Bispecific antibody comprising Anti-CLDN18.2 antibody, and pharmaceutical composition and use thereof

Abstract

FIELD: biotechnology.SUBSTANCE: invention relates to recombinant bispecific antibodies, and can be used in medicine for the treatment or prevention of CD47- and / or CLDN18.2-positive tumours. Provided is an anti-CLDN18.2 / anti-CD47 bispecific antibody comprising first and second protein functional domains, wherein the first protein functional domain is an anti-CLDN18.2 (claudin 18.2) antibody or antigen-binding fragment thereof, and the second protein functional domain is an anti-CD47 (integrin-associated protein) antibody or antigen-binding fragment thereof.EFFECT: production of a bispecific antibody capable of binding to tumour cells expressing CD47 and / or CLDN18.2 with high specificity, without causing agglutination or damage to red blood cells, demonstrating good safety and having good anti-tumour application prospects.27 cl, 21 dwg, 11 tbl, 11 ex

Description

[0001] FIELD OF INVENTION

[0002] The present invention belongs to the field of biomedicine and relates to an anti-CLDN18.2 antibody, a bispecific antibody comprising an anti-CLDN18.2 antibody or an antigen-binding fragment thereof, a pharmaceutical composition thereof, and a use thereof. Specifically, the bispecific antibody is an anti-CLDN18.2 / anti-CD47 bispecific antibody.

[0003] PRIOR ART

[0004] In today's world, tumors, especially malignant tumors, are a serious health threat and, when combined with other diseases, the second leading cause of death. In recent years, the incidence of cancer-related morbidity has increased significantly. Malignant tumors are characterized by a poor response to treatment, a high rate of late metastasis, and a poor prognosis. Although traditional treatments (such as radiation therapy, chemotherapy, and surgery) currently in clinical use significantly alleviate pain and prolong survival, these methods have significant limitations, and further improvement of their effectiveness is difficult.

[0005] CD47 is also known as integrin-associated protein (IAP). CD47 is a transmembrane protein with five transmembrane domains and a molecular weight of approximately 50 kDa, belonging to the immunoglobulin superfamily. Its extracellular N-terminus is an IgV domain and associates with α integrins. V β3 (CD51 / CD61) and α IIb β3 (CD41 / CD61). CD47 is involved in a variety of physiological functions, such as cellular transport, T cell and dendritic cell (DC) activation, and axonal development.

[0006] CD47 is expressed on all cell types, including red blood cells, and its expression is increased on almost all tumor cell types. It is also associated with an unfavorable prognosis. CD47 has two ligands, namely, signal regulatory protein α (SIRPα) and thrombin-sensitive protein 1 (TSP1). SIRPα is a receptor transmembrane glycoprotein containing an immunoglobulin domain, belonging to the SIRP family and expressed primarily on phagocytes and nerve cells. Within the CD47-SIRPα pathway, CD47 protein binds to SIRPα, phosphorylates its immunoreceptor tyrosine-based inhibitory motif (ITIM), and leads to subsequent intracellular recruitment of SHP-1 protein and a series of cascade interactions that inhibit macrophage phagocytosis (Matozaki T, Murata Y, Okazawa H, et al., Functions and molecular mechanisms of the CD47 SIRPα signaling pathway. Trends in cell biology, 2009, 19(2): 72–80.). Normal erythrocytes are not subject to phagocytosis due to an inhibitory signal resulting from the binding of CD47 on the surface of their cell membranes to SIPRα of macrophages (Oldenborg P A, Zheleznyak A, Fang YF, et al., Role of CD47 as a marker of self on red blood cells. Science, 2000, 288(5473): 2051–2054). TSP1, a homotrimer consisting of 3 peptide chains, is involved in cell proliferation, apoptosis, adhesion, migration, angiogenesis and other processes through interactions with other cell surface receptors, matrix components and growth factors (Jiang P, Lagenaur CF, Narayanan V. Integrin-associated Protein Is a Ligand for the P84 Neural Adhesion Molecule. Journal of Biological Chemistry 1999, 274: 559-62).

[0007] Macrophages originate from monocytes, which in turn originate from bone marrow progenitor cells. Their primary functions are to phagocytose cellular debris and pathogens and to activate lymphocytes or other immune cells to respond to pathogens in the form of fixed or free cells. Research currently indicates a mechanism that allows tumor cells to evade macrophage phagocytosis. As tumor cells grow, certain proteins, such as calreticulin, appear on their surface. These proteins help identify tumor cells, allowing attracted macrophages to phagocytose them.However, SIRPα-expressing macrophages mistakenly recognize CD47-high tumor cells as normal cells, causing them to avoid macrophage phagocytosis because the CD47-SIRPα pathway results in active inhibition of macrophage phagocytosis (CD47 is Upregulated on Circulating Hematopoietic Stem Cells and Leukemia Cells to Avoid Phagocytosis. Jaiswal S, Jamieson C HM, Pang WW, et al., Cell, 2009, 138(3): 271-285).

[0008] CLDN18.2 protein is an integral membrane protein present in epithelial and endothelial tight junctions. It consists of 261 amino acids and is a member of the claudin (CLDNs) family, with both N- and C-termini located intracellularly. This protein is expressed entirely on the cell membrane. CLDN18.2 has 4 transmembrane domains, 2 extracellular loops, and 1 intracytoplasmic loop and is involved in the formation of the structure of tight junctions between cells (Gunzel, D.; Yu, ASL Claudins and the Modulation of Tight Junction Permeability [J]. Physiological Reviews. 2013, 93(2), 525-569).

[0009] The extracellular loop 2 of CLDN18.2 protein is a helix-turn-helix structure and forms a tight contact with the extracellular loop of CLDN18.2 protein of the adjacent cell through hydrophobic bonds between aromatic residues. The extracellular loops of CLDN18.2 proteins of adjacent cells can maintain tight contact between epithelial cells and endothelial cells through their interactions, regulate intercellular osmotic pressure, maintain the polarity of epithelial cells and endothelial cells, and participate in cell proliferation, and may participate in various signaling pathways through the C-termini with high serine, threonine, and tyrosine content (Cao Chenxin, Research Progress of Transmembrane CLDN18.2 in Targeted Cancer Therapy [J]. International Journal of Biologicals, 2020(01): 35-36-37-38-39-40).

[0010] CLDN18.2 protein expression is highly restricted to normal healthy tissues and is detected only in differentiated epithelial cells of the gastric mucosa, where it is beneficial for maintaining the barrier function of the gastric mucosa. However, during the development and progression of malignant tumors, abnormal changes in CLDN18.2 protein often occur. For example, during malignant transformation of gastric epithelial tissue, disruption of cell polarity will lead to the appearance of CLDN18.2 protein epitopes on the cell surface. At the same time, abnormal activation of the CLDN18 gene also occurs.2 with its highly selective and stable expression in certain tumor tissues, such as gastric cancer, which affects intercellular adhesion and cell adhesion to the extracellular matrix and the maintenance of barrier function and cell polarity, thereby leading to changes in intercellular permeability to ions and cytokines, disruption of barrier function and proliferative transformation of cells (Hashimoto Itaru, Oshima Takashi, Claudins and Gastric Cancer: An Overview. [J]. Cancers (Basel), 2022, 14: undefined).

[0011] Both CD47 and CLDN18.2 are tumor membrane antigens. Antibodies targeting CD47 or CLDN18.2 have demonstrated antitumor activity. However, CD47 is also expressed on many cells and blood components, including platelets and red blood cells. Drugs containing CD47 antibodies, by binding to red blood cells, platelets, and other blood cells, can damage red blood cells and platelets through ADCC, ADCP, and other mechanisms, resulting in severe hematological toxicity and side effects such as anemia and / or platelet deficiency.

[0012] SUMMARY OF THE INVENTION

[0013] Through intensive research and creative efforts, the inventors obtained an anti-CLDN18.2 antibody and, based on this, developed an anti-CLDN18.2 / anti-CD47 bispecific antibody. The inventors unexpectedly found that the anti-CLDN18.2 antibody (also referred to as the antibody of the present invention for short) and the anti-CLDN18.2 / anti-CD47 bispecific antibody (also referred to as the bispecific antibody or the bispecific antibody of the present invention for short) of the present invention have excellent affinity and / or specificity, can bind to tumor cells expressing CD47 and / or CLDN18.2 with high specificity without causing agglutination or damage to red blood cells, exhibit good safety, and have good prospects for antitumor application. The present invention is described in detail below.

[0014] One aspect of the present invention relates to an antibody against CLDN18.2 or an antigen-binding fragment thereof, wherein the antibody against CLDN18.2 comprises a heavy chain variable region comprising HCDR1-HCDR3 and a light chain variable region comprising LCDR1-LCDR3, wherein:

[0015] the amino acid sequence of HCDR1 is set forth in SEQ ID NO:31, the amino acid sequence of HCDR2 is set forth in SEQ ID NO:32, and the amino acid sequence of HCDR3 is set forth in SEQ ID NO:33; and

[0016] The amino acid sequence of LCDR1 is set forth in SEQ ID NO:34, the amino acid sequence of LCDR2 is set forth in SEQ ID NO:35, and the amino acid sequence of LCDR3 is set forth in SEQ ID NO:36.

[0017] In some embodiments, the present invention provides an anti-CLDN18.2 antibody or antigen-binding fragment thereof, wherein:

[0018] the amino acid sequence of the heavy chain variable region of the anti-CLDN18.2 antibody is selected from SEQ ID NO:28, SEQ ID NO:37, SEQ ID NO:39 and SEQ ID NO:41; and

[0019] the amino acid sequence of the light chain variable region of the anti-CLDN18.2 antibody is selected from SEQ ID NO:30, SEQ ID NO:43, SEQ ID NO:45 and SEQ ID NO:47.

[0020] In some embodiments, the present invention provides an anti-CLDN18.2 antibody or antigen-binding fragment thereof, wherein:

[0021] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:28, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:30;

[0022] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:37, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:43;

[0023] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:37, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:45;

[0024] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:37, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:47;

[0025] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:39, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:43;

[0026] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:39, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:45;

[0027] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:39, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:47;

[0028] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:41, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:43;

[0029] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:41, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:45;

[0030] or

[0031] The amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:41, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:47.

[0032] In some embodiments, the present invention provides an anti-CLDN18.2 antibody or an antigen-binding fragment thereof, wherein: the heavy chain constant region of the antibody is the C region of an Ig gamma-1 chain (e.g., having the amino acid sequence set forth in SEQ ID NO:61) or the C region of an Ig gamma-4 chain (e.g., having NCBI accession number P01861.1); the light chain constant region of the antibody is the C region of an Ig kappa chain (e.g., having NCBI accession number P01834).

[0033] In some embodiments, the present invention provides an anti-CLDN18.2 antibody or antigen-binding fragment thereof selected from Fab, Fab', F(ab')2, Fd, Fv, dAb, complementarity determining region fragment, single-chain variable fragment, humanized antibody, chimeric antibody, or diabody.

[0034] In some embodiments, the present invention provides an anti-CLDN18.2 antibody or antigen-binding fragment thereof, wherein

[0035] the antibody comprises a non-CDR region that is derived from a species other than a mouse or rat, such as a human antibody.

[0036] In some embodiments, the present invention provides an anti-CLDN18.2 antibody or antigen-binding fragment thereof, wherein the EC 50 antibodies against CLDN18.2, with respect to binding to a cell expressing CLDN18.2, less than or equal to 15 nM, less than or equal to 10 nM, less than or equal to 9 nM, less than or equal to 8 nM, less than or equal to 7 nM, less than or equal to 6 nM, or less than or equal to 5 nM; preferably, the EU 50 determined by FACS (flow cytometry). In one embodiment of the present invention, the cell expressing CLDN18.2 is a CHO-K1 cell expressing CLDN18.2. In one embodiment of the present invention, the cell expressing CLDN18.2 is a CHO-K1 cell overexpressing CLDN18.2.

[0037] In some embodiments, the present invention provides an anti-CLDN18.2 antibody or antigen-binding fragment thereof, wherein the EC 50 the CLDN18.2 antibody has an EC of less than or equal to 10 nM, less than or equal to 5 nM, or less than or equal to 2 nM with respect to binding to a cell expressing both CLDN18.2 and CD47; preferably, 50 determined by FACS. In one embodiment of the present invention, a cell expressing both CLDN18.2 and CD47 is a CHO-K1 cell expressing both CLDN18.2 and CD47. In one embodiment of the present invention, a cell expressing both CLDN18.2 and CD47 is a CHO-K1 cell overexpressing both CLDN18.2 and CD47.

[0038] In some embodiments of the present invention, the anti-CLDN18.2 antibody is an anti-CLDN18.2 monoclonal antibody.

[0039] The present invention also relates to an antibody against CLDN18.2 or an antigen-binding fragment thereof, wherein the antibody against CLDN18.2 is a monoclonal antibody produced by the hybridoma cell line LT020 deposited at the China Center for Type Culture Collection (CCTCC) under the accession number C2022124.

[0040] The present invention also relates to a hybridoma cell line LT020, deposited in the China Center for Type Culture Collection (CCTCC) under the accession number C2022124.

[0041] Another aspect of the present invention relates to an isolated nucleic acid molecule encoding an anti-CLDN18.2 antibody or antigen-binding fragment thereof according to any aspect of the present invention.

[0042] Another aspect of the present invention relates to a recombinant vector comprising an isolated nucleic acid molecule of the present invention.

[0043] Another aspect of the present invention relates to a host cell comprising an isolated nucleic acid molecule of the present invention or a recombinant vector of the present invention.

[0044] Another aspect of the present invention relates to an antibody-drug conjugate comprising an antibody or an antigen-binding fragment thereof and a small molecule drug, wherein the antibody or an antigen-binding fragment thereof is an anti-CLDN18.2 antibody or an antigen-binding fragment thereof according to any aspect of the present invention; preferably, the small molecule drug is a small molecule cytotoxic drug; more preferably, the small molecule drug is an antitumor chemotherapeutic drug.

[0045] The chemotherapeutic drug may be a traditional antitumor chemotherapeutic drug such as an alkylating agent, antimetabolite, antitumor antibiotic, herbal anticancer agent, hormone and immunological agent.

[0046] In one or more embodiments, the present invention provides an antibody-drug conjugate, wherein the antibody or antigen-binding fragment thereof is linked to a small molecule drug via a linker; the linker may be a linker known to those skilled in the art; for example, the linker is a hydrazone bond, a disulfide bond, or a peptide bond.

[0047] In one or more embodiments, the present invention provides an antibody-drug conjugate, wherein the molar ratio of the antibody or antigen-binding fragment thereof to the small molecule drug is 1:(2-4), such as 1:2, 1:3, or 1:4.

[0048] Another aspect of the present invention relates to a pharmaceutical composition comprising an effective amount of an anti-CLDN18.2 antibody or antigen-binding fragment thereof according to any aspect of the present invention or an antibody-drug conjugate according to any aspect of the present invention, wherein, optionally, the pharmaceutical composition further comprises one or more pharmaceutically acceptable excipients.

[0049] In some embodiments of the present invention, the pharmaceutical composition further comprises an effective amount of an anti-CD47 antibody or antigen-binding fragment thereof, wherein,

[0050] preferably, the anti-CD47 antibody comprises a heavy chain variable region comprising HCDR1-HCDR3 and a light chain variable region comprising LCDR1-LCDR3, wherein:

[0051] the amino acid sequence of HCDR1 is set forth in SEQ ID NO:5, the amino acid sequence of HCDR2 is set forth in SEQ ID NO:6, and the amino acid sequence of HCDR3 is set forth in SEQ ID NO:7; and

[0052] The amino acid sequence of LCDR1 is set forth in SEQ ID NO:8, the amino acid sequence of LCDR2 is set forth in SEQ ID NO:9, and the amino acid sequence of LCDR3 is set forth in SEQ ID NO:10.

[0053] In some embodiments, the present invention provides a pharmaceutical composition, wherein:

[0054] the amino acid sequence of the heavy chain variable region of the anti-CD47 antibody is selected from SEQ ID NO:2, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:20 and SEQ ID NO:24; and

[0055] the amino acid sequence of the light chain variable region of the anti-CD47 antibody is selected from SEQ ID NO:4, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:22 and SEQ ID NO:26;

[0056] wherein, preferably, in the anti-CD47 antibody:

[0057] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:2, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:4;

[0058] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:12, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:14;

[0059] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:12, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:18;

[0060] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:12, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:22;

[0061] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:12, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:26;

[0062] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:16, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:14;

[0063] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:16, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:18;

[0064] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:16, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:22;

[0065] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:16, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:26;

[0066] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:20, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:14;

[0067] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:20, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:18;

[0068] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:20, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:22;

[0069] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:20, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:26;

[0070] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:24, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:14;

[0071] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:24, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:18;

[0072] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:24, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:22;

[0073] or

[0074] The amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:24, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:26.

[0075] In some embodiments, the present invention provides a pharmaceutical composition, wherein: the constant region of the heavy chain of the anti-CD47 antibody is the C region of the Ig gamma-1 chain (e.g., NCBI Accession Number P01857) or the C region of the Ig gamma-4 chain (e.g., NCBI Accession Number P01861.1); the constant region of the light chain of the anti-CD47 antibody is the C region of the Ig kappa chain (e.g., NCBI Accession Number P01834).

[0076] Another aspect of the present invention relates to a bispecific antibody comprising:

[0077] the first protein functional region targeting CLDN18.2; and

[0078] a second protein functional region directed to a target other than CLDN18.2 (e.g., CD47);

[0079] where

[0080] the first protein functional region is an anti-CLDN18.2 antibody or antigen-binding fragment thereof according to any aspect of the present invention.

[0081] Unless otherwise indicated, the bispecific antibody of the present invention is an anti-CLDN18.2 / anti-CD47 bispecific antibody.

[0082] In some embodiments, the present invention provides a bispecific antibody, wherein the second protein functional region is an anti-CD47 antibody or an antigen-binding fragment thereof, wherein

[0083] anti-CD47 antibody comprises a heavy chain variable region containing HCDR1-HCDR3 and a light chain variable region containing LCDR1-LCDR3, wherein:

[0084] the amino acid sequence of HCDR1 is set forth in SEQ ID NO:5, the amino acid sequence of HCDR2 is set forth in SEQ ID NO:6, and the amino acid sequence of HCDR3 is set forth in SEQ ID NO:7; and

[0085] The amino acid sequence of LCDR1 is set forth in SEQ ID NO:8, the amino acid sequence of LCDR2 is set forth in SEQ ID NO:9, and the amino acid sequence of LCDR3 is set forth in SEQ ID NO:10.

[0086] In some embodiments, the present invention provides a bispecific antibody, wherein:

[0087] the amino acid sequence of the heavy chain variable region of the anti-CD47 antibody is selected from SEQ ID NO:2, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:20 and SEQ ID NO:24; and

[0088] the amino acid sequence of the light chain variable region of the anti-CD47 antibody is selected from SEQ ID NO:4, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:22 and SEQ ID NO:26.

[0089] In some embodiments, the present invention provides a bispecific antibody, wherein in the anti-CD47 antibody:

[0090] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:2, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:4;

[0091] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:12, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:14;

[0092] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:12, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:18;

[0093] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:12, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:22;

[0094] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:12, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:26;

[0095] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:16, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:14;

[0096] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:16, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:18;

[0097] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:16, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:22;

[0098] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:16, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:26;

[0099] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:20, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:14;

[0100] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:20, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:18;

[0101] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:20, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:22;

[0102] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:20, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:26;

[0103] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:24, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:14;

[0104] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:24, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:18;

[0105] the amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:24, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:22;

[0106] or

[0107] The amino acid sequence of the heavy chain variable region is set forth in SEQ ID NO:24, and the amino acid sequence of the light chain variable region is set forth in SEQ ID NO:26.

[0108] In some embodiments, the present invention provides a product in the form of a pharmaceutical composition, wherein: the constant region of the heavy chain of the anti-CD47 antibody is the C region of the Ig gamma-1 chain (e.g., NCBI accession number P01857) or the C region of the Ig gamma-4 chain (e.g., NCBI accession number PO1861.1); the constant region of the light chain of the anti-CD47 antibody is the C region of the Ig kappa chain (e.g., NCBI accession number P01834).

[0109] In some embodiments, the present invention provides a bispecific antibody, wherein the first protein functional region and the second protein functional region are independently a single-chain variable fragment fusion protein or a half-molecular monovalent antibody (IgG half molecule, IgG-HM).

[0110] In some embodiments, the present invention provides a bispecific antibody, wherein:

[0111] the first protein functional region is a single-chain variable fragment fusion protein, and the second protein functional region is a semi-molecular monovalent antibody (half an IgG molecule, IgG-HM);

[0112] or

[0113] The first protein functional region is a semi-molecular monovalent antibody (half molecule of IgG, IgG-HM), and the second protein functional region is a single-chain variable fragment fusion protein.

[0114] In some embodiments, the present invention provides a bispecific antibody, wherein:

[0115] the first protein functional region is a single-chain variable fragment fusion protein targeting CLDN18.2, and the second protein functional region is a semi-molecular monovalent antibody (half an IgG molecule, IgG-HM) targeting CD47;

[0116] or

[0117] The first protein functional region is a semi-molecular monovalent antibody (half IgG molecule, IgG-HM) targeting CLDN18.2, and the second protein functional region is a single-chain variable fragment fusion protein targeting CD47.

[0118] In some embodiments, the present invention provides a bispecific antibody, wherein the single-chain variable fragment fusion protein consists of a single-chain variable fragment, a hinge region, and an Fc region, or consists of a single-chain variable fragment and a heavy chain constant region,

[0119] wherein: preferably, the hinge region and the Fc region are a hinge region and an Fc region of human IgG1; preferably, the hinge region and the Fc region have the amino acid sequence set forth in SEQ ID NO:62;

[0120] the heavy chain constant region is a heavy chain constant region of human IgG1; preferably, the heavy chain constant region has the amino acid sequence set forth in SEQ ID NO:63.

[0121] In some embodiments, the present invention provides a bispecific antibody, wherein:

[0122] The Fc fragment in the fusion protein or the heavy chain constant region of the single-chain variable fragment has a knob mutation (e.g., S354C and T366W mutations); and

[0123] The heavy chain constant region of the semi-molecular monovalent antibody is the heavy chain constant region of human IgG1 and has a hole mutation (such as Y349C, T366S, L368A, and Y407V mutations).

[0124] In some embodiments of the present invention, the bispecific antibody consists of the peptide chain set forth in SEQ ID NO:53, the peptide chain set forth in SEQ ID NO:56, and the peptide chain set forth in SEQ ID NO:59,

[0125] wherein: preferably, the peptide chain set forth in SEQ ID NO:53 and the peptide chain set forth in SEQ ID NO:56 are linked by one or more disulfide bonds of the hinge region, and the peptide chain set forth in SEQ ID NO:56 and the peptide chain set forth in SEQ ID NO:59 are linked by one or more disulfide bonds;

[0126] preferably, the peptide chain set forth in SEQ ID NO:53 and the peptide chain set forth in SEQ ID NO:56 are linked by two disulfide bonds of the hinge region, and the peptide chain set forth in SEQ ID NO:56 and the peptide chain set forth in SEQ ID NO:59 are linked by one disulfide bond.

[0127] In some embodiments, the present invention provides a bispecific antibody, wherein:

[0128] EU 50 the bispecific antibody, with respect to binding to a cell expressing CLDN18.2, is less than or equal to 20 nM, less than or equal to 15 nM, or less than or equal to 12 nM; preferably, the EC 50 determined by FACS;

[0129] and / or

[0130] EC 50 the bispecific antibody has an EC of greater than or equal to 20 nM, greater than or equal to 40 nM, or greater than or equal to 50 nM in relation to binding to CD47 on the surface of the red blood cell membrane; preferably, 50 determined by FACS. In one embodiment of the present invention, the cell expressing CLDN18.2 is a CHO-K1 cell expressing CLDN18.2. In one embodiment of the present invention, the cell expressing CLDN18.2 is a CHO-K1 cell overexpressing CLDN18.2.

[0131] In some embodiments, the present invention provides a bispecific antibody, wherein:

[0132] EC 50 the bispecific antibody, with respect to binding to a cell expressing both CLDN18.2 and CD47, is less than or equal to 10 nM, less than or equal to 5 nM, or less than or equal to 2 nM; preferably, the EC 50determined by FACS. In one embodiment of the present invention, a cell expressing both CLDN18.2 and CD47 is a CHO-K1 cell expressing both CLDN18.2 and CD47. In one embodiment of the present invention, a cell expressing both CLDN18.2 and CD47 is a CHO-K1 cell overexpressing both CLDN18.2 and CD47.

[0133] In some embodiments of the present invention, the bispecific antibody does not induce red blood cell agglutination at a concentration of 3000 nM or less.

[0134] In some embodiments of the present invention, the bispecific antibody has ADCP activity, ADCC activity and CDC activity.

[0135] In some embodiments of the present invention, the anti-CD47 antibody is an anti-CD47 monoclonal antibody.

[0136] Another aspect of the present invention relates to an isolated nucleic acid molecule encoding a bispecific antibody according to any aspect of the present invention.

[0137] Another aspect of the present invention relates to a vector comprising an isolated nucleic acid molecule of the present invention.

[0138] Another aspect of the present invention relates to a host cell comprising an isolated nucleic acid molecule of the present invention or a vector of the present invention.

[0139] Another aspect of the present invention relates to a pharmaceutical composition comprising an effective amount of a bispecific antibody according to any aspect of the present invention and one or more pharmaceutically acceptable excipients.

[0140] Another aspect of the present invention relates to the use of an anti-CLDN18.2 antibody or antigen-binding fragment thereof according to any aspect of the present invention or a bispecific antibody according to any aspect of the present invention in the manufacture of a medicament for the treatment or prevention of a tumor,

[0141] wherein, preferably, the tumor is a CD47- and / or CLDN18.2-positive tumor;

[0142] Preferably, the tumor is one or more selected from biliary tract cancer, bronchogenic carcinoma, lymphoma, ovarian cancer, esophageal cancer, melanoma, hematological malignancy, glioblastoma, lung cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, liver cancer, stomach cancer, breast cancer, brain cancer, pancreatic cancer, thyroid cancer, head and neck cancer, and kidney cancer.

[0143] An anti-CLDN18.2 antibody or antigen-binding fragment thereof according to any aspect of the present invention or a bispecific antibody according to any aspect of the present invention is used in the treatment or prevention of a tumor,

[0144] wherein, preferably, the tumor is a CD47- and / or CLDN18.2-positive tumor;

[0145] Preferably, the tumor is one or more selected from biliary tract cancer, bronchogenic carcinoma, lymphoma, ovarian cancer, esophageal cancer, melanoma, hematological malignancy, glioblastoma, lung cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, liver cancer, stomach cancer, breast cancer, brain cancer, pancreatic cancer, thyroid cancer, head and neck cancer, and kidney cancer.

[0146] Another aspect of the present invention relates to a method for treating or preventing a tumor, comprising the step of administering to a subject in need thereof an effective amount of an anti-CLDN18.2 antibody or antigen-binding fragment thereof according to any aspect of the present invention or a bispecific antibody according to any aspect of the present invention,

[0147] wherein, preferably, the tumor is a CD47- and / or CLDN18.2-positive tumor;

[0148] Preferably, the tumor is one or more selected from biliary tract cancer, bronchogenic carcinoma, lymphoma, ovarian cancer, esophageal cancer, melanoma, hematological malignancy, glioblastoma, lung cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, liver cancer, stomach cancer, breast cancer, brain cancer, pancreatic cancer, thyroid cancer, head and neck cancer, and kidney cancer.

[0149] In some embodiments, the present invention provides a method for treating or preventing a tumor, wherein the drug is administered before or after surgery and / or before or after radiation therapy.

[0150] In some embodiments, the present invention provides a method for treating or preventing a tumor, wherein:

[0151] the anti-CLDN18.2 antibody or antigen-binding fragment thereof or the bispecific antibody is administered at a standard dose of 0.1-100 mg / kg body weight, preferably 5-50 mg or 5-15 mg / kg body weight;

[0152] Preferably, the drug is administered once every 3 days, 4 days, 5 days, 6 days, 10 days, 1 week, 2 weeks, or 3 weeks;

[0153] Preferably, the administration route is intravenous drip infusion or intravenous injection.

[0154] In the present invention, unless otherwise defined, scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, laboratory techniques related to cell culture, molecular genetics, nucleic acid chemistry, and immunology employed herein are standard techniques widely used in the relevant fields. However, to better understand the present invention, definitions and explanations of important terms are provided below.

[0155] When used here, the term EU 50 refers to the concentration for 50% of the maximum effect, that is, the concentration that can lead to 50% of the maximum effect.

[0156] As used herein, the term "antibody" refers to an immunoglobulin molecule typically consisting of two pairs of polypeptide chains (each pair has one "light" (L) chain and one "heavy" (H) chain). The light chains of antibodies are classified as κ and λ light chains. Heavy chains are classified as μ, δ, γ, α, or ε. Antibody isotypes are defined as IgM, IgD, IgG, IgA, and IgE. In light chains and heavy chains, the variable region and constant region are joined by a "J region" of approximately 12 or more amino acids, and the heavy chain further comprises a "D region" of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (V H ) and the constant region of the heavy chain (C H ). The constant region of the heavy chain consists of 3 domains (C H 1, C H 2 and C H 3). Each light chain consists of a light chain variable region (V L ) and the constant region of the light chain (C L). The constant region of the light chain consists of a single C domain L The constant region of an antibody can mediate the binding of immunoglobulins to tissues or host factors, including the binding of various cells of the immune system (e.g., effector cells) to the first component (C1q) of the classical complement system. Regions V H and V L can be further divided into hypervariable regions (called complementarity determining regions (CDRs)) interspersed with conserved regions called framework regions (FRs). Each V H and V L consists of 3 CDRs and 4 FRs, arranged from N-terminus to C-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. Variable regions (V H and V L) of each heavy chain / light chain pair form the binding site of the antibody. The assignment of amino acids to regions or domains is based on Bethesda Md, Kabat Sequences of Proteins of Immunological Interest {National Institutes of Health (1987 and 1991)), or Chothia & Lesk J. Mol. Biol, 1987, 196:901–917; Chothia et al., Nature, 1989; 342: 878–883, or the definition of the IMGT numbering system, see definition in Ehrenmann F, Kaas Q, Lefranc M P., IMGT / 3Dstructure-DB and IMGT / DomainGapAlign: a database and a tool for immunoglobulins or antibodies, T cell receptors, MHC, IgSF, and MhcSF[J]., Nucleic acids research, 2009; 38(suppl_1): D301-D307.

[0157] The term "antibody" is not limited to any particular method of producing the antibody. For example, an antibody includes a recombinant antibody, a monoclonal antibody, and a polyclonal antibody. Antibodies can be of various isotypes, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtype), IgA1, IgA2, IgD, IgE, or IgM.

[0158] As used herein, the terms "mAb" and "monoclonal antibody" refer to an antibody or antibody fragment derived from a group of highly homogeneous antibodies, that is, from a group of identical antibody molecules excluding natural mutations that may occur spontaneously. A monoclonal antibody has high specificity for a single epitope on an antigen. A polyclonal antibody, in contrast to a monoclonal antibody, typically comprises at least two or more different antibodies, which typically recognize different epitopes on the antigen. Monoclonal antibodies can typically be produced using hybridoma technology, first described by Kohler et al. Milstein C. Continuous cultures of fused cells secreting antibody of predefined specificity [J]. Nature, 1975; 256(5517): 495), but can also be obtained using recombinant DNA technology (see, e.g., US Patent 4,816,567).

[0159] As used herein, the term "humanized antibody" refers to an antibody or antibody fragment obtained by replacing all or part of the CDR regions of a human immunoglobulin (recipient antibody) with the CDR regions of a non-human antibody (donor antibody), wherein the donor antibody may be a non-human antibody (e.g., mouse, rat, or rabbit) having the intended specificity, affinity, or reactivity. In addition, some amino acid residues of the framework regions (FRs) of the recipient antibody may also be replaced with amino acid residues of the corresponding non-human antibodies or with amino acid residues of other antibodies to further improve or optimize the activity of the antibody. Further details of humanized antibodies are provided in, for example, Jones et al., Nature, 1986; 321: 522-525; Reichmann et al., Nature, 1988; 332: 323-329; Presta, Curr. Op.Struct.Biol. 1992; 2: 593–596; and Clark, Immunol. Today 2000; 21: 397–402. In some cases, the antigen-binding fragment of an antibody is a diabody in which the Vh and Vl domains are expressed on a single polypeptide chain. However, the linker used is too short to pair the two domains located on the same chain. Therefore, the domains can only pair with complementary domains of another chain, and this creates two antigen-binding sites (see, e.g., Holliger P et al., Proc. Natl. Acad. Sci. USA 1993; 90: 6444–6448 and Poljak RJ et al., Structure 1994; 2: 1121–1123).

[0160] As used herein, the term “single chain variable fragment (scFv)” refers to a molecule comprising the variable region of an antibody heavy chain (V H ) and the variable region of the light chain of the antibody (V L ), connected via a linker. V domains L and V Hpair to form a monovalent molecule through a linker that allows them to form a single polypeptide chain (see, e.g., Bird et al., Science, 1988; 242:423-426 and Huston et al., Proc. Natl. Acad. Sci. USA, 1988; 85:5879-5883). Such scFv molecules may have the following general structures: NH2-V L -linker fragment-V H -COOH or NH2-V H -linker fragment-V L-COOH. In the prior art, a suitable linker consists of repeats of the amino acid sequence GGGGS or a variant thereof. For example, a linker having the amino acid sequence (GGGGS)4 can be used, but variants thereof are also possible (Holliger et al., Proc. Natl. Acad. Sci. USA, 1993; 90: 6444-6448). Other linkers that can be used in the present invention are described in Alfthan et al., Protein Eng., 1995; 8: 725-731, Choi et al., Eur. J. Immunol, 2001; 31:94-106, Hu et al., Cancer Res., 1996; 56: 3055-3061, Kipriyanov et al., J. Mol. Biol, 1999; 293: 41-56 and Roovers et al., Cancer Immunology, Immunotherapy, 2001, 50(1): 51-59.

[0161] As used herein, the term "isolated" refers to being obtained artificially from a natural state. If a particular "isolated" substance or component occurs in nature, its natural environment may be modified and / or it may be separated from its natural environment. For example, a particular non-isolated polynucleotide or polypeptide occurs naturally in a particular animal, and the same polynucleotide or polypeptide isolated with a high degree of purity from such a natural state is called an isolated polynucleotide or polypeptide. The term "isolated" does not exclude the presence of artificial or synthetic substances or other impurities that do not affect the activity of the substance.

[0162] As used herein, the term "vector" refers to a carrier nucleic acid into which a polynucleotide can be introduced. When a vector allows for the expression of a protein encoded by the introduced polynucleotide, such a vector is called an expression vector. The vector can be introduced into a host cell by transformation, transduction, or transfection such that elements of the genetic material carried by the vector can be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), or P1-derived artificial chromosome (PAC); phages such as λ phages or M13 phages; and animal viruses.Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papovaviruses (such as SV40). The vector may contain various expression control elements, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Furthermore, the vector may further contain an origin of replication.

[0163] As used herein, the term "host cell" refers to cells into which vectors can be introduced, including, but not limited to, prokaryotic cells such as E. coli or Bacillus subtilis, fungal cells such as yeast or Aspergillus cells, insect cells such as Drosophila S2 or Sf9 cells, or animal cells such as fibroblasts, CHO cells, GS cells, COS cells, NS0 cells, HeLa cells, BHK cells.

[0164] As used herein, the term “specific binding” refers to a non-random interaction involving the binding of two molecules, such as the interaction of an antibody with the antigen to which it is directed. In some embodiments, an antibody specifically binding to an antigen (or an antibody specific for an antigen) means that the antibody binds to the antigen with an affinity (K D ) less than approximately 10 -5 M, for example, less than approximately 10 -6 M, 10 -7 M, 10 -8 M, 10 -9M or 10 -10 M or less.

[0165] When used here, the term "K D " refers to the equilibrium dissociation constant for the specific interaction of an antibody with an antigen and is used to describe the binding affinity of an antibody to an antigen. The lower the equilibrium dissociation constant, the stronger the binding of the antibody to the antigen and the higher the affinity of the antibody for the antigen. Antibodies typically bind to antigens (e.g., the CLDN18.2 protein) with an equilibrium dissociation constant (K D ) less than approximately 10 -5 M, such as less than approximately 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M or 10 -10 M or less. K D can be determined using methods known to those skilled in the art, such as the Fortebio molecular interaction assessment tool.

[0166] As used herein, the terms "monoclonal antibody" and "mAb" have the same meaning and are used interchangeably; the terms "polyclonal antibody" and "pAb" have the same meaning and are used interchangeably. Furthermore, as used herein, amino acids are typically abbreviated using single-letter and three-letter abbreviations known in the art. For example, alanine may be abbreviated as A or Ala.

[0167] As used herein, the term "pharmaceutically acceptable carrier and / or excipient" refers to a carrier and / or excipient that is pharmacologically and / or physiologically compatible with the subject and the active ingredient. Such carriers and / or excipients are well known in the art (see, e.g., Remington's Pharmaceutical Sciences, edited by Gennaro AR, 19 thEd., Pennsylvania, Mack Publishing Company, 1995), including, but not limited to, pH adjusters, surfactants, adjuvants, and ionic strength increasing agents. For example, pH adjusters include, but are not limited to, phosphate buffer; surfactants include, but are not limited to, cationic, anionic, or nonionic surfactants such as Tween-80; ionic strength increasing agents include, but are not limited to, sodium chloride.

[0168] As used herein, the term "effective amount" refers to an amount sufficient to produce, or at least partially produce, a desired effect. For example, a prophylactically effective amount for a disease (e.g., a tumor) refers to an amount sufficient to prevent, arrest, or delay the onset of the disease (e.g., a tumor); a therapeutically effective amount refers to an amount sufficient to cure or at least partially arrest the disease and its complications in patients suffering from the disease. Those skilled in the art are undoubtedly able to determine such an effective amount.For example, the amount effective for therapeutic purposes will depend on the severity of the disease being treated, the general state of the patient's own immune system, the patient's general condition parameters such as age, body weight and gender, the route of administration, the concomitant use of other treatments, and so on.

[0169] When used herein, the reference to the amino acid sequence of the CD47 protein (NCBI GenBank: NP_001768.1) includes the full-length CD47 protein, or the extracellular fragment of CD47 (CD47 ECD), or a fragment containing CD47 ECD, and also includes a fusion protein of the full-length CD47 protein or a fusion protein of CD47 ECD, such as a fragment fused with the Fc protein fragment of mouse or human IgG (mEc or hFc). However, it will be clear to those skilled in the art that it is possible for natural or artificial mutations or changes (including, but not limited to, substitutions, deletions and / or additions) to occur in the amino acid sequence of the CD47 protein without affecting its biological functions. Therefore, in the present invention, the term "CD47 protein" includes all such sequences, including natural or artificial variants thereof.In addition, when describing a fragment of the CD47 protein sequence, the CD47 protein also includes the corresponding fragments of the sequence in its natural or artificial variants.

[0170] When used herein, the reference to the amino acid sequence of the CLDN18.2 protein (NCBI GenBank: NP_001002026.1) includes the full-length CLDN18.2 protein, or the extracellular fragment of CLDN18.2 (CLDN18.2 ECD), or a fragment containing CLDN18.2 ECD, and also includes a fusion protein of the full-length CLDN18.2 protein or a fusion protein of CLDN18.2 ECD, such as a fragment fused with the Fc protein fragment of mouse or human IgG (mFc or hFc). However, it will be clear to those skilled in the art that it is possible for natural or artificial mutations or changes (including, but not limited to, substitutions, deletions and / or additions) to occur in the amino acid sequence of the CLDN18.2 protein without affecting its biological functions. Therefore, in the present invention, the term "CLDN18.2 protein" includes all such sequences, including their natural or artificial variants. Furthermore, the description of a fragment of the CLDN18 protein sequence is not provided.2 also includes the corresponding sequence fragments in their natural or artificial variants.

[0171] In the present invention, the term "ADCP" refers to antibody-dependent cellular phagocytosis. The Fc fragment of an antibody that binds to an antigen on the cell surface binds to the Fc receptor of a phagocytically active cell, such as a macrophage, which in turn mediates phagocytosis of the cells to which the antibody is bound by phagocytic cells.

[0172] In the present invention, the term "ADCC" refers to antibody-dependent cell-mediated cytotoxicity. The Fab fragment of the antibody binds to an epitope on a virus-infected cell or tumor cell, and the Fc fragment of the antibody binds to the Fc receptor (FcR) on the surface of a killer cell (NK cell, macrophage, etc.), mediating direct killing of target cells by the killer cells.

[0173] In the present invention, the term "CDC" refers to complement-dependent cytotoxicity. When an antibody specifically binds to a corresponding antigen on the surface of a cell membrane, a complex is formed, activating the complement system and subsequently forming a MAC on the surface of the target cell, leading to subsequent lysis of the target cell. Components of the complement system can lead to cell lysis of various bacteria and other pathogenic microorganisms and are an important defense mechanism against infections caused by pathogenic microorganisms.

[0174] Unless otherwise specified, in the present invention, the terms "first" (e.g., a first protein functional region or a first pharmaceutical product) and "second" (e.g., a second protein functional region or a second pharmaceutical product) are used for distinction or clarity of expression, but not to denote a typical sequence.

[0175] In the present invention, the term “half-molecular monovalent antibody (IgG half molecule, IgG-HM)” is an antibody molecule composed of 1 heavy chain and 1 light chain of an IgG antibody (e.g., IgG1, IgG2, IgG3, or IgG4), which is a monovalent antibody (see, for example, FENG Yi-fan et al., Construction and activity analysis of HIV specific monovalent mAb 2G12, Chinese Journal of Viral Diseases, May 2015, 5(3): 171-175).

[0176] Beneficial effects of the present invention

[0177] The present invention can achieve one or more of the following effects:

[0178] (1) The anti-CLDN18.2 antibody of the present invention has excellent affinity and specificity;

[0179] (2) The bispecific antibody of the present invention, for example, can bind well and specifically to CLDN18.2, exerting an effect of antibody-mediated killing of cells expressing CLDN18.2;

[0180] (3) The bispecific antibody of the present invention, for example, can bind well and specifically to cells expressing both CD47 and CLDN18.2, exerting an effect of antibody-mediated killing of cells expressing both CD47 and CLDN18.2;

[0181] (3) The bispecific antibody of the present invention can specifically bind to CD47 and effectively block the binding of CD47 to SIRPα, thereby specifically reducing CD47 / SIRPα-mediated immunosuppression and activating the immune response;

[0182] (4) the first protein functional region and the second protein functional region of the bispecific antibody of the present invention act synergistically;

[0183] (5) The bispecific antibody of the present invention does not induce erythrocyte agglutination, has high safety, and can exert ADCP, ADCC, and CDC effects.

[0184] BRIEF DESCRIPTION OF GRAPHIC MATERIALS

[0185] FIG. 1: FACS analysis of the binding activity of 8C5.1H3L3, 6F7H1L1(hG4), and AsAb-8C4703 to CHO-K1-CLDN18.2-CD47 cells.

[0186] FIG. 2: FACS analysis of the binding activity of 8C5.1H3L3 and AsAb-8C4703 to CHO-K1-CLDN18.2 cells.

[0187] FIG. 3: FACS analysis of AsAb-8C4703 binding activity to erythrocytes.

[0188] FIG. 4: FACS analysis of competition between 6F7H1L1(hG4) and AsAb-8C4703 for binding to CHO-K1-CLDN18.2-CD47 surface antigens.

[0189] FIG. 5: Agglutination of normal human erythrocytes induced by 6F7H1L1(hG4) and AsAb-8C4703.

[0190] FIG. 6: Phagocytosis of KATOIII-CLDN18.2 cells by HPMM mediated by 8C5.1H3L3, 6F7H1L1(hG4) and AsAb-8C4703.

[0191] FIG. 7: ADCC activity analysis of 8C5.1H3L3, AsAb-8C4703, and 6F7H1L1(hG4) on CHO-K1-CLDN18.2-CD47 cells.

[0192] FIG. 8: Analysis of CDC activity of 8C5.1H3L3, AsAb-8C4703, and 6F7H1L1(hG4) against CHO-K1-CLDN18.2-CD47 cells.

[0193] FIG. 9: Phagocytosis of CHO-K1-CLDN18.2 cells by HPMM mediated by 8C5.1H3L3 and IMAB362.

[0194] FIG. 10: Analysis of the binding activity of AsAb-8C4703, 8C5.1H3L3, and IMAB362 to human CLDN18.2 antigen by indirect ELISA.

[0195] FIG. 11: Analysis of the binding activity of AsAb-8C4703 and 6F7H1L1(hG4) to CD47-IgV-TEV-His antigen by indirect ELISA.

[0196] FIG. 12: Results of determination of the kinetic parameters of binding of 8C5.1H3L3 to CLDN18.2 His.

[0197] FIG. 13: Results of determination of the kinetic parameters of binding of AsAb-8C4703 to CLDN18.2 His.

[0198] FIG. 14: Results of determination of the kinetic parameters of binding of 6F7H1L1(hG4) to CD47 ECD-His.

[0199] FIG. 15: Results of determination of the kinetic parameters of AsAb-8C4703 binding to CD47 ECD-His.

[0200] FIG. 16: Analysis of binding activity of AsAb-8C4703 and Hu5F9-G4 to T cells.

[0201] FIG. 17: Analysis of the binding activity of AsAb-8C4703 and Hu5F9-G4 to B cells.

[0202] FIG. 18: Efficacy results of AsAb-8C4703, 6F7H1L1(hG4), and 8C5.1H3L3 as monotherapy in MC38-CD47-CLDN18.2 tumor xenograft model in C57BL / 6 mice.

[0203] FIG. 19: Efficacy results of 6F7H1L1(hG4) in combination with 8C5.1H3L3 in MC38-CD47-CLDN18.2 tumor xenograft model in C57BL / 6 mice.

[0204] FIG. 20: Effects of AsAb-8C4703, 6F7H1L1(hG4), and 8C5.1H3L3 as monotherapy on body weight in MC38-CD47-CLDN18.2 tumor xenograft model in C57BL / 6 mice.

[0205] FIG. 21: Effects of 6F7H1L1(hG4) in combination with 8C5.1H3L3 on body weight in MC38-CD47-CLDN18.2 tumor xenograft model in C57BL / 6 mice.

[0206] The hybridoma cell line LT012 was deposited with the China Center for Type Culture Collection (CCTCC) on June 21, 2018, under the accession number C2018135; the depository address is Wuhan University, Wuhan, China, postal code 430072.

[0207] The hybridoma cell line LT020 was deposited with the China Center for Type Culture Collection (CCTCC) on May 19, 2022, under the accession number C2022124; the depository address is Wuhan University, Wuhan, China, postal code 430072.

[0208] Below are some sequences related to the present invention.

[0209]

[0210]

[0211]

[0212]

[0213]

[0214]

[0215]

[0216]

[0217] DETAILED DESCRIPTION OF THE INVENTION

[0218] Next, embodiments of the present invention will be described in detail by means of examples. However, it will be clear to those skilled in the art that the following examples are provided solely for illustrative purposes and should not be construed as limiting the scope of the present invention. In these examples, experimental procedures without specified conditions were used under conventional conditions or conditions recommended by the manufacturer. Unless the manufacturers of the reagents or instruments used are indicated, all such reagents or instruments are commonly available commercially.

[0219] In the following Experimental Examples of the present invention, the isotope control antibodies used, i.e., hIgG1 and hIgG4, were human anti-hen egg lysozyme (HEL) antibodies, and the variable region sequences of these antibodies were adopted from the study of Acierno et al. entitled “Affinity maturation increases the stability and plasticity of the Fv domain of anti-protein antibodies” (Acierno et al., J Mol Biol., 2007; 374(1): 130-46). In the constant region fragment of hIgG1, the constant region of the heavy chain is the C region of the Ig gamma-1 chain, accession number P01857, and the constant region of the light chain is the C region of the Ig kappa chain, accession number P01834. In hIgG4, the heavy chain constant region is the C region of the Ig gamma-4 chain, accession number P01861.1, with the S228P mutation introduced to improve stability, and the light chain constant region is the C region of the Ig kappa chain, accession number P01834. Both hIgG1 and hIgG4 were obtained by Akeso Biopharma Inc.

[0220] Production Example 1: Production of Anti-Human CD47 Antibody 6F7

[0221] 1. Production of hybridoma cell line LT012

[0222] The antigen used was CD47-IgV-TEV-His (containing the mature peptide of human CD47, positions 19-141, Genbank identifier NP 942088.1, and a fusion protein with a TEV-His tag) and 3T3-CD47 cells (NIH / 3T3, manufacturer ATCC, catalog number CRL-1658; the cells were transfected with the mature peptide of human CD47 based on NIH / 3T3 to obtain a stably expressing 3T3-CD47 cell line). Spleen cells of immunized mice were fused with mouse myeloma cells to obtain hybridoma cells. Using CD47-IgV-TEV-His and 3T3-CD47 cells as individual antigens, hybridoma cells were screened by indirect ELISA to obtain hybridoma cells capable of secreting antibodies capable of specifically binding to CD47.The hybridoma cells obtained from this ELISA screening were screened by a competitive ELISA to obtain a hybridoma cell line capable of secreting a monoclonal antibody capable of competing for binding to human CD47-IgV-TEV-His with the human SIRPαECD-hFc-Biotin receptor (SIRPαECD refers to the extracellular region of SIRPα, positions 31-373 of the protein with the GenBank accession number NP_542970.1; hFc refers to the Fc purification tag of human IgG, specifically the C region of the Ig gamma-1 chain, positions 114-330, GenBank identifier P01857), which was then subjected to serial dilution analysis to obtain a stable hybridoma cell line. The hybridoma cell line generated above was named hybridoma cell line LT012, and the monoclonal antibody secreted by this cell line was named 6F7.

[0223] The hybridoma cell line LT012 was deposited with the China Center for Type Culture Collection (CCTCC) on June 21, 2018, under the accession number C2018135; the depository address is Wuhan University, Wuhan, China, postal code 430072.

[0224] 2. Obtaining the 6F7 antibody against CD47

[0225] The LT012 cell line obtained as described above was cultured in a chemically defined medium (CD medium containing 1% penicillin-streptomycin, GlutaMAX (Gibco 35050079)) in an incubator with 5% CO2 at 37°C. After 7 days, the supernatant was collected, subjected to high-speed centrifugation and vacuum filtration through a microfiltration membrane, and purified using a HiTrap Protein A HP column to obtain the 6F7 antibody.

[0226] Production Example 2: Sequence Analysis of Anti-CD47 Antibody 6F7

[0227] mRNA was isolated from the LT012 cell line cultured in Preparation Example 1 following the method described in the instruction manual of the RNAprep Pure Cell / Bacteria Kit (Tiangen, catalog number DP430).

[0228] cDNA was synthesized according to the instructions of the Superscript® III First-Strand Synthesis System from Invitrogen for RT-PCR kit and amplified by PCR.

[0229] PCR amplified products were directly subjected to TA cloning following the instruction of the pEASY-T1 Cloning Kit (Transgen, CT101).

[0230] The TA cloning product was subjected to direct sequencing, and the following results were obtained.

[0231] The nucleotide sequence of the heavy chain variable region is set forth in SEQ ID NO:1, length 351 bp.

[0232] The encoded amino acid sequence is set forth in SEQ ID NO:2, length 117 aa.

[0233] The nucleotide sequence of the light chain variable region is set forth in SEQ ID NO:3, length 321 bp.

[0234] The encoded amino acid sequence is set forth in SEQ ID NO:4, length 107 aa.

[0235] The 6 CDRs of antibody 6F7 are assigned according to the IMGT numbering system as follows. The sequences of HCDR1, HCDR2, and HCDR3 of the heavy chain are set forth in SEQ ID NOs: 5, 6, and 7, respectively.

[0236] The sequences of the light chain LCDR1, LCDR2 and LCDR3 are set forth in SEQ ID NO: 8, 9 and 10, respectively.

[0237] Production Example 3: Development of Light and Heavy Chains and Production of Humanized Anti-Human CD47 Antibodies 6F7H1L1(hG4), 6F7H2L2(hG4), 6F7H3L3(hG4), and 6F7H4L4(hG4)

[0238] 1. Development of light and heavy chains of humanized antibodies 6F7H1L1(hG4), 6F7H2L2(hG4), 6F7H3L3(hG4) and 6F7H4L4(hG4) against human CD47

[0239] Based on the three-dimensional crystal structure of human CD47 protein (Hage T, Reinemer P, Sebald W., Crystals of a 1:1 Complex Between Human Interleukin-4 and the Extracellular Domain of Its Receptor Alpha Chain, Eur. J. Biochem., 1998; 258(2): 831-6.) and the sequence of 6F7 antibody obtained in Production Example 2, the variable region sequences of 6F7H1L1, 6F7H2L2, 6F7H3L3 and 6F7H4L4 antibodies were obtained through computer modeling and mutation engineering (antibody constant region sequences from the NCBI database: the constant region of the heavy chain is the C region of the Ig gamma-4 chain, accession number P01861.1; the constant region of the light chain is the C region of the Ig kappa chain, accession number P01834).

[0240] The designed variable region sequences are as follows.

[0241] (1) Sequences of the variable regions of the heavy and light chains of the humanized monoclonal antibody 6F7H1L1

[0242] The nucleotide sequence of the heavy chain variable region is set forth in SEQ ID NO:11, length 351 bp.

[0243] The encoded amino acid sequence is set forth in SEQ ID NO:12, length 117 aa.

[0244] The nucleotide sequence of the light chain variable region is set forth in SEQ ID NO:13, length 321 bp.

[0245] The encoded amino acid sequence is set forth in SEQ ID NO:14, length 107 aa.

[0246] (2) Sequences of the variable regions of the heavy and light chains of the humanized monoclonal antibody 6F7H2L2

[0247] The nucleotide sequence of the heavy chain variable region is set forth in SEQ ID NO:15, length 351 bp.

[0248] The encoded amino acid sequence is set forth in SEQ ID NO:16, length 117 aa.

[0249] The nucleotide sequence of the light chain variable region is set forth in SEQ ID NO:17, length 321 bp.

[0250] The encoded amino acid sequence is set forth in SEQ ID NO:18, length 107 aa.

[0251] (3) Sequences of the variable regions of the heavy and light chains of the humanized monoclonal antibody 6F7H3L3

[0252] The nucleotide sequence of the heavy chain variable region is set forth in SEQ ID NO:19, length 351 bp.

[0253] The encoded amino acid sequence is set forth in SEQ ID NO:20, length 117 aa.

[0254] The nucleotide sequence of the light chain variable region is set forth in SEQ ID NO:21, length 321 bp.

[0255] The encoded amino acid sequence is set forth in SEQ ID NO:22, length 107 aa.

[0256] (4) Sequences of the variable regions of the heavy and light chains of humanized monoclonal antibody 6F7H4L4

[0257] The nucleotide sequence of the heavy chain variable region is set forth in SEQ ID NO:23, length 351 bp.

[0258] The encoded amino acid sequence is set forth in SEQ ID NO:24, length 117 aa.

[0259] The nucleotide sequence of the light chain variable region is set forth in SEQ ID NO:25, length 321 bp.

[0260] The encoded amino acid sequence is set forth in SEQ ID NO:26, length 107 aa.

[0261] Moreover, 6F7H1L1, 6F7H2L2, 6F7H3L3 and 6F7H4L4 have the following identical HCDR1-HCDR3 and LCDR1-LCDR3:

[0262] the HCDR1 sequence is set forth in SEQ ID NO:5, the HCDR2 sequence is set forth in SEQ ID NO:6, and the HCDR3 sequence is set forth in SEQ ID NO:7;

[0263] The sequence of LCDR1 is set forth in SEQ ID NO:8, the sequence of LCDR2 is set forth in SEQ ID NO:9, and the sequence of LCDR3 is set forth in SEQ ID NO:10.

[0264] 2. Production of humanized antibodies 6F7H1L1(hG4), 6F7H2L2(hG4), 6F7H3L3(hG4) and 6F7H4L4

[0265] All the constant regions of the heavy chain were the C region of the Ig gamma-4 chain, accession number P01861.1; all the constant regions of the light chain were the C region of the Ig kappa chain, accession number P01834.

[0266] The heavy chain cDNA and light chain cDNA of 6F7H1L1(hG4), the heavy chain cDNA and light chain cDNA of 6F7H2L2(hG4), the heavy chain cDNA and light chain cDNA of 6F7H3L3(hG4), and the heavy chain cDNA and light chain cDNA of 6F7H4L4(hG4) were individually cloned into pUC57simple vectors (provided by GenScript) to obtain pUC57simple-6F7H1 and pUC57simple-6F7L1, pUC57simple-6F7H2 and pUC57simple-6F7L2, pUC57simple-6F7H3 and pUC57simple-6F7L3, and pUC57simple-6F7H4 and pUC57simple-6F7L4, respectively. Using standard techniques described in Molecular Cloning: A Laboratory Manual (Second Edition), full-length heavy and light chain genes synthesized by digestion of the genes with EcoRI and HindIII were subcloned into the expression vector pcDNA3.1 by digestion with restriction enzymes (EcoRI and HindIII), generating expression plasmids pcDNA3.1-6F7H1, pcDNA3.1-6F7L1, pcDNA3.1-6F7H2, pcDNA3.1-6F7L2, pcDNA3.1-6F7H3, pcDNA3.1-6F7L3, pcDNA3.1-6F7H4, and pcDNA3.1-6F7L4, followed by sequencing of the heavy and light chain genes from the resulting recombinant expression plasmids. The developed gene combinations, including the corresponding light and heavy chains, were then used individually as recombinant plasmids (pcDNA3.1-6F7H1 / pcDNA3.1-6F7L1, pcDNA3.1-6F7H2 / pcDNA3.1-6F7L2, pcDNA3.1-6F7H3 / pcDNA3.1-6F7L3, and pcDNA3.1-6F7H4 / pcDNA3.1-6F7L4) to cotransfect 293F cells, followed by harvesting and purification of the culture solutions. After sequence verification, endotoxin-free expression plasmids were obtained and transiently transfected into HEK293 cells for antibody expression. Cell cultures were harvested after 7 days of cultivation and subjected to affinity purification on a protein A column (MabSelect SuRe (GE)) to obtain humanized antibodies.

[0267] Production Example 4: Production of 8C5.1 Antibody against CLDN18.2

[0268] 1. Production of hybridoma cell line LT020

[0269] The immunogen used was the 3T3 cell line (Chinese Academy of Sciences) overexpressing human claudin 18.2 (Claudin18.2, Genbank ID NP_001002026.1) (3T3 hClaudin18.2). Spleen cells from immunized mice were fused with mouse myeloma cells to produce hybridoma cells. Using the CHO-K1 cell line overexpressing human claudin 18.2 (Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) (CHO-K1-hClaudin18.2) as an antigen, hybridoma cells were screened by indirect ELISA to obtain hybridoma cells capable of secreting antibodies specifically binding to CLDN18.2. The hybridoma cells obtained from the screening were analyzed by serial dilution to obtain a stable hybridoma cell line.The hybridoma cell line generated above was named hybridoma cell line LT020, and the monoclonal antibody secreted by this hybridoma cell line was named 8C5.1.

[0270] The hybridoma cell line LT020 was deposited with the China Center for Type Culture Collection (CCTCC) on May 19, 2022, under the accession number C2022124; the depository address is Wuhan University, Wuhan, China, postal code 430072.

[0271] 2. Production of 8C5.1 antibody against CLDN18.2

[0272] The LT020 cell line obtained as described above was cultured in CD medium (Chemical Defined Medium) containing 4% GlutaMAX (Gibco 35050079) and 1% penicillin-streptomycin (Gibco 15140163)) at 5% CO2 and 37°C. After 7 days, the cell culture supernatant was collected, subjected to high-speed centrifugation and vacuum filtration through a microfiltration membrane, and purified using a HiTrap Protein A HP column to obtain 8C5.1 antibody.

[0273] Production Example 5: Sequence Analysis of Anti-CLDN18.2 Antibody 8C5.1

[0274] mRNA was isolated from the LT020 cell line cultured in Preparation Example 4 following the method described in the instruction manual of the RNAprep Pure Cell / Bacteria Kit (Tiangen, catalog number DP430).

[0275] cDNA was synthesized according to the instructions of the Superscript® III First-Strand Synthesis System from Invitrogen for RT-PCR kit and amplified by PCR.

[0276] The PCR amplified product was directly subjected to TA cloning following the instruction of the pEASY-T1 Cloning Kit (Transgen, CT101).

[0277] The TA cloning product was subjected to direct sequencing, and the following results were obtained.

[0278] The nucleotide sequence of the heavy chain variable region is set forth in SEQ ID NO:27, length 354 bp.

[0279] The encoded amino acid sequence is set forth in SEQ ID NO:28, length 118 amino acids.

[0280] The nucleotide sequence of the light chain variable region is set forth in SEQ ID NO:29, length 339 bp.

[0281] The encoded amino acid sequence is set forth in SEQ ID NO:30, length 113 amino acids.

[0282] 6 CDRs of antibody 8C5.1 are assigned according to the IMGT numbering system as follows.

[0283] The sequences of HCDR1, HCDR2 and HCDR3 of the heavy chain are set forth in SEQ ID NO:31, 32 and 33, respectively.

[0284] The sequences of the light chain LCDR1, LCDR2, and LCDR3 are set forth in SEQ ID NO:34, 35, and 36, respectively.

[0285] Production Example 6: Development of Light and Heavy Chains and Production of Humanized Anti-Human CLDN18.2 Antibodies

[0286] 1. Development of light and heavy chains of humanized antibodies 8C5.1H1L1, 8C5.1H1L2, 8C5.1H2L1, 8C5.1H2L2, 8C5.1H2L3, 8C5.1H3L2 and 8C5.1H3L3 against human CLDN18.2

[0287] Based on the three-dimensional crystal structure of the human CLDN18.2 protein and the sequence of the 8C5 antibody obtained in Production Example 5, variable region sequences of the antibodies 8C5.1H1L1, 8C5.1H1L2, 8C5.1H2L1, 8C5.1H2L2, 8C5.1H2L3, 8C5.1H3L2 and 8C5.1H3L3 were obtained through computer modeling and subsequent mutation engineering (antibody constant region sequences from the NCBI database: the heavy chain constant region is the C region of the Ig gamma-1 chain, SEQ ID NO: 61; the light chain constant region is the C region of the Ig kappa chain, accession number P01834).

[0288] The designed variable region sequences are shown in Table A

[0289] below.

[0290]

[0291] Each of the above 7 antibodies, 8C5.1H1L1, 8C5.1H1L2, 8C5.1H2L1, 8C5.1H2L2, 8C5.1H2L3, 8C5.1H3L2 and 8C5.1H3L3, had a nucleotide sequence length of the heavy chain variable region of 354 bp with an encoded amino acid sequence length of 118 aa, and a nucleotide sequence length of the light chain variable region of 339 bp with an encoded amino acid sequence length of 113 aa.

[0292] In addition, the 7 antibodies above had the same HCDRT-HCDR3 and LCDR1-LCDR3 shown below.

[0293] The sequences of HCDR1, HCDR2 and HCDR3 are set forth in SEQ ID NO:31, 32 and 22, respectively.

[0294] The sequences of LCDR1, LCDR2 and LCDR3 are set forth in SEQ ID NO:34, 35 and 36, respectively.

[0295] 2. Production of humanized antibodies 8C5.1H1L1, 8C5.1H1L2, 8C5.1H2L1, 8C5.1H2L2, 8C5.1H2L3, 8C5.1H3L2 and 8C5.1H3L3

[0296] All the constant regions of the heavy chain were the C region of the Ig gamma-1 chain, SEQ ID NO:61; all the constant regions of the light chain were the C region of the Ig kappa chain, accession number P01834.

[0297] The heavy chain cDNA and light chain cDNA of each of 8C5.1H1L1, 8C5.1H1L2, 8C5.1H2L1, 8C5.1H2L2, 8C5.1H2L3, 8C5.1H3L2, and 8C5.1H3L3 were cloned into the pUC57simple vector (provided by GenScript) to obtain pUC57simple-8C5.1Hl, pUC57simple-8C5.1L1, respectively;

[0298] pUC57simple-8C5.1H1, pUC57simple-8C5.1L2;

[0299] pUC57simple-8C5.1H2, pUC57simple-8C5.1L1;

[0300] pUC57simple-8C5.1H2, pUC57simple-8C5.1L2;

[0301] pUC57simple-8C5.1H2, pUC57simple-8C5.1L3;

[0302] pUC57simple-8C5.1H3, pUC57simple-8C5.1L2; And

[0303] pUC57simple-8C5.1H3, pUC57simple-8C5.1L3.

[0304] According to the standard procedures described in Molecular Cloning: A Laboratory Manual (Second Edition), EcoRI and HindIII were used to digest the synthesized full-length heavy and light chain genes. The genes were subcloned into the expression vector pcDNA3.1 by digestion with restriction enzymes (EcoRI and HindIII), yielding expression plasmids pcDNA3.1-8C5.1H1, pcDNA3.1-8C5.1L1, pcDNA3.1-8C5.1H2, pcDNA3.1-8C5.1L2, pcDNA3.1-8C5.1H3, and pcDNA3.1-8C5.1L3, and then the heavy / light chain genes from the resulting recombinant expression plasmids were sequenced. Then, each of the developed gene combinations, including the corresponding light and heavy chains, was produced in the form of recombinant plasmids (pcDNA3.1-8C5.1H1 / pcDNA3.1-8C5.1L1,

[0305] pcDNA3.1-8C5.1H1 / pcDNA3.1-8C5.1L2,

[0306] pcDNA3.1-8С5.1H2 / pcDNA3.1-8С5.1L1,

[0307] pcDNA3.1-8C5.1H2 / pcDNA3.1-8C5.1L2,

[0308] pcDNA3.1-8C5.1H2 / pcDNA3.1-8C5.1L3,

[0309] pcDNA3.1-8C5.1H3 / pcDNA3.1-8C5.1L2 and

[0310] pcDNA3.1-8C5.1H3 / pcDNA3.1-8C5.1L3) were used to co-transfect 293F cells, after which the culture solutions were collected and purified. After sequence accuracy was verified by sequencing, endotoxin-free expression plasmids were obtained and transiently transfected into HEK293 cells for antibody expression. After 7 days, the cell culture solutions were collected and affinity purified on a protein A column to produce humanized antibodies.

[0311] Production Example 7: Development and Production of Anti-CLDN18.2 / Anti-CD47 Bispecific Antibody

[0312] 1. Development of the structure

[0313] In this example, the bispecific antibody was named AsAb-8C4703, and its structure is shown in Table 1 below.

[0314]

[0315] In Table 1 above:

[0316] the linker moiety, i.e., the linker, has the amino acid sequence set forth in SEQ ID NO:50;

[0317] The positions of Knob and Hole mutations are numbered according to the EU numbering system.

[0318] The bispecific antibody AsAb-8C4703 contains three peptide chains, from left to right, named HC1, HC2 and L3, respectively, and has the following composition.

[0319] (1) НС1 composition: scFv (VH 6F7H4 + linker sequence + VL 6F7L4) + hinge region + CH2 hIgG1 + CH3 hIgG1 with Knob mutation sites (S354C mutation with replacement of serine at position 354 with cysteine ​​and T366W mutation with replacement of threonine at position 366 with tryptophan).

[0320] The amino acid sequence of scFv is as follows (where the linker sequence is underlined):

[0321]

[0322] Hinge region, CH2 domain and CH3 domain (where the sites of Knob mutations S354C and T366W are highlighted with double underlining):

[0323]

[0324] The full-length amino acid sequence of HC1 is as follows (476 aa, where the linker sequence is underlined and the Knob mutation sites are double underlined):

[0325]

[0326] The full-length nucleotide sequence of HC1 is as follows:

[0327]

[0328]

[0329] (2) НС2 composition: VH 8С5.1Н3 + hIgG1 constant region with Hole mutation sites (Y349C mutation with tyrosine at position 349 being replaced by cysteine, T366S mutation with threonine at position 366 being replaced by serine, L368A mutation with leucine at position 368 being replaced by alanine, and Y407V mutation with tyrosine at position 407 being replaced by valine).

[0330] The amino acid sequence of 8C5.1H3VH is set forth in SEQ ID NO:41.

[0331] Constant region sequence (where the Hole mutation sites Y349C, T366S, L368A and Y407V are highlighted with double underlining):

[0332]

[0333] The full-length amino acid sequence of HC2 is as follows (447 aa, where the sites of Hole mutations are highlighted by double underlining):

[0334]

[0335]

[0336] The full-length nucleotide sequence of HC2 is as follows:

[0337]

[0338] (3) Composition of L3: VL 8C5.1L3 + constant region of human Ig kappa.

[0339] The amino acid sequence of 8C5.1L3VL is set forth in SEQ ID NO:47.

[0340] Amino acid sequence of the constant region of human Ig kappa:

[0341]

[0342] The full-length amino acid sequence of L3 is as follows:

[0343]

[0344] The full-length nucleotide sequence of L3 is as follows:

[0345]

[0346] The peptide chains of HC1 and HC2 of the bispecific antibody AsAb-8C4703 are linked by two disulfide bonds in the hinge region, and HC2 and L3 are linked by one disulfide bond.

[0347] 2. Molecular construction, expression and purification of antibodies

[0348] The cDNA sequences encoding the three polypeptide chains of the bispecific antibody AsAb-8C4703 were individually cloned into pUC57simple vectors (provided by Genscript) to generate plasmids pUC57simple-HC1, pUC57simple-HC2, and pUC57simple-L3, respectively.

[0349] Each of the pUC57simple-HC1, pUC57simple-HC2, and pUC57simple-L3 plasmids was digested with HindIII and EcoRI and, after electrophoresis, subcloned into pcDNA3.1 vectors. Recombinant plasmids were isolated and cotransfected into 293F cells. After 7 days of cell culture, the culture solution was centrifuged at high speed. The supernatant was concentrated and loaded onto a HiTrap MabSelect SuRe column. Protein was eluted in a single step using elution buffer. The target sample was isolated and the buffer was exchanged with PBS.

[0350] The obtained anti-CLDN18.2 / anti-CD47 bispecific antibody was used in the following experiments.

[0351] Example 1: FACS analysis of the binding activity of anti-CLDN18.2 / anti-CD47 bispecific antibody

[0352] 1. FACS (fluorescence-activated cell sorting) analysis of the binding activity of anti-CLDN18.2 / anti-CD47 bispecific antibody to CHO-K1-CLDN18.2-CD47 cells

[0353] CHO-K1-CLDN18.2-CD47 cells (CHO-K1 cells overexpressing CLDN18.2 and CD47 antigens and obtained from the Cell Resource Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) in the logarithmic growth phase were collected and transferred into a 96-well V-bottom plate at 1 × 10 5cells per well. 100 μl of 1% PBSA (PBS + 1% BSA) were added and the resulting mixture was centrifuged at 1000 g for 5 min, followed by removal of the supernatant. 100 μl of antibodies diluted in 1% PBSA were added (at final concentrations of 900 nM, 300 nM, 100 nM, 33.3 nM, 11.1 nM, 3.7 nM, 1.23 nM, 0.41 nM, 0.041 nM, and 0.0041 nM, respectively). The resulting mixture was gently and thoroughly mixed and then incubated on ice for 40 minutes. 150 μl of 1% PBSA were added to each well and the resulting mixture was centrifuged at 1000 g for 5 min. The supernatant was removed, and the plate was washed three times with 200 µl each time. For resuspension, a 300-fold dilution of anti-human IgG Fc antibody with PE (BioLegend, catalog number 409304) was added, and the resulting mixture was thoroughly mixed.The diluted antibody was added to the respective samples at 100 μl per well, the cell pellet was resuspended and incubated on ice in the dark for 0.5 h. 150 μl of 1% PBSA was added to each well, and the resulting mixture was centrifuged at 1000 g for 5 min, followed by removal of the supernatant. The plate was then washed twice with 200 μl of 1% PBSA. 200 μl of 1% PBSA was added to each well to resuspend the cell pellet, and the resulting mixture was transferred to a flow cytometry tube. Binding activity was assessed using a FACS Calibur.

[0354] The experimental results are shown in Table 2 and FIG. 1.

[0355]

[0356] The results obtained showed that under the same experimental conditions of the EU 50The binding efficiencies of 8C5.1H3L3, 6F7H1L1(hG4) and AsAb-8C4703 to CHO-K1-CLDN18.2-CD47 cells were 1.220 nM, 1.295 nM and 1.245 nM, respectively.

[0357] The results obtained show that under the same experimental conditions, 8C5.1H3L3, 6F7H1L1(hG4) and AsAb-8C4703 exhibit comparable binding activity to CHO-K1-CLDN18.2-CD47 cells, indicating that AsAb-8C4703 has the activity of effectively binding to cells expressing both CLDN18.2 and CD47.

[0358] 2. FACS analysis of the binding activity of anti-CLDN18.2 / anti-CD47 bispecific antibody to CHO-K1-CLDN18.2 cells

[0359] CHO-K1-CLDN18.2 cells (CHO-K1 cells overexpressing CLDN18.2 antigen and obtained from the Cell Resource Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences) in the logarithmic growth phase were collected and transferred into a 96-well V-bottom plate at 2.5 × 10 5cells per well. 100 μl of 1% PBSA (PBS + 1% BSA) were added and the resulting mixture was centrifuged at 1000 g for 5 min, followed by removal of the supernatant. 100 μl of antibodies diluted in 1% PBSA were added (at final concentrations of 900 nM, 300 nM, 100 nM, 33.3 nM, 11.1 nM, 3.7 nM, 1.23 nM, 0.123 nM, 0.0123 nM, and 0.00123 nM, respectively). The resulting mixture was gently and thoroughly mixed and then incubated on ice for 40 minutes. 150 µl of 1% PBSA was added to each well, and the resulting mixture was centrifuged at 1000 g for 5 minutes. The supernatant was discarded, and the plate was washed three times, with 200 µl each time. For resuspension, a 300-fold dilution of anti-human IgG Fc antibody with PE (BioLegend, catalog number 409304) was added, and the resulting mixture was thoroughly mixed.The diluted antibody was added to the respective samples at 100 μl per well, the cell pellet was resuspended, and the mixture was incubated on ice in the dark for 0.5 h. 150 μl of 1% PBSA was added to each well, and the resulting mixture was centrifuged at 1000 g for 5 min, followed by removal of the supernatant. The plate was then washed twice with 200 μl of 1% PBSA. 200 μl of 1% PBSA was added to each well to resuspend the cell pellet, and the resulting mixture was transferred to a flow cytometry tube for analysis on a FACS Calibur.

[0360] The experimental results are shown in Table 3 and FIG. 2.

[0361]

[0362] The results obtained showed that under the same experimental conditions of the EU 50 8C5.1H3L3 and AsAb-8C4703 for binding to CHO-K1-CLDN18.2 cells was 4.439 nM and 10.59 nM, respectively.

[0363] The obtained results show that under the same experimental conditions, 8C5.1H3L3 and AsAb-8C4703 exhibit comparable binding activity to CHO-K1-CLDN18.2 cells, indicating that AsAb-8C4703 has the activity of efficiently binding to CLDN18.2 on the surface of CHO-K1-CLDN18.2 cell membrane.

[0364] 3. FACS analysis of the binding activity of bispecific anti-CLDN18.2 / anti-CD47 antibody to red blood cells

[0365] To obtain red blood cells, fresh blood was collected (from healthy volunteers after obtaining informed consent). The red blood cells were washed twice with an appropriate amount of PBS, after which the cell density was adjusted. The resulting cell suspension was added to a 96-well V-bottom plate, 1 x 10 6cells per sample, and an appropriate amount of 1% PBSA (PBS + 1% BSA) was added to each well. The resulting mixture was centrifuged at 750 g for 5 min, and the supernatant was removed. Antibodies were diluted to concentrations of 900 nM, 300 nM, 100 nM, 33.33 nM, 11.11 nM, 3.70 nM, 1.23 nM, 0.41 nM, 0.041 nM, and 0.0041 nM using 1% PBSA, and the diluted antibodies were added to the respective samples at 100 μl per well. The cell pellet was resuspended and incubated on ice for 40 min. 150 µl of 1% PBSA were added to each well, and the resulting mixture was centrifuged at 750 g for 5 min. The supernatant was discarded, and the plate was washed three times with 200 µl each time. Anti-human IgG Fc antibody with PE (BioLegend, catalog number 409304) was diluted in 1% PBSA, and the diluted antibody was added to the corresponding samples at 100 µl per well. The cell pellet was resuspended and incubated on ice in the dark for 30 min.150 µl of 1% PBSA was added to each well, and the resulting mixture was centrifuged at 350 g for 5 min. The supernatant was discarded, and the plate was washed twice, with 200 µl each time. 200 µl of 1% PBSA was added to each well, the cell pellet was resuspended, and then transferred to a loading tube for analysis.

[0366] The experimental results are shown in Table 4 and FIG. 3.

[0367]

[0368] The results obtained showed that the EU 50 The binding activity of the bispecific antibody AsAb-8C4703 against CLDN18.2 / against CD47 to erythrocytes was 50.68 nM.

[0369] These results indicate that the anti-CLDN18.2 / anti-CD47 bispecific antibody AsAb-8C4703 has only very weak binding activity to red blood cells.

[0370] Example 2: Competitive binding of anti-CLDN18.2 / anti-CD47 bispecific antibody to cell membrane surface antigen CD47

[0371] The competition of a bispecific antibody against CLDN18.2 / anti-CD47 and hSIRPαV2 for binding to the cell membrane surface antigen CD47 was analyzed by competitive flow cytometry. The method was as follows.

[0372] CHO-K1-CLDN18.2-CD47 cells were treated routinely and transferred into a 96-well V-bottom plate at 3 × 10 5cells per well. Then, 100 µl of 1% PBSA was added to each well, and the resulting mixture was centrifuged and washed. 50 µl of hSIRPαV2-hFc-mFc (manufacturer Akeso Biopharma Inc., lot number 20210120) was added to each well, the resulting mixture was thoroughly mixed to a final concentration of 3 nM and incubated on ice for 30 min. The corresponding serially diluted antibodies (at final concentrations of 900 nM, 300 nM, 100 nM, 33.3 nM, 11.1 nM, 3.7 nM, 1.23 nM, 0.41 nM, 0.041 nM, and 0.0041 nM, respectively) were added at 50 μl per sample, and the resulting mixture was incubated on ice for 30 min. 150 μl of 1% PBSA was added to each well, and the resulting mixture was centrifuged at 1000 g for 5 min. The supernatant was discarded, after which the plate was washed three times, with 200 μl each time. APC Streptavidin (BioLegend, catalog number 405207) was diluted in 1% PBSA (300-fold dilution) and the diluted antibody was added to the respective samples at 100 µl per well.The cell pellet was resuspended and incubated on ice in the dark for 30 min. 150 µl of 1% PBSA was added to each well, and the resulting mixture was centrifuged at 1000 g for 5 min. The supernatant was discarded, and the plate was washed twice, with 200 µl each time. 200 µl of 1% PBSA was added to each well, the cell pellet was resuspended, and then transferred to a loading tube for analysis.

[0373] The obtained results are shown in FIG. 4, and the EC values 50 in the analyzed samples are shown in Table 5. Based on the results of quantitative fluorescence analysis and curve fitting, it was calculated that, in relation to competitive binding, the EC values 50 antibodies 6F7H1L1(hG4) and AsAb-8C4703 were 5.003 nM and 10.05 nM, respectively.

[0374]

[0375] The results obtained show that 6F7H1L1(hG4) and AsAb-8C4703 antibodies can effectively block the binding of hSIRPαV2 to CD47 on the surface of CHO-K1-CLDN18.2-CD47 host cells in a dose-dependent manner.

[0376] Example 3: Effect of anti-CLDN18.2 / anti-CD47 bispecific antibody on agglutination of normal human red blood cells (RBC)

[0377] Dextran T500 solution, 0.2 mg / ml, was prepared. To obtain red blood cells, fresh blood was collected (from healthy volunteers after obtaining informed consent), the red blood cells were washed twice with an appropriate amount of PBS, and then the density of the obtained cells was adjusted. The cells were seeded in a 96-well plate at 50 µl per well (cell count 1 × 10 7per well) and 50 μl of the corresponding antibodies were added to each well (antibody working concentrations were 3000 nM, 1000 nM, 333.3 nM, 111.1 nM, 37.0 nM, 12.3 nM, 4.1 nM, 1.4 nM, 0.14 nM and 0.014 nM). In the positive control group, 50 μl of Dextran T500 (0.1 mg / ml) were added, in the negative control group, 50 μl of PBS were added, and in the isotype control group, hIgG1 antibody (manufacturer Akeso Biopharma Inc., lot number 20190410) and hIgG4 antibody (manufacturer Akeso Biopharma Inc., lot number 20190910) were added. The system was incubated at 37°C for 2-4 hours and erythrocyte agglutination was analyzed and photographed.

[0378] Agglutination of normal human erythrocytes induced by 6F7H1L1(hG4) and AsAb-8C4703 is shown in FIG. 5.

[0379] The results showed that 6F7H1L1(hG4) and AsAb-8C4703 had no effect on red blood cell agglutination at a concentration of 3000 nM or less, indicating good safety.

[0380] Example 4: Phagocytosis of tumor cells by macrophages stimulated by anti-CLDN18.2 / anti-CD47 bispecific antibody

[0381] In this example, HPMM were used as the effector cells, KATOIII-CLDN18.2 were used as the target cells, and the antibody-dependent cellular phagocytosis (ADCP) activity mediated by 8C5.1H3L3, 6F7H1L1(hG4), and AsAb-8C4703 antibodies was analyzed. The method was as follows.

[0382] KATOIII-CLDN18.2 cells (KATOIII cells overexpressing CLDN18.2, obtained from the Cell Resource Center, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) were washed once with PBS and centrifuged at 170 g for 5 min. The cells were counted and their viability was analyzed. CFSE (BioLegend, catalog no. 423801) was diluted to a concentration of 2.5 μM with PBS, and an appropriate amount of the diluted CFSE was used to resuspend the cells (staining density of 10 million cells / mL). The cells were incubated in the incubator for 20 min, and 6 mL of complete 1640 medium (containing 10% FBS) was added to stop the staining. The cells were centrifuged at 170 g for 5 min, and the supernatant was removed. To resuspend the cells, 1 ml of complete 1640 medium was added.The cells were incubated in an incubator for 10 min and their density was adjusted to 150,000 cells per tube.

[0383] Antibodies (working concentrations of 1 nM, 10 nM, 100 nM, and 300 nM) were diluted with 1640 complete medium, and isotype control antibodies hIgG1 (manufactured by Akeso Biopharma Inc., lot number 20190410) and hIgG4 (manufactured by Akeso Biopharma Inc., lot number 20190910) (both at a working concentration of 300 nM) were prepared.

[0384] HRMN (isolated from peripheral blood obtained from healthy volunteers after obtaining informed consent) were collected, counted, and analyzed for viability. The cell suspension was placed in a 1.5 ml EP tube, 50,000 cells per tube, centrifuged at 1200 g for 5 min, and the supernatant was removed. Fifty microliters of the target cell suspension and 50 microliters of antibodies were placed in a 1.5 ml EP tube, and the resulting mixture was incubated in an incubator for 2 hours.

[0385] 700 μl of 1% PBSA (PBS + 1% BSA) was added to each well, the resulting mixture was centrifuged at 1200 g for 5 min, and the supernatant was removed. Anti-mouse / human CD11b APC antibody (BioLegend, catalog number 101212) was diluted 500-fold in 1% PBSA, and the diluted antibody was added to the respective samples at 100 μl per well. The cell pellet was resuspended and incubated on ice for 30 min. 800 μl of 1% PBSA was added to each well, and the resulting mixture was centrifuged at 1200 g for 5 min. The supernatant was removed, and the plate was then washed twice with 800 μl each time. 200 μl of 1% PBSA was added to each well, the cell pellet was resuspended and then transferred to a loading tube for analysis.

[0386] In this system, macrophages were APC-CD11b-positive, and macrophages involved in phagocytosis were both APC-CD11b- and CFSE-positive. The phagocytic index was determined as the ratio of the number of cells with a double-positive phenotype to the number of APC-positive cells, and antibody-mediated ADCP activity was assessed. In each group, ADCP activity, represented as P%, was calculated using the following formula:

[0387]

[0388] The obtained results are shown in FIG. 6.

[0389] The results showed that the phagocytic indices of 8C5.1H3L3, 6F7H1L1(hG4) and AsAb-8C4703 were significantly higher than those of the negative control and isotype control, indicating that 8C5.1H3L3, 6F7H1L1(hG4) and AsAb-8C4703 had ADCP activity, and the ADCP activity of AsAb-8C4703 was higher than the ADCP activity of the 8C5.1H3L3 group, the 6F7H1L1(hG4) group and the combined group of 8C5.1H3L3 and 6F7H1L1(hG4).

[0390] Example 5: ADCC activity assay of anti-CLDN18.2 / anti-CD47 bispecific antibody

[0391] CHO-K1-CLDN18.2-CD47 cells were collected routinely and centrifuged at 170 g for 5 min, after which the cells were resuspended in medium (RPMI-1640 + 1% FBS) and washed twice. The cells were resuspended in the medium and then counted and adjusted for cell density. The target cell suspension was added to a 96-well plate at 100 μl per well (approximately 3 × 10 4 target cells per well), antibodies 8C5.1H3L3, 6F7H1L1(hG4) and AsAb-8C4703 (for all working concentrations 3 nM, 1 nM, 0.33 nM, 0.11 nM, 0.037 nM, 0.0123 nM and 0.00123 nM) were diluted in the medium (RPMI-1640 + 1% FBS) and the diluted antibodies were added to the corresponding wells at 50 μl per well. The system was incubated in an incubator at 37°C for 1 h and the medium for the negative control and hIgG1 for the isotype control (manufacturer Akeso Biopharma Inc., lot number 20190410) were prepared.

[0392] Effector PBMCs were collected as usual, centrifuged at 170 g for 5 min, and the supernatant was removed. The cells were washed twice, resuspended in medium (RPMI-1640 + 1% FBS), and then counted. Density adjustment of the resulting cells was performed. 50 μl of effector cell suspension (containing approximately 9 × 10 5 effector cells per well) were added to the pre-incubated target cells and the resulting mixture was thoroughly mixed. The cells were incubated in an incubator with 5% CO2 at 37°C for 4 h and centrifuged at 250 g for 5 min. 100 μl of the culture supernatant was carefully pipetted and transferred to a new 96-well flat-bottomed plate, and 100 μl of freshly prepared reaction solution were added to each well. The resulting mixture was incubated in the dark at room temperature for 30 min, and OD values ​​were measured at 490 nm and 650 nm. The OD value in each group was equal to the difference in OD490nm and OD 650nm .

[0393] The obtained results are shown in FIG. 7.

[0394] The results show that, compared with the isotope control, the bispecific anti-CLDN18.2 / anti-CD47 antibody AsAb-8C4703 has ADCC activity, can specifically kill CLDN18.2- and CD47-positive cells, and its cell killing activity is better than that of the corresponding monoclonal antibodies 8C5.1H3L3 and 6F7H1L1(hG4).

[0395] Example 6: Assay for CDC activity of anti-CLDN18.2 / anti-CD47 bispecific antibody

[0396] CHO-K1-CLDN18.2-CD47 cells were collected by routine processing and centrifuged at 170 g for 5 min. The cells were then resuspended in the medium (RPMI-1640 + 1% FBS) and washed twice. The cells were resuspended in the medium (RPMI-1640 + 1% FBS) and then counted. The density of the resulting cells was adjusted. The target cell suspension was added to a 96-well plate at 100 μl per well (approximately 3 × 104 The antibodies (working concentrations of 100 nM, 10 nM, 1 nM, and 0.1 nM for all cells per well) were diluted in the medium (RPMI-1640 + 1% FBS). The diluted antibodies were added to the corresponding wells at 50 μl per well, and the system was pre-incubated at room temperature for 10 min. After pre-incubation, 50 μl of serum with complement (at a final concentration of 2%) were added to the target cells, and the resulting mixture was thoroughly mixed. The system was incubated in an incubator with 5% CO2 at 37°C for 4 h and centrifuged at 250 g for 5 min. One hundred microliters of the culture supernatant was carefully pipetted and transferred to a new 96-well flat-bottomed plate. 100 microliters of freshly prepared reaction solution was added to each well. The resulting mixture was incubated in the dark at room temperature for 30 min, and the OD values ​​were measured at 490 nm and 650 nm. The OD value in each group was equal to the difference in OD 490nm and OD650nm .

[0397] As shown in FIG. 8, compared with the hIgG1 isotype control (manufacturer: Akeso Biopharma Inc., lot number: 20190410), the anti-CLDN18.2 / anti-CD47 bispecific antibody AsAb-8C4703 has CDC activity, is able to kill tumor cells through complement, and has an activity comparable to that of 8C5.1H3L3.

[0398] Example 7: Phagocytosis of tumor cells by macrophages stimulated by anti-CLDN18.2 antibody

[0399] In this example, antibody-dependent cellular phagocytosis (ADCP) activity mediated by the 8C5.1H3L3 antibody was detected using mouse bone marrow-derived macrophages (MBMM) as effector cells and CHO-K1-CLDN18.2 as target cells.

[0400] Claudiximab (IMAB362, zolbetuximab) is a human-mouse chimeric monoclonal antibody targeting CLDN18.2, developed by Ganymed, Germany. Claudiximab can specifically recognize and bind to the first extracellular domain (ECD1) of CLDN18.2 protein outside the cell membrane, mediating antibody-dependent cellular phagocytosis (ADCP) and so on (Singh P, Toom S, Huang Y. Anti-claudinl8.2 antibody as a new targeted therapy for advanced gastric cancer [J]. Journal of Hematology & Oncology, 2017, 10(1)).

[0401] The specific methodology of this example is as follows.

[0402] CHO-K1-CLDN18.2 cells were harvested as usual, centrifuged at 170 g for 5 min, resuspended, counted, analyzed for viability, and washed once with PBS. CFSE was diluted to 2.5 μM with PBS, cells were resuspended with the appropriate amount of diluted CFSE (staining density 10 million cells / mL), and incubated in an incubator for 20 min.

[0403] To stop staining, 6 ml of complete DMEM medium (containing 10% FBS) was added. The cells were centrifuged at 170 g for 5 min, and the supernatant was removed. 1 ml of complete DMEM medium was added, and the cells were incubated in an incubator for 10 min. The antibody was diluted with complete DMEM medium (10 μg / ml, 1 μg / ml, and 0.1 μg / ml), and isotype control and reference antibodies were prepared.

[0404] Macrophages were collected and centrifuged at 170 g for 5 min. The supernatant was removed, and the cells were counted. The cells were transferred to a 1.5 ml EP tube, centrifuged at 1200 g for 5 min, and the supernatant was removed. The tumor cell and antibody suspension, incubated for 30 min, was added to the EP tube containing the macrophages, and the cells were resuspended. The resulting mixture was thoroughly mixed and incubated in an incubator at 37°C for 2 hours. 800 μl of 1% PBSA at normal temperature was added to each tube, and the resulting mixture was centrifuged at 1200 g for 5 min. The supernatant was removed, after which the cells were washed once using 800 μl of PBSA. APC anti-mouse / human CD11b antibody (BioLegend, catalog number 101212) was added to the respective samples at a 600-fold dilution of 100 µl per sample, and the resulting mixture was mixed thoroughly and incubated on ice for 40 min.Eight hundred microliters of 1% PBSA were added to each tube, and the resulting mixture was centrifuged at 1200 g for 5 min. The supernatant was removed, and each tube was washed once with 200 μl of PBSA. Another 200 μl of 1% PBSA was added to each tube for resuspension. The resulting mixture was then transferred to a flow cytometry tube for analysis.

[0405] The obtained results are shown in FIG. 9.

[0406] The results obtained show that the phagocytic indices of 8C5.1H3L3 and IMAB362 (reference antibody) (manufacturer Akeso Biopharma Inc., lot number 20190704) were significantly higher than those of the negative control and the isotype control, indicating that both 8C5.1H3L3 and the reference antibody IMAB362 have ADCP activity and that 8C5.1H3L3 has better ADCP activity than IMAB362.

[0407] Example 8: Analysis of the binding activity of anti-CLDN18.2 / anti-CD47 bispecific antibody to antigens by ELISA

[0408] 1. The binding activity of AsAb-8C4703, 8C5.1H3L3, and IMAB362 to human CLDN18.2 antigen was analyzed separately using indirect ELISA. The method was as follows.

[0409] Human CLDN18.2 (2 μg / mL) was coated on an ELISA plate and incubated at 4°C overnight. The ELISA plate containing the coated antigen was then washed once with PBST and then blocked with a PBST solution containing 1% BSA as a blocking solution at 37°C for 2 h. After blocking, the ELISA plate was washed three times with PBST. Antibodies serially diluted in PBST were added (antibody dilution gradients are shown in Table 6). The ELISA plate containing the antibodies to be analyzed was incubated at 37°C for 30 min and then washed three times with PBST. After washing, a working solution of secondary HRP-labeled goat anti-human IgG Fc antibody (H+L) (Jackson, catalog number 109-035-098) diluted 1:5000 was added, and the plate was then incubated at 37°C for 30 min.After incubation, the plate was washed three times with PBST, TMB (Neogen, 308177) was added to develop color in the dark for 5 min, and then a stop solution was added to terminate the chromogenic reaction. The ELISA plate was immediately placed in a microplate reader, and the OD value in each well of the ELISA plate was read at 450 nm. The resulting data were analyzed and processed using SoftMax Pro 6.2.1.

[0410] The analysis results are shown in Table 6 and FIG. 10.

[0411]

[0412] FIG. 10 shows that AsAb-8C4703, 8C5.1H3L3, and IMAB362 can effectively bind to human CLDN18.2 in a dose-dependent manner. Based on the results of quantitative analysis of the optical density of the bound antibodies, the binding efficiency values ​​EC 50The ELISAs of AsAb-8C4703, 8C5.1H3L3, and IMAB362 (as a control) antibodies obtained by curve fitting calculations were 0.021 nM, 0.011 nM, and 0.014 nM, respectively.

[0413] The results obtained show that under the same experimental conditions, AsAb-8C4703 and 8C5.1H3L3 exhibit effective binding activity to human CLDN18.2.

[0414] 2. The binding activity of AsAb-8C4703 and 6F7H1L1(hG4) to the CD47-IgV-TEV-His antigen was analyzed separately using indirect ELISA. The method was as follows.

[0415] CD47-IgV-TEV-His, 2 μg / mL, was adsorbed onto an ELISA plate and incubated at 4°C overnight. The ELISA plate containing the adsorbed antigen was then washed once with PBST and then blocked with a PBST solution containing 1% BSA as a blocking solution at 37°C for 2 h. After blocking, the ELISA plate was washed 3 times with PBST. Antibodies serially diluted in PBST were added (antibody dilution gradients are shown in Table 7). The ELISA plate containing the analyzed antibodies was incubated at 37°C for 30 min and then washed 3 times with PBST. After washing, a working solution of secondary HRP-labeled goat anti-human IgG Fc antibody (H+L) (Jackson, catalog number 109-035-098) diluted 1:5000 was added, and the plate was then incubated at 37°C for 30 min.After incubation, the plate was washed three times with PBST, TMB (Neogen, 308177) was added to develop color in the dark for 5 min, and then a stop solution was added to terminate the chromogenic reaction. The ELISA plate was immediately placed in a microplate reader, and the OD value in each well of the ELISA plate was read at 450 nm. The resulting data were analyzed and processed using SoftMax Pro 6.2.1.

[0416] The analysis results are shown in Table 7 and FIG. 11.

[0417]

[0418] FIG. 11 shows that AsAb-8C4703 and 6F7H1L1(hG4) can effectively bind to CD47-IgV-TEV-His in a dose-dependent manner. According to the results of quantitative analysis of the optical density of the bound antibodies, the binding efficiency values ​​of EC 50 The concentrations of AsAb-8C4703 and 6F7H1L1(hG4) antibodies obtained by curve fitting calculations were 0.164 nM and 0.012 nM, respectively.

[0419] The results obtained show that under the same experimental conditions, AsAb-8C4703 and 6F7H1L1(hG4) exhibit effective binding activity to human CD47-IgV-TEV-His.

[0420] Example 9: Determination of kinetic parameters of humanized anti-CLDN18.2 / anti-CD47 bispecific antibody

[0421] The affinity constant of the antibody for human CLDN18.2 and CD47 ECD-His was determined using the ForteBio Octet Molecular Interaction Instrument. The sample dilution buffer was PBS (0.02% Tween-20, 0.1% BSA, pH 7.4). The method for determining the affinity of the antibody for human CLDN18.2 is as follows. The antibody, 20 nM, was immobilized on the ANS sensor for 60 s. The sensor was equilibrated in the buffer for 60 s, and the binding of the antibody immobilized on the sensor to CLDN18.2 His at concentrations of 6.25-100 nM (two-fold dilution) was determined for 180 s. Protein dissociation was carried out in the buffer for 360 s. The method for determining the affinity of an antibody for human CD47 ECD-His is as follows. The antibody, 30 nM, was immobilized on the ANS sensor for 120 s.The sensor was equilibrated in the buffer for 60 s, and the binding of the antibody immobilized on the sensor to the CD47 ECD-His protein at concentrations of 4.94–400 nM (three-fold dilution) was determined for 50 s. The protein was dissociated in the buffer for 120 s. The sample plate was shaken at 1000 rpm at 30°C and 5.0 Hz. Data were analyzed using a 1:1 fit to the model to obtain affinity constants. Fortebio Data Acquisition 12.0 was used for data acquisition, and Fortebio Data Analysis HT 12.0 was used for data analysis.

[0422] The binding kinetic parameters of antibodies 8C5.1H3L3 and AsAb-8C4703 to CLDN18.2 His are shown in Table 8, and the results of determining the characteristic kinetic parameters are shown in FIG. 12 and FIG. 13, respectively.

[0423]

[0424] K D represents the affinity constant; kon represents the rate of binding of antigen and antibody; k dis represents the rate of dissociation of antigen and antibody; K D equal to the ratio k dis to k on .

[0425] The results obtained show that both 8C5.1H3L3 antibody and AsAb-8C4703 antibody have good affinity for CLDN18.2 His antigen.

[0426] The binding kinetic parameters of antibodies 6F7H1L1(hG4) and AsAb-8C4703 to CD47 ECD-His are shown in Table 9, and the results of determining the characteristic kinetic parameters are shown in FIG. 14 and FIG. 15, respectively.

[0427]

[0428] K D represents the affinity constant; k on represents the rate of binding of antigen and antibody; k dis represents the rate of dissociation of antigen and antibody; K D equal to the ratio k dis to k on .

[0429] The results obtained show that both the 6F7H1L1(hG4) antibody and the AsAb-8C4703 antibody have good affinity for the antigen in question.

[0430] Example 10: Binding of anti-CLDN18.2 / anti-CD47 bispecific antibody to human immune cells

[0431] PBMC cells were collected routinely, washed twice with appropriate amount of PBS, counted and analyzed for viability. The PBMC suspension was placed in a 96-well plate, 3 × 10 5cells per sample, centrifuged at 750 g for 5 min, and the supernatant removed. Mouse IgG isotype control (Thermo Fisher, catalog number 10400C) was added at a final concentration of 5 μg / ml, the resulting mixture was blocked on ice for 20 min and centrifuged at 750 g for 5 min. The supernatant was removed. 100 μl of serially diluted antibody (working concentrations of 900 nM, 300 nM, 100 nM, 33.3 nM, 11.1 nM, 3.7 nM, 0.37 nM, 0.037 nM, and 0.0037 nM) were added, and isotype control antibodies hIgG1 (manufacturer Akeso Biopharma Inc., lot number 20170424) and hIgG4 (manufacturer Akeso Biopharma Inc., lot number 20190910) were prepared (both at a working concentration of 900 nM). The cell pellet was resuspended, incubated on ice for 40 min, and 1% PBSA (PBS + 1% BSA) was added. The resulting mixture was centrifuged at 750 g for 5 minutes, and the supernatant was removed. The plate was then washed three times.100 µl of anti-human CD3 antibody with FITC (BioLegend, catalog #344804), anti-human CD19 antibody with Brilliant Violet 421™ (BioLegend, catalog #302234), and mouse anti-human IgG Fc antibody with AF647 (Southern Biotech, catalog #9040-31) were added, the cell pellet was resuspended, and incubated on ice in the dark for 30 min. 1% PBSA was added to each well, and the resulting mixture was centrifuged at 750 g for 5 min. The supernatant was discarded, and the plate was washed twice. 1% PBSA was added to each well, the cell pellet was resuspended, and then transferred to a loading tube for analysis on a FACS Calibur. The Hu5F9-G4 reference antibody is a high-affinity, drug-grade humanized IgG4 antibody developed by Forty Seven that targets CD47.

[0432] The obtained results are shown in Table 10 and FIGS. 16 and 17.

[0433]

[0434] The experimental results showed that the MFI values ​​of AsAb-8C4703 binding to both CD3+CD19- T cells and CD3-CD19+ B cells were smaller, and the corresponding EC values 50 were higher than those of the reference antibody Hu5F9-G4 (manufacturer Akeso Biopharma Inc., lot number 20220303), indicating that AsAb-8C4703 has much weaker binding activity to both CD3+ CD19- T cells and CD3- CD19+ B cells than Hu5F9-G4.

[0435] Example 11: Tumor growth inhibition experiment of anti-CLDN18.2 / anti-CD47 bispecific antibody in vivo

[0436] To determine the tumor inhibitory activity of the anti-CLDN18.2 / anti-CD47 bispecific antibody in vivo, MC38-CD47-CLDN18.2 cells (a murine colon cancer cell line MC38 transfected with human CD47 and human CLDN18.2, obtained from Akeso Biopharma Inc., where MC38 was obtained from Shanghai Model Organisms Center, Inc.) were first injected into the right flank of 5-7-week-old female C57BL / 6 mice (purchased from GemPharmatech). The model used and the specific administration route are shown in Table 11. After administration, the tumor length and width were measured in each group, and its volume was calculated.

[0437] After the groups were formed, the tumor size was measured twice a week using a caliper, and the tumor volume was calculated using the formula TV = 0.5 × ab 2, where a is the long diameter of the tumor, b is the short diameter of the tumor, and TV is the tumor volume. TGI (%) (tumor growth inhibition) was calculated based on the tumor volume using the formula:

[0438] TGI (%) = (1 - mean RTV treat / mean RTV vehicle ) x 100%,

[0439] where RTV = Vt / V0, RTV is the relative tumor volume, mean RTV treat and mean RTV vehicle represent the average relative tumor volume in the treatment and model groups, respectively, and Vt and V0 represent the tumor volume on day t and day 0, respectively. GraphPad Prism software was used to plot the graphs, and drug efficacy results were assessed using the TGI.

[0440]

[0441]

[0442] Note: In Table 11, “IP” means “intraperitoneal injection” and “BIW” means “twice a week”.

[0443] The obtained results are shown in FIGS. 18, 19, 20 and 21.

[0444] These results showed the following:

[0445] On the 21st day of administration after the formation of groups, the tumor growth inhibition, TGI (%), when using AsAb-8C4703, 17 mg / kg, was 85.43%, which is better than the 6F7H1L1(hG4) monotherapy group, 20 mg / kg (TGI 18.28%), the 8C5.1H3L3 monotherapy group, 20 mg / kg (TGI 23.09%), and the combination group of 6F7H1L1(hG4), 20 mg / kg, and 8C5.1H3L3, 20 mg / kg (TGI 23.78%), at equimolar doses;

[0446] Tumor growth inhibition, TGI (%), with AsAb-8C4703, 5.7 mg / kg, was 83.62%, which was better than that of the combination group of 6F7H1L1(hG4), 6.7 mg / kg, and 8C5.1H3L3, 6.7 mg / kg (TGI 51.22%) at an equimolar dose;

[0447] Tumor growth inhibition, TGI (%), when using AsAb-8C4703, 1.9 mg / kg, was 44.26%, which was better than the combination group of 6F7H1L1(hG4), 2.2 mg / kg, and 8C5.1H3L3, 2.2 mg / kg (TGI 3.74%), at an equimolar dose.

[0448] In addition, as shown in FIGS. 20 and 21, the study drugs showed good tolerability in tumor-bearing mice, and no effect on the body weight of tumor-bearing mice was found in the analyzed groups, indicating good safety.

[0449] While specific embodiments of the present invention have been described in detail, those skilled in the art will appreciate that various modifications and substitutions are possible with respect to the details of all disclosed concepts, and that such modifications are within the scope of protection of the present invention. The full scope of the present invention is defined by the appended claims and any equivalents thereto.

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[0541] <insdfeature>

[0542] <INSDFeature_key>source< / INSDFeature_key>

[0543] <INSDFeature_location>1..321< / INSDFeature_location>

[0544] <INSDFeature_quals>

[0545] <insdqualifier>

[0546] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0547] <INSDQualifier_value>other DNA< / INSDQualifier_value>

[0548] < / insdqualifier>

[0549] <insdqualifier id="q135">

[0550] <INSDQualifier_name> organism< / INSDQualifier_name>

[0551] <INSDQualifier_value> synthetic construct< / INSDQualifier_value>

[0552] <NonEnglishQualifier_value> synthetic

[0553] design< / NonEnglishQualifier_value>

[0554] < / insdqualifier>

[0555] < / INSDFeature_quals>

[0556] < / insdfeature>

[0557] < / INSDSeq_feature-table>

[0558] <INSDSeq_sequence>aacatcgtgatgacccagtcccccaagtctatgagcatgtccctgggcg

[0559] agagggtgaccctgtcctgtaaggcctctgagatcgtgggcacatacgtgtcttggtttcagcagaagcc

[0560] acaccagagccccaagctgctgatctacggcgcctccaatcggtatacaggcgtgcctgacagattcacc

[0561] ggctctggcagcgccacagacttcaccctgacaatctctaacgtgcaggccgaggacctggccgattatc

[0562] actgcggccagagctacaatttcccttatacctttggcggcggcacaaagctggagatcaag< / INSDSe

[0563] q_sequence>

[0564] < / insdseq>

[0565] < / sequencedata>

[0566] <sequencedata sequenceidnumber="4">

[0567] <insdseq>

[0568] <INSDSeq_length>107< / INSDSeq_length>

[0569] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[0570] <INSDSeq_division>PAT< / INSDSeq_division>

[0571] <INSDSeq_feature-table>

[0572] <insdfeature>

[0573] <INSDFeature_key>source< / INSDFeature_key>

[0574] <INSDFeature_location>1..107< / INSDFeature_location>

[0575] <INSDFeature_quals>

[0576] <insdqualifier>

[0577] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0578] <INSDQualifier_value>protein< / INSDQualifier_value>

[0579] < / insdqualifier>

[0580] <insdqualifier id="q136">

[0581] <INSDQualifier_name> organism< / INSDQualifier_name>

[0582] <INSDQualifier_value> synthetic construct< / INSDQualifier_value>

[0583] <NonEnglishQualifier_value> synthetic

[0584] design< / NonEnglishQualifier_value>

[0585] < / insdqualifier>

[0586] < / INSDFeature_quals>

[0587] < / insdfeature>

[0588] < / INSDSeq_feature-table>

[0589] <INSDSeq_sequence>NIVMTQSPKