ANTI-TNF-ANTI-IL-17 BISPECIFIC ANTIBODIES
The present invention is in the field of medicine, particularly in the novel field of
bispecific antibodies directed against Tumor Necrosis Factor alpha (TNFa) and
Interleukin-17 (IL-17A). The bispecific antibodies of the present invention are expected
to be useful in treating Rheumatoid Arthritis (RA), Psoriatic Arthritis (PsA), and
Ankylosing Spondylitis (AS).
RA is a systemic, chronic, inflammatory disease. The inflammation is primarily
driven by a multitude of cytokines, including TNFa and IL-17. Current FDA approved
bioproducts (e.g., HUMIRA® that bind to and neutralize TNFa have demonstrated
efficacy in reducing signs and symptoms of RA and in slowing progression of RA in a
subset of patients. IL-17 antibodies are also being studied in clinical trials (secukinumab,
ixekizumab, and brodalumab) for various autoimmune diseases, such as rheumatoid
arthritis. However, because inflammation is driven by multiple cytokines, it would be
advantageous to target two cytokines in a single antibody. It would therefore be
advantageous to target both TNFa and IL-17 simultaneously to alleviate inflammation
and reduce the immune response in RA patients to a minimum.
Currently, co-administration of a TNFa antibody and anIL-17 antibody requires
either injections of two separate products or a single injection of a co-formulation of two
different antibodies. Two injections would permit flexibility of dose amount and timing,
but are inconvenient to patients both for compliance and for pain. A co-formulation
might also provide some flexibility of dose amounts, but it is quite challenging or
impossible to find formulation conditions that permit chemical and physical stability of
both antibodies due to different molecular characteristics of the two different antibodies.
Furthermore, co-administration or co-formulation involves the additive costs of two
different drug therapies, which can increase patient and/or payor costs, whereas a single
bispecific antibody allows the price to be optimized for the benefit delivered.
WO2010/102251 discloses a dual variable domain immunoglobulin ("DVD-Ig")
that binds TNFa and IL-17. A DVD-Ig is a multispecific immunoglobulin that has two
identical antigen binding arms with identical specificity and identical CDR sequences,
and is bivalent for each antigen to which it binds. Each antigen binding arm has two
different variable domains linked in tandem without an intervening constant region
between the variable domains, and each variable domain has specificity for a different
antigen. WO1995/09917 discloses a method for producing bispecific, tetravalent
antibodies using recombinant DNA technology by producing a single chain antibody
fused to a complete antibody having a different specificity. This gene fusion is expressed
by transfection resulting in a tetravalent antibody having dual specificity. U.S. Patent No.
6,090,382 discloses human antibodies that bind to and neutralize hTNFa.
WO2007/070750 discloses anti-IL-17 antibodies that bind and neutralize human IL-17.
Despite the disclosures above, significant problems associated with chemical and
physical stability were encountered when building a bispecific antibody of the present
invention. Many changes were required in the starting bispecific antibody to sufficiently
overcome myriad issues, including stabilizing the VH/VL interface of the single chain
fragment variable region, increasing thermal stability, decreasing aggregation, and
rebalancing the electrostatic distribution in the binding surfaces of the bispecific antibody,
all while maintaining binding affinity for both antigens.
Therefore, a need still exists for a single bispecific antibody that neutralizes both
human TNFa and human IL-17. It is desirable to provide a bispecific antibody that is
thermally stable, physically stable, exhibits low aggregation, and neutralizes human
TNFa and human IL-17. It is also desirable to provide a pharmaceutical composition
including a single bispecific antibody that neutralizes both human TNFa and human IL-
17, thereby avoiding the challenges of finding formulation conditions that must satisfy the
different molecular characteristics of two different, separate antibodies. The present
invention therefore seeks to address one or more of the above mentioned problems.
The present invention provides a bispecific antibody comprising a first
polypeptide and a second polypeptide, wherein the first polypeptide has amino acid
sequence of SEQ ID NO: 1, and the second polypeptide has an amino acid sequence of
SEQ ID NO: 2.
The present invention provides a bispecific antibody comprising two first
polypeptides and two second polypeptides, wherein the first polypeptide has amino acid
sequence of SEQ ID NO: 1, and the second polypeptide has an amino acid sequence of
SEQ ID NO: 2.
The present invention also provides a DNA molecule comprising a polynucleotide
sequence encoding the first polypeptide.
The present invention further provides a DNA molecule comprising a
polynucleotide sequence encoding the second polypeptide.
The present invention provides a DNA molecule comprising a polynucleotide
sequence encoding the first and the second polypeptide.
The present invention also provides a mammalian cell transformed with DNA
molecule(s) wherein the cell is capable of expressing a bispecific antibody comprising the
first polypeptide and the second polypeptide.
The present invention provides a process for producing a bispecific antibody
comprising two first polypeptides and two second polypeptides, the process comprising
cultivating the mammalian cell under conditions such that the bispecific antibody is
expressed.
The present invention further provides a bispecific antibody produced by said
process.
The present invention also provides a method of treating rheumatoid arthritis,
psoriatic arthritis, or ankylosing spondylitis comprising administering to a patient in need
thereof a therapeutically effective amount of a bispecific antibody according to the
present disclosure.
The present invention provides a bispecific antibody according to the present
disclosure for use in therapy.
The present invention further provides the use of a bispecific antibody according
to the present disclosure for the manufacture of a medicament for use in treatment of
rheumatoid arthritis, psoriatic arthritis, or ankylosing spondylitis.
The present invention further provides a bispecific antibody according to the
present disclosure for use in the treatment of rheumatoid arthritis, psoriatic arthritis, or
ankylosing spondylitis.
The present invention also provides a pharmaceutical composition comprising the
bispecific antibody of the present invention and one or more pharmaceutically acceptable
carriers, diluents, or excipients.
As used herein, the term "human IL-17" is understood to encompass a
homodimeric protein comprising two 15 kD human IL-17A proteins (also known as
"human IL-17A"), as well as a heterodimeric protein comprising a 15kD human IL-17A
protein and a 15kD human IL-17F protein (also known as "human IL-17A/F").
As used herein, the term "bispecific antibody" is understood to comprise two first
polypeptides and two second polypeptides as described herein. The bispecific antibody
binds two different antigens with specificity for each antigen. The bispecific antibody is
capable of binding each antigen alone or each antigen simultaneously. It is further
understood that the term encompasses any cellular post-translational modifications to the
bispecific antibody including, but not limited to, glycosylation profiles.
The bispecific antibodies of the present invention comprise two first polypeptides
and two second polypeptides. One of the first polypeptides forms an inter-chain disulfide
bond with one of the second polypeptides. Each of the two first polypeptides forms two
inter-chain disulfide bonds with each other, and each of the first polypeptides forms at
least one intra-chain disulfide bond. The relationship of the polypeptides and the disulfide
bonds are shown in the following schematic for illustrative purposes only:
Second polypeptide
First polypeptide
Second polypeptide
The amino acid sequence of the first polypeptide is:
EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITW
NSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSL
DYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKR
VESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEV
QFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSN
KGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEW
ESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNH
YTQKSLSLSLGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYKF
TDYHIHWVRQAPGQCLEWMGVINPTYGTTDYNQRFKGRVTITADESTSTAYMEL
SSLRSEDTAVYYCARYDYFTGTGVYWGQGTLVTVSSGGGGSGGGGSGGGGSGG
GGSDIVMTQTPLSLSVTPGQPASISCRSSRSLVHSRGETYLHWYLQKPGQSPQLLI
YKVSNRFIGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHLPFTFGCGTKL
EIK (SEQ ID NO: 1).
An expression vector containing the DNA sequence of SEQ ID NO: 3 encodes a
first polypeptide having the amino acid sequence of SEQ ID NO: 1.
The amino acid sequence of the second polypeptide is:
DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQ
SGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKRTVA
APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ
DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ
ID NO: 2)
An expression vector containing the DNA sequence of SEQ ID NO: 4 encodes a
second polypeptide having the amino acid sequence of SEQ ID NO: 2.
An inter-chain disulfide bond of one of the first polypeptides and one of the
second polypeptides forms between cysteine residue 135 of SEQ ID NO: 1 and cysteine
residue 214 of SEQ ID NO: 2. One first polypeptide forms two inter-chain disulfide
bonds with the other first polypeptide. The first inter-chain disulfide bond forms between
cysteine residue 227 of the first polypeptide of SEQ ID NO: 1 and cysteine residue 227 of
the other first polypeptide of SEQ ID NO: 1. The second inter-chain disulfide bond forms
between cysteine residue 230 of the first polypeptide of SEQ ID NO: 1 and cysteine
residue 230 of the other first polypeptide of SEQ ID NO: 1.
At least one intra-chain disulfide bond is formed between cysteine residue 505 of
SEQ ID NO: 1 and cysteine residue 705 of SEQ ID NO: 1 in each of the first
polypeptides.
The first polypeptides comprise a first heavy chain variable region (HCVR1), a
heavy chain constant region (CH), a second heavy chain variable region (HCVR2), and a
second light chain variable region (LCVR2). The second polypeptides comprise a first
light chain variable region (LCVR1) and a light chain constant region (CL). The HCVR
and LCVR regions can be further subdivided into regions of hypervariability, termed
complementarity determining regions (CDR), interspersed with framework regions (FR).
Each HCVR and LCVR is composed of three CDRs and four FRs, arranged from aminoterminus
to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,
CDR3, FR4.
The 3 CDRs of HCVR1 are herein referred to as CDRHl-1, CDRHl-2, and
CDRHl-3. The 3 CDRs of HCVR2 are referred to as CDRH2-1, CDRH2-2, and CDRH2-
3. Likewise, the 3 CDRs of LCVR1 are referred to as CDRLl-1, CDRLl-2, and CDRL1-
3, and the 3 CDRs of LCVR2 are referred to as CDRL2-1, CDRL2-2, and CDRL2-3.
The CH is fused to HCVR2 by an amino acid linker (LI). HCVR2 is fused to
LCVR2 by an amino acid linker (L2).
The present invention also encompasses diabodies. Diabodies are bispecific
antibodies 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 chain, the variable domains pair with complementary domains of the other chain.
For example, if the bispecific antibody comprises two first polypeptides (for convenience,
1A and IB) and two second polypeptides (for convenience, 2A and 2B), HCVR2 of the
1A polypeptide pairs with complementary domains of LCVR2 of the IB polypeptide
instead of pairing with LCVR2 of the 1A polypeptide, and vice versa. Bispecific
diabodies as described herein maintain binding affinity and neutralization capacity for
both human TNFa and human IL-17.
Alternatively, it may be beneficial to purify out diabodies from the bispecific
antibodies described above. Diabody content can be up to 17% after cellular expression
and can be reduced to less than 1% after purification.
The relationship of the various regions and linkers is as follows, arranged from
amino-terminus to carboxy-terminus, according to the Kabat numbering convention:
Polypeptide 1 - SEQ ID NO:l Polypeptide 2 - SEQ ID NO:2
Region Positions Region Positions
FRHl-1 1-25 FRLl-1 1-23
CDRHl-1 26-35 CDRLl-1 24-34
HCVR1 FRH1-2 36-49 LCVR1 FRL1-2 35-49
TNF CDRHl-2 50-66 TNF CDRLl-2 50-56
FRH1-3 67-98 FRL1-3 57-88
CDRHl-3 99-110 CDRLl-3 89-97
FRH1-4 111-121 FRL1-4 98-107
Constant CH 122-447 Constant CL 108-214
Linker LI 448-461
FRH2-1 462-486
CDRH2-1 487-496
FRH2-2 497-510
HCVR2
CDRH2-2 511-527
IL-17
FRH2-3 528-559
CDRH2-3 560-569
FRH2-4 570-580
Linker L2 581-600
FRL2-1 601-623
CDRL2-1 624-639
FRL2-2 640-654
LCVR2
CDRL2-2 655-661
IL-17
FRL2-3 662-693
CDRL2-3 694-702
FRL2-4 703-712
Bispecific Antibody En2ineerin2
Significant problems associated with chemical and physical stability were
encountered when constructing a bispecific antibody of the present invention. For
example, the parental IL- 17 antibody exhibited physical stability limitations (e.g. , phase
separation) at high concentration. Additionally, a bispecific antibody constructed from
the parental IL- 17 antibody exhibited concentration-dependent self-aggregation.
Chemical modifications were therefore made in the CDRL2- 1 and CDRH2-2 portions of
the bispecific antibody to improve chemical and physical stability and reduce
concentration-dependent aggregation. Extensive protein stability and solubility studies in
combination with LC/MS identified chemically unstable residues in CDRL2- 1 and
CDRH2-2. These labile residues were replaced with charge neutral amino acids using
targeted libraries constructed by codon depletion. Replacing these labile residues led to
improved chemical stability. Additionally, the electrostatic surface of the bispecific
antibody was calculated and charged patches were identified. Disrupting these charged
patches led to a decrease in protein self-association. Thus, mutations were identified in
the CDRH2-1 and CDRL2-1 portions of the bispecific antibody that rebalanced the
surface electrostatic distribution, improved thermal stability, reduced aggregation, and
improved chemical stability (eliminating specific deamidation and oxidation sites). None
of the above modifications were identified in initial characterizations of the parental
single antibodies. These changes were encountered only in the context of constructing a
bispecific antibody, suggesting that the local environment around the mutated areas of the
single antibody differed in the context of a bispecific antibody.
Further chemical modifications were made to reduce bispecific antibody
aggregation. In particular, chemical modifications were made to stabilize the VH/VL
interface in the IL- 17 portion of the bispecific antibody. Studies conducted to determine
the driving force behind bispecific antibody aggregation showed that the observed protein
self-association was not driven by conformational instability of the individual VH or VL
domains, but rather by the opening or "breathing" of the VH-VL interface, leading to
intermolecular protein interactions. Thus, various intra-chain disulfide bonds were
introduced into the VH-VL interface of the IL-17 portion of the bispecific antibody. One
such intra-chain disulfide bond occurs in each of the first polypeptides between cysteine
residue 505 of SEQ ID NO: 1 and cysteine residue 705 of SEQ ID NO: 1. This disulfide
bond covalently connects the VH and VL interface in the IL- 17 portion of the bispecific
antibody, which stabilizes the VH-VL interface and reduces intermolecular protein
interactions that can lead to physical instability and unfavorable formulation limitations.
Out of the nine different disulfide bonds tested, 8 of which expressed functional protein,
the magnitude of affinity loss ranged from about 2 to about 35-fold. The intra-chain
disulfide bond in each of the first polypeptides between cysteine residue 505 of SEQ ID
NO: 1 and cysteine residue 705 of SEQ ID NO: 1 best stabilized the VH/VL interface
while maintaining optimal binding affinity for IL-17.
In addition, studies indicated that linker length for LI affected functional activity
of the bispecific antibody, particularly binding kinetics. Kinetic analysis (by surface
plasmon resonance) showed that a 10 amino acid linker caused a 2-fold slower Kon rate
compared to 15 amino acid and 20 amino acid linkers. Thus, a minimum linker length of
15 was introduced into the bispecific antibody of the present invention.
The bispecific antibody of the present invention was also engineered to reduce or
eliminate activation of the immune system via interaction with Fey receptors. Immune
activation is not part of the intended mechanism of action of the bispecific antibody of the
present invention. To that end, the bispecific antibody of the present invention was
constructed as an IgG4 isotype, which is known to have low binding ability to Fey
receptors or components of the complement system. In addition, two alanine mutations
were made in the lower hinge region to further reduce this binding potential.
Bispecific Antibody Binding
The bispecific antibodies of the present invention bind both human TNFa and
human IL-17. The bispecific antibodies of the present invention neutralize at least one
human TNFa bioactivity and at least one human IL-17 bioactivity in vitro or in vivo. The
bispecific antibodies of the present invention are potent inhibitors of IL-17 in vitro, and of
both soluble and membrane-bound TNFa in vitro.
The bispecific antibodies of the present invention have a binding affinity (KD) for
human TNFa in the range of about 30 pM to about 1 pM, and for human IL-17A in the
range of about 40 pM to about 1 pM. Further, the bispecific antibodies of the present
invention have a K for human IL-17A/F heterodimer of in the range of about 50 pM to
about 1 pM. In an aspect, the bispecific antibodies of the present invention have a K for
human TNFa ranging from about 2 1 pM to about 3 pM. In another aspect, the bispecific
antibodies of the present invention have a KD for human IL-17A ranging from about 8 pM
to about 10 pM.
Bispecific Antibody Expression
Expression vectors capable of directing 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
and the second polypeptide may be expressed independently from different promoters to
which they are operably linked in one vector or, alternatively, the first polypeptide and
the second polypeptide may be expressed independently from different promoters to
which they are operably linked in two vectors - one expressing the first polypeptide and
one expressing the second polypeptide.
A host cell includes cells stably or transiently transfected, transformed,
transduced, or infected with one or more expression vectors expressing a first
polypeptide, a second polypeptide, or both a first polypeptide and a second polypeptide 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 of the present invention 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 Fc region of the antibody at a highly
conserved N-glycosylation site. N-glycans typically attach to asparagine. Each of the
first polypeptides is glycosylated at asparagine residue 300 of SEQ ID NO: 1.
