), the HCVR2 of each
scFv is: a) linked, at its N-terminus, to the C-terminus of one HC of the IgG via a
polypeptide linker (L1); and b) L1 is linked, at its C-terminus, to theN-terminus ofthe
LCVR2 of the same scFv via a second polypeptide linker (L2). Further, each scFv of the
present invention includes a disulfide bond formed between a cysteine residue of HCVR2
and a cysteine residue ofLCVR2 of the same polypeptide chain (as represented in the
following schematic):
IgG Light Chain
I i s I
IgG Heavy Chain- Linker (Ll)- scFv HCVR2- Linker (L2) -scFv LCVR2
I I
s s
IgG klavy Chain- Linker (Ll)- scFv HCVR2-Linker (L2) -scFv LCVR2
I s I
s
I
IgG Light Chain
A "parent antibody" or "parental antibody," as used interchangeably herein, is an
antibody encoded by an amino acid sequence which is used in the preparation of one of
the IgG and scFv of the bispecific antibody, for example through amino acid substitutions
and structural alteration. The parent antibody may be a murine, chimeric, humanized or
human antibody.
The terms "Kabat numbering" or "Kabat labeling" are used interchangeably
herein. These terms, which are recognized in the art, refer to a system of numbering
amino acid residues which are more variable (i.e., hypervariable) than other amino acid
residues in the heavy and light chains variable regions of an antibody (Kabat, et al., Ann.
14
NY Acad. Sci. 190:382-93 (1971); Kabat et al., Sequences of Proteins of Immunological
Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication
No. 91-3242 (1991)).
The terms "North numbering" or "North labeling" are used interchangeably
herein. These terms, which are recognized in the art, refer to a system of numbering
amino acid residues which are more variable (i.e., hypervariable) than other amino acid
residues in the heavy and light chains variable regions of an antibody and is based, at
least in part, on affinity propagation clustering with a large number of crystal structures,
as described in (North et al., A New Clustering of Antibody CDR Loop Conformations,
Journal of Molecular Biology, 406:228-256 (20 ll ).
The terms "patient," "subject," and "individual," used interchangeably herein,
refer to an animal, preferably the term refers to humans. In certain embodiments, the
subject, preferably a human, is further characterized with a disease or disorder or
condition (e.g., an autoimmune disorder) that would benefit from a decreased level or
decreased bioactivity of both IL-23 and CORP. In another embodiment the subject,
preferably a human, is further characterized as being at risk of developing a disorder,
disease or condition that would benefit from a decreased level or decreased bioactivity of
both IL-23 and CORP.
Bispccific Antibody Engineering
Significant problems associated with chemical and physical stability were
encountered when constructing a bispecific antibody of the present invention. Problems
encountered included poor to no expression, poor purification recovery, low
thermostability, high salt-dependent aggregation, diabody formation (and challenges in
reducing diabodies through purification), high solution viscosity, low binding affinity and
cross-reactivity.
For example, initial attempts in constructing an IgO-scFv formatted bispecific
antibody included constructs in which a parental IL-23 antibody (the IL-23 antibody
described in U.S. Patent No. 9,023,358) comprised the IgO antibody portion and a
parental CORP antibody (see for example U.S. Patent No. 9,073,991) comprised the scFv
15
portion of the bispecific antibody. Other initially attempted constructs included the
parental IL-23 antibody comprising the scFv portion while the CGRP antibody comprised
the IgG portion of the bispecific antibody. Additionally, initial constructs included the
scFv portion being conjugated to the IgG portion in various configurations, including at
the amino-terminus or the carboxyl terminus for both the heavy and light chains,
respectively. Moreover, initial constructs included the scFv portion varying in
arrangement of the HCVR2 and LCVR2 (e.g., lgG portion (CorN terminus) -linker 1-
LCVR2 or HCVR2 - linker 2 -the other of LCVR2 or HCVR2). Further, parental IL-23
antibody constructs included combinations of heavy chain germline frameworks VH 5-51
and 1-69, and light chain germline frameworks VK 02, VK 12 and VK B3. Parental
CGRP antibody constructs (when comprising the IgG portion) included an IgG4 subclass
structure having three amino acid mutations (from native IgG4) within the constant region
(CH). Initial constructs were cloned into a human IgG4-Fc mammalian expression
vector. However, initially produced bispecific constructs as (described above) exhibited
one or more chemical and I or physical problem(s) described above. For instance,
constructs wherein the scFv portion is positioned at theN-terminus exhibit multiple
stability issues when compared to constructs wherein the scFv portion is positioned at the
C-terminus.
Electrostatic surface of the bispecific antibody was calculated and charged patches
were identified and disrupted. Extensive protein stability studies were performed and the
constructed bispecific antibodies were screened for thermostability properties as well as
CGRP and IL-23 binding (relative to the respective parental antibody) properties.
Chemical and physical modifications were therefore made to improve chemical
and physical stability of the bispecific antibody of the present invention. Modifications to
the parental IL-23 antibody, in scFv format, were made in HCDR4, HCDR5, LCDR4,
LCDR5 and LCDR6 to improve chemical and physical stability. Constructed HCVR and
LCVR were combined into the IL-23 scFv format according to the following formula:
CGRP IgG (C-term.) - L1- HCVR2- L2- LCVR2. A disulfide bond, for stabilizing the
IL-23 scFv, was engineered between the HCVR2 (G503C) and the LCVR2 (G694C) of
the IL-23 scFv (numbering of amino acids applies linear numbering based on exemplified
16
bispecific antibody presented in Tables l(a) and (b)). Additionally, the parental CORP
antibody, in an IgO portion of the bispecific antibody, was engineered to an Ig04 subclass
because of a reduced ability to engage Fe receptor-mediated inflammatory mechanisms or
to activate complement resulting in reduced effector function. The engineered IL-23 scFv
construct and CORP IgO construct, comprising these chemical and physical
modifications, were inserted into an expression vector.
More specifically, the bispecific antibody of the present invention contains an
Ig04-PAA Fe portion. The Ig04-PAA Fe portion has a serine to proline mutation at
position 227 (S227P; SEQ ID NO: 1 or SEQ 10 NO: 2), a phenylalanine to alanine
mutation at position 233 (F233A; SEQ 10 NO: 1 or SEQ ID NO: 2) and a leucine to
alanine mutation at position 234 (L234A; SEQ ID NO: 1 ). The S227P mutation is a hinge
mutation that prevents half-antibody formation (phenomenon of dynamic exchange of
half-molecules in lg04 antibodies). The F233A and L234A mutations further reduce
effector function of the already low human lg04 isotype.
A bispecific antibody containing a CORP lgO, as an lg04 subclass, and an IL-23
scFv with six CDR mutations (relative to the parental IL-23 antibody described in U.S.
Patent No. 9,023,358: HCVR2 at K28P and T58V (SEQ ID NO: 6); and LCVR2 at L300,
L54K, E55L and M90Q/M90T (SEQ 10 NO: 8 or SEQ ID NO: 9)(as represented in the
exemplified bispecific antibody reflected in Tables 1 (a) and (b): HCVR2 at K487P and
T517V; and LCVR2 at L6240, L648K, E649L and M684Q/M684T, numbering of amino
acids applies linear numbering based on exemplified bispecific antibody presented in
Tables 1(a) and (b)) was identified as improving the expression, affinity (for IL-23
relative to the parental molecule) and thermostability issues demonstrated in initial
constructs. The M90T mutation (SEQ ID NO: 9; (relative to the parental IL-23 antibody
described in U.S. Patent No. 9,023,358) has been found to improve the photostability of
the molecule without adversely affecting binding to CORP and IL-23pl9. Additionally,
these mutations resulted in a significantly reduced clearance rate in cynolomolgus
monkeys. None ofthe above modifications were identified in initial characterizations of
the parental single antibodies.
17
Bispecific Antibody Binding and Activity
The bispecific antibodies of the present invention bind both human CGRP and
human IL23p19 and neutralize at least one human CGRP bioactivity and at least one
human IL-23 bioactivity in vitro or in vivo. The bispecific antibodies of the present
invention are inhibitors of IL-23 in the presence and absence of CGRP in vitro. The
bispecific antibodies of the present invention are inhibitors of CGRP in the presence or
absence ofiL-23 in vitro.
The first exemplified bispecific antibody of the present invention (Bispecific
Antibody 1) is characterized as having a binding affinity (Ko) for human CGRP in the
range of26.0 ± 26.0 pM and human IL23p19 in the range of213.0 ± 184.0 pM at 37°C.
The second exemplified bispecific antibody of the present invention (Bispecific
Antibody 2) is characterized as having a binding affinity (Ko) for human CGRP of
approximately 77.0 pM and human IL23p 19 of 215 pM at 3 7°C.
The bispecific antibodies effectively neutralize CGRP and this neutralization is
not affected by the presence of saturating amounts of human IL-23. The bispecific
antibodies effectively neutralize human IL-23 and this neutralization is not affected by the
presence of saturating amounts of human CGRP.
Bispecific Antibody Expression
Expression vectors capable of direct expression of genes to which they are
operably linked are well known in the art. Expression vectors can encode a signal peptide
that facilitates secretion of the polypeptide(s) from a host cell. The signal peptide can be
an immunoglobulin signal peptide or a heterologous signal peptide. The first polypeptide
chain (comprising a HC, scFv, Ll and L2) and the second polypeptide chain (comprising
a LC) may be expressed independently from different promoters to which they are
operably linked in one vector or, alternatively, the first and second polypeptide chains
may be expressed independently from different promoters to which they are operably
linked in two vectors - one expressing the first polypeptide chain and one expressing the
second polypeptide chain.
18
A host cell includes cells stably or transiently transfected, transformed, transduced
or infected with one or more expression vectors expressing a first polypeptide chain, a
second polypeptide chain or both a first and a second polypeptide chain of the invention.
Creation and isolation of host cell lines producing a bispecific antibody of the invention
can be accomplished using standard techniques known in the art. Mammalian cells are
preferred host cells for expression of bispecific antibodies. Particular mammalian cells
are HEK 293, NSO, DG-44, and CHO. Preferably, the bispecific antibodies are secreted
into the medium in which the host cells are cultured, from which the bispecific antibodies
can be recovered or purified.
It is well known in the art that mammalian expression of antibodies results in
glycosylation. Typically, glycosylation occurs in the Fe region of the antibody at a highly
conserved N-glycosylation site. N-glycans typically attach to asparagine. By way of
example, each HC of exemplified bispecific antibody presented in Tables 1 (a) and (b) is
glycosylated at asparagine residue 296 of SEQ ID NO: 1 or SEQ ID NO: 2.
Medium, into which a bispecific antibody has been secreted, may be purified by
conventional techniques. For example, the medium may be applied to and eluted from a
Protein A or G column using conventional methods. Soluble aggregate and multimers
may be effectively removed by common techniques, including size exclusion,
hydrophobic interaction, ion exchange, or hydroxyapatite chromatography. The product
may be immediately frozen, for example at -70°C, refrigerated, or may be lyophilized.
In some instances, a process for producing a bispecific antibody of the present
invention may result in the formation of diabodies. Diabodies are bivalent formations of
scFv in which HCVR2 and LCVR2 regions are expressed on a single polypeptide chain,
but instead of the variable domains pairing with complementary domains of the same
polypeptide chain, the variable domains pair with complementary domains of the other
polypeptide chain or a different molecule. For example, if the bispecific antibody
comprises two first polypeptides (for convenience, 1A and 1B, where each of 1A and 1B
comprise a HC, a scFv, L 1 and L2), and two second polypeptides (for convenience, 2A
and 2B, where each of2A and 2B comprise a LC), HCVR2 of 1A pairs with
complementary domains of LCVR2 of 1 B instead of pairing with LCVR2 of 1 A.
19
Therapeutic Uses
As used herein, "treatment" and/or "treating" are intended to refer to all processes
wherein there may be a slowing, interrupting, arresting, controlling, or stopping of the
progression ofthe disorders described herein, but does not necessarily indicate a total
elimination of all disorder symptoms. Treatment includes administration of a bispecific
antibody of the present invention for treatment of a disease or condition in a mammal,
particularly in a human, that would benefit from a decreased level of CGRP and I or IL-
23 or decreased bioactivity ofCGRP and I or IL-23, and includes: (a) inhibiting further
progression of the disease, i.e., arresting its development; and (b) relieving the disease,
i.e., causing regression of the disease or disorder or alleviating symptoms or
complications thereof.
The bispecific antibody of the present invention is expected to treat autoimmune
diseases, including IBD (such as CD and UC), PsA and ankylosing spondylitis.
Pharmaceutical Composition
A bispecific antibody of the invention can be incorporated into a pharmaceutical
composition suitable for administration to a patient. A bispecific antibody of the
invention may be administered to a patient alone or with a pharmaceutically acceptable
carrier and I or diluent in single or multiple doses. Such pharmaceutical compositions are
designed to be appropriate for the selected mode of administration, and pharmaceutically
acceptable diluents, carrier, and I or excipients such as dispersing agents, buffers,
surfactants, preservatives, solubilizing agents, isotonicity agents, stabilizing agents and
the like are used as appropriate. Said compositions can be designed in accordance with
conventional techniques disclosed in, e.g., Remington, The Science and Practice of
Pharmacy, 22nd Edition, Loyd V, Ed., Pharmaceutical Press, 2012, which provides a
compendium of formulation techniques as are generally known to practitioners. Suitable
carriers for pharmaceutical compos_itions include any material which, when combined
with a bispecific antibody of the invention, retains the molecule's activity and is nonreactive
with the patient's immune system. A pharmaceutical composition of the present
20
invention comprises a bispecific antibody and one or more pharmaceutically acceptable
carriers, diluents or excipients.
A pharmaceutical composition comprising a bispecific antibody of the present
invention can be administered to a patient at risk for or exhibiting diseases or disorders as
described herein using standard administration techniques.
A pharmaceutical composition of the invention contains an "effective" amount of
a bispecific antibody of the invention. An effective amount refers to an amount necessary
(at dosages and for periods of time and for the means of administration) to achieve the
desired therapeutic result. An effective amount of the bispecific antibody may vary
according to factors such as the disease state, age, sex, and weight of the individual, and
the ability of the antibody or antibody portion to elicit a desired response in the
individual. An effective amount is also one in which any toxic or detrimental effect of
the bispecific antibody, are outweighed by the therapeutically beneficial effects.
Examples
Except as noted otherwise, the exemplified bispecific antibody referred to
throughout the Examples refers to the exemplified bispecific antibodies of the present
invention set forth in Tables 1(a) and (b) above.
Bispecific Antibody Expression and Purification
An exemplified bispecific antibody of the present invention set forth in Tables
1 (a) and (b) above (Bispecific Antibody 1) is expressed and purified essentially as
follows. A glutamine synthetase (GS) expression vector containing a DNA
polynucleotide sequence encoding for a polypeptide comprising the IgG HC-linker LlscFv
HCVR2-linker L2-scFv LCVR2 (polypeptide of SEQ ID NO: 1) and a second DNA
polynucleotide sequence encoding a polypeptide comprising the IgG LC (polypeptide of
SEQ ID NO: 3) is transfected into a GS-knockout Chinese hamster cell line (CHO) by
electroporation. The expression vector encodes an SV Early promoter (Simian Virus 40E)
and the gene for GS. Expression of GS allows for the biochemical synthesis of
glutamine, an amino acid required by the CHO cells. Post-transfection cells undergo bulk
21
selection with 50!-!M L-methionine sulfoximine (MSX). The inhibition ofGS by MSX is
utilized to increase the stringency of selection. Cells with integration of the expression
vector eDNA into transcriptionally active regions of the host cell genome can be selected
against CHO wild type cells, which express an endogenous level of GS. Transfected
pools are plated at low density to allow for close-to-clonal outgrowth of stable expressing
cells. These masterwells are screened for bispecific antibody expression and then scaled
up in serum-free suspension cultures to be used for production. Clarified medium, into
which the exemplified bispecific antibody has been secreted, is applied to a Protein A
affinity column that has been equilibrated with a compatible buffer such as 20mM TRIS
(pH 8.0). The column is washed to remove nonspecific binding components. The bound
bispecific antibody is eluted, for example, by pH step or gradient such as 20mM citrate
(pH 3.0) and neutralized with Tris (pH 8) buffer. Bispecific antibody fractions are
detected, such as by SDS-PAGE or analytical size-exclusion, and then are pooled.
Soluble aggregate and multimers may be effectively removed by common techniques
including size exclusion, hydrophobic interaction, ion exchange, or hydroxyapatite
chromatography. Cation-exchange chromatography is used for Bispecific Antibody 1.
Bispecific Antibody 1 is concentrated and I or sterile filtered using common techniques.
The purity of Bispecific Antibody 1 after these chromatography steps is greater than 97%
(monomer). The bispecific antibody may be immediately frozen at -70°C or stored at 4°C
for several months.
The second exemplified bispecific antibody of the present invention (hereinafter
referred to as Bispecific Antibody 2), which, relative to Bispecific Antibody 1,
incorporates an engineered single amino acid change that substitutes threonine (T) for
glutamine (Q) at position 684 (Q684T)(SEQ ID NO: 1 vs. SEQ ID NO: 2), is expressed in
transiently transfected CHO and by Protein A and hydrophobic interaction
chromatography. A glutamine synthetase (GS) expression vector containing a DNA
polynucleotide sequence encoding for a polypeptide comprising the IgG HC-linker L 1-
scFv HCVR2-linker L2-scFv LCVR2 (polypeptide of SEQ ID NO: 2) and a second DNA
polynucleotide sequence encoding a polypeptide comprising the IgG LC (polypeptide of
22
SEQ 10 NO: 3) is transiently transfected into GS-knockout Chinese hamster cell line
(CHO) by chemical treatment with polyethyleimine. The remaining expression and
purification steps are the same as for Bispecific Antibody 1. The purity of Bispecific
Antibody 2 after these chromatography steps is greater than 98% (monomer).

We Claim:
1. A bispecific antibody comprising an immunoglobulin G (IgG) antibody
and two single chain variable fragments (scFv) wherein,
(a) said lgG comprises two heavy chains (HC) and two light chains (LC), each
HC comprises a heavy chain variable region (HCVRl) comprising heavy
chain CDRs (HCDR) 1-3 and each light chain comprises a light chain
variable region (LCVR1) comprising light chain CDRs (LCDR) 1-3,
wherein the amino acid sequence ofHCDR1 is SEQ ID NO: 10, the amino
acid sequence ofHCDR2 is SEQ ID NO: 11, the amino acid sequence of
HCDR3 is SEQ ID NO: 12, the amino acid sequence ofLCDR1 is SEQ ID
NO: 16, the amino acid sequence ofLCDR2 is SEQ ID NO: 17, and the
amino acid sequence ofLCDR3 is SEQ ID NO: 18; and
(b) each scFv comprises a heavy chain variable region (HCVR2) and a light
chain variable region (LCVR2), the HCVR2 comprising HCDRs 4-6, and
the LCVR2 comprising LCDRs 4-6, wherein the amino acid sequence of
HCDR4 is SEQ ID NO: 13, the amino acid sequence of HCDR5 is SEQ
ID NO: 14, the amino acid sequence ofHCDR6 is SEQ ID NO: 15, the
amino acid sequence ofLCDR4 is SEQ ID NO: 19, the amino acid
sequence ofLCDR5 is SEQ ID NO: 20, and the amino acid sequence of
LCDR6 is SEQ ID NO: 21 or SEQ ID NO: 22,
wherein each scFv is linked at theN-terminus of HCVR2 of each scFv to
said IgG antibody at the C-terminus of each lgG HC via a polypeptide linker (L 1 ),
wherein the HCVR2 of each scFv is linked at the C-terminus ofthe
HCVR2 to the LCVR2 ofthe same scFv at theN-terminus of the LCVR2 ofthe
same scFv via a second polypeptide linker (L2)
and wherein the bispecific antibody binds to human calcitonin gene related
peptide (CGRP) and the p19 subunit of human IL-23.
43
2. A bispecific antibody according to claim 1, wherein the amino acid
sequence ofLCDR6 is SEQ ID NO: 21.
3. A bispecific antibody according to claim 1, wherein the amino acid
sequence of LCDR6 is SEQ ID NO: 22.
4. A bispecific antibody according to claim 1, wherein the amino acid
sequence ofHCVR1 of each HC is SEQ ID NO: 5, the amino acid sequence of
LCVR1 of each LC is SEQ ID NO: 7, the amino acid sequence ofHCVR2 of each
scFv is SEQ ID NO: 6 and the amino acid sequence ofLCVR2 of each scFv is
SEQ ID NO: 8 or SEQ ID NO: 9.
5. A bispecific antibody according to claim 4, wherein the amino acid
sequence ofLCVR2 of each scFv is SEQ ID NO: 8.
6. A bispecific antibody according to claim 4, wherein the amino acid
sequence ofLCVR2 of each scFv is SEQ ID NO: 9.
7. A bispecific antibody according to claim 1 or claim 4, wherein the amino
acid sequence of each HC is SEQ ID NO: 4, the amino acid sequence of each LC
is SEQ ID NO: 3, the amino acid sequence ofHCVR2 of each scFv is SEQ ID
NO: 6 and the amino acid sequence of LCVR2 of each scFv is SEQ ID NO: 8 or
SEQ IDNO: 9.
8. A bispecific antibody according to claim 7, wherein the amino acid
sequence ofLCVR2 of each scFv is SEQ ID NO: 8.
9. A bispecific antibody according to claim 7, wherein the amino acid
sequence ofLCVR2 of each scFv is SEQ ID NO: 9.
44
10. A bispecific antibody according to any one ofthe preceding claims,
wherein the amino acid sequence of L 1 is SEQ ID NO: 23 and the amino acid
sequence of L2 is SEQ ID NO: 24.
11. A bispecific antibody according to any one of claims 1, 4 or 7, wherein the
amino acid sequence of each HC is SEQ ID NO: 4, the amino acid sequence of
each LC is SEQ ID NO: 3, the amino acid sequence of HCVR2 of each scFv is
SEQ ID NO: 6, the amino acid sequence of LCVR2 of each scFv is SEQ ID NO:
8, the amino acid sequence ofL1 is SEQ ID NO: 23 and the amino acid sequence
of L2 is SEQ ID NO: 24.
12. A bispecific antibody according to any one of claims 1, 4 or 7, wherein the
amino acid sequence of each HC is SEQ ID NO: 4, the amino acid sequence of
each LC is SEQ ID NO: 3, the amino acid sequence of HCVR2 of each scFv is
SEQ ID NO: 6, the amino acid sequence ofLCVR2 of each scFv is SEQ ID NO:
9, the amino acid sequence ofL1 is SEQ ID NO: 23 and the amino acid sequence
ofL2 is SEQ ID NO: 24.
13. A DNA molecule comprising a polynucleotide sequence encoding a
polypeptide chain comprising a HC, a scFv, a first polypeptide linker L1 and a
second polypeptide linker L2 of the bispecific antibody of any one of claims 1-12.
14. A DNA molecule according to claim 13, wherein the amino acid sequence
of the encoded polypeptide chain is SEQ ID NO: 1 or SEQ ID NO: 2.
15. An expression vector comprising a DNA molecule according to claim 13
or claim 14 and a DNA molecule comprising a polynucleotide sequence encoding
a polypeptide chain comprising a LC of the hi specific antibody of any one of
claims 1-12, wherein the amino acid sequence of the LC is SEQ ID NO: 3.
45
16. A recombinant host cell comprising the DNA molecule of claim 14 and a
DNA molecule comprising a polynucleotide sequence encoding a polypeptide
chain comprising a LC of the bispecific antibody of any one of claims 1-12,
wherein the amino acid sequence of the LC is SEQ ID NO: 3, which cell is
capable of expressing a bispecific antibody according to claim 11 or claim 12.
1 7. A process for producing a bispecific antibody according to claim 11 or
claim 12, said process comprising the steps of:
a) cultivating a recombinant host cell of claim 16, under conditions such that said
bispecific antibody is expressed; and
b) recovering from said host cell the expressed bispecific antibody.
18. A bispecific antibody produced by the process of claim 17.
19. A pharmaceutical composition comprising a bispecific antibody according
to any one of claims 1-12 and 18 and one or more pharmaceutically acceptable
carriers, diluents or excipients.
20. A method of treating autoimmune diseases comprising administering to a
patient in need thereof an effective amount of a bispecific antibody according to
any of claims 1-12 and 18.
21. A method according to claim 20, wherein the autoimmune disease is an
inflammatory bowel disease.
22. A method according to claim 21, wherein the inflammatory bowel disease
is Crohn's Disease or Ulcerative Colitis.
46
23. A method according to claim 20, wherein the autoimmune disease is
psoriatic arthritis or ankylosing spondylitis.
24. A bispecific antibody according to any one of claims 1-12 and 18 for use
in therapy.
25. A bispecific antibody according to any one of claims 1-12 and 18 for use
in the treatment of autoimmune diseases.
26. A bispecific antibody for use according to claim 25, wherein the
autoimmune disease is an inflammatory bowel disease.
27. A bispecific antibody for use according to claim 26, wherein the
inflammatory bowel disease is Crohn's Disease or Ulcerative Colitis.
28. A bispecific antibody for use according to claim 25, wherein the
autoimmune disease is psoriatic arthritis or ankylosing spondylitis.