CN116265487A - anti-ANG 2-VEGF bispecific antibodies and uses thereof - Google Patents

anti-ANG 2-VEGF bispecific antibodies and uses thereof Download PDF

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CN116265487A
CN116265487A CN202111541547.9A CN202111541547A CN116265487A CN 116265487 A CN116265487 A CN 116265487A CN 202111541547 A CN202111541547 A CN 202111541547A CN 116265487 A CN116265487 A CN 116265487A
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郎国竣
刘婵娟
王立燕
曹静丽
司远青
李文
苏飞
闫闰
张震
张文海
胡宇豪
孙兴鲁
陈克让
江茹兰
林紫绮
戴珊珊
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Sanyou Biopharmaceuticals Co Ltd
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Sanyou Biopharmaceuticals Co Ltd
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Priority to PCT/CN2022/139203 priority patent/WO2023109888A1/en
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Abstract

The invention belongs to the field of biological medicine. In particular, the invention provides anti-ANG 2-VEGF bispecific antibodies, pharmaceutical compositions comprising the bispecific antibodies, and uses thereof. The invention further provides polynucleotides and expression vectors encoding the bispecific antibodies and methods of producing the bispecific antibodies.

Description

anti-ANG 2-VEGF bispecific antibodies and uses thereof
Technical Field
The invention belongs to the field of biological medicine. In particular, the invention provides anti-ANG 2-VEGF bispecific antibodies, pharmaceutical compositions comprising the bispecific antibodies, and uses thereof. The invention further provides polynucleotides and expression vectors encoding the bispecific antibodies and methods of producing the bispecific antibodies.
Background
Vascular endothelial growth factor A (VEGF-A, also abbreviated as VEGF) is Sub>A key cytokine that promotes angiogenesis. The receptors for VEGF include VEGFR1 and VEGFR2, which are expressed in vascular endothelial cells. VEGFR2 is the primary receptor tyrosine kinase receptor mediating angiogenesis, and VEGF activates VEGFR2 to promote vascular endothelial cell mitosis and increased vascular permeability, thereby promoting cardiovascular sprouting. Thus, targeting VEGF or VEGFR2 is effective in inhibiting abnormal vascular proliferation (Ferrara, N. (2010). Mol Biol Cell 21 (5): 687-690.).
Angiogenin (angiogenin) is part of the vascular growth factor family. ANG2 (also known as ANGPT 2) belongs to the angiogenin family and is expressed by endothelial cells and is usually stored in the Wei Boxiao body (Weibel-Palade bodies) of endothelial cells, when hypoxia, inflammation and the like are encountered to promote neovascularization, ANG2 is released from Wei Boxiao bodies to compete with ANG1, and acts as an antagonist of Tie2 to inhibit the stability of blood vessels, so that peripheral cells are separated from endothelial cells, the permeability of endothelial cells is increased, and VEGF plays a better role in promoting neovascularization (saharrinen, p., et al (2017) Nat Rev Drug Discov (9): 635-661). Meanwhile, many studies have shown that ANG2 overexpression plays a key role in promoting lymph node metastasis of tumors, and thus ANG2 is an important target for pathological angiogenesis-related diseases and tumor therapy (Holash, j., et al (1999). Science 284 (5422): 1994-1998; gengengenbacher, n., (2021). Cancer discover 11 (2): 424-445). In 2004, blocking ANG2 activity was demonstrated to be effective in inhibiting tumor neovascularization and tumor growth by neutralizing antibodies that ANG2 interacted with its receptor Tie2 for the first time (Oliner, j., et al (2004). Cancer Cell 6 (5): 507-516).
Complementary coordination and co-action of VEGF and ANG2 in abnormal angiogenesis was demonstrated in vivo functional experiments, showing remarkable tumor inhibiting effects in animal models in ANG 2-targeting antibody combinations and bispecific antibodies and superior to the efficacy of any target monoclonal antibody (Hashizume, h., et al (2010). Cancer Res 70 (6): 2213-2223.Kienast, y., et al (2013). Clin Cancer Res 19 (24): 6730-6740). Therefore, the VEGF and the ANG2 can be blocked at the same time to inhibit abnormal angiogenesis more effectively, so that the aim of treating diseases related to angiogenesis is fulfilled.
Several bispecific antibodies blocking both VEGF and ANG2 signaling pathways and their uses are currently reported in the art, including Vanucizumab (see e.g., WO2010040508 A1), faricimab (see e.g., WO2014009465 A1), and BI836880 (see e.g., US9527925B 2), in the clinical stage, but there is still a need to develop new highly potent anti-ANG 2-VEGF bispecific antibodies.
Disclosure of Invention
In one aspect, the invention provides a bispecific antibody comprising a first antigen binding portion that binds ANG2 comprising a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 and a light chain variable region comprising LCDR1, LCDR2 and LCDR3, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequence is selected from any of (1) - (3), and a second antigen binding portion that binds VEGF: (1) the HCDR1 sequence shown in SEQ ID NO. 25; the HCDR2 sequence shown in SEQ ID NO. 26; the HCDR3 sequence shown in SEQ ID NO. 27; the LCDR1 sequence shown in SEQ ID NO. 28; the LCDR2 sequence shown in SEQ ID NO. 29; and the LCDR3 sequence shown in SEQ ID NO. 30; (2) the HCDR1 sequence shown in SEQ ID NO. 33; the HCDR2 sequence shown in SEQ ID NO. 34; an HCDR3 sequence shown in SEQ ID NO. 35; the LCDR1 sequence shown in SEQ ID NO. 36; the LCDR2 sequence shown in SEQ ID NO. 37; and the LCDR3 sequence shown in SEQ ID NO. 38; (3) the HCDR1 sequence shown in SEQ ID NO. 41; an HCDR2 sequence shown in SEQ ID NO. 42; an HCDR3 sequence shown in SEQ ID NO. 43; the LCDR1 sequence shown in SEQ ID NO. 44; the LCDR2 sequence shown in SEQ ID NO. 45; and the LCDR3 sequence shown in SEQ ID NO. 46.
In one embodiment, the heavy chain variable region and the light chain variable region are selected from any one of (1) - (3): (1) A heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 31; a light chain variable region comprising the amino acid sequence of SEQ ID NO. 32; (2) A heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 39; a light chain variable region comprising the amino acid sequence of SEQ ID NO. 40; (3) A heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 47; a light chain variable region comprising the amino acid sequence of SEQ ID No. 48.
In one embodiment, the second antigen binding portion comprises an immunoglobulin single variable domain (VHH) that binds VEGF. In a preferred embodiment, the immunoglobulin single variable domain comprises: CDR1 sequence shown in SEQ ID NO. 49, CDR2 sequence shown in SEQ ID NO. 50, and CDR3 sequence shown in SEQ ID NO. 51.
In one embodiment, the first antigen binding portion and the second antigen binding portion are linked by a linker.
In one embodiment, the bispecific antibody of the present invention further comprises a heavy chain constant region of human IgG1 and a human kappa light chain constant region. In a preferred embodiment, the heavy chain constant region of human IgG1 comprises a leucine to alanine mutation at positions 234 and 235 according to EU numbering.
The invention also provides a polynucleotide encoding a bispecific antibody of the invention.
The invention also provides an expression vector comprising a polynucleotide of the invention.
The invention also provides a host cell comprising a polynucleotide or expression vector of the invention.
The invention further provides a method of producing a bispecific antibody of the invention comprising:
culturing a host cell of the invention under suitable conditions to express said bispecific antibody, an
(ii) isolating the bispecific antibody from the host cell or culture thereof.
The invention also provides a pharmaceutical composition comprising a bispecific antibody of the invention, and a pharmaceutically acceptable carrier.
The invention also provides the use of a bispecific antibody or pharmaceutical composition of the invention in the manufacture of a medicament for the treatment of: (1) angiogenesis-related eye diseases; or (2) cancer.
Drawings
FIGS. 1A-1E show schematic structural diagrams of bispecific antibodies against ANG 2-VEGF: FIG. 1A, bsAb1, bsAb13 and BsAb21; FIG. 1B, bsAb2, bsAb14 and BsAb22; FIG. 1C, bsAb3, bsAb15 and BsAb23; FIG. 1D, bsAb4, bsAb16 and BsAb24; FIG. 1E, bsAb29, bsAb30 and BsAb31.
FIGS. 2A-2E show the binding activity of an anti-ANG 2-VEGF bispecific antibody to the recombinant protein hVEGF-His.
FIGS. 3A-3E show the binding activity of an anti-ANG 2-VEGF bispecific antibody to the recombinant protein hANG 2-His.
FIGS. 4A-4C show the activity of anti-ANG 2-VEGF bispecific antibodies to neutralize VEGF.
FIGS. 5A-5B show the blocking activity of anti-ANG 2-VEGF bispecific antibodies against ANG2/Tie 2.
FIGS. 6A-6C show the activity of anti-ANG 2-VEGF bispecific antibodies in inhibiting HUVEC cell proliferation.
FIGS. 7A-7C show the effect of anti-ANG 2-VEGF bispecific antibodies on inhibiting human colon cancer cell subcutaneous engraftment growth: FIG. 7A shows the mean tumor volume of each group of mice over time; FIG. 7B shows the average body weight of each group of mice over time; figure 7C shows the average tumor weights of the groups of mice at the end of the experiment.
Detailed Description
Definition of the definition
In the present invention, unless otherwise indicated, scientific and technical terms used herein have the meanings commonly understood by one of ordinary skill in the art. Also, protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, immunology-related terms and laboratory procedures as used herein are terms and conventional procedures that are widely used in the corresponding arts. Meanwhile, in order to better understand the present invention, definitions and explanations of related terms are provided below.
As used herein, the terms "comprises," "comprising," "includes," "including," "having" and "containing" are open-ended, meaning the inclusion of the stated elements, steps or components, but not the exclusion of other non-recited elements, steps or components. The expression "consisting of … …" does not include any elements, steps or components not specified. The expression "consisting essentially of … …" means that the scope is limited to the specified elements, steps, or components, plus any optional elements, steps, or components that do not significantly affect the basic and novel properties of the claimed subject matter. It should be understood that the expressions "consisting essentially of … …" and "consisting of … …" are encompassed within the meaning of the expression "comprising".
As used herein, "bispecific antibody" refers to an antibody that specifically binds to two different antigens. The bispecific antibodies of the invention comprise an antigen binding portion that specifically binds to two different antigens of ANG2 and VEGF.
As used herein, "antibody" refers to an immunoglobulin or fragment thereof that specifically binds an epitope through at least one antigen binding site. The term "antibody" includes multispecific antibodies (e.g., bispecific antibodies), fully human antibodies, non-human antibodies, humanized antibodies, chimeric antibodies, single domain antibodies, and antigen-binding fragments. Antibodies may be synthetic (e.g., produced by chemical or biological coupling), enzymatically treated, or recombinantly produced. Antibodies include any immunoglobulin class (e.g., igG, igM, igD, igE, igA and IgY), any class (e.g., igG1, igG2, igG3, igG4, igA1, and IgA 2), or subclass (e.g., igG2a and IgG2 b). Antibodies may be multivalent, i.e. comprise multiple antigen binding sites. For example, antibodies can be bivalent, trivalent, tetravalent, pentavalent, and hexavalent, comprising two, three, four, five, and six antigen binding sites, respectively.
As used herein, an "antigen binding fragment" refers to a portion of a full-length antibody that is less than full length, but that comprises at least a portion of the variable region of the full-length antibody (e.g., comprises one or more CDRs and/or one or more antigen binding sites), and thus retains at least a portion of the ability of the full-length antibody to specifically bind an antigen. Examples of antigen binding fragments include, but are not limited to, sdabs (e.g., variable domains of heavy chain antibodies))、Fv、scFv、dsFv、scdsFv、Fab、scFab、Fab'、F(ab') 2 Diabodies, fd and Fd' fragments, and other fragments (e.g., fragments comprising modifications).
As used herein, a "full length antibody" generally comprises four polypeptides: two Heavy Chains (HC) and two Light Chains (LC). Each light chain comprises a light chain variable region (VL) and a light chain constant region (CL). Each heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region (CH). In general, the heavy chain constant region of a full length antibody may comprise a CH 1-hinge region-CH 2-CH3 from the N-terminus to the C-terminus. In certain immunoglobulin types (e.g., igM and IgE), the heavy chain constant region may comprise, from N-terminus to C-terminus, a CH 1-hinge region-CH 2-CH3-CH4.
The light chain variable region and the heavy chain variable region may each comprise three highly variable "Complementarity Determining Regions (CDRs)" and four relatively conserved "Framework Regions (FR)", and are connected from the N-terminus to the C-terminus in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR 4. Herein, CDRs of the light chain variable region (CDRL or LCDR) may be referred to as LCDR1, LCDR2 and LCDR3, and CDRs of the heavy chain variable region (CDRH or HCDR) may be referred to as HCDR1, HCDR2 and HCDR3.
The CDRs of antibodies can be defined in the art by a variety of methods, such as Chothia (see, e.g., chothia, c.et Al, nature,342,877-883 (1989)), and Al-Lazikani, b.et Al, j. Mol. Biol.,273,927-948 (1997)), kabat (see, e.g., kabat, e.a.et Al (1991) Sequences of Proteins of Immunological Interest, fifth Edition, u.s.Department of Health and Human Services, NIH Publication No. 91-3242), abM (Martin, a.c.r.and j. Allen (2007) "Bioinformatics tools for antibody engineering," in s.d. Ben (ed.), handbook of Therapeutic antibodies.wein. Wiley-VCH veriag, pp.95-118), act (MacCallum, R.M.et j. (1996), contact (1996) and hc. 6, and by clustering of the topology of the CDR loops, and by means of, e.g., kabat, e.a.g., et Al, e.g., 1991) Sequences of Proteins of Immunological Interest, fifth Edition, u.s.d. Part of Health and Human Services, NIH Publication No. 91-3242), abM (Martin, a.c.r.and j.al (2007) "Bioinformatics tools for antibody engineering), in s.d. Ibm (ed.), 35 anti-antibodies.wein.wiley-VCH vering, pp.95-118), contact (1996), contact (6), and by the use of the clustering of the structures of the nearest neighbor (i.g., n. It will be appreciated by those skilled in the art that unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or region thereof (e.g., variable region) are to be understood as encompassing complementarity determining regions defined in any of the above known schemes as described by the present invention. Although the scope of the claimed invention is based on the sequences shown by the AbM definition rules, the amino acid sequences corresponding to the definition rules according to other CDRs should also fall within the scope of the claimed invention.
Thus, when referring to defining antibodies with a particular CDR sequence as defined herein, the scope of the antibodies also encompasses antibodies whose variable region sequences comprise the particular CDR sequence, but whose purported CDR boundaries differ from the particular CDR boundaries defined herein by the application of different protocols (e.g., different assignment system rules or combinations).
As used herein, the terms "framework region" and "framework region" are used interchangeably. As used herein, the term "framework region", "framework region" or "FR" residues refer to those amino acid residues in the variable region of an antibody other than the CDR sequences as defined above.
Generally, an "Fv" fragment consisting of one VH and one VL by non-covalent interactions is considered to be the smallest antigen-binding fragment that contains an antigen-binding site. But single variable domains (single domain antibodies) also have antigen binding capacity. "Single chain Fv (scFv)" may be obtained by linking VH and VL via a peptide linker. "disulfide stabilized Fv (dsFv)" or "single chain disulfide stabilized Fv (scdsFv or dsscFv)", respectively, can be obtained by introducing disulfide bonds into Fv or scFv.
As used herein, "Fab" comprises one complete antibody light chain (VL-CL) and antibody heavy chain variable region and one heavy chain constant region (VH-CH 1, also known as Fd). A single chain "Fab (scFab)" can be obtained by ligating CL and CH1 in "Fab" with a peptide linker. "F (ab') 2 "essentially comprising two Fab fragments linked by a disulfide bridge of the hinge region. "Fab '" is F (ab') 2 Is capable of reducing F (ab') 2 Disulfide bonds of the hinge region are obtained.
As used herein, "single domain antibody (sdAb)" or "nanobody" refers to an antibody that comprises a single immunoglobulin variable domain (single variable domain) as a functional antigen-binding fragment. Like the variable regions of full length antibodies, single variable domains typically comprise CDR1, CDR2, and CDR3, which form the antigen binding site, and a framework region that serves as a support. The single variable domain may be, for example, a variable domain of a heavy chain antibody (variable domain of heavy-chain antibody, VHH), a shark IgNAR variable domain, a human light chain antibody variable domain, and a heavy chain antibody variable domain.
As used herein, "percent (%) sequence identity" of amino acid sequences, sequence identity "has art-recognized definitions that refer to the percentage of identity between two polypeptide sequences as determined by sequence alignment (e.g., by manual inspection or by a known algorithm). The determination may be made using methods known to those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, clustal Omega and FASTA software.
Polypeptides (e.g., CDR regions, framework regions, and constant regions of antibodies) may be modified, e.g., by substitution, addition, and/or deletion of one or more amino acids, without altering the function of the polypeptide. Substitutions are preferably conservative substitutions of amino acids. Suitable conservative substitutions are well known to those skilled in the art. In addition, antibodies can be modified to alter their properties using methods known in the art, such as altering the type of antibody glycosylation modification, altering the ability to form interchain disulfide bonds, altering the effector function of an antibody (e.g., enhancing or reducing antibody-dependent cell-mediated cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC)), or providing reactive groups for the preparation of antibody conjugates. Such modified antibodies are also encompassed within the scope of the antibodies of the invention.
"affinity" or "binding affinity" is used to measure the strength of the interaction between an antibody and an antigen through non-covalent interactions. The magnitude of the "affinity" can be reported as the equilibrium dissociation constant, K D Or EC (EC) 50 。K D Can be calculated by measuring the equilibrium association constant (ka) and equilibrium dissociation constant (kd)And (3) calculating: k (K) D =kd/ka. Affinity can be determined using conventional techniques known in the art, such as biofilm interference techniques (e.g., forteBio Octet or gate detection systems can be employed), surface plasmon resonance, enzyme-linked immunosorbent assay (ELISA) or flow cytometry (FACS), and the like.
As used herein, the expression "isolated" means that a substance (e.g., a polynucleotide or polypeptide) is isolated from the source or environment in which it is present, i.e., does not substantially comprise any other components. The bispecific antibodies of the invention or polynucleotides encoding the same may be isolated.
The terms "polynucleotide" and "nucleic acid" are used interchangeably herein to refer to an oligomer or polymer comprising at least two linked nucleotides or nucleotide derivatives. Polynucleotides may include deoxyribonucleic acid (DNA) and ribonucleic acid (RNA).
Herein, a "vector" is a vector used to introduce an exogenous polynucleotide into a host cell, which is amplified or expressed when the vector is transformed into an appropriate host cell. As used herein, the definition of vector encompasses plasmids, linearized plasmids, viral vectors, cosmids, phage vectors, phagemids, artificial chromosomes (e.g., yeast artificial chromosomes and mammalian artificial chromosomes), and the like. Viral vectors include, but are not limited to, retroviral vectors (including lentiviral vectors), adenoviral vectors, adeno-associated viral vectors, herpes viral vectors, poxviral vectors, and baculovirus vectors, among others. As used herein, an "expression vector" refers to a vector capable of expressing a polypeptide of interest. Expression vectors may generally comprise a polynucleotide sequence encoding a polypeptide of interest and regulatory sequences (e.g., promoters and ribosome binding sites) operably linked thereto.
As used herein, a "host cell" is a cell that is used to receive, hold, replicate, or amplify a vector. Host cells may also be used to express polypeptides encoded by polynucleotides or vectors. The host cell may be a eukaryotic cell or a prokaryotic cell. Prokaryotic cells such as E.coli (E.coli) or B.subtilis (Bacillus subtilis), fungal cells such as yeast cells or Aspergillus, insect cells such as S2 drosophila cells or Sf9, and animal cells such as fibroblasts, CHO cells, COS cells, heLa cells, NSO cells or HEK293 cells.
As used herein, the term "treatment" refers to an improvement in a disease/symptom, e.g., alleviation or disappearance of a disease/symptom, prevention or slowing of the occurrence, progression, and/or worsening of a disease/symptom.
As used herein, "effective amount" means an amount of an active substance (e.g., an antibody or pharmaceutical composition of the invention) that induces a biological or medical response or desired therapeutic effect against a tissue, system, animal, mammal, or human. Thus, an "effective amount" may be that amount required to prevent, cure, ameliorate, block or partially block a disease or condition (e.g., cancer). The effective amount can be determined by one skilled in the art based on factors such as the age, physical condition, sex, severity of symptoms, particular composition or route of administration of the subject, and the like.
As used herein, the term "pharmaceutically acceptable carrier" refers to a carrier that is pharmacologically and/or physiologically compatible with the subject and the active ingredient, as is well known in the art (see, e.g., remington's Pharmaceutical sciences. Mediated by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995).
Examples of mammals, as used herein, include, but are not limited to, humans, non-human primates, rats, mice, cows, horses, pigs, sheep, alpacas, dogs, cats, and the like. As used herein, the term "subject" refers to a mammal, such as a human. In some embodiments, the subject is a patient, e.g., a cancer patient.
Bispecific antibodies against ANG2-VEGF
In one aspect, the invention provides a bispecific antibody comprising a first antigen binding portion that binds ANG2 comprising a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 and a light chain variable region comprising LCDR1, LCDR2 and LCDR3, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequence is selected from any of (1) - (3), and a second antigen binding portion that binds VEGF: (1) the HCDR1 sequence shown in SEQ ID NO. 25; the HCDR2 sequence shown in SEQ ID NO. 26; the HCDR3 sequence shown in SEQ ID NO. 27; the LCDR1 sequence shown in SEQ ID NO. 28; the LCDR2 sequence shown in SEQ ID NO. 29; and the LCDR3 sequence shown in SEQ ID NO. 30; (2) the HCDR1 sequence shown in SEQ ID NO. 33; the HCDR2 sequence shown in SEQ ID NO. 34; an HCDR3 sequence shown in SEQ ID NO. 35; the LCDR1 sequence shown in SEQ ID NO. 36; the LCDR2 sequence shown in SEQ ID NO. 37; and the LCDR3 sequence shown in SEQ ID NO. 38; (3) the HCDR1 sequence shown in SEQ ID NO. 41; an HCDR2 sequence shown in SEQ ID NO. 42; an HCDR3 sequence shown in SEQ ID NO. 43; the LCDR1 sequence shown in SEQ ID NO. 44; the LCDR2 sequence shown in SEQ ID NO. 45; and the LCDR3 sequence shown in SEQ ID NO. 46.
In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO. 31, SEQ ID NO. 39 or SEQ ID NO. 47. In some embodiments, the heavy chain variable region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to SEQ ID NO. 31, SEQ ID NO. 39 or SEQ ID NO. 47. In some embodiments, the heavy chain variable region polypeptide comprises an amino acid sequence having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid substitutions, additions, and/or deletions compared to SEQ ID No. 31, SEQ ID No. 39, or SEQ ID No. 47. Preferably, the amino acid substitutions, additions and/or deletions do not occur in the CDR regions.
In some embodiments, the light chain variable region comprises the amino acid sequence of SEQ ID NO. 32, SEQ ID NO. 40 or SEQ ID NO. 48. In some embodiments, the light chain variable region comprises an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to SEQ ID NO. 32, SEQ ID NO. 40 or SEQ ID NO. 48. In some embodiments, the light chain variable region comprises an amino acid sequence having one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10) amino acid substitutions, additions, and/or deletions compared to SEQ ID NO. 32, SEQ ID NO. 40, or SEQ ID NO. 48. Preferably, the amino acid substitutions, additions and/or deletions do not occur in the CDR regions.
In some embodiments, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO. 31, SEQ ID NO. 39 or SEQ ID NO. 47; the light chain variable region comprises the amino acid sequence of SEQ ID NO. 32, SEQ ID NO. 40 or SEQ ID NO. 48.
In a specific embodiment, the heavy chain variable region and the light chain variable region are selected from any one of (1) - (3): (1) A heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 31; a light chain variable region comprising the amino acid sequence of SEQ ID NO. 32; (2) A heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 39; a light chain variable region comprising the amino acid sequence of SEQ ID NO. 40; (3) A heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 47; a light chain variable region comprising the amino acid sequence of SEQ ID No. 48.
The first antigen binding portion and the second antigen binding portion may comprise any form of antigen binding fragment. In some embodiments, the first antigen binding portion is scFv, fab, fab ', F (ab') 2 Fv fragments or disulfide stabilized Fv (dsFv). In one embodiment, the first antigen binding portion is a fully human antibody or antigen binding portion thereof.
In some embodiments, the second antigen binding portion comprises an immunoglobulin single variable domain that binds VEGF. In one embodiment, the single variable domain is a VHH. In one embodiment, the single variable domain is a humanized VHH. In one embodiment, the immunoglobulin single variable domain comprises: CDR1 sequence shown in SEQ ID NO. 49, CDR2 sequence shown in SEQ ID NO. 50, and CDR3 sequence shown in SEQ ID NO. 51. In a specific embodiment, the immunoglobulin single variable domain comprises the amino acid sequence of SEQ ID NO. 11. In yet another specific embodiment, the immunoglobulin single variable domain comprises an amino acid sequence that has at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO. 11.
In some embodiments, the first antigen binding portion and the second antigen binding portion are connected by a linker. The linker may be a peptide linker or a chemical bond, preferably a peptide linker (also referred to as "linker peptide"). The peptide linker may comprise no more than 30 amino acids. Exemplary peptide linkers may include, but are not limited to, poly glycine (G), poly alanine (a), poly serine (S), or combinations thereof, such as GGAS, GGGS, GGGSG or (G) 4 S) n Wherein n is an integer of 1 to 20. Preferably, n is an integer from 1 to 5. In a specific embodiment, the peptide linker comprises the amino acid sequence of SEQ ID NO. 12.
In some embodiments, the bispecific antibodies of the invention further comprise an immunoglobulin constant region. Immunoglobulin constant regions may be derived from the heavy chain constant region (CH) and the light chain constant region (CL) of immunoglobulins of any species.
The heavy chain constant region may be derived from the heavy chain constant region of immunoglobulins of any subclass (e.g., igA, igD, igE, igG and IgM), class (e.g., igG1, igG2, igG3, igG4, igA1 and IgA 2) or subclass (e.g., igG2a and IgG2 b), or a combination thereof. In a preferred embodiment, the heavy chain constant region comprises at least an Fc region. In a preferred embodiment, the heavy chain constant region is a heavy chain constant region of human IgG1, an exemplary amino acid sequence of which is set forth in SEQ ID NO. 1. In one embodiment, the bispecific antibody of the present invention further comprises an Fc region of human IgG1, an exemplary amino acid sequence of which is shown in SEQ ID NO. 3.
In some embodiments, bispecific antibodies of the invention have reduced ADCC and CDC effector function, e.g., by inclusion of amino acid substitutions (mutations) in the heavy chain constant region. In some embodiments, bispecific antibodies of the invention may comprise a heavy chain constant region of human IgG 1. In an exemplary embodiment, the heavy chain constant region of human IgG1 may comprise a mutation selected from the group consisting of: leucine at position 234 (corresponding to position 117 of SEQ ID NO: 1) is mutated to alanine (L234A), leucine at position 235 (corresponding to position 118 of SEQ ID NO: 1) is mutated to alanine (L235A), aspartic acid at position 265 (corresponding to position 148 of SEQ ID NO: 1) is mutated to alanine (D265A), asparagine at position 297 (corresponding to position 180 of SEQ ID NO: 1) is mutated to alanine (N297A) or glycine (N297G) or glutamine (N297Q), lysine at position 322 (corresponding to position 205 of SEQ ID NO: 1) is mutated to alanine (K322A), proline at position 329 (corresponding to position 212 of SEQ ID NO: 1) is mutated to alanine (P329A), and combinations thereof. In some embodiments, bispecific antibodies of the invention may comprise a heavy chain constant region of human IgG 4. In an exemplary embodiment, the heavy chain constant region of human IgG4 may comprise a mutation selected from the group consisting of: serine at position 228 is mutated to proline (S228P), phenylalanine at position 234 is mutated to alanine (F234A), leucine at position 235 is mutated to alanine (L235A), and combinations thereof, according to EU numbering.
As used herein, "antibody-dependent cell-mediated cytotoxicity (ADCC)" refers to the binding of an antibody (via its Fc region) to an Fc receptor (FcR) on the surface of a cytotoxic cell (e.g., natural Killer (NK) cell, neutrophil, and macrophage) to target these cytotoxic cells to a target cell, and then killing the target cell with a cytotoxin. The ADCC activity of an antibody may be assessed by methods known in the art (see, e.g., US5821337 a).
As used herein, "Complement Dependent Cytotoxicity (CDC)" refers to the activation of the complement system by antibodies that bind to antigens on target cells, thereby causing lysis of the target cells. Antibodies can be evaluated for CDC activity by methods known in the art (see, e.g., gazzano-Santoro et al, J.Immunol. Methods 202:163 (1996)).
The light chain constant region may be derived from a Lambda (Lambda) light chain or a Kappa (Kappa) light chain constant region. In a preferred embodiment, the light chain constant region is a human kappa light chain constant region. In one embodiment, the light chain constant region comprises the amino acid sequence of SEQ ID NO. 2.
In some embodiments, the bispecific antibodies of the invention further comprise a heavy chain constant region (CH) and a light chain constant region (CL) of an immunoglobulin. In one embodiment, the heavy chain constant region is a human IgG1 or human IgG4 heavy chain constant region and the light chain constant region is a human kappa light chain constant region. In one embodiment, the heavy chain constant region of human IgG1 comprises a leucine to alanine mutation at positions 234 and 235 (L234A/L235A) according to EU numbering. In one embodiment, the heavy chain constant region of human IgG4 comprises serine to proline, phenylalanine to alanine, and leucine to alanine mutations at positions 228, 234, and 235, respectively, according to EU numbering (S228P/F234A/L235A).
In one embodiment, the VH and VL of the first antigen binding portion are fused to the N-terminus of CH and CL, respectively, and a single variable domain (e.g., VHH) of the second antigen binding portion is fused to the N-terminus of the VH, the N-terminus of the VL, the C-terminus of CH, and/or the C-terminus of CL, optionally via a linker.
In some embodiments, a bispecific antibody of the invention comprises a first polypeptide and a second polypeptide. In one embodiment, the first polypeptide comprises a heavy chain variable region (VH) and a heavy chain constant region (CH) of a first antigen binding portion, the second polypeptide comprises a light chain variable region (VL) and a light chain constant region (CL) of a first antigen binding portion, and a single variable domain (e.g., VHH) of a second antigen binding portion is optionally fused to: (1) the N-terminus of the VH; (2) the N-terminus of VL; (3) the N-terminus of the VH and VL; (4) the C-terminus of the CH; (5) the C-terminus of the CL; (6) the C-terminus of the CH and CL; (8) the N-terminus of the VH and the C-terminus of the CH; or (9) the N-terminus of the VL and the C-terminus of the CH.
In one embodiment, the first polypeptide and the second polypeptide are selected from any one of (1) - (5):
(1) The first polypeptide has the structure of formula (I), and the second polypeptide has the structure of formula (II)
VH-CH-Linker-VHH of formula (I),
VL-CL formula (II);
(2) The first polypeptide has the structure of formula (III), and the second polypeptide has the structure of formula (II)
VHH-Linker-VH-CH formula (III);
(3) The first polypeptide has the structure of formula (III), and the second polypeptide has the structure of formula (IV)
VHH-Linker-VL-CL formula (IV);
(4) The first polypeptide has the structure of formula (V), and the second polypeptide has the structure of formula (IV)
VH-CH formula (v);
(5) The first polypeptide has the structure of formula (V) and the second polypeptide has the structure of formula (VI)
VL-CL-Linker-VHH of formula (VI);
wherein the method comprises the steps of
VH and VL are the heavy chain variable region and the light chain variable region, respectively, of the first antigen-binding portion;
VHH is a single variable domain of a second antigen binding portion;
CH and CL are the heavy and light chain constant regions of an immunoglobulin, respectively;
linker is the Linker.
In a specific embodiment, the first polypeptide and the second polypeptide are selected from any one of (1) - (15):
(1) A first polypeptide comprising the amino acid sequence of SEQ ID NO. 13; a second polypeptide comprising the amino acid sequence of SEQ ID NO. 6;
(2) A first polypeptide comprising the amino acid sequence of SEQ ID NO. 14; a second polypeptide comprising the amino acid sequence of SEQ ID NO. 6;
(3) A first polypeptide comprising the amino acid sequence of SEQ ID NO. 5; a second polypeptide comprising the amino acid sequence of SEQ ID NO. 15;
(4) A first polypeptide comprising the amino acid sequence of SEQ ID NO. 5; a second polypeptide comprising the amino acid sequence of SEQ ID NO. 16;
(5) A first polypeptide comprising the amino acid sequence of SEQ ID NO. 17; a second polypeptide comprising the amino acid sequence of SEQ ID NO. 8;
(6) A first polypeptide comprising the amino acid sequence of SEQ ID NO. 18; a second polypeptide comprising the amino acid sequence of SEQ ID NO. 8;
(7) A first polypeptide comprising the amino acid sequence of SEQ ID NO. 7; a second polypeptide comprising the amino acid sequence of SEQ ID NO. 19;
(8) A first polypeptide comprising the amino acid sequence of SEQ ID NO. 7; a second polypeptide comprising the amino acid sequence of SEQ ID NO. 20;
(9) A first polypeptide comprising the amino acid sequence of SEQ ID NO. 21; a second polypeptide comprising the amino acid sequence of SEQ ID NO. 10;
(10) A first polypeptide comprising the amino acid sequence of SEQ ID NO. 22; a second polypeptide comprising the amino acid sequence of SEQ ID NO. 10;
(11) A first polypeptide comprising the amino acid sequence of SEQ ID NO. 9; a second polypeptide comprising the amino acid sequence of SEQ ID NO. 23;
(12) A first polypeptide comprising the amino acid sequence of SEQ ID NO. 9; a second polypeptide comprising the amino acid sequence of SEQ ID NO. 24;
(13) A first polypeptide comprising the amino acid sequence of SEQ ID NO. 14; a second polypeptide comprising the amino acid sequence of SEQ ID NO. 15;
(14) A first polypeptide comprising the amino acid sequence of SEQ ID NO. 18; a second polypeptide comprising the amino acid sequence of SEQ ID NO. 19;
(15) A first polypeptide comprising the amino acid sequence of SEQ ID NO. 22; a second polypeptide comprising the amino acid sequence of SEQ ID NO. 23.
In one embodiment, the first polypeptide comprises a leucine to alanine mutation at the following amino acid positions: positions 238 and 239 (L238A/L239A) of SEQ ID NO. 5 or SEQ ID NO. 13; positions 242 and 243 (L242A/L243A) of SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 17 or SEQ ID NO. 21; bits 382 and 383 of SEQ ID NO. 14 (L382A/L383A); or positions 386 and 387 (L386A/L387A) of SEQ ID NO:18 or SEQ ID NO: 22.
In a specific embodiment, the first polypeptide and the second polypeptide are selected from any one of (1) - (15):
(1) A first polypeptide comprising an amino acid sequence having a mutation of L238A and L239A compared to SEQ ID NO. 13; a second polypeptide comprising the amino acid sequence of SEQ ID NO. 6;
(2) A first polypeptide comprising an amino acid sequence having mutations L382A and L383A compared to SEQ ID No. 14; a second polypeptide comprising the amino acid sequence of SEQ ID NO. 6;
(3) A first polypeptide comprising an amino acid sequence having a mutation of L238A and L239A compared to SEQ ID No. 5; a second polypeptide comprising the amino acid sequence of SEQ ID NO. 15;
(4) A first polypeptide comprising an amino acid sequence having a mutation of L238A and L239A compared to SEQ ID No. 5; a second polypeptide comprising the amino acid sequence of SEQ ID NO. 16;
(5) A first polypeptide comprising an amino acid sequence having an L242A and L243A mutation compared to SEQ ID NO. 17; a second polypeptide comprising the amino acid sequence of SEQ ID NO. 8;
(6) A first polypeptide comprising an amino acid sequence having an L386A and an L387A mutation compared to SEQ ID No. 18; a second polypeptide comprising the amino acid sequence of SEQ ID NO. 8;
(7) A first polypeptide comprising an amino acid sequence having an L242A and L243A mutation compared to SEQ ID NO. 7; a second polypeptide comprising the amino acid sequence of SEQ ID NO. 19;
(8) A first polypeptide comprising an amino acid sequence having an L242A and L243A mutation compared to SEQ ID NO. 7; a second polypeptide comprising the amino acid sequence of SEQ ID NO. 20;
(9) A first polypeptide comprising an amino acid sequence having an L242A and L243A mutation compared to SEQ ID NO. 21; a second polypeptide comprising the amino acid sequence of SEQ ID NO. 10;
(10) A first polypeptide comprising an amino acid sequence having an L386A and an L387A mutation compared to SEQ ID No. 22; a second polypeptide comprising the amino acid sequence of SEQ ID NO. 10;
(11) A first polypeptide comprising an amino acid sequence having an L242A and an L243A mutation compared to SEQ ID No. 9; a second polypeptide comprising the amino acid sequence of SEQ ID NO. 23;
(12) A first polypeptide comprising an amino acid sequence having an L242A and an L243A mutation compared to SEQ ID No. 9; a second polypeptide comprising the amino acid sequence of SEQ ID NO. 24;
(13) A first polypeptide comprising an amino acid sequence having mutations L382A and L383A compared to SEQ ID No. 14; a second polypeptide comprising the amino acid sequence of SEQ ID NO. 15;
(14) A first polypeptide comprising an amino acid sequence having an L386A and an L387A mutation compared to SEQ ID No. 18; a second polypeptide comprising the amino acid sequence of SEQ ID NO. 19;
(15) A first polypeptide comprising an amino acid sequence having an L386A and an L387A mutation compared to SEQ ID No. 22; a second polypeptide comprising the amino acid sequence of SEQ ID NO. 23.
Bispecific antibodies of the invention may be multivalent antibodies, such as bivalent, trivalent, tetravalent, pentavalent, and hexavalent. In some embodiments, the bispecific antibodies of the invention are tetravalent antibodies comprising two first antigen binding portions and two second antigen binding portions. In one embodiment, the bispecific antibody of the present invention is a hexavalent antibody comprising two first antigen binding moieties and four second antigen binding moieties.
In one embodiment, the bispecific antibody of the present invention comprises two first polypeptides and two second polypeptides, wherein the first polypeptides are the same or different and the second polypeptides are the same or different. In a specific embodiment, the bispecific antibody of the present invention comprises two identical first polypeptides and two identical second polypeptides. The first polypeptide and the second polypeptide are as described above.
In some embodiments, the bispecific antibodies of the invention specifically bind ANG2 and VEGF. In some embodiments, the bispecific antibodies of the invention block the binding of ANG2 and VEGF to their respective receptors. In some embodiments, the bispecific antibodies of the invention specifically bind ANG2, but do not bind or do not substantially bind ANG1. The expression "unbound" or "substantially unbound" as used herein means that the binding capacity of the bispecific antibody of the invention to ANG1 is significantly lower than to ANG2, e.g. as described in example 4.
Antibodies or antigen binding fragments thereof may be prepared and produced using methods known in the art. Such methods may include, for example, preparing and isolating nucleic acids encoding antibodies or antigen binding fragments from phage display libraries, yeast display libraries, immortalized B cells (e.g., mouse B cell hybridoma cells or EBV immortalized B cells). It is also possible to immunize an animal, for example, an animal (e.g., a humanized mouse) with an antigen or a DNA encoding the antigen, and then isolate B cells expressing the antibody from the immunized animal. Polynucleotides encoding antibodies or antigen-binding fragments thereof may also be isolated from immunized animals or humans or prepared by chemical synthesis, and then used to construct expression vectors for expressing the antibodies or antigen-binding fragments.
Polynucleotides, vectors and host cells
In another aspect, the invention provides a polynucleotide encoding a bispecific antibody of the invention.
Polynucleotides of the invention may be obtained using methods known in the art. For example, polynucleotides of the invention may be isolated from humans, phage display libraries, yeast display libraries, immunized animals, immortalized cells (e.g., mouse B cell hybridoma cells, EBV-mediated immortalized B cells), or chemically synthesized. The polynucleotide may be codon optimized for the host cell used for expression.
In yet another aspect, the invention also provides an expression vector comprising a polynucleotide of the invention. The expression vector may further comprise additional polynucleotide sequences, such as transcriptional regulatory sequences and antibiotic resistance genes.
The invention also provides a host cell comprising a polynucleotide or expression vector of the invention. The polynucleotides or expression vectors of the invention may be introduced into a suitable host cell using a variety of methods known in the art. Such methods include, but are not limited to, viral transduction, liposome transfection, electroporation, and calcium phosphate transfection, among others. In a preferred embodiment, the host cell is used to express a bispecific antibody of the invention. Examples of host cells include, but are not limited to, prokaryotic cells (e.g., bacteria, e.g., E.coli) and eukaryotic cells (e.g., yeast, insect cells, mammalian cells). Mammalian host cells suitable for antibody expression include, but are not limited to, myeloma cells, heLa cells, HEK293 cells, chinese Hamster Ovary (CHO) cells, and other mammalian cells suitable for expression of antibodies.
The invention also provides a method of producing a bispecific antibody of the invention comprising:
Culturing a host cell of the invention under suitable conditions to express a bispecific antibody of the invention, an
(ii) isolating the bispecific antibody from the host cell or culture thereof.
Pharmaceutical composition
The invention also provides a pharmaceutical composition comprising a bispecific antibody of the invention, and a pharmaceutically acceptable carrier.
Pharmaceutically acceptable carriers can include, but are not limited to: diluents, binders and adhesives, lubricants, disintegrants, preservatives, vehicles, dispersants, glidants, sweeteners, coatings, excipients, preservatives, antioxidants (such as ascorbic acid, cysteine hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite, ascorbyl palmitate, butylated Hydroxyanisole (BHA), butylated Hydroxytoluene (BHT), lecithin, propyl gallate, alpha-tocopherol, citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like), solubilizing agents, gelling agents, softeners, solvents (such as water, alcohols, acetic acid, and syrups), buffers (such as phosphate buffers, histidine buffers, and acetate buffers), surfactants (such as nonionic surfactants, such as polysorbate 80, polysorbate 20, poloxamer, or polyethylene glycol), antibacterial agents, antifungal agents, isotonic agents (such as trehalose, sucrose, mannitol, sorbitol, lactose, glucose), absorption retarders, chelating agents, and emulsifiers. For compositions comprising an antibody or antibody conjugate, a suitable carrier may be selected from buffers (e.g., citrate buffer, acetate buffer, phosphate buffer, histidine salt buffer), isotonic agents (e.g., trehalose, sucrose, mannitol, sorbitol, lactose, glucose), nonionic surfactants (e.g., polysorbate 80, polysorbate 20, poloxamers), or combinations thereof.
The pharmaceutical compositions provided herein may be in a variety of dosage forms including, but not limited to, solid, semi-solid, liquid, powder, or lyophilized forms. Preferably, the pharmaceutical composition is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal or epidermal administration (e.g., by injection or infusion).
The pharmaceutical compositions provided herein may be administered by a variety of routes. Routes of administration include, but are not limited to, parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular, or intracavity), topical (e.g., intratumoral), epidural, or mucosal (e.g., intranasal, oral, vaginal, rectal, sublingual, or topical). The method of administration may be, for example, injection or infusion.
As a general guideline, bispecific antibodies of the invention can be administered in a dosage range of about 0.0001 to 100mg/kg, more typically 0.01 to 20mg/kg of subject body weight. For example, the dosage may be 0.3mg/kg body weight, 1mg/kg body weight, 3mg/kg body weight, 5mg/kg body weight, 10mg/kg body weight or 20mg/kg body weight, or in the range of 1-20 mg/kg. Exemplary treatment regimens require weekly dosing, biweekly dosing, tricyclically dosing, weekly dosing, monthly dosing, 3 months dosing, 3-6 months dosing, or slightly shorter initial dosing intervals followed by longer post dosing intervals.
Treatment of
Without wishing to be bound by any theory, the bispecific antibodies of the invention block ANG2 and VEGF signaling pathways by binding to ANG2 and VEGF, including blocking angiogenesis associated with ANG2 and VEGF signaling pathways. As used herein, "angiogenesis" refers to the formation of new blood vessels. Studies have shown that angiogenesis is associated with a variety of diseases, such as cancer and angiogenesis-related eye diseases.
In a general aspect, bispecific antibodies or pharmaceutical compositions of the invention are provided for use in cancer and angiogenesis-related eye diseases. The invention also provides the use of a bispecific antibody or pharmaceutical composition of the invention in the manufacture of a medicament for the treatment of cancer and angiogenesis-related eye diseases. The invention also provides a method of treating cancer and angiogenesis-related eye diseases in a subject comprising administering to the subject an effective amount of a bispecific antibody or pharmaceutical composition of the invention.
The angiogenesis-related eye disease may be an eye disease associated with choroidal and retinal vascular diseases, including but not limited to choroidal neovascularization, retinal neovascularization, and diseases associated with vascular leakage. In one embodiment, the angiogenesis-related eye disease is macular degeneration (e.g., dry or wet age-related macular degeneration (AMD)), retinal vein occlusion, retinopathy of prematurity (ROP), diabetic retinopathy, neovascular glaucoma, pathological myopia, macular edema, retinal edema, diabetic Macular Edema (DME), or choroidal neovascularization disease.
As used herein, the term "cancer" or "tumor" refers to or describes a physiological condition in an individual that is generally characterized by unregulated cell growth. Preferably, the cancer is a solid tumor associated with angiogenesis. Cancers may include primary and metastatic cancers. Non-limiting examples of cancers include lung cancer (e.g., non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma), liver cancer (e.g., hepatocellular carcinoma), pancreatic cancer, skin cancer, head and neck cancer, melanoma, ovarian cancer, colorectal cancer, gastric cancer, breast cancer, prostate cancer, uterine cancer, hodgkin's lymphoma, esophageal cancer, anal cancer, cancer of the endocrine system, thyroid cancer, parathyroid cancer, renal cancer (e.g., renal cell carcinoma, renal pelvis cancer, and adrenal cancer), soft tissue sarcoma, bladder cancer, central Nervous System (CNS) tumors, mesothelioma, glioma, meningioma, and pituitary adenoma. In a preferred embodiment, the cancer is colorectal cancer, lung cancer, breast cancer, ovarian cancer, gastric cancer or hepatocellular carcinoma.
Kit for detecting a substance in a sample
The invention also provides a kit comprising an anti-ANG 2-VEGF bispecific antibody or pharmaceutical composition of the invention. In one embodiment, the kit further comprises instructions for use of the bispecific antibodies or pharmaceutical compositions of the invention. The kit may also comprise a suitable container, such as an ampoule. In some embodiments, the kit further comprises a device for administering the drug. The kit may also comprise a label for indicating the intended use and/or method of use of the kit contents. The term "label" includes any written or recorded material provided on or with or otherwise with the kit.
Advantageous effects
The anti-ANG 2-VEGF bispecific antibody of the invention achieves at least one of the following beneficial effects:
(1) Specifically binds ANG2, but does not bind or does not substantially bind ANG1;
(2) Specifically bind to and neutralize VEGF;
(3) Blocking the binding of ANG2 to Tie 2;
(4) Inhibiting angiogenesis associated with ANG2 and VEGF activity; and
(5) Inhibit tumor growth.
Examples
The invention generally described herein will be understood more readily by reference to the following examples, which are provided by way of illustration and are not intended to limit the invention. The experimental procedures, which are not specified in the following examples, were carried out according to conventional methods and conditions, or according to the commercial specifications.
Example 1 preparation of raw materials
1.1 antigen preparation
The expression vectors for the recombinant proteins hVEGF-Fc, hANG2-His and hVEGF-His were prepared by PCR amplification of the coding sequences for the antigen VEGF (hVEGF, uniProt ID: P15692-4, SEQ ID NO: 52) and antigen Ang2 (hANG 2, unipro ID: O15123-1, SEQ ID NO: 53) (synthesized by general Biotechnology Co., ltd.) linked at the 3' end to the coding sequence of the human IgG1 Fc fragment (SEQ ID NO: 3) or His tag and then constructed by homologous recombination to eukaryotic expression vector pcDNA3.4 (Invitrogen). The recombinant protein expression vectors were transformed into E.coli DH 5. Alpha. Respectively, cultured overnight at 37℃and then subjected to plasmid extraction using endotoxin-free plasmid extraction kit (OMEGA, D6950-01), and passed through an Expi293 TM Expression system kit(ThermoFisher, A14635) expression, transient methods see the kit instructions.
After 7 days of transfection, the cell expression supernatants were centrifuged at high speed for 10min, and the hANG2-Fc and hVEGF-Fc expression supernatants were affinity purified with COLUMN XK16/20 (from Cytiva), then the protein of interest was eluted with 100mM sodium acetate (pH 3.0), followed by neutralization with 1M Tris-HCl; the hANG2-His or hVEGF-His expression supernatants were affinity purified using Ni Smart Beads 6FF (Hemsl and Biotechnology Co., ltd., SA 036050) and then the target protein was eluted with a gradient concentration of imidazole. The eluted proteins were each transferred to PBS buffer through ultrafiltration concentrators (Millipore, UFC 901096). And (5) after passing SDS-PAGE identification and activity identification, freezing at-80 ℃ for standby.
1.2 preparation of positive control antibodies
The anti-VEGF positive control antibody used was Bevacizumab (synthesized from the antibody sequence information provided in patent US9527925B 2) and P30-10-26 (VHH) developed independently by the applicant VEGF huFc, amino acid sequence SEQ ID NO: 4); the positive control antibodies against ANG2 used were Nesvocumab (synthesized according to the sequence disclosed in patent application US20170355762A 1), fully human antibody 17 of the IgG1 type (heavy chain amino acid sequence shown in SEQ ID NO:5, light chain amino acid sequence shown in SEQ ID NO: 6), 78A14 (heavy chain amino acid sequence shown in SEQ ID NO:7, light chain amino acid sequence shown in SEQ ID NO: 8) and 78A74-7 (heavy chain amino acid sequence shown in SEQ ID NO:9, light chain amino acid sequence shown in SEQ ID NO: 10) which were developed independently of the applicant; the anti-ANG 2-VEGF positive control antibody used was Faricimab (synthesized from antibody sequence information provided in patent WO2016073157A1, available from the bio-tech company of baiying, thailand).
The remaining control antibodies were expressed using an ExpiCHO transient expression system (Gibco, A29133) except for Faricimab, and the resulting supernatant was filtered through a 0.22 μm filter and purified using Protein A/G affinity chromatography column affinity method, followed by elution of the target Protein with 100mM sodium acetate (pH 3.0), followed by neutralization with 1M Tris-HCl, and finally displacement of the resulting Protein into PBS buffer by ultrafiltration concentration tube (Millipore, UFC 901096). Purified antibodies were subjected to SDS-PAGE and activity assay and then sub-stored at-80 ℃.
1.3 construction of cell lines
HEK293 cell strain (hereinafter abbreviated as huTie2-HEK293 cell strain) which overexpresses human Tie2 and HEK293 cell strain (hereinafter abbreviated as VEGFR2/NFAT-HEK293 cell strain) which overexpresses VEGFR2 and NFAT luciferase reporter genes are constructed, and the cell strain construction methods are as follows:
1.3.1 Construction of huTie2-HEK293 cell line
A DNA fragment encoding the human Tie2 protein (NCBI Gene ID: 7010) was synthesized by a Gene synthesis technique and cloned into the expression vector pLVX-Puro (Clontech, 632164) to obtain a Tie2 expression plasmid. HEK293 cells were cultured using Gibco's DMEM serum-free Medium (cat# 12634010)
Figure BDA0003414384290000141
CRL-1573 TM ). Tie2 expression plasmids were introduced into HEK293 cells using Invitrogen's electrotransfer kit (cat# MPK 10096) and electrotransfer apparatus (cat# MP 922947), and monoclonal cell lines were screened by medium of puromycin at a final concentration of 2. Mu.g/mL and huTie2-HEK293 cell lines were obtained by FACS identification.
1.3.2 Construction of VEGFR2/NFAT-HEK293 cell line
Firstly, constructing an NFAT-HEK293 cell strain: pGL4.30 plasmid (promega, #E8481) containing the luciferase gene whose transcription and expression is driven by the NFAT response element (NFAT-RE) was passed through an electrotransport apparatus (Invitrogen, neon) TM Transfection System, MP 922947) were electrotransformed into HEK293 cells
Figure BDA0003414384290000142
CRL-1573 TM ) The monoclonal cell line was then screened for identification by Hygromycin B (source culture, S160J 7) at a final concentration of 500. Mu.g/mL. After qualification of the NFAT-HEK293 cell line, plasmids expressing VEGFR2 (Gene sequence see NCBI Gene ID:3791, from general biosynthesis) were electrotransferred into the NFAT-HEK293 cell line by the method of example 1.3.1, and the VEGFR2/NFAT-HEK293 cell line was obtained by FACS identification.
EXAMPLE 2 construction of anti-ANG 2-VEGF bispecific antibody
This example describes the construction of an anti-ANG 2-VEGF bispecific antibody (BsAb) in which the first antigen binding portion uses the heavy chain variable region (VH) and the light chain variable region (VL) of an anti-ANG 2 fully human antibody 17, 78A14 or 78A74-7 (amino acid sequences of HCDR1-3 and LCDR1-3 and VH and VL defined by AbM (SEQ ID NO: shown in Table 1A)), and the second antigen binding portion uses the VHH of a humanized anti-VEGF single domain antibody P30-10-26 (amino acid sequences of CDR1, CDR2 and CDR defined by AbM are shown in SEQ ID NO:49, SEQ ID NO:50 and SEQ ID NO:51, respectively; and the amino acid sequence of VHH is shown in SEQ ID NO: 11). The bispecific antibodies of the invention further comprise a human IgG1 heavy chain constant region (SEQ ID NO: 1) or a human IgG1 heavy chain constant region mutant (abbreviated as LALA; leucine to alanine at positions 117 and 118 of SEQ ID NO: 1) and a human Kappa light chain constant region (SEQ ID NO: 2), fused to the C-terminus of the VH and VL, respectively, of the first antigen-binding portion; and a connecting peptide (SEQ ID NO: 12). FIGS. 1A-1E show schematic structural diagrams of candidate bispecific antibodies. The candidate bispecific antibody contains two identical first polypeptides and two identical second polypeptides, the amino acid sequences of which are shown in table 1B.
The specific method for preparing the expression vector of the first polypeptide and the second polypeptide is as follows: amplifying target fragments by a PCR method, connecting all fragments by an overlap extension PCR method, and constructing the fragments on a modified eukaryotic expression vector plasmid pcDNA3.4 (Invitrogen) respectively by a homologous recombination method to obtain expression vectors of the first polypeptide and the second polypeptide. The expression vectors of the first polypeptide and the second polypeptide are respectively transformed into escherichia coli DH5 alpha, and then plasmid extraction is carried out by utilizing an endotoxin-free plasmid extraction kit (OMEGA, D6950-01) to obtain the expression plasmids of the first polypeptide chain and the second polypeptide chain without endotoxin for eukaryotic expression.
TABLE 1A
Antibody name HCDR1 HCDR2 HCDR3 LCDR1 LCDR2 LCDR3 VH VL
17 25 26 27 28 29 30 31 32
78A14 33 34 35 36 37 38 39 40
78A74-7 41 42 43 44 45 46 47 48
TABLE 1B
Figure BDA0003414384290000151
EXAMPLE 3 expression and purification of anti-ANG 2-VEGF bispecific antibodies
The anti-ANG 2-VEGF bispecific antibody of example 2 was expressed by the expcho transient expression system (Thermo Fisher, a 29133) as follows: on the day of transfection, cell density was confirmed to be 7×10 6 Up to 1X 10 7 Cell viability about living cells/mL>98 at this time, the cells were adjusted to a final concentration of 6X 10 using fresh ExpiCHO expression medium pre-warmed at 37 ℃ 6 Individual cells/mL. OptiPRO pre-cooled at 4deg.C TM SFM dilution of plasmid of interest (1. Mu.g plasmid was added to 1mL of the medium), while using OptiPRO TM SFM dilution of Expifectamine TM CHO, mixing the two materials in equal volume, and gently stirring to obtain the product TM The CHO/plasmid DNA mixture was incubated at room temperature for 1-5min, slowly added to the prepared cell suspension while gently shaking, and finally placed in a cell culture shaker at 37℃and 8% CO 2 Culturing under the condition. 18-22h after transfection, expiCHO was added to the culture broth TM Enhance and ExpiCHO TM Feed, shake flask placed on a shaker at 32℃and 5% CO 2 Culturing was continued under the conditions. On day 5 post transfection, the same volume of ExpiCHO was added TM Feed, slowly add while gently mix the cell suspension. After 7-15 days of transfection, the cells expressing the protein of interest are culturedThe supernatant was centrifuged at 15000g for 10min, and the resulting supernatant was affinity purified with COLUMN XK16/20 (available from cytova), followed by elution of the protein of interest with 100mM sodium acetate (ph 3.0), followed by neutralization with 1m tris-HCl, and finally displacement of the resulting protein into PBS buffer by ultrafiltration concentrate (Millipore, UFC 901096). Purified antibodies were greater than 90% pure as identified by SDS-PAGE and SEC-HPLC, and stored in aliquots at-80 ℃.
Example 4 antigen binding Activity assay of anti-ANG 2-VEGF bispecific antibody
4.1 ELISA method for detecting binding activity of anti-ANG 2-VEGF bispecific antibody to recombinant protein hVEGF-His
The recombinant protein hVEGF-His was coated on 96-well ELISA plates overnight at 4 ℃. The next day, the well plate was blocked with 5% skim milk for 2h after 3 washes with PBST, and incubated with different concentrations of antibody to be tested for 1h after 3 washes with PBST. After 3 washes with PBST, secondary Anti-human-IgG-Fc-HRP (abcam, ab 79225) was added and incubated for 1h. After incubation, PBST plates were washed six times and developed with TMB (SurModics, TMBS-1000-01). Based on the color development, the reaction was stopped by adding 2M HCl and the OD450 value was read by a microplate reader (Molecular Devices, specterMax 190). By PRISM TM (GraphPad Software, san Diego, calif.) analysis data and calculation of EC 50 Values.
ELISA binding assay results are shown in FIGS. 2A-2E, candidate bispecific antibodies showed comparable binding activity to recombinant protein hVEGF-His to antibodies P30-10-26 and Faricimab.
4.2 ELISA method for detecting binding activity of candidate bispecific antibody to recombinant protein hANG2-His
The recombinant protein hANG2-His was coated on 96-well ELISA plates overnight at 4 ℃. The next day, the well plate was blocked with 5% skim milk for 2h after 3 washes with PBST, and incubated with different concentrations of antibody to be tested for 1h after 3 washes with PBST. After 3 washes with PBST, secondary Anti-human-IgG-Fc-HRP (abcam, ab 79225) was added and incubated for 1h. After incubation, PBST plates were washed six times and developed with TMB (SurModics, TMBS-1000-01). According to the color development result, 2M HCl is added to stop the reaction, and OD450 value is read by a microplate reader (Molecular Devices, specterMax 190) . By PRISM TM (GraphPad Software, san Diego, calif.) analysis data and calculation of EC 50 Values.
The ELISA binding assay results are shown in FIGS. 3A-3E, and the candidate bispecific antibodies showed comparable binding activity to recombinant protein hANG2-His to antibodies 17, 78A14, 78A74-7, and were significantly superior to the positive bispecific antibody Faricimab.
4.3 ELISA-based detection of binding Activity of anti-ANG 2 fully human antibodies to hANG1-Fc
A fusion protein of 30. Mu.L/well of 2. Mu.g/mL hANG1-Fc (hANG 1 (Uniprot ID: Q15389-1) with the Fc fragment of human IgG1 (SEQ ID NO: 3) was coated on a 96-well ELISA plate, see example 1.1 for preparation, overnight at 4 ℃. The next day, the well plate was blocked with 5% skim milk for 2h after 3 washes with PBST, and after 3 washes with PBST, the test antibody was added in gradient dilutions and incubated for 1h. After 3 washes with PBST, anti-human Kappa HRP (Millipore, AP 502P) was added and incubated for 1h. After incubation, PBST was washed six times, TMB was added for color development, 2M HCl was added according to the color development result to terminate the reaction, and OD450 value was read by an ELISA reader.
The results showed that none of antibodies 17, 78A14 and 78A74-7 bound to hANG 1.
Example 5 anti-ANG 2-VEGF bispecific antibody neutralization VEGF Activity assay
The VEGFR2/NFAT-HEK293 cell line constructed in example 1 was used to screen candidate bispecific antibodies capable of neutralizing VEGF activity. Under the cell strain culture system, VEGF protein activates the transcription activity of intracellular transcription factor NFAT through VEGF-VEGFR2 signal axis, thereby starting the transcription and expression of luciferase reporter gene; when the candidate bispecific antibody is added, the candidate bispecific antibody neutralizes VEGF and blocks its binding to VEGFR2, thereby blocking luciferase reporter expression.
The specific method comprises the following steps: resuscitates VEGFR2/NFAT-HEK293 cell strain, uses cells with good growth state after passage 2-4 times for experiment, resuspends cells after pancreatin digestion, removes supernatant by centrifugation at normal temperature and 300g, resuspends cells with DMEM medium, and adjusts cell density to 4×10 after counting 5 Individual cells/mL, 100. Mu.L per well was added to a new 96-well platePlacing in a cell incubator at 37 ℃. Meanwhile, the antibody to be detected is diluted in a gradient way by using a DMEM culture medium, 60ng/mL of recombinant protein hVEGF-Fc is added, and the mixture is incubated for 30min at room temperature after mixing. Subsequently, the co-incubated antibody/hVEGF-Fc mixture was added to a 96-well plate and incubated in an incubator at 37℃for 18h. After the incubation, 30. Mu.L of luciferase substrate Bright-Lite (Vazyme, DD 1204-03) was added to each well, and after shaking for 2min, the fluorescence value of the 96-well plate was measured and IC was calculated 50 。
The results are shown in FIGS. 4A-4C and tables 2A-2C, and the activity of candidate bispecific antibodies BsAb3, bsAb14, bsAb30, bsAb31, bsAb22 and BsAb29 to neutralize VEGF is comparable to that of P30-10-26; the effect of candidate bispecific antibodies BsAb14-LALA and BsAb22-LALA on neutralizing VEGF was comparable to P30-10-26 and Faricimab.
TABLE 2A
Antibody name Neutralizing VEGF Activity IC 50 (nM)
BsAb3 0.194
BsAb14 0.213
BsAb22 0.418
P30-10-26 0.292
TABLE 2B
Antibody name Neutralizing VEGF Activity IC 50 (nM)
BsAb29 1.130
BsAb30 0.608
BsAb31 0.754
P30-10-26 0.878
TABLE 2C
Antibody name Neutralizing VEGF Activity IC 50 (nM)
BsAb14-LALA 0.469
BsAb22-LALA 0.401
Faricimab 0.582
P30-10-26 0.482
Example 6 detection of anti-ANG 2-VEGF bispecific antibody Activity blocking ANG2 binding to Tie2 based on FACS method
Candidate antibodies were evaluated for blocking binding activity of ANG2 and the cell surface expression receptor Tie2 based on FACS methods. The specific method comprises the following steps: antibodies to be tested, which were diluted in a gradient in FACS buffer (1×pbs+2% fbs), were added to 96-well round bottom plates at 100 μl per well. To the 96-well plate was then added 2. Mu.g/mL biotin-labeled hANG2-Fc protein (biotin-hANG 2-Fc) in 100. Mu.L of FACS buffer, followed by incubation at 4℃for 1h.
huTie2-HEK293 cells passaged 2-4 times and well grown were used for the experiments. Will be 1X 10 6 Cells in FACS buffer at 100 μl/well were added to the new 96-well round bottom plate, centrifuged at 4deg.C/300 g and the supernatant removed; subsequently, 180. Mu.L per well of pre-incubated antibody/biotin-hANG 2-Fc mixture was added to the cells, followed by incubation at 4℃for 30min; cells were then centrifuged at 4 ℃/300g and the supernatant removed, followed by 200 μl/well FACS buffer and resuspension of cells, centrifugation at 4 ℃/300g to remove supernatant; this procedure was repeated twice. PE-labeled strepitavidins (Invitrogen, 12-4317-87;1:200 diluted in FACS buffer) were added to the wells at 200. Mu.L/well and resuspended cells were gently blown, followed by incubation at 4℃for 30min in the absence of light. After the incubation, the cells were centrifuged at 300g at 4℃to remove the supernatant, and the cells were resuspended in FACS buffer and the procedure was repeated twice. Finally the amount of biotin-hANG2-Fc bound to the cells (expressed as the Mean Fluorescence Intensity (MFI) of PE) was detected by flow cytometry (Beckman, cytoFLEX AOO-1-1102).
The results are shown in FIGS. 5A-5B and tables 3A-3B. Wherein, fig. 5A and table 3A show that the blocking effect of the candidate bispecific antibodies BsAb14, bsAb30, bsAb31, and BsAb22 is comparable to antibodies 78a14 and 78a 74-7; fig. 5B and table 3B show that the blocking effect of the candidate bispecific antibodies BsAb14-LALA and BsAb22-LALA was comparable to antibodies 78a14 and 78a74-7, significantly better than Faricimab.
TABLE 3A
Antibody name Blocking IC 50 Value (nM)
BsAb14 0.415
BsAb22 0.350
BsAb30 0.362
BsAb31 0.299
78A14 0.390
78A74-7 0.226
TABLE 3B
Antibody name Blocking IC 50 Value (nM)
BsAb14-LALA 0.411
BsAb22-LALA 0.320
78A14 0.464
78A74-7 0.440
Faricimab 17.76
EXAMPLE 7 detection of efficacy of anti-ANG 2-VEGF bispecific antibody in inhibiting HUVEC proliferation
In this example, the inhibitory effect of anti-ANG 2-VEGF bispecific antibodies BsAb14 and BsAb22 on VEGF-induced endothelial cell proliferation was demonstrated. The specific implementation mode is as follows: resuscitates human umbilical vein endothelial cell line (HUVEC) (from North Nanoorganism), uses cells with good growth state after passage 2-4 times for experiment, resuspends cells after pancreatin digestion, removes supernatant by centrifugation at normal temperature and 300g, resuspends cells with EBM-2 medium of 0.5% FBS, and adjusts cell density to 5×10 after counting 4 Each cell/mL was added at 50. Mu.L per well to a fresh 96-well cell culture plate and incubated overnight in a 37℃cell incubator. On the next day, the antibody to be tested was diluted in a gradient using EBM-2 medium, 400ng/mL of recombinant protein hVEGF-Fc was added, and after mixing, incubated at room temperature for 30min. Subsequently, the co-incubated antibody/hVEGF-Fc mixture was added to 96-well cell plates and incubated in an incubator at 37℃for 72h. After incubation, 20. Mu.L MTS (Progema, G3581) was added to each well and after shaking for 2min, the OD492 value of the 96-well plate was measured. Proliferation inhibition was calculated from the respective OD492, proliferation inhibition (%) = [1- (OD 492) Sample of -OD492 Cell only )/(OD492 VEGF -OD492 Cell only )]×100％。
The results are shown in FIGS. 6A-6C, with candidate bispecific antibodies BsAb14 and BsAb22 significantly inhibiting VEGF-induced HUVEC proliferation and with activity comparable to P30-10-26; at higher antibody concentrations, e.g., at antibody concentrations up to 100nM, P30-10-26 has better activity in inhibiting VEGF-induced HUVEC proliferation than Bevacizumab.
Example 8 detection of efficacy of anti-ANG 2-VEGF bispecific antibody against Colo205 tumor cell animal model
The tumor inhibiting effect of anti-ANG 2-VEGF bispecific antibodies BsAb3, bsAb14, and BsAb22 in the human colon cancer cell Colo205 engraftment model was verified and compared to antibodies Nesvacumab, bevacizumab and P30-10-26. The specific method comprises the following steps: will be 5X 10 6 Colo205 cells (from North Nanoorganism) were inoculated subcutaneously into male nude mice (Beijing Vietnam laboratory animal technologies Co., ltd.) of 6-8 weeks old. The experimental mice were kept under standard conditions. The tumor waiting volume reaches 130mm 3 When the medicine is left and right, the operation of dividing the medicine into component cages and administering medicine is carried out. Each group of 8 tumor-bearing nude mice contained 7 groups, including 3 candidate bispecific antibody groups (BsAb 3 3.4952mg/kg, bsAb14 3.5224mg/kg and BsAb22 3.528mg/kg; equimolar dosages), 3 positive control antibody groups (Nesvocumab 3mg/kg and Bevacizumab 3 mg/kg; equimolar dosages to bispecific antibodies; P30-10-26 3mg/kg, molar dosages 2.3 fold greater than bispecific antibodies), and PBS negative control groups. The administration was by intraperitoneal injection, and the doses were as shown in fig. 7A to 7C, administered once every 3 to 4 days, 2 times per week and 2 tumor volumes were measured (v= (l×w) 2 ) 2, wherein L is the longest of the tumor diameters and W is the shortest of the tumor diameters (mm)), were administered 9 times. Tumors were taken 10 days after the end of the administration and the tumor weights were measured. The average tumor volume, tumor weight and change in body weight of each group of mice were analyzed, and tumor inhibition rate was calculated, and the results are shown in fig. 7A to 7C and table 4.
As shown in fig. 7B, there was no significant difference between the weights of the mice in each group, indicating that the mice were well tolerated by the antibodies. As shown in fig. 7A, 7C and table 4, PBS group mice developed the fastest tumor; compared with PBS group, all antibodies have obvious tumor inhibiting effect; at equimolar doses, the tumor inhibiting effect of the candidate bispecific antibodies BsAb3, bsAb14 and BsAb22 was superior to that of Nesvacumab and Bevacizumab; unexpectedly, bsAb14 and BsAb22 exhibited excellent antitumor activity, comparable to the antitumor effect of the anti-VEGF antibody P30-10-26 up to 2.3 times the molar number thereof.
TABLE 4 Table 4
Figure BDA0003414384290000191
Those skilled in the art will further recognize that the invention may be embodied in other specific forms without departing from the spirit or central characteristics thereof. Since the foregoing description of the present disclosure discloses only exemplary embodiments thereof, it should be understood that other variations are considered to be within the scope of the invention. Therefore, the present invention is not limited to the specific embodiments described in detail herein. Rather, reference should be made to the appended claims for indicating the scope and content of the invention.
Sequence listing
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Sequence listing
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Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
195 200 205
Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys
210 215 220
Ser Leu Ser Leu Ser Pro Gly Lys
225 230
<210> 4
<211> 361
<212> PRT
<213> Artificial Sequence
<220>
<223> P30-10-26 VHH-huFc
<400> 4
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Gly Leu Asp Tyr Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Gly Ser Ser Ser Lys Glu Thr Asn Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Ala Gly Ser Pro Leu Cys Leu Ile Ser Leu Gln Asp His Tyr Gly
100 105 110
Leu Tyr Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro
130 135 140
Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys
145 150 155 160
Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
165 170 175
Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
180 185 190
Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu
195 200 205
Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His
210 215 220
Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys
225 230 235 240
Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln
245 250 255
Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu
260 265 270
Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
275 280 285
Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn
290 295 300
Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu
305 310 315 320
Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val
325 330 335
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln
340 345 350
Lys Ser Leu Ser Leu Ser Pro Gly Lys
355 360
<210> 5
<211> 451
<212> PRT
<213> Artificial Sequence
<220>
<223> 17/BsAb3/BsAb4 heavy chain
<400> 5
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Lys Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Lys Tyr
20 25 30
Thr Ile Asn Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Leu Val
35 40 45
Ala Asp Ile Ser Ser Gly Gly Asp Tyr Val Asn Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asp Ser Val Tyr
65 70 75 80
Leu Leu Met Asn Gly Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asp Ala Leu Thr Tyr Ser Asn Asn Trp Leu Asp Ser Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
115 120 125
Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
130 135 140
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
145 150 155 160
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
165 170 175
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
180 185 190
Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
195 200 205
Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
210 215 220
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
225 230 235 240
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
245 250 255
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
260 265 270
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
275 280 285
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
290 295 300
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
305 310 315 320
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
325 330 335
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
340 345 350
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
355 360 365
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
370 375 380
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
385 390 395 400
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
405 410 415
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
420 425 430
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
435 440 445
Pro Gly Lys
450
<210> 6
<211> 214
<212> PRT
<213> Artificial Sequence
<220>
<223> 17/BsAb1/BsAb2 light chain
<400> 6
Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Gly Gly Tyr Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Glu Ser Tyr Pro Leu
85 90 95
Thr Phe Gly Gly Gly Thr Arg Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 7
<211> 455
<212> PRT
<213> Artificial Sequence
<220>
<223> 78A14/BsAb15/BsAb16 heavy chain
<400> 7
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Val Tyr
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Ala Thr Leu Asp Gly Tyr Thr Ala Gly Tyr Tyr Tyr Gly Met
100 105 110
Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr
115 120 125
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
130 135 140
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
145 150 155 160
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
165 170 175
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
180 185 190
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
195 200 205
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
210 215 220
Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
225 230 235 240
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
245 250 255
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
260 265 270
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
275 280 285
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
290 295 300
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
305 310 315 320
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
325 330 335
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
340 345 350
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
355 360 365
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
370 375 380
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
385 390 395 400
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
405 410 415
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
420 425 430
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
435 440 445
Leu Ser Leu Ser Pro Gly Lys
450 455
<210> 8
<211> 214
<212> PRT
<213> Artificial Sequence
<220>
<223> 78A14/BsAb13/BsAb14 light chain
<400> 8
Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Gly Pro Ser Asn Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly
50 55 60
Gly Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Tyr Gly Val Ser Gln His
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 9
<211> 455
<212> PRT
<213> Artificial Sequence
<220>
<223> 78A74-7/BsAb23/BsAb24 heavy chain
<400> 9
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Ala Thr Leu Asp Gly Tyr Thr Ala Gly Tyr Tyr Tyr Gly Met
100 105 110
Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr
115 120 125
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
130 135 140
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
145 150 155 160
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
165 170 175
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
180 185 190
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
195 200 205
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
210 215 220
Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
225 230 235 240
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
245 250 255
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
260 265 270
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
275 280 285
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
290 295 300
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
305 310 315 320
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
325 330 335
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
340 345 350
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
355 360 365
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
370 375 380
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
385 390 395 400
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
405 410 415
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
420 425 430
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
435 440 445
Leu Ser Leu Ser Pro Gly Lys
450 455
<210> 10
<211> 216
<212> PRT
<213> Artificial Sequence
<220>
<223> 78A74-7/BsAb21/BsAb22 light chain
<400> 10
Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Leu Met Ser Asn Trp Pro Pro
85 90 95
Glu Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val
100 105 110
Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys
115 120 125
Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg
130 135 140
Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
145 150 155 160
Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser
165 170 175
Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
180 185 190
Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
195 200 205
Lys Ser Phe Asn Arg Gly Glu Cys
210 215
<210> 11
<211> 129
<212> PRT
<213> Artificial Sequence
<220>
<223> P30-10-26 VHH
<400> 11
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Gly Leu Asp Tyr Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Gly Ser Ser Ser Lys Glu Thr Asn Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Ala Gly Ser Pro Leu Cys Leu Ile Ser Leu Gln Asp His Tyr Gly
100 105 110
Leu Tyr Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser
<210> 12
<211> 15
<212> PRT
<213> Artificial Sequence
<220>
<223> connecting peptide
<400> 12
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10 15
<210> 13
<211> 595
<212> PRT
<213> Artificial Sequence
<220>
<223> BsAb1 heavy chain
<400> 13
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Lys Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Lys Tyr
20 25 30
Thr Ile Asn Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Leu Val
35 40 45
Ala Asp Ile Ser Ser Gly Gly Asp Tyr Val Asn Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asp Ser Val Tyr
65 70 75 80
Leu Leu Met Asn Gly Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asp Ala Leu Thr Tyr Ser Asn Asn Trp Leu Asp Ser Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
115 120 125
Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
130 135 140
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
145 150 155 160
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
165 170 175
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
180 185 190
Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
195 200 205
Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
210 215 220
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
225 230 235 240
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
245 250 255
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
260 265 270
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
275 280 285
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
290 295 300
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
305 310 315 320
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
325 330 335
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
340 345 350
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
355 360 365
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
370 375 380
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
385 390 395 400
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
405 410 415
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
420 425 430
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
435 440 445
Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
450 455 460
Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
465 470 475 480
Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Gly Leu Asp
485 490 495
Tyr Tyr Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu
500 505 510
Gly Val Ser Cys Ile Gly Ser Ser Ser Lys Glu Thr Asn Tyr Ala Asp
515 520 525
Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr
530 535 540
Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr
545 550 555 560
Tyr Cys Thr Ala Gly Ser Pro Leu Cys Leu Ile Ser Leu Gln Asp His
565 570 575
Tyr Gly Leu Tyr Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr
580 585 590
Val Ser Ser
595
<210> 14
<211> 595
<212> PRT
<213> Artificial Sequence
<220>
<223> BsAb2/BsAb29 heavy chain
<400> 14
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Gly Leu Asp Tyr Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Gly Ser Ser Ser Lys Glu Thr Asn Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Ala Gly Ser Pro Leu Cys Leu Ile Ser Leu Gln Asp His Tyr Gly
100 105 110
Leu Tyr Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Lys Pro Gly Gly
145 150 155 160
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Lys Tyr
165 170 175
Thr Ile Asn Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Leu Val
180 185 190
Ala Asp Ile Ser Ser Gly Gly Asp Tyr Val Asn Tyr Ala Asp Ser Val
195 200 205
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asp Ser Val Tyr
210 215 220
Leu Leu Met Asn Gly Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
225 230 235 240
Ala Arg Asp Ala Leu Thr Tyr Ser Asn Asn Trp Leu Asp Ser Trp Gly
245 250 255
Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser
260 265 270
Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala
275 280 285
Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val
290 295 300
Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala
305 310 315 320
Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val
325 330 335
Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His
340 345 350
Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys
355 360 365
Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
370 375 380
Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met
385 390 395 400
Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His
405 410 415
Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val
420 425 430
His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr
435 440 445
Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly
450 455 460
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
465 470 475 480
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val
485 490 495
Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser
500 505 510
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu
515 520 525
Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro
530 535 540
Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
545 550 555 560
Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met
565 570 575
His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser
580 585 590
Pro Gly Lys
595
<210> 15
<211> 358
<212> PRT
<213> Artificial Sequence
<220>
<223> BsAb3/BsAb29 light chain
<400> 15
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Gly Leu Asp Tyr Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Gly Ser Ser Ser Lys Glu Thr Asn Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Ala Gly Ser Pro Leu Cys Leu Ile Ser Leu Gln Asp His Tyr Gly
100 105 110
Leu Tyr Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140
Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly
145 150 155 160
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Tyr
165 170 175
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
180 185 190
Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
195 200 205
Gly Gly Tyr Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
210 215 220
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Glu Ser Tyr Pro Leu
225 230 235 240
Thr Phe Gly Gly Gly Thr Arg Leu Glu Ile Lys Arg Thr Val Ala Ala
245 250 255
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
260 265 270
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
275 280 285
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
290 295 300
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
305 310 315 320
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
325 330 335
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
340 345 350
Phe Asn Arg Gly Glu Cys
355
<210> 16
<211> 358
<212> PRT
<213> Artificial Sequence
<220>
<223> BsAb4 light chain
<400> 16
Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Gly Gly Tyr Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Glu Ser Tyr Pro Leu
85 90 95
Thr Phe Gly Gly Gly Thr Arg Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
210 215 220
Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
225 230 235 240
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
245 250 255
Gly Leu Asp Tyr Tyr Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys
260 265 270
Glu Arg Glu Gly Val Ser Cys Ile Gly Ser Ser Ser Lys Glu Thr Asn
275 280 285
Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala
290 295 300
Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
305 310 315 320
Ala Val Tyr Tyr Cys Thr Ala Gly Ser Pro Leu Cys Leu Ile Ser Leu
325 330 335
Gln Asp His Tyr Gly Leu Tyr Glu Tyr Asp Tyr Trp Gly Gln Gly Thr
340 345 350
Leu Val Thr Val Ser Ser
355
<210> 17
<211> 599
<212> PRT
<213> Artificial Sequence
<220>
<223> BsAb13 heavy chain
<400> 17
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Val Tyr
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Ala Thr Leu Asp Gly Tyr Thr Ala Gly Tyr Tyr Tyr Gly Met
100 105 110
Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr
115 120 125
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
130 135 140
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
145 150 155 160
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
165 170 175
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
180 185 190
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
195 200 205
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
210 215 220
Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
225 230 235 240
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
245 250 255
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
260 265 270
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
275 280 285
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
290 295 300
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
305 310 315 320
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
325 330 335
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
340 345 350
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
355 360 365
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
370 375 380
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
385 390 395 400
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
405 410 415
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
420 425 430
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
435 440 445
Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly
450 455 460
Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly
465 470 475 480
Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
485 490 495
Phe Gly Leu Asp Tyr Tyr Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly
500 505 510
Lys Glu Arg Glu Gly Val Ser Cys Ile Gly Ser Ser Ser Lys Glu Thr
515 520 525
Asn Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
530 535 540
Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
545 550 555 560
Thr Ala Val Tyr Tyr Cys Thr Ala Gly Ser Pro Leu Cys Leu Ile Ser
565 570 575
Leu Gln Asp His Tyr Gly Leu Tyr Glu Tyr Asp Tyr Trp Gly Gln Gly
580 585 590
Thr Leu Val Thr Val Ser Ser
595
<210> 18
<211> 599
<212> PRT
<213> Artificial Sequence
<220>
<223> BsAb14/BsAb30 heavy chain
<400> 18
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Gly Leu Asp Tyr Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Gly Ser Ser Ser Lys Glu Thr Asn Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Ala Gly Ser Pro Leu Cys Leu Ile Ser Leu Gln Asp His Tyr Gly
100 105 110
Leu Tyr Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
145 150 155 160
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Val Tyr
165 170 175
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
180 185 190
Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val
195 200 205
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
210 215 220
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
225 230 235 240
Ala Lys Ala Thr Leu Asp Gly Tyr Thr Ala Gly Tyr Tyr Tyr Gly Met
245 250 255
Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr
260 265 270
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
275 280 285
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
290 295 300
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
305 310 315 320
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
325 330 335
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
340 345 350
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
355 360 365
Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
370 375 380
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
385 390 395 400
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
405 410 415
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
420 425 430
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
435 440 445
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
450 455 460
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
465 470 475 480
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
485 490 495
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
500 505 510
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
515 520 525
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
530 535 540
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
545 550 555 560
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
565 570 575
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
580 585 590
Leu Ser Leu Ser Pro Gly Lys
595
<210> 19
<211> 358
<212> PRT
<213> Artificial Sequence
<220>
<223> BsAb15/BsAb30 light chain
<400> 19
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Gly Leu Asp Tyr Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Gly Ser Ser Ser Lys Glu Thr Asn Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Ala Gly Ser Pro Leu Cys Leu Ile Ser Leu Gln Asp His Tyr Gly
100 105 110
Leu Tyr Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140
Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
145 150 155 160
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr
165 170 175
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
180 185 190
Tyr Gly Pro Ser Asn Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly
195 200 205
Gly Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro
210 215 220
Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Tyr Gly Val Ser Gln His
225 230 235 240
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
245 250 255
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
260 265 270
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
275 280 285
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
290 295 300
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
305 310 315 320
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
325 330 335
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
340 345 350
Phe Asn Arg Gly Glu Cys
355
<210> 20
<211> 358
<212> PRT
<213> Artificial Sequence
<220>
<223> BsAb16 light chain
<400> 20
Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Gly Pro Ser Asn Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly
50 55 60
Gly Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Tyr Gly Val Ser Gln His
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
210 215 220
Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
225 230 235 240
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe
245 250 255
Gly Leu Asp Tyr Tyr Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys
260 265 270
Glu Arg Glu Gly Val Ser Cys Ile Gly Ser Ser Ser Lys Glu Thr Asn
275 280 285
Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala
290 295 300
Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr
305 310 315 320
Ala Val Tyr Tyr Cys Thr Ala Gly Ser Pro Leu Cys Leu Ile Ser Leu
325 330 335
Gln Asp His Tyr Gly Leu Tyr Glu Tyr Asp Tyr Trp Gly Gln Gly Thr
340 345 350
Leu Val Thr Val Ser Ser
355
<210> 21
<211> 599
<212> PRT
<213> Artificial Sequence
<220>
<223> BsAb21 heavy chain
<400> 21
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Ala Thr Leu Asp Gly Tyr Thr Ala Gly Tyr Tyr Tyr Gly Met
100 105 110
Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr
115 120 125
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
130 135 140
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
145 150 155 160
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
165 170 175
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
180 185 190
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
195 200 205
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
210 215 220
Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
225 230 235 240
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
245 250 255
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
260 265 270
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
275 280 285
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
290 295 300
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
305 310 315 320
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
325 330 335
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
340 345 350
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
355 360 365
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
370 375 380
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
385 390 395 400
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
405 410 415
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
420 425 430
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
435 440 445
Leu Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly
450 455 460
Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly
465 470 475 480
Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly
485 490 495
Phe Gly Leu Asp Tyr Tyr Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly
500 505 510
Lys Glu Arg Glu Gly Val Ser Cys Ile Gly Ser Ser Ser Lys Glu Thr
515 520 525
Asn Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
530 535 540
Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
545 550 555 560
Thr Ala Val Tyr Tyr Cys Thr Ala Gly Ser Pro Leu Cys Leu Ile Ser
565 570 575
Leu Gln Asp His Tyr Gly Leu Tyr Glu Tyr Asp Tyr Trp Gly Gln Gly
580 585 590
Thr Leu Val Thr Val Ser Ser
595
<210> 22
<211> 599
<212> PRT
<213> Artificial Sequence
<220>
<223> BsAb22/BsAb31 heavy chain
<400> 22
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Gly Leu Asp Tyr Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Gly Ser Ser Ser Lys Glu Thr Asn Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Ala Gly Ser Pro Leu Cys Leu Ile Ser Leu Gln Asp His Tyr Gly
100 105 110
Leu Tyr Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
145 150 155 160
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
165 170 175
Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
180 185 190
Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val
195 200 205
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
210 215 220
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
225 230 235 240
Ala Lys Ala Thr Leu Asp Gly Tyr Thr Ala Gly Tyr Tyr Tyr Gly Met
245 250 255
Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr
260 265 270
Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
275 280 285
Gly Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu
290 295 300
Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His
305 310 315 320
Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser
325 330 335
Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys
340 345 350
Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu
355 360 365
Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro
370 375 380
Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
385 390 395 400
Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val
405 410 415
Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp
420 425 430
Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr
435 440 445
Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp
450 455 460
Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu
465 470 475 480
Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg
485 490 495
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys
500 505 510
Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp
515 520 525
Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys
530 535 540
Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser
545 550 555 560
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser
565 570 575
Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
580 585 590
Leu Ser Leu Ser Pro Gly Lys
595
<210> 23
<211> 360
<212> PRT
<213> Artificial Sequence
<220>
<223> BsAb23/BsAb31 light chain
<400> 23
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Gly Leu Asp Tyr Tyr
20 25 30
Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val
35 40 45
Ser Cys Ile Gly Ser Ser Ser Lys Glu Thr Asn Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Thr Ala Gly Ser Pro Leu Cys Leu Ile Ser Leu Gln Asp His Tyr Gly
100 105 110
Leu Tyr Glu Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
115 120 125
Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
130 135 140
Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
145 150 155 160
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr
165 170 175
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
180 185 190
Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
195 200 205
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro
210 215 220
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Leu Met Ser Asn Trp Pro Pro
225 230 235 240
Glu Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val
245 250 255
Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys
260 265 270
Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg
275 280 285
Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
290 295 300
Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser
305 310 315 320
Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
325 330 335
Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
340 345 350
Lys Ser Phe Asn Arg Gly Glu Cys
355 360
<210> 24
<211> 360
<212> PRT
<213> Artificial Sequence
<220>
<223> BsAb24 light chain
<400> 24
Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Leu Met Ser Asn Trp Pro Pro
85 90 95
Glu Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val
100 105 110
Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys
115 120 125
Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg
130 135 140
Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn
145 150 155 160
Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser
165 170 175
Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys
180 185 190
Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr
195 200 205
Lys Ser Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser Gly Gly Gly
210 215 220
Gly Ser Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly
225 230 235 240
Gly Leu Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser
245 250 255
Gly Phe Gly Leu Asp Tyr Tyr Ala Ile Gly Trp Phe Arg Gln Ala Pro
260 265 270
Gly Lys Glu Arg Glu Gly Val Ser Cys Ile Gly Ser Ser Ser Lys Glu
275 280 285
Thr Asn Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp
290 295 300
Asn Ala Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu
305 310 315 320
Asp Thr Ala Val Tyr Tyr Cys Thr Ala Gly Ser Pro Leu Cys Leu Ile
325 330 335
Ser Leu Gln Asp His Tyr Gly Leu Tyr Glu Tyr Asp Tyr Trp Gly Gln
340 345 350
Gly Thr Leu Val Thr Val Ser Ser
355 360
<210> 25
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> 17 HCDR1
<400> 25
Gly Phe Thr Phe Asn Lys Tyr Thr Ile Asn
1 5 10
<210> 26
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> 17 HCDR2
<400> 26
Asp Ile Ser Ser Gly Gly Asp Tyr Val Asn
1 5 10
<210> 27
<211> 12
<212> PRT
<213> Artificial Sequence
<220>
<223> 17 HCDR3
<400> 27
Asp Ala Leu Thr Tyr Ser Asn Asn Trp Leu Asp Ser
1 5 10
<210> 28
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> 17 LCDR1
<400> 28
Arg Ala Ser Gln Gly Ile Ser Ser Tyr Leu Ala
1 5 10
<210> 29
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> 17 LCDR2
<400> 29
Ala Ala Ser Thr Leu Gln Ser
1 5
<210> 30
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> 17 LCDR3
<400> 30
Gln Gln Phe Glu Ser Tyr Pro Leu Thr
1 5
<210> 31
<211> 121
<212> PRT
<213> Artificial Sequence
<220>
<223> 17 VH
<400> 31
Glu Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Lys Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Lys Tyr
20 25 30
Thr Ile Asn Trp Val Arg Gln Thr Pro Gly Lys Gly Leu Glu Leu Val
35 40 45
Ala Asp Ile Ser Ser Gly Gly Asp Tyr Val Asn Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asp Ser Val Tyr
65 70 75 80
Leu Leu Met Asn Gly Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Arg Asp Ala Leu Thr Tyr Ser Asn Asn Trp Leu Asp Ser Trp Gly
100 105 110
Gln Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 32
<211> 107
<212> PRT
<213> Artificial Sequence
<220>
<223> 17 VL
<400> 32
Asp Ile Gln Leu Thr Gln Ser Pro Ser Phe Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Gly Gly Tyr Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Glu Ser Tyr Pro Leu
85 90 95
Thr Phe Gly Gly Gly Thr Arg Leu Glu Ile Lys
100 105
<210> 33
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> 78A14 HCDR1
<400> 33
Gly Phe Thr Phe Asn Val Tyr Gly Met His
1 5 10
<210> 34
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> 78A14 HCDR2
<400> 34
Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr
1 5 10
<210> 35
<211> 16
<212> PRT
<213> Artificial Sequence
<220>
<223> 78A14 HCDR3
<400> 35
Ala Thr Leu Asp Gly Tyr Thr Ala Gly Tyr Tyr Tyr Gly Met Asp Val
1 5 10 15
<210> 36
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> 78A14 LCDR1
<400> 36
Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala
1 5 10
<210> 37
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> 78A14 LCDR2
<400> 37
Gly Pro Ser Asn Arg Ala Thr
1 5
<210> 38
<211> 9
<212> PRT
<213> Artificial Sequence
<220>
<223> 78A14 LCDR3
<400> 38
Gln His Tyr Gly Val Ser Gln His Thr
1 5
<210> 39
<211> 125
<212> PRT
<213> Artificial Sequence
<220>
<223> 78A14 VH
<400> 39
Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Val Tyr
20 25 30
Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Ala Thr Leu Asp Gly Tyr Thr Ala Gly Tyr Tyr Tyr Gly Met
100 105 110
Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
115 120 125
<210> 40
<211> 107
<212> PRT
<213> Artificial Sequence
<220>
<223> 78A14 VL
<400> 40
Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Gly Pro Ser Asn Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser Gly
50 55 60
Gly Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu Pro
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Tyr Gly Val Ser Gln His
85 90 95
Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys
100 105
<210> 41
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> 78A74-7 HCDR1
<400> 41
Gly Phe Thr Phe Ser Ser Tyr Ser Met Asn
1 5 10
<210> 42
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> 78A74-7 HCDR2
<400> 42
Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr
1 5 10
<210> 43
<211> 16
<212> PRT
<213> Artificial Sequence
<220>
<223> 78A74-7 HCDR3
<400> 43
Ala Thr Leu Asp Gly Tyr Thr Ala Gly Tyr Tyr Tyr Gly Met Asp Val
1 5 10 15
<210> 44
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> 78A74-7 LCDR1
<400> 44
Arg Ala Ser Gln Ser Val Ser Ser Tyr Leu Ala
1 5 10
<210> 45
<211> 7
<212> PRT
<213> Artificial Sequence
<220>
<223> 78A74-7 LCDR2
<400> 45
Asp Ala Ser Asn Arg Ala Thr
1 5
<210> 46
<211> 11
<212> PRT
<213> Artificial Sequence
<220>
<223> 78A74-7 LCDR3
<400> 46
Gln Leu Met Ser Asn Trp Pro Pro Glu Trp Thr
1 5 10
<210> 47
<211> 125
<212> PRT
<213> Artificial Sequence
<220>
<223> 78A74-7 VH
<400> 47
Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Lys Pro Gly Gly
1 5 10 15
Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr
20 25 30
Ser Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ala Lys Ala Thr Leu Asp Gly Tyr Thr Ala Gly Tyr Tyr Tyr Gly Met
100 105 110
Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser
115 120 125
<210> 48
<211> 109
<212> PRT
<213> Artificial Sequence
<220>
<223> 78A74-7 VL
<400> 48
Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly
1 5 10 15
Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr
20 25 30
Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile
35 40 45
Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly
50 55 60
Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro
65 70 75 80
Glu Asp Phe Ala Val Tyr Tyr Cys Gln Leu Met Ser Asn Trp Pro Pro
85 90 95
Glu Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 49
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> P30-10-26 CDR1
<400> 49
Gly Phe Gly Leu Asp Tyr Tyr Ala Ile Gly
1 5 10
<210> 50
<211> 10
<212> PRT
<213> Artificial Sequence
<220>
<223> P30-10-26 CDR2
<400> 50
Cys Ile Gly Ser Ser Ser Lys Glu Thr Asn
1 5 10
<210> 51
<211> 20
<212> PRT
<213> Artificial Sequence
<220>
<223> P30-10-26 CDR3
<400> 51
Gly Ser Pro Leu Cys Leu Ile Ser Leu Gln Asp His Tyr Gly Leu Tyr
1 5 10 15
Glu Tyr Asp Tyr
20
<210> 52
<211> 191
<212> PRT
<213> Homo sapiens
<400> 52
Met Asn Phe Leu Leu Ser Trp Val His Trp Ser Leu Ala Leu Leu Leu
1 5 10 15
Tyr Leu His His Ala Lys Trp Ser Gln Ala Ala Pro Met Ala Glu Gly
20 25 30
Gly Gly Gln Asn His His Glu Val Val Lys Phe Met Asp Val Tyr Gln
35 40 45
Arg Ser Tyr Cys His Pro Ile Glu Thr Leu Val Asp Ile Phe Gln Glu
50 55 60
Tyr Pro Asp Glu Ile Glu Tyr Ile Phe Lys Pro Ser Cys Val Pro Leu
65 70 75 80
Met Arg Cys Gly Gly Cys Cys Asn Asp Glu Gly Leu Glu Cys Val Pro
85 90 95
Thr Glu Glu Ser Asn Ile Thr Met Gln Ile Met Arg Ile Lys Pro His
100 105 110
Gln Gly Gln His Ile Gly Glu Met Ser Phe Leu Gln His Asn Lys Cys
115 120 125
Glu Cys Arg Pro Lys Lys Asp Arg Ala Arg Gln Glu Asn Pro Cys Gly
130 135 140
Pro Cys Ser Glu Arg Arg Lys His Leu Phe Val Gln Asp Pro Gln Thr
145 150 155 160
Cys Lys Cys Ser Cys Lys Asn Thr Asp Ser Arg Cys Lys Ala Arg Gln
165 170 175
Leu Glu Leu Asn Glu Arg Thr Cys Arg Cys Asp Lys Pro Arg Arg
180 185 190
<210> 53
<211> 496
<212> PRT
<213> Homo sapiens
<400> 53
Met Trp Gln Ile Val Phe Phe Thr Leu Ser Cys Asp Leu Val Leu Ala
1 5 10 15
Ala Ala Tyr Asn Asn Phe Arg Lys Ser Met Asp Ser Ile Gly Lys Lys
20 25 30
Gln Tyr Gln Val Gln His Gly Ser Cys Ser Tyr Thr Phe Leu Leu Pro
35 40 45
Glu Met Asp Asn Cys Arg Ser Ser Ser Ser Pro Tyr Val Ser Asn Ala
50 55 60
Val Gln Arg Asp Ala Pro Leu Glu Tyr Asp Asp Ser Val Gln Arg Leu
65 70 75 80
Gln Val Leu Glu Asn Ile Met Glu Asn Asn Thr Gln Trp Leu Met Lys
85 90 95
Leu Glu Asn Tyr Ile Gln Asp Asn Met Lys Lys Glu Met Val Glu Ile
100 105 110
Gln Gln Asn Ala Val Gln Asn Gln Thr Ala Val Met Ile Glu Ile Gly
115 120 125
Thr Asn Leu Leu Asn Gln Thr Ala Glu Gln Thr Arg Lys Leu Thr Asp
130 135 140
Val Glu Ala Gln Val Leu Asn Gln Thr Thr Arg Leu Glu Leu Gln Leu
145 150 155 160
Leu Glu His Ser Leu Ser Thr Asn Lys Leu Glu Lys Gln Ile Leu Asp
165 170 175
Gln Thr Ser Glu Ile Asn Lys Leu Gln Asp Lys Asn Ser Phe Leu Glu
180 185 190
Lys Lys Val Leu Ala Met Glu Asp Lys His Ile Ile Gln Leu Gln Ser
195 200 205
Ile Lys Glu Glu Lys Asp Gln Leu Gln Val Leu Val Ser Lys Gln Asn
210 215 220
Ser Ile Ile Glu Glu Leu Glu Lys Lys Ile Val Thr Ala Thr Val Asn
225 230 235 240
Asn Ser Val Leu Gln Lys Gln Gln His Asp Leu Met Glu Thr Val Asn
245 250 255
Asn Leu Leu Thr Met Met Ser Thr Ser Asn Ser Ala Lys Asp Pro Thr
260 265 270
Val Ala Lys Glu Glu Gln Ile Ser Phe Arg Asp Cys Ala Glu Val Phe
275 280 285
Lys Ser Gly His Thr Thr Asn Gly Ile Tyr Thr Leu Thr Phe Pro Asn
290 295 300
Ser Thr Glu Glu Ile Lys Ala Tyr Cys Asp Met Glu Ala Gly Gly Gly
305 310 315 320
Gly Trp Thr Ile Ile Gln Arg Arg Glu Asp Gly Ser Val Asp Phe Gln
325 330 335
Arg Thr Trp Lys Glu Tyr Lys Val Gly Phe Gly Asn Pro Ser Gly Glu
340 345 350
Tyr Trp Leu Gly Asn Glu Phe Val Ser Gln Leu Thr Asn Gln Gln Arg
355 360 365
Tyr Val Leu Lys Ile His Leu Lys Asp Trp Glu Gly Asn Glu Ala Tyr
370 375 380
Ser Leu Tyr Glu His Phe Tyr Leu Ser Ser Glu Glu Leu Asn Tyr Arg
385 390 395 400
Ile His Leu Lys Gly Leu Thr Gly Thr Ala Gly Lys Ile Ser Ser Ile
405 410 415
Ser Gln Pro Gly Asn Asp Phe Ser Thr Lys Asp Gly Asp Asn Asp Lys
420 425 430
Cys Ile Cys Lys Cys Ser Gln Met Leu Thr Gly Gly Trp Trp Phe Asp
435 440 445
Ala Cys Gly Pro Ser Asn Leu Asn Gly Met Tyr Tyr Pro Gln Arg Gln
450 455 460
Asn Thr Asn Lys Phe Asn Gly Ile Lys Trp Tyr Tyr Trp Lys Gly Ser
465 470 475 480
Gly Tyr Ser Leu Lys Ala Thr Thr Met Met Ile Arg Pro Ala Asp Phe
485 490 495

Claims (16)

1. A bispecific antibody comprising a first antigen-binding portion that binds ANG2 comprising a heavy chain variable region comprising HCDR1, HCDR2 and HCDR3 and a light chain variable region comprising LCDR2 and LCDR3, wherein the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 sequence is selected from any one of (1) - (3), and a second antigen-binding portion that binds VEGF:
(1) The HCDR1 sequence shown in SEQ ID NO. 25; the HCDR2 sequence shown in SEQ ID NO. 26; the HCDR3 sequence shown in SEQ ID NO. 27; the LCDR1 sequence shown in SEQ ID NO. 28; the LCDR2 sequence shown in SEQ ID NO. 29; and the LCDR3 sequence shown in SEQ ID NO. 30;
(2) The HCDR1 sequence shown in SEQ ID NO. 33; the HCDR2 sequence shown in SEQ ID NO. 34; an HCDR3 sequence shown in SEQ ID NO. 35; the LCDR1 sequence shown in SEQ ID NO. 36; the LCDR2 sequence shown in SEQ ID NO. 37; and the LCDR3 sequence shown in SEQ ID NO. 38;
(3) The HCDR1 sequence shown in SEQ ID NO. 41; an HCDR2 sequence shown in SEQ ID NO. 42; an HCDR3 sequence shown in SEQ ID NO. 43; the LCDR1 sequence shown in SEQ ID NO. 44; the LCDR2 sequence shown in SEQ ID NO. 45; and the LCDR3 sequence shown in SEQ ID NO. 46.
2. The bispecific antibody of claim 1, wherein the heavy chain variable region comprises:
(1) The amino acid sequence of SEQ ID NO. 31, SEQ ID NO. 39 or SEQ ID NO. 47; or alternatively
(2) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to SEQ ID No. 31, SEQ ID No. 39 or SEQ ID No. 47.
3. The bispecific antibody of claim 1 or 2, wherein the light chain variable region comprises:
(1) The amino acid sequence of SEQ ID NO. 32, SEQ ID NO. 40 or SEQ ID NO. 48; or alternatively
(2) An amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% sequence identity to SEQ ID No. 32, SEQ ID No. 40 or SEQ ID No. 48.
4. The bispecific antibody of any one of claims 1-3, wherein the heavy chain variable region and light chain variable region are selected from any one of (1) - (3)
(1) A heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 31; a light chain variable region comprising the amino acid sequence of SEQ ID NO. 32;
(2) A heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 39; a light chain variable region comprising the amino acid sequence of SEQ ID NO. 40;
(3) A heavy chain variable region comprising the amino acid sequence of SEQ ID NO. 47; a light chain variable region comprising the amino acid sequence of SEQ ID No. 48.
5. The bispecific antibody of any one of claims 1-4, wherein the second antigen-binding portion comprises an immunoglobulin single variable domain (VHH) that binds VEGF;
preferably, the immunoglobulin single variable domain comprises: CDR1 sequence shown in SEQ ID NO. 49, CDR2 sequence shown in SEQ ID NO. 50, and CDR3 sequence shown in SEQ ID NO. 51;
More preferably, the immunoglobulin single variable domain comprises: 1) The amino acid sequence of SEQ ID NO. 11; or 2) an amino acid sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO. 11.
6. The bispecific antibody of any one of claims 1-5, wherein the first antigen binding moiety and the second antigen binding moiety are linked by a linker.
7. The bispecific antibody of any one of claims 1-6, further comprising a heavy chain constant region (CH) and a light chain constant region (CL) of an immunoglobulin;
preferably, the heavy chain constant region is a human IgG1 heavy chain constant region and the light chain constant region is a human kappa light chain constant region;
more preferably, the heavy chain constant region of human IgG1 comprises a leucine to alanine mutation at positions 234 and 235 according to EU numbering.
8. The bispecific antibody of claim 7, comprising a first polypeptide comprising the heavy chain variable region (VH) and the heavy chain constant region (CH) and a second polypeptide comprising the light chain variable region (VL) and the light chain constant region (CL), the single variable domain of the second antigen-binding portion optionally being fused to: (1) the N-terminus of the VH; (2) the N-terminus of VL; (3) the N-terminus of the VH and VL; (4) the C-terminus of the CH; (5) the C-terminus of the CL; (6) the C-terminus of the CH and CL; (8) the N-terminus of the VH and the C-terminus of the CH; or (9) the N-terminus of the VL and the C-terminus of the CH.
9. The bispecific antibody of claim 8, wherein the first and second polypeptides are selected from any one of (1) - (15):
(1) A first polypeptide comprising the amino acid sequence of SEQ ID No. 13 or an amino acid sequence having the L238A and L239A mutations compared to SEQ ID No. 13; a second polypeptide comprising the amino acid sequence of SEQ ID NO. 6;
(2) A first polypeptide comprising the amino acid sequence of SEQ ID No. 14 or an amino acid sequence having the L382A and L383A mutations compared to SEQ ID No. 14; a second polypeptide comprising the amino acid sequence of SEQ ID NO. 6;
(3) A first polypeptide comprising the amino acid sequence of SEQ ID No. 5 or an amino acid sequence having the L238A and L239A mutations compared to SEQ ID No. 5; a second polypeptide comprising the amino acid sequence of SEQ ID NO. 15;
(4) A first polypeptide comprising the amino acid sequence of SEQ ID No. 5 or an amino acid sequence having the L238A and L239A mutations compared to SEQ ID No. 5; a second polypeptide comprising the amino acid sequence of SEQ ID NO. 16;
(5) A first polypeptide comprising the amino acid sequence of SEQ ID No. 17 or an amino acid sequence having the L242A and L243A mutations compared to SEQ ID No. 17; a second polypeptide comprising the amino acid sequence of SEQ ID NO. 8;
(6) A first polypeptide comprising the amino acid sequence of SEQ ID No. 18 or an amino acid sequence having the L386A and L387A mutations compared to SEQ ID No. 18; a second polypeptide comprising the amino acid sequence of SEQ ID NO. 8;
(7) A first polypeptide comprising the amino acid sequence of SEQ ID No. 7 or an amino acid sequence having the L242A and L243A mutations compared to SEQ ID No. 7; a second polypeptide comprising the amino acid sequence of SEQ ID NO. 19;
(8) A first polypeptide comprising the amino acid sequence of SEQ ID No. 7 or an amino acid sequence having the L242A and L243A mutations compared to SEQ ID No. 7; a second polypeptide comprising the amino acid sequence of SEQ ID NO. 20;
(9) A first polypeptide comprising the amino acid sequence of SEQ ID No. 21 or an amino acid sequence having the L242A and L243A mutations compared to SEQ ID No. 21; a second polypeptide comprising the amino acid sequence of SEQ ID NO. 10;
(10) A first polypeptide comprising the amino acid sequence of SEQ ID No. 22 or an amino acid sequence having L386A and L387A mutations compared to SEQ ID No. 22; a second polypeptide comprising the amino acid sequence of SEQ ID NO. 10;
(11) A first polypeptide comprising the amino acid sequence of SEQ ID No. 9 or an amino acid sequence having the L242A and L243A mutations compared to SEQ ID No. 9; a second polypeptide comprising the amino acid sequence of SEQ ID NO. 23;
(12) A first polypeptide comprising the amino acid sequence of SEQ ID No. 9 or an amino acid sequence having the L242A and L243A mutations compared to SEQ ID No. 9; a second polypeptide comprising the amino acid sequence of SEQ ID NO. 24;
(13) A first polypeptide comprising the amino acid sequence of SEQ ID No. 14 or an amino acid sequence having the L382A and L383A mutations compared to SEQ ID No. 14; a second polypeptide comprising the amino acid sequence of SEQ ID NO. 15;
(14) A first polypeptide comprising the amino acid sequence of SEQ ID No. 18 or an amino acid sequence having the L386A and L387A mutations compared to SEQ ID No. 18; a second polypeptide comprising the amino acid sequence of SEQ ID NO. 19;
(15) A first polypeptide comprising the amino acid sequence of SEQ ID No. 22 or an amino acid sequence having L386A and L387A mutations compared to SEQ ID No. 22; a second polypeptide comprising the amino acid sequence of SEQ ID NO. 23.
10. The bispecific antibody of claim 8 or 9 comprising two of said first polypeptides and two of said second polypeptides, wherein said first polypeptides are the same or different and said second polypeptides are the same or different.
11. A polynucleotide encoding the bispecific antibody of any one of claims 1-10.
12. An expression vector comprising the polynucleotide of claim 11.
13. A host cell comprising the polynucleotide of claim 11 or the expression vector of claim 12.
14. A method of producing the bispecific antibody of any one of claims 1-10, comprising:
culturing the host cell of claim 13 under suitable conditions to express said bispecific antibody, an
(ii) isolating the bispecific antibody from the host cell or culture thereof.
15. A pharmaceutical composition comprising the bispecific antibody of any one of claims 1-10, and a pharmaceutically acceptable carrier.
16. Use of a bispecific antibody according to any one of claims 1 to 10 or a pharmaceutical composition according to claim 15 for the preparation of a medicament for the treatment of: (1) angiogenesis-related eye diseases; or (2) cancer;
preferably, the method comprises the steps of,
the angiogenesis-related eye disease is macular degeneration, retinal vein occlusion, retinopathy of prematurity, diabetic retinopathy, neovascular glaucoma, pathological myopia, macular edema, retinal edema, diabetic macular edema or choroidal neovascularization disease;
The cancer is lung cancer, liver cancer, pancreatic cancer, skin cancer, head and neck cancer, melanoma, ovarian cancer, colorectal cancer, gastric cancer, breast cancer, prostate cancer, uterine cancer, hodgkin's lymphoma, esophageal cancer, anal cancer, cancer of the endocrine system, thyroid cancer, parathyroid cancer, renal cancer, soft tissue sarcoma, bladder cancer, central Nervous System (CNS) tumor, mesothelioma, glioma, meningioma or pituitary adenoma.
CN202111541547.9A 2021-12-16 2021-12-16 anti-ANG 2-VEGF bispecific antibodies and uses thereof Pending CN116265487A (en)

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PCT/CN2022/139203 WO2023109888A1 (en) 2021-12-16 2022-12-15 Anti-ang2-vegf bispecific antibody and use thereof

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8268314B2 (en) * 2008-10-08 2012-09-18 Hoffmann-La Roche Inc. Bispecific anti-VEGF/anti-ANG-2 antibodies
SI3495387T1 (en) * 2012-07-13 2021-12-31 Roche Glycart Ag Bispecific anti-vegf/anti-ang-2 antibodies and their use in the treatment of ocular vascular diseases
KR20150063728A (en) * 2013-12-02 2015-06-10 삼성전자주식회사 Anti-VEGF-C/anti-Ang2 bispecific antibody
EP3504237A1 (en) * 2016-08-23 2019-07-03 Medimmune Limited Anti-vegf-a and anti-ang2 antibodies and uses thereof
US20220185875A1 (en) * 2019-03-18 2022-06-16 Jiangsu Hengrui Medicine Co., Ltd. Bispecific antibody specifically bound to vegf and ang2

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