A particular DNA polynucleotide sequence encoding the first polypeptide having
an amino acid sequence of SEQ ID NO: 1 is:
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGAGGTCCC
TGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGATGACTATGCCATGCAC
TGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGTCAGCTATTACTT
GGAATAGTGGTCACATAGACTACGCAGACTCCGTGGAGGGCCGGTTCACCAT
CTCCAGAGACAATGCCAAGAACTCCCTGTATCTGCAAATGAACAGCCTGAGA
GCCGAGGACACGGCCGTATATTACTGTGCGAAAGTGAGCTACCTGAGTACTG
CCTCCAGCCTGGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCCTCAGCC
TCCACCAAGGGCCCATCGGTCTTCCCGCTAGCGCCCTGCTCCAGGAGCACCTC
CGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCG
GTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCC
CGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTG
CCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGC
CCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATG
CCCACCCTGCCCAGCACCTGAGGCCGCCGGGGGACCATCAGTCTTCCTGTTCC
CCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTG
CGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTAC
GTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAG
TTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTG
GCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCC
TCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGG
TGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCT
GACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAA
AGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACT
CCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTG
GCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAAC
CACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGAGGCGGAGGATCCGGGG
GAGGGGGTTCCGGAGGAGGGGGCTCGCAGGTGCAGCTGGTGCAGTCTGGGGC
TGAGGTGAAGAAGCCTGGGTCCTCAGTGAAGGTTTCCTGCAAGGCATCTGGT
TACAAGTTCACTGACTACCATATTCATTGGGTGCGACAGGCCCCTGGACAATG
CCTTGAGTGGATGGGAGTAATTAATCCTACTTATGGTACTACTGACTACAATC
AGCGGTTCAAAGGCCGTGTCACCATTACCGCGGACGAATCCACGAGCACAGC
CTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGT
GCGAGATATGATTACTTTACTGGGACGGGTGTGTACTGGGGCCAAGGAACCC
TGGTCACCGTCTCCTCAGGTGGCGGAGGATCTGGTGGAGGTGGCTCAGGAGG
TGGCGGAAGCGGCGGAGGTGGAAGTGATATTGTGATGACTCAGACTCCACTC
TCCCTGTCCGTCACCCCTGGACAGCCGGCCTCCATCTCCTGCAGATCTAGTAG
GAGCCTTGTACACAGTCGTGGAGAAACCTATTTACATTGGTATCTGCAGAAGC
CAGGCCAATCTCCACAGCTCCTAATTTATAAAGTTTCCAACCGGTTTATTGGG
GTCCCAGACAGATTCAGCGGCAGTGGGTCAGGCACAGATTTCACACTGAAAA
TCAGCAGGGTGGAGGCCGAAGATGTTGGGGTTTATTACTGCTCTCAAAGTAC
ACATCTTCCATTCACGTTTGGCTGCGGGACCAAGCTGGAGATCAAA
(SEQ ID NO: 3)
A particular DNA polynucleotide sequence encoding the second polypeptide
having an amino acid sequence of SEQ ID NO: 2 is:
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAG
AGTCACCATCACTTGCCGGGCGAGTCAGGGCATTCGCAATTATTTAGCCTGGT
ATCAGCAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATGCTGCATCCAC
TTTGCAATCAGGGGTCCCATCTCGGTTCAGTGGCAGTGGATCTGGGACAGATT
TCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATTACTGT
CAACGCTATAACCGTGCCCCTTACACGTTCGGCCAAGGGACCAAGGTGGAAA
TCAAACGGACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAG
CAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCC
CAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAAC
TCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCA
GCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGC
CTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAAC
AGGGGAGAGTGC (SEQ ID NO: 4)
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, or may be lyophilized.
There may be a need to reduce the level of diabody present in the medium.
For example, the medium containing the diabody may be applied to and eluted from
strong cation exchange resin. For example, SP-Sepharose HP strong cation exchange
resin is used to purify correctly-folded bispecific antibody from diabody. The pH of the
medium containing the diabody is adjusted to pH 8.1 using 20 mM Bicine. The medium
is loaded onto an SP-Sepharose HP column, washed with 2 column volumes of 20 mM
Bicine (pH 8.1), and eluted with 20 mM Bicine and 100 mM NaCl (pH 8.1) over 20
column volumes (10-90 mM NaCl). The collected pools can be assessed for high
molecular weight versus main peak. A typical result is an improvement from about 17%
diabody to less than 1% diabody with about 68% recovery.
Optionally, diabody may be purified according to the following non-limiting
procedure: Clarified medium into which the bispecific antibody and diabody have been
secreted can be applied to a Protein A affinity column that has been equilibrated with a
compatible buffer, such as phosphate buffered saline (pH 7.4). The column can be
washed to remove nonspecific binding components. The bound bispecific antibody and
diabody can be eluted, for example, by pH gradient (such as 0.1 M sodium phosphate
buffer pH 6.8 to 0.1 M sodium citrate buffer pH 2.5). The bispecific diabody fractions
can be detected by limited lysyl endopeptidase (LysC) digestion to cut between the Fc
region and the ScFv/diabody region, followed by reverse phase HPLC quantitative
analysis. Briefly, 15 g of sample can be digested for approximately 20 hours at 37°C
with 0.2 g of LysC (Wako, P/N 125-05061) in 20 mM Tris pH 8.0 + 0.1 mg/mL
iodoacetamide in a total volume of 50 m . Samples can be analyzed by injecting 20 m
(6 g) on a PLRP-S 50x2.1 mm reversed phase column (Varian P/N PL1912-1802).
Flow rate can be 0.6 mL/min, column temperature can be 80°C, detection can be at 214
nm, Buffer A can be 0.05% TFA in water, and Buffer B can be 0.04% TFA in
acetonitrile. ScFv and diabody peaks (previously identified by LC-MS) can be
determined by integrating the appropriate peaks. Material from cation exchange (CEX)
chromatography containing the bispecific diabody can be pooled and di-filtered into PBS,
pH 7.0. To remove high molecular weight aggregates, the CEX pool can be placed over a
Superdex 200 50/60 SEC column run at 7 mL/min in PBS, pH7. The bispecific diabody
pool can be determined by SDS-PAGE and analytical SEC analysis. SEC pool can be
then diluted 5 fold into the following buffer system: 3.3 mM MES, 3.3 mM Hepes, 3.3
mM Tris, 3.3 mM Bis-Tris Propane, 3.3 mM CHES, 3.3 mM CAPS, pH 5.8. The diluted
protein pool can be then loaded onto a preparative ProPAC WCX-10 BioLC cation
exchange column (22 x 250 mm prep scale) at 15 mL/min. Using the buffer system
described previously, the bispecific diabody can be separated from bispecific antibody by
elution using a linear pH gradient from pH 8.4 to pH 11 at 15 mL/min over 45 minutes
collecting 7.5 mL fractions. The ProPac CEX pool made can be based on analytical SEC
(TSK3000), analytical CEX (ProPac WCX-10), gel analysis (NuPAGE with MES buffer
system), and Lys C digest to measure diabody content in each fraction. The final ProPac
CEX pool can be dialyzed into PBS, pH7.
This purification process can remove reduce the diabody content from up to 12% diabody
to less than 5% diabody.
Pharmaceutical Compositions and Therapeutic Uses
The bispecific antibody of the invention is expected to treat rheumatoid arthritis,
psoriatic arthritis, and ankylosing spondylitis. A "patient" refers to a mammal, preferably
a human with a disease, disorder, or condition that would benefit from a decreased level
of TNF and/or IL-17 or decreased bioactivity of TNF and/or IL-17.
"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 of
the 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 a
human, 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 invention can be incorporated into pharmaceutical
compositions suitable for administration to a subject. Typically the pharmaceutical
composition comprises a bispecific antibody of the invention and a pharmaceutically
acceptable carrier. As used herein, "pharmaceutically acceptable carrier" includes any
and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic
and absorption delaying agents, and the like that are physiologically compatible.
Pharmaceutically acceptable carriers may further comprise minor amounts of auxiliary
substances that enhance the shelf life or effectiveness of the bispecific antibody.
The compositions of this invention may be in a variety of forms. The preferred
form depends on the intended mode of administration and therapeutic application.
Typical preferred compositions are in the form of injectable or infusible solutions, such as
compositions similar to those used for passive immunization of humans with other
antibodies. The preferred mode of administration is parenteral (e.g., intravenous,
subcutaneous, intraperitoneal, intramuscular). In anembodiment, the bispecific antibody
is administered by subcutaneous injection. However, as will be appreciated by the
skilled artisan, the route and/or mode of administration will vary depending upon the
desired results
The pharmaceutical compositions of the invention may include a "therapeutically
effective amount" of a bispecific antibody of the invention. A "therapeutically effective
amount" refers to an amount effective, at dosages and for periods of time necessary, to
achieve the desired therapeutic result. A therapeutically 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 bispecific antibody to elicit a desired
response in the individual. A therapeutically effective amount is also one in which any
toxic or detrimental effects of the bispecific antibody are outweighed by the
therapeutically beneficial effects.
Dosage regimens may be adjusted to provide the optimum desired response (e.g.,
a therapeutic response). For example, a single bolus may be administered, several
divided doses may be administered over time, or the dose may be proportionally reduced
or increased as indicated by the exigencies of the therapeutic situation.
Dosage values may vary with the type and severity of the condition to be
alleviated. It is further understood that for any particular subject, specific dosage
regimens should be adjusted over time according to the individual need and the
professional judgment of the person administering or supervising the administration of
the compositions.
In another embodiment, the invention provides a method for treating autoimmune
diseases, particularly those associated with inflammation, for example rheumatoid
arthritis, psoriatic arthritis, and ankylosing spondylitis. Typically, the bispecific antibody
is administered systemically, although for certain disorders, local administration of the
bispecific antibody at a site of inflammation may be beneficial.
This invention is further illustrated by the following non-limiting example.
EXAMPLE
Expression and Purification of the Bispecific Antibody
The bispecific antibody can be expressed and purified essentially as follows. A
glutamine synthetase (GS) expression vector containing the DNA of SEQ ID NO: 3
(encoding the first polypeptide having amino acid sequence of SEQ ID NO: 1) and SEQ
ID NO: 4 (encoding the light chain amino acid sequence of SEQ ID NO: 2) is used to
transfect the Chinese hamster cell line, CHOK1SV (Lonza Biologies PLC, Slough,
United Kingdom) by electroporation. The expression vector encodes an SV Early
(Simian Virus 40E) promoter and the gene for GS. Expression of GS allows for the
biochemical synthesis of glutamine, an amino acid required by the CHOK1SV cells.
Post-transfection, cells undergo bulk selection with 50 mM L-methionine sulfoximine
(MSX). The inhibition of GS by MSX is utilized to increase the stringency of selection.
Cells with integration of the expression vector cDNA into transcriptionally active regions
of the host cell genome are selected against CHOK1SV wild type cells, which express an
endogenous level of GS. Transfected pools are plated at low density to allow for closeto-
clonal outgrowth of stable expressing cells. The 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 bispecific antibody has been
secreted, is applied to a Protein A affinity column that has been equilibrated with a
compatible buffer, such as phosphate buffered saline (pH 7.4). The column is washed to
remove nonspecific binding components. The bound bispecific antibody is eluted, for
example, by pH gradient (such as 0.1 M sodium phosphate buffer pH 6.8 to 0.1 M sodium
citrate buffer pH 2.5). 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. The bispecific antibody
may be concentrated and/or sterile filtered using common techniques. The purity of the
bispecific antibody after these chromatography steps is greater than 98%. The bispecific
antibody may be immediately frozen at -70°C or stored at 4°C for several months.
Binding affinity to TNF and IL-17
TNFa
Binding affinity of the bispecific antibody to human TNFa is determined using a
solution equilibrium binding assay on a Sapidyne KinExA 3000 instrument at 37°C using
Blocker Casein in PBS (Pierce) for running buffer and sample diluent. Human TNFa is
immobilized on NHS sepharose through standard amine coupling chemistry. Samples are
prepared by mixing the bispecific antibody at a fixed concentration of 20 pM with human
TNFa at concentrations of 200, 100, 50, 25, 12.5, 6.25, 3.13, 1.56, 0.78, 0.39, and 0
(blank) M. Samples are incubated for 18 hours at 37 °C to reach equilibrium prior to
analysis. Each analysis cycle consists of (1) packing a column of human TNFa beads by
injecting 367 m of beads at 1 mL/min, (2) injecting 10 mL (20 minute) of bispecific
antibody/human TNFa complex over the column at 0.5 mL/min, (3) injecting 0.5 mL (2
minute) of buffer at 0.25 mL/min to wash out unbound sample, (4) injecting 1mL (30
sec) of 500 ng/mL DyLight-649 Rabbit Anti-Human IgG detection antibody (Jackson
ImmunoResearch), (5) injecting 2.25 mL (90 sec) of buffer at 1.5 mL/min to wash out
unbound detection antibody, and (6) cleaning the system with a 1mL (60 sec) injection of
IN NaOH followed by a backflush. Data are fit using N-curve analysis of two replicate
experiments using the KinExA Pro Software, version 2.0.1.14. The equilibrium
dissociation constant (¾) is calculated from the percent free bispecific antibody. The
bispecific antibody of the present invention showed a ¾ for human TNFa of 4.4 pM
(95% confidence interval of 0.6 to 16.3 pM).
IL-17
Binding affinity of the bispecific antibody to human IL-17 is determined using a
surface plasmon resonance assay on a Biacore T200 instrument primed with HBS-EP+
(GE Healthcare, 10 mM Hepes pH7.4 + 150 mM NaCl + 3 mM EDTA + 0.05%
surfactant P20) running buffer and analysis temperature set at 37°C. A CM4 chip
containing immobilized protein A (generated using standard NHS-EDC amine coupling)
on all four flow cells (Fc) is used to employ a capture methodology. Antibody samples
are prepared at 4 g/mL by dilution into running buffer. Human IL-17 is prepared at final
concentrations of 80.0, 40.0, 20.0, 10.0, 5.0, 2.5, 1.25, and 0 (blank) nM by dilution into
running buffer. Each analysis cycle consists of (1) capturing antibody samples on
separate flow cells (Fc2, Fc3, and Fc4), (2) injecting 200 m (120 sec) of human IL-17
over all flow cells at 100 m /ih h, (3) returning buffer flow for 20 min to monitor
dissociation phase, (4) regeneration of chip surfaces with a 10 m (20 sec) injection of
glycine, pH 2.0. Data are processed using standard double-referencing and fit to a 1:1
binding model using Biacore T200 Evaluation software, version 1.0, to determine the
association rate (kon) and dissociation rate (k ff) . The equilibrium dissociation constant
(KD) is calculated as from the relationship KD = k0g kon.
Table 1: Binding affinity to human IL-17 by the bispecific antibody.
These results demonstrate that the bispecific antibody of the present invention
separately can bind human TNFa and human IL-17.
Simultaneous binding to human TNF and human IL-17
A Biacore T200 instrument is used to determine whether human TNFa and human
IL17 can bind to the bispecific antibody simultaneously. All Biacore reagents and
materials are purchased from Biacore unless otherwise noted. All measurements are
performed at 25°C. HBS-EP+ buffer (150 mM sodium chloride, 3 mM EDTA, 0.05%
(w/v) surfactant P-20, and 10 mM Hepes, pH 7.4) is used as the running buffer and
sample buffer. Protein A is immobilized on flow cells 1 and 2 of a CM4 sensor chip
using an amine coupling kit. The bispecific antibody diluted to 3 g/mL is first captured
on flow cell 2 with a 35 second injection at 30 mI7ih h yielding 165 resonance units (RU)
of antibody captured. This capture is followed by a 35 second injection of buffer. The
flow rate is then increased to 100 mI7ih h and flow is directed over flow cell 1 (Fcl) and
flow cell 2 (Fc2). To saturate TNFa binding, 50 nM of human TNFa is injected for 2
minutes. Reference- subtracted data are collected as Fc2-Fcl. A binding signal of 45 RU
is observed. After human TNFa injection, 80 nM of human IL-17 is injected for an
additional 2 minutes to saturate IL-17 binding. Again, reference-subtracted data are
collected as Fc2-Fcl. An additional binding signal of 37 RU is observed. The chip
surface is then regenerated using 10 mM Glycine, pH 1.5. These results demonstrate that
the bispecific antibody of the present invention can bind human TNFa and human IL17
simultaneously, as shown by the increase in resonance units (initial 45 RU from TNFa,
then additional 37 RU from human IL-17) from the two ligands binding to the bispecific
antibody.
Inhibition of IL-17-induced CXCL1 production in vitro from HT-29 cells
HT-29 cells are human colorectal adenocarcinoma epithelial cells that naturally
express the IL-17 receptor. Incubation of HT-29 cells with human IL-17 results in the
production of CXCL1, which can be measured using a commercially available ELISA.
A dose range of the bispecific antibody from 20 pM to 10 nM is evaluated (MW
of bispecific antibody is 200 kDa). Each test concentration of bispecific antibody is
added (50 E) to wells containing 50 E of 2 nM (final concentration) recombinant IL-
17. Testing is carried out in duplicate wells per treatment. Assay medium is used for
"medium alone" and "IL-17 alone" controls. An IL-17 neutralizing antibody (U.S. Patent
No. 7,838,638) is used as positive control in the assay. Control antibodies are tested at the
same molar range as the bispecific antibody. Plates containing IL-17 and antibody
mixtures are incubated for 60 to 90 minutes (at 37°C, 95% relative humidity, 5% C0 2) in
tissue-culture treated 96-well plates.
HT-29 cells are routinely cultured in assay medium (McCoy's 5A containing 10%
FBS, penicillinG (0.2 U/mL) and streptomycin (0.2 g/mL)). The cells are harvested one
day before the day of the assay. The cells are rinsed with l x PBS and detached from the
culture flasks with Cell Dissociation buffer, enzyme-free, PBS. Complete assay medium
is added to the detached cells. The cells are then centrifuged at 310Xg for 5 minutes at
room temperature. The cell pellet is resuspended in assay medium. Cell density is
measured with Invitrogen Countess, and 20,000 HT-29 cells (in 100 ) are added to
each of the 96-well plates. The 96-well plates are placed in a tissue culture incubator
(37°C, 95% relative humidity, 5% C0 2) overnight. The antibody/IL-17 mixtures (100
m ) are added to the HT-29 cells and incubated (37°C, 95% relative humidity, 5% C0 2)
for 24-48 hours.
At the end of the assay, the plates are centrifuged (500Xg for 5 minutes at room
temperature), and the cell culture medium is transferred to polypropylene 96-well plates,
which are sealed and frozen at -80°C. On the day of measuring CXCL1 by ELISA, the
plates are thawed at room temperature. CXCL1 levels in medium are measured with a
CXCL1 sandwich ELISA (R&D Systems DuoSet #DY275), as per the manufacturer's
instructions. At the end of the ELISA reactions, plates are read at 450 nm on a microplate
reader (Molecular Devices VersaMax Tunable). Results are expressed as the
concentration where 50% of the IL-17-induced response is inhibited (IC 50) by either
bispecific antibody or the positive control is calculated using a 4 parameter sigmoidal fit
of the data (GraphPad Prism).
The results demonstrate that the bispecific antibody of the present invention
inhibited IL-17-induced secretion of CXCLl by HT-29 cells in a concentration-dependent
manner. The inhibition was comparable to that observed with the positive control
antibody [with an IC50 for bispecific antibody of 0.628 + 0.072 nM versus 0.614 + 0.099
nM for the positive control antibody (average of 3 independent experiments + SEM)],
whereas the negative control antibody did not inhibit CXCLl production. The bispecific
antibody of the present invention effectively neutralized IL-17.
Inhibition of TNF-induced CXCLl production in vitro from HT-29 cells
HT-29 cells are human colorectal adenocarcinoma epithelial cells that naturally
express the TNF receptor. Incubation of HT-29 cells with human TNFa results in the
production of CXCLl, which can be measured using a commercially available ELISA.
A dose range of the bispecific antibody from 0.5 pM to 10 nM is evaluated (MW
of bispecific antibody is 200 kDa). Each test concentration of bispecific antibody is then
added (50 ) to wells containing 50 m of 30 pM (final concentration) recombinant
TNFa. Testing is carried out in duplicate wells per treatment. Assay medium is used for
"medium alone" and "TNF alone" controls. A TNF neutralizing antibody (adalimumab)
is used as positive control in the assay. Control antibodies are tested at the same molar
range as the bispecific antibody. Plates containing TNFa and antibody mixtures are
incubated for 60 to 90 minutes (at 37°C, 95% relative humidity, 5% C0 2) in tissue-culture
treated 96-well plates.
HT-29 cells are routinely cultured in assay medium (McCoy's 5A containing 10%
FBS, penicillinG (0.2 U/mL) and streptomycin (0.2 mcg/mL)). The cells are harvested
one day before the day of the assay. The cells are rinsed with l x PBS and detached from
the culture flasks with Cell Dissociation buffer, enzyme-free, PBS. Complete assay
medium is added to the detached cells. The cells are then centrifuged at 310Xg for 5
minutes at room temperature. The cell pellet is resuspended in assay medium. Cell
density is measured with Invitrogen Countess, and 20,000 HT-29 cells (in 100 ) are
added to each of the 96-well plates. The 96-well plates are placed in a tissue culture
incubator (37°C, 95% relative humidity, 5%C0 2) overnight. The antibody/TNFa
mixtures are added to the HT-29 cells and incubated (37°C, 95% relative humidity, 5%
C0 2) for 24 hours.
At the end of the assay, the plates are centrifuged (500xg for 5 minutes at room
temperature), and the cell culture medium is transferred to polypropylene 96-well plates,
which are sealed and frozen at -80°C. On the day of measuring CXCLl by ELISA, the
plates are thawed at room temperature. CXCLl levels in medium are measured with a
CXCLl sandwich ELISA (R&D Systems DuoSet #DY275), as per the manufacturer's
instructions. At the end of the ELISA reactions, plates are read at 450 nm on a microplate
reader (Molecular Devices VersaMax Tunable). Results are expressed as the
concentration where 50% of the TNF-induced response is inhibited (IC 50) by either
bispecific antibody or the positive control is calculated using a 4 parameter sigmoidal fit
of the data (GraphPad Prism).
The results demonstrate that the bispecific antibody of the present invention
inhibits TNF-induced secretion of CXCLl by HT-29 cells in a concentration-dependent
manner. The inhibition was comparable to that observed with the positive control
antibody [with an IC50 for bispecific antibody of 18.8 + 1 pM versus 14.0 + 2 pM for the
positive control antibody (average of 3 independent experiments + SEM)], whereas the
negative control antibody did not inhibit CXCLl production. The bispecific antibody of
the present invention effectively neutralized TNFa.
Inhibition of CXCLl production from HT-29 cells induced by combination of IL-17
and TNF
As described above, HT-29 cells are human colorectal adenocarcinoma epithelial
cells that naturally express the IL-17 and TNF receptors. Incubation of HT-29 cells with
human TNFa and human IL-17 results in the production of CXCLl, which can be
measured using a commercially available ELISA.
The antibodies are tested at a fixed dose of 4 nM (MW of bispecific antibody is
200 kDa). The bispecific antibody is then added (50 ) to wells containing 50 m of 3
pM recombinant TNFa and 50 m of 200 pM recombinant IL-17. Testing is carried out in
five replicate wells per treatment. Assay medium is used for "medium alone" and "IL-
17+TNF alone". Anti-IL-17 antibody (U.S. Patent No. 7,838,638); anti-TNFa antibody
(adalimumab); and combination of anti-IL-17antibody/anti-TNF antibody are used as
controls in the assay. Control antibodies are tested at the same molar range as the
bispecific antibody. Plates containing TNF+IL-17 and antibody mixtures are incubated
for 60 to 90 minutes (at 37°C, 95% relative humidity, and 5% C0 2) in tissue-culture
treated 96-well plates.
HT-29 cells are routinely cultured in assay medium [McCoy's 5A containing 10%
FBS, penicillinG (0.2 U/mL) and streptomycin (0.2 mcg/mL)]. The cells are harvested
one day before the day of the assay. The cells are rinsed with l x PBS and detached from
the culture flasks with Cell Dissociation buffer, enzyme-free, PBS. Complete assay
medium is added to the detached cells. HT-29 cells are then centrifuged at 310Xg for 5
minutes at room temperature. The cell pellet is resuspended in assay medium. Cell
density is measured with Invitrogen Countess, and 20,000 HT-29 cells (in 100 ) are
added to each of the 96-well plates. The 96-well plates are placed in a tissue culture
incubator (37°C, 95% relative humidity, 5%C0 2) overnight. The bispecific antibody/IL-
17/TNF mixtures are added to the HT-29 cells and incubated (37°C, 95% relative
humidity, 5% C0 2) for 24-48 h.
At the end of the assay, the plates are centrifuged (500xg for 5 minutes at room
temperature), and the cell culture medium is transferred to polypropylene 96-well plates,
which are sealed and frozen at -80°C. On the day of measuring CXCL1 by ELISA, the
plates are thawed at room temperature. CXCL1 levels in medium are measured with a
CXCL1 sandwich ELISA (R&D Systems DuoSet #DY275), as per the manufacturer's
instructions. At the end of the ELISA reactions, plates are read at 450 nm on a microplate
reader (Molecular Devices VersaMax Tunable). The results are expressed as percent
human CXCL1 (with TNF + IL-17 alone being 100%) left after incubation with various
antibodies: bispecific antibody 0.85 +/- 0.12 %; anti-TNFa 8.97 +/- 2.65%; anti-IL-17 27
+/- 2.07 %; anti-TNFa + anti-IL-17 0.59 +/- 1.23 %. The results demonstrated that the
bispecific antibody of the present invention inhibited simultaneous TNFa- and IL-17-
induced secretion of CXCL1 by HT-29 cells better than the single agents alone.
Inhibition of soluble TNFa-induced cytotoxicity in L929 cells in vitro
L929 cells are mouse fibrosarcoma cells that naturally express the TNF receptor.
Incubation of L929 cells with human TNFa results in rapid cell death due to excessive
formation of reactive oxygen intermediates. The cell death can be measured using an
MTT cytotoxicity assay, where mitochondrial succinate dehydrogenase in viable cells
reduces tetrazolium salt into formazan product, which can be detected with a fluorescence
plate reader.
A dose range of the bispecific antibody from 20 nM to 10 pM is evaluated (MW
of bispecific antibody is 200 kDa). Each test concentration of bispecific antibody (100
), 200 pg/mL recombinant human TNFa (100 ), and 6.25 g/mL Actinomycin-D
(100 ) are added to wells containing L929 cells. Testing is carried out in duplicate
wells per treatment. A TNFa neutralizing antibody (adalimumab with IgG4 isotype) is
used as a positive control in the assay. Plates containing antibody mixtures are incubated
for 60 minutes at room temperature.
L929 cells are routinely cultured in assay medium (lxDMEM Cellgro, 10% FBS,
1% Pen-Strep, 1% MEM essential amino acids, 1% L-glutamine, 1% sodium pyruvate).
On the day of the assay, the cells are rinsed with l x PBS (no Ca++ or Mg++) and detached
from the culture flasks with 0.25% trypsin + EDTA. The trypsin is inactivated with assay
medium. L929 cells are centrifuged at 215xg for 5 minutes at room temperature. The
cell pellet is resuspended in assay medium. Cell density is measured with a
hemocytometer, and 10,000 L929 cells (in 100 mE) are added to the 96-well plates and
placed in a tissue culture incubator (37°C, 95% relative humidity, 5% C0 2) over
night. The antibody/TNFa/actinomycin-D mixture is transferred to the 96 well plates
with L929 adherent cells and incubated 18 hrs at 37°C, 95% relative humidity, 5% C0 2.
The assay medium is removed and the MTT substrate mixture is added to the wells (120
mE) . The plates are placed at 37°C, 95% relative humidity, 5% C0 2 for 3 hours. The cell
death is determined by reading the plates at 490 nm on a microplate reader (Molecular
Devices SpectraMax 190). Results are expressed as the concentration where 50% of the
TNFa induced response is inhibited (IC 50) (average of four independent experiments +/-
SEM) by either the bispecific antibody or the positive control antibody calculated using a
4 parameter sigmoidal fit of the data (GraphPad Prism).
The results demonstrate that the bispecific antibody of the present invention
inhibited TNFa-induced killing of L929 cells in a dose-dependent manner with an IC50 of
226 +/- 52 pM. This inhibition was comparable to that observed with the positive control
antibody (IC 50 = 243 +/- 49 pM), whereas the negative control antibody did not inhibit
human TNFa. The bispecific antibody of the present invention effectively neutralized
human TNFa.
Inhibition of membrane bound human TNFa induced cytotoxicity in vitro in
L929 cells
In order to study the ability of the bispecific antibody to inhibit membrane bound
TNFa, known cleavage sites of TNFa are inactivated using a set of mutations that were
previously demonstrated to allow expression of bioactive TNFa on cell surface (Mueller
et. al. 1999) in the absence of TNF cleavage. The non-cleavable TNFa construct is stably
transfected to Chinese hamster ovary (CHO) cells. These cells express membrane bound
TNFa as shown by flow cytometry. Incubation of L929 cells with CHO cells expressing
human non-cleavable membrane bound TNFa results in rapid L929 cell death.
CHO cells expressing membrane bound human TNFa are routinely maintained in
selection medium (AM2001 media, an internal CHO growth media without MSX, 8 mM
glutamine, GS supplement, HT supplement with 500 g/mL G418). On the day of the
assay, the cells are counted, rinsed with l x PBS (no Ca - or Mg++), centrifuged at 215xg
for 5 min and re-suspended at 50,000 cells/mL in L929 assay medium together with
Actinomycin-D (6.25 g/mL). 500 cells (in 10 ) of cell suspension are added to each
concentration of antibody mixtures that were incubated for 60 minutes at 37°C, 95%
relative humidity, 5% C0 2. The mixtures containing bispecific antibody, human noncleavable
membrane bound TNFa CHO cells, and Actinomycin-D are transferred to 96-
well plates with L929 adherent cells and incubated 18 hours at 37°C, 95% relative
humidity, 5% C0 2. The cell death is measured using an MTT cytotoxicity assay as
described above for soluble TNFa L929 assay. Results are expressed as the concentration
where 50% of the TNFa induced response is inhibited (IC 50) (average of 3 independent
experiments +/- SEM) by either the bispecific antibody or the positive control antibody.
The results demonstrate that the bispecific antibody of the present invention
inhibited killing of L929 cells by human non-cleavable membrane bound TNFa CHO
cells in a dose-dependent manner with an IC50 of 646 +/- 89.5 pM. This inhibition was
comparable to that observed with the positive control antibody (adalimumab with IgG4
isotype) (IC 50 = 669 +/- 134 pM), whereas the negative control antibody did not inhibit
human TNFa. The bispecific antibody of the present invention effectively neutralized
membrane bound human TNFa.
Inhibition of human IL-17 or TNFa -induced production of CXCL1 in vivo
Injection of human IL-17 or TNFa leads to a rapid and transient increase in mouse
CXCL1 in circulation. Regular C57BI/6J mice (n= 7 per group) are injected
subcutaneously (16.7 nmol/kg) with the following: (a) bispecific antibody, (b) positive
control anti-IL-17 antibody (BAFF/IL-17 bispecific antibody), (c) positive control anti-
TNFa antibody (adalimumab with IgG4 isotype); or (d) negative control antibody (human
IgG4). Two days later, mice receive a single intraperitoneal injection of human IL-17 (3
g/mouse) or human TNFa ( 1 g/mouse). Two hours after cytokine challenge, the mice
are sacrificed and plasma is analyzed for CXCL1 using a commercial ELISA.
Table 2: Average %inhibition of human IL-17- or TNFa-induced CXCL1
production in vivo.
The results demonstrate that the bispecific antibody of the present invention
significantly inhibited human IL-17- and TNFa-induced CXCL1 production relative to
animals that received the negative control antibody (p<0.001, calculated by ANOVA
followed by Tukey's Multiple Comparison test). The reduction in CXCL1 production
with the bispecific antibody was comparable to that observed with the positive control
antibodies. Thus, the bispecific antibody of the present invention effectively neutralized
biological effects induced by human IL-17 and TNFa in mouse.
Binding Assays
CD16a, CD32a, and Clq
A 96-well microplate is coated with 100 / \\ of CD32a with a C-terminal 10-
His tag (R&D Systems) or recombinant human CD16a with a C-terminal 6-His Tag
(R&D Systems) at 1 g/mL in Phosphate Buffered Saline (PBS). A 96-well microplate is
coated with 100 of human Clq (MP Biologicals) at 2 g/mL in PBS. The plate is
sealed and incubated overnight at 4° C. The coating reagent is removed from each well,
and 200 / \\ of casein blocking reagent (Thermo) is added. The plate is sealed and
incubated for 1 hour at room temperature (RT). Each well is washed two times with wash
buffer (20 mM Tris, 0.15 M NaCl, 0.1% Tween-20, pH 7.5). Serial dilutions of the
bispecific antibody of the present invention, human IgGl positive control, or human IgG4
negative control, all diluted in casein blocking reagent, are added to each well (100
i \\) and incubated for 2 hours at RT (antibodies are tested with a concentration range
of 6.25 to 200 g/mL in two-fold serial dilutions). Testing is performed in duplicate
wells. The plate is then washed three times with wash buffer before 100 / \\ of a
1:12,500 dilution of HRP-conjugated Goat Anti-Human IgG, F(ab')2 (Jackson
ImmunoResearch Catalog 109-036-097) in casein blocking reagent is added and
incubated for 1 hour at RT. This polyclonal antibody recognizes both human IgGl and
IgG4 (data not shown). The plate is washed four times with wash buffer and TMB
Substrate (Pierce, 100 / \\) is added. Incubation times are 4.5 minutes for CD16a, 9
minutes for CD32a, and 30 minutes for Clq, all in the dark and at RT. Lastly, 100 of
1.0 N HC1 is added to each well. Optical density is immediately measured using a
colormetric microplate reader set to 450 nm.
CD64
A 96-well microplate is coated with 100 / \\ of CD64 with a C-terminal 6-His
Tag (R&D Systems) at 1 g/mL in PBS. The plate is sealed and incubated overnight at 4
C. The coating reagent is removed from each well, and 200 / \\ of casein blocking
reagent is added. The plate is sealed and incubated for 1 hour at RT. Each well is washed
two times with wash buffer. Serial dilutions of the bispecific antibody of the present
invention, human IgGl positive control, or human IgG4 negative control, all diluted in
casein blocking reagent, are added to each well (100 / \\) and incubated for 1 hour at
RT (antibodies are tested with a concentration range of 0.001 to 300 mg/mL in 4-fold
serial dilutions). Testing is performed in duplicate wells. The plate is then washed three
times with wash buffer before 100 / \\ of a 1:12,500 dilution of HRP-conjugated
Goat Anti-Human IgG, F(ab')2 in casein blocking reagent is added and incubated for 1
hour at RT. The plate is washed four times with wash buffer, and 100 / \\ of TMB
Substrate is added and incubated for 4.5 minutes in the dark at RT, at which time 100
of 1.0 N HC1 is added to each well. Optical density is immediately measured using a
colormetric microplate reader set to 450 nm.
The results of the in vitro binding experiments show the bispecific antibody of the
present invention binding to any of CD16a, CD32a, CD64, or Clq is equal to that
observed with the human IgG4 negative control antibody. The human IgGl positive
control antibody binds to all four molecules tested, demonstrating the validity of the
assays.
Inhibition of CD4 T cell and rheumatoid arthritis synoviocyte co-culture -induced
MMP-1. MMP-3. IL-8 and G-CSF production in vitro
Incubation of activated human CD4 T cells with human fibroblast-like
synoviocytes from patients with Rheumatoid Arthritis (RA-FLS) results in the production
of inflammatory mediators, such as, MMP-1, MMP-3, IL-8 and G-CSF, and destruction
of cartilage and bone. The bispecific antibody of the present invention (Bispecific)
(30nM (based on a MW or 200kDa)) or a control antibody (Ab) is added in 50 to
wells containing 50 CD4 T Cells (50,000 T cells activated with CD3/CD28
Dynabeads at a 1:1 bead/cell ratio). 100 of activated CD4 T cells/ with or without an
Ab is then added onto RA-FLS plated in 100 the night before. Testing is carried out in
8-9 replicate wells per treatment. Human IgG4 Isotype is used as a negative control. IL-
17 neutralizing Ab and TNF neutralizing Ab are used as positive controls in the assay.
Control Abs were tested at the same molar concentration as the bispecific antibody.
Human PBMC's are isolated using Ficoll-Paque method from a buffy coat
[Leuko Reduction System (LRS) chamber], obtained from San Diego Blood Bank. 7 mL
LRS product is brought up to 140 mL with PBS. 35 mL of the buffy coat/PBS is overlaid
onto 15 mL Ficoll/Histopaque Plus (GE Healthcare). The tubes are balanced and spun at
900xg for 30 minutes at room temperature (RT) without brake. The cell interphase is
collected with a serological pipet and washed twice with PBS. Isolated PBMC's are
stored at 4°C overnight in Iscoves Modified Dulbecco's Medium containing 10% FBS,
penicillin (100 U/mL), streptomycin (100 U/mL), L-glutamine (100 units/mL) and 5xl0 5
M 2-beta mercaptoethanol. CD4 T cells are isolated by negative selection (Miltenyi
Biotec isolation kit) as per manufacturer's instructions.
RA-FLS cells from Cell Applications, Inc. are routinely cultured in Complete
Synoviocyte Growth Medium from Cell Applications, Inc. RA-FLS are harvested one
day before the day of the assay. The cells are rinsed with l x PBS and detached from the
culture flasks with Trypsin-EDTA. Complete assay medium is added to the detached
cells. RA-FLS are centrifuged at 310xg for 5 minutes at RT. The cell pellet is
resuspended in assay medium [Assay medium Dulbecco's Modified Eagle's Medium
containing 10% FBS, penicillin G (100 U/mL) and streptomycin (lOOU/mL)]. Cell
density is measured with Invitrogen Countess, and 10,000 RA-FLS cells (in 100 ) are
added to each well of 96 well plates. The 96 well flat bottom plates are placed in a tissue
culture incubator (37°C, 5%).
T cell activation is achieved with Dynabeads coated with anti-CD3 and anti-CD28
(Gibco, Life Technologies). Prior to use, the Dynabeads are washed with an equal
amount of wash buffer (PBS with 0.1% bovine serum albumin and 2 mM EDTA, pH 7.4).
The beads are placed on a Dynamagnet and after a minute, the supernatant is removed.
The beads are removed from the Dynamagnet and resuspended in PBMC media [Iscoves
Modified Dulbecco's Medium containing 10% FBS, penicillin (100 U/mL), streptomycin
(100 U/mL), L-glutamine (100 units/mL) and 5xl0 ~5 M 2-beta mercaptoethanol] to obtain
the original bead concentration of 4xl0 beads/mL. 50,000 washed beads in 1.25 are
added to 50,000 T cells. The bispecific antibody of the present invention or a control
antibody (Ab) is added onto CD4 T Cells with CD3/CD28 Dynabeads. The dynabead
activated CD4 T cells with or without antibodies are added onto the 96 well plates
containing the RA-FLSs. The plates are placed in a tissue culture incubator (37°C, 5%
C0 2 ) for 6 days.
At the end of the assay, the plates are centrifuged (500xg for 5 minutes at RT),
and the cell culture media is transferred to polypropylene 96-well plates and frozen at -
80°C. On the day of measuring MMP-1, MMP-3, IL-8 and G-CSF by ELISA, the plates
are thawed at RT. MMP-1, MMP-3, IL-8 and G-CSF levels in media were measured by
sandwich ELISA (R&D Systems DuoSet No. DY901, DY513, DY208, DY214,
respectively), as per manufacturer's instructions. At the end of the ELISA reactions,
plates are read at 450 nm on a microplate reader (Molecular Devices VersaMax Tunable).
Results are expressed as cytokine production in ng/mL. Cytokine inhibition with the
bispecific antibody of the present invention in activated CD4 T cell: RA-FLS coculture is
shown as mean % cytokine left compared to activated CD4 T cell: RA-FLS coculture in
the absence of Ab treatment.
IL-17 Ab Bispecific TNF Ab Neg Ctrl Ab
IL-8 35.6 ±17.5 12.5 +2.6 53.4 +3.7 86.5 +18.2
MMP-1 61.0 +17.5 25.1 +9.9 44.6 +13.4 83.5 +12.7
MMP-3 11.9 +2.5 6.0 +4.4 56.3 +26.1 76.1 +17.1
G-CSF 8.8 + 3.0 4.2 +0.9 62.2 +12.1 80.3 +12.8
The bispecific antibody of the present invention inhibited activated human CD4 T
cell: RA-FLS co-culture induced production of MMP-1, MMP-3, IL-8 and G-CSF
relative to control Abs. The assays were performed three times with similar results.
In vivo Testin2 in a Humanized Arthritis Mouse Model
Transgenic expression of human TNF causes spontaneous, progressive
inflammatory arthritis in mice (Hayward M.D., et al. BMC Physiology. Dec 10;7:13
(2007)). Additional expression of human IL-17 with an Adeno-Associated Virus (AAV)
in these mice will further exacerbate spontaneous, progressive polyarthritis. Male human
TNF-transgenic mice ((B6.Cg(SJL)-Tg(TNF) N21,Taconic Farms, Georgetown, NY,
model 1006) carry the entire human TNFa gene including a promoter and a stabilized
3'UTR that results in low constitutive expression of human TNFa in all tissues. Animals
are housed 2/cage with free access to food and water. A standardized scoring system is
used to score their arthritic disease in front and hind paws (Front Paw: 0 = no evidence of
distortion or swelling, 1 = mild swelling of the ankle, 2 = moderate swelling or mild
distortion, 3 = severe swelling or severe distortion, 4 = severe swelling and severe
distortion. Hind paw: 0 = no evidence of distortion or swelling, 1 = mild
distortion/inability to spread toes straight, 2 = moderate distortion/inability to spread toes,
3 = severe medial contortion/mild swelling, 4 = severe medial contortion with marked
swelling). At 8 weeks of age mice are intravenously injected (100 uL/mouse via tail vein)
with lxlO genomic copies of Adeno-associated virus (AAV) carrying a gene for human
IL-17 (n=32) or an irrelevant gene (lacz, n=8). Viral expression of human IL-17 is
detected in mouse plasma obtained from tail snips using a commercial ELISA kit (Meso
Scale Discovery, Rockville, MD) according to the manufacturer's instructions. The
average plasma levels are about 500 pg/ml human IL-17. At 12 weeks of age the mice
are randomized into study groups based on their clinical arthritis score, human IL-17
plasma levels, and body weight. Treatment with the different antibodies is initiated on the
day of grouping. Animals are dosed weekly, subcutaneously for 9 weeks with 2 different
doses (20 and 3.3 nmol/kg) of the bispecifc antibody of the present invention (Bispecific)
or TNF neutralizing antibody (TNF Ab) or an isotype control antibody (Neg Ctrl Ab) (20
nmol/kg). Clinical arthritis scores are determined routinely in a blinded fashion. At
termination plasma is obtained by cardiac puncture, hind legs are fixed in 10% formalin.
The hind legs are demineralized in EDTA, trimmed, processed in the routine manner,
embedded in paraffin, sectioned, and stained with hematoxylin and eosin. Arthritis
scoring is conducted for the following categories: Inflammation, bone resorption,
cartilage damage, and pannus formation on a scale of 0-5: 0= normal, 1= minimal, 2=
mild, 3= moderate, 4= marked, 5= severe for a potential total of 20.
All antibodies are formulated in PBS at an appropriate concentration to result in a
200 uL/mouse subcutaneous dose.
Average Histology Scores of Hindpaws of Human TNF transgenic/IL-17 Mice after
Treatment
*** p<0.001, ** p< 0.01 versus Neg Ctrl Ab (one way ANOVA). Histology scores are
cumulative of the four different parameters (Inflammation, bone resorption, cartilage
damage, and pannus formation scored at a scale from 0-5).
This data demonstrates the bispecific antibody of the present invention is effective
in a disease model of human cytokine-driven disease.
In Vivo Testin2 of a Humanized Psoriasis Mouse Model
A humanized mouse model of psoriasis is a model that involves grafting of human
non-lesional skin biopsies from psoriasis patients onto the back skin of immunodeficient
mice. After the human skin has grafted (3 to 4 weeks later), T-cell activated human
peripheral blood mononuclear cells (PBMCs) from the same donor are intradermally
injected into the graft to induce psoriasis-like epidermal thickening (Wrone-Smith and
Nickoloff J., Clin Invest.l5;98(8): 1878-87 (1996)).
Mice (10-27/group) are treated once weekly with the bispecific antibody of the
present invention (Bispecific) (66.6, 3.3 or 0.67 nmol/kg), TNF neutralizing antibody
(TNF Ab) (66.6 or 3.3 nmol/kg), PBS or betamethasone (twice daily topical), starting the
day before the PBMC injection. After three weeks the mice are euthanized, the grafted
skin was isolated, and the thickness of the epidermis is measured.
The bispecific antibody of the present invention (66.6 nmol/kg) significantly
reduced epidermal thickening in the human skin grafts compared to PBS control
(p=0.047). The bispecific antibody of the present invention (66.6 nmol/kg) was able to
reduce the epidermal thickening in the human skin grafts better than TNF Ab (66.6
nmol/kg) (p=0.0057). These results demonstrate efficacy of the bispecific antibody of the
present invention in a humanized mouse model of psoriasis.
Mean epidermal thickness
Stability Analysis
The bispecific antibody is formulated in 10 mM citrate + 150 mM NaCl, pH 6.
The bispecific antibody is concentrated at 100 mg/mL using Amicon Ultra-4 30,000
MWCO concentrators (Millipore). Tween-80 is added to a final concentration of 0.02%
(v/v). Concentrated samples are stored at 25°C over a period of 4 weeks. Samples are
analyzed for percent high molecular weight ( HMW) with size exclusion
chromatography (SEC) at time zero, after 1 week, and after 4 weeks. SEC is performed
on an Agilent 1100 system using a TSK G3000SW-XL (Tosoh Bioscience) column. 50
mM sodium phosphate + 0.35 M NaCl, pH 7.0 is used as the mobile phase running at 0.5
niL/min for 35 minutes. A volume of 1 uL of the concentrated bispecific antibody is
injected into the column and monitored at 280 nm. Chromatograms are analyzed using
ChemStation, and HMW is calculated using the ratio of AUC of the peaks eluted before
the monomer peak to total AUC. Samples stored at 25°C at different time points are
analyzed for HMW. At time zero, HMW was 1.52; at 1 week, HMW was 2.01; and
at 4 weeks, HMW was 2.37.
The results demonstrate that the bispecific antibody of the present invention is
stable as there was no significant change in soluble aggregate after 4 weeks.
Sequences
HC-ScFv: SEP ID NO: 1
EVQLVESGGGLVQPGRSLRLSCAASGFTFDDYAMHWVRQAPGKGLEWVSAITW
NSGHIDYADSVEGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCAKVSYLSTASSL
DYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWN
SGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKR
VESKYGPPCPPCPAPEAAGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEV
QFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSN
KGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEW
ESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNH
YTQKSLSLSLGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYKF
TDYHIHWVRQAPGQCLEWMGVINPTYGTTDYNQRFKGRVTITADESTSTAYMEL
SSLRSEDTAVYYCARYDYFTGTGVYWGQGTLVTVSSGGGGSGGGGSGGGGSGG
GGSDIVMTQTPLSLSVTPGQPASISCRSSRSLVHSRGETYLHWYLQKPGQSPQLLI
YKVSNRFIGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCSQSTHLPFTFGCGTKL
EIK
LC: SEP ID NO: 2
DIQMTQSPSSLSASVGDRVTITCRASQGIRNYLAWYQQKPGKAPKLLIYAASTLQ
SGVPSRFSGSGSGTDFTLTISSLQPEDVATYYCQRYNRAPYTFGQGTKVEIKRTVA
APSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQ
DSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
HC-ScFv: SEP ID NO: 3
GAGGTGCAGCTGGTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGAGGTCCC
TGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTGATGACTATGCCATGCAC
TGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTGTCAGCTATTACTT
GGAATAGTGGTCACATAGACTACGCAGACTCCGTGGAGGGCCGGTTCACCAT
CTCCAGAGACAATGCCAAGAACTCCCTGTATCTGCAAATGAACAGCCTGAGA
GCCGAGGACACGGCCGTATATTACTGTGCGAAAGTGAGCTACCTGAGTACTG
CCTCCAGCCTGGACTACTGGGGCCAAGGAACCCTGGTCACCGTCTCCTCAGCC
TCCACCAAGGGCCCATCGGTCTTCCCGCTAGCGCCCTGCTCCAGGAGCACCTC
CGAGAGCACAGCCGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCG
GTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCC
CGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTG
CCCTCCAGCAGCTTGGGCACGAAGACCTACACCTGCAACGTAGATCACAAGC
CCAGCAACACCAAGGTGGACAAGAGAGTTGAGTCCAAATATGGTCCCCCATG
CCCACCCTGCCCAGCACCTGAGGCCGCCGGGGGACCATCAGTCTTCCTGTTCC
CCCCAAAACCCAAGGACACTCTCATGATCTCCCGGACCCCTGAGGTCACGTG
CGTGGTGGTGGACGTGAGCCAGGAAGACCCCGAGGTCCAGTTCAACTGGTAC
GTGGATGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAG
TTCAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTG
GCTGAACGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGGCCTCCCGTCC
TCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAGCCACAGG
TGTACACCCTGCCCCCATCCCAGGAGGAGATGACCAAGAACCAGGTCAGCCT
GACCTGCCTGGTCAAAGGCTTCTACCCCAGCGACATCGCCGTGGAGTGGGAA
AGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACT
CCGACGGCTCCTTCTTCCTCTACAGCAGGCTAACCGTGGACAAGAGCAGGTG
GCAGGAGGGGAATGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAAC
CACTACACACAGAAGAGCCTCTCCCTGTCTCTGGGAGGCGGAGGATCCGGGG
GAGGGGGTTCCGGAGGAGGGGGCTCGCAGGTGCAGCTGGTGCAGTCTGGGGC
TGAGGTGAAGAAGCCTGGGTCCTCAGTGAAGGTTTCCTGCAAGGCATCTGGT
TACAAGTTCACTGACTACCATATTCATTGGGTGCGACAGGCCCCTGGACAATG
CCTTGAGTGGATGGGAGTAATTAATCCTACTTATGGTACTACTGACTACAATC
AGCGGTTCAAAGGCCGTGTCACCATTACCGCGGACGAATCCACGAGCACAGC
CTACATGGAGCTGAGCAGCCTGAGATCTGAGGACACGGCCGTGTATTACTGT
GCGAGATATGATTACTTTACTGGGACGGGTGTGTACTGGGGCCAAGGAACCC
TGGTCACCGTCTCCTCAGGTGGCGGAGGATCTGGTGGAGGTGGCTCAGGAGG
TGGCGGAAGCGGCGGAGGTGGAAGTGATATTGTGATGACTCAGACTCCACTC
TCCCTGTCCGTCACCCCTGGACAGCCGGCCTCCATCTCCTGCAGATCTAGTAG
GAGCCTTGTACACAGTCGTGGAGAAACCTATTTACATTGGTATCTGCAGAAGC
CAGGCCAATCTCCACAGCTCCTAATTTATAAAGTTTCCAACCGGTTTATTGGG
GTCCCAGACAGATTCAGCGGCAGTGGGTCAGGCACAGATTTCACACTGAAAA
TCAGCAGGGTGGAGGCCGAAGATGTTGGGGTTTATTACTGCTCTCAAAGTAC
ACATCTTCCATTCACGTTTGGCTGCGGGACCAAGCTGGAGATCAAA
LC: SEP ID NO: 4
GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAG
AGTCACCATCACTTGCCGGGCGAGTCAGGGCATTCGCAATTATTTAGCCTGGT
ATCAGCAGAAACCAGGGAAAGCTCCTAAGCTCCTGATCTATGCTGCATCCAC
TTTGCAATCAGGGGTCCCATCTCGGTTCAGTGGCAGTGGATCTGGGACAGATT
TCACTCTCACCATCAGCAGCCTGCAGCCTGAAGATGTTGCAACTTATTACTGT
CAACGCTATAACCGTGCCCCTTACACGTTCGGCCAAGGGACCAAGGTGGAAA
TCAAACGGACTGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAG
CAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTATCC
CAGAGAGGCCAAAGTACAGTGGAAGGTGGATAACGCCCTCCAATCGGGTAAC
TCCCAGGAGAGTGTCACAGAGCAGGACAGCAAGGACAGCACCTACAGCCTCA
GCAGCACCCTGACGCTGAGCAAAGCAGACTACGAGAAACACAAAGTCTACGC
CTGCGAAGTCACCCATCAGGGCCTGAGCTCGCCCGTCACAAAGAGCTTCAAC
AGGGGAGAGTGC
WE CLAIM:
1. A bispecific antibody comprising a first polypeptide and a second polypeptide,
wherein the first polypeptide has amino acid sequence of SEQ ID NO: 1, and the
second polypeptide has an amino acid sequence of SEQ ID NO: 2.
2. The bispecific antibody of claim 1 comprising two first polypeptides and two
second polypeptides.
3. A DNA molecule comprising a polynucleotide sequence encoding a polypeptide
having the amino acid sequence of SEQ ID NO: 1.
4. A DNA molecule comprising a polynucleotide sequence encoding a polypeptide
having the amino acid sequence of SEQ ID NO: 2.
5. A DNA molecule comprising a polynucleotide sequence encoding a polypeptide
having the amino acid sequence of SEQ ID NO: 1, and comprising a
polynucleotide sequence encoding a polypeptide having the amino acid sequence
of SEQ ID NO: 2.
6. A mammalian cell comprising the DNA molecule of Claim 3 and the DNA
molecule of Claim 4, wherein the cell is capable of expressing a bispecific
antibody comprising a first polypeptide having an amino acid sequence of SEQ ID
NO: 1 and a second polypeptide having an amino acid sequence of SEQ ID NO: 2.
7. A process for producing a bispecific antibody comprising cultivating the
mammalian cell of Claim 6 under conditions such that the bispecific antibody is
expressed, and recovering the expressed bispecific antibody.
8. A bispecific antibody produced by the process of Claim 7.
9. A method of treating rheumatoid arthritis, psoriatic arthritis, or ankylosing
spondylitis comprising administering to a patient in need thereof a therapeutically
effective amount of a bispecific antibody of Claims 2 or 8.
10. A bispecific antibody of Claims 2 or 8 for use in therapy.
11. A bispecific antibody of Claims 2 or 8 for use in the treatment of rheumatoid
arthritis, psoriatic arthritis, or ankylosing spondylitis.
12. A pharmaceutical composition comprising a bispecific antibody of Claims 2 or 8 and
one or more pharmaceutically acceptable carriers, diluents, or excipients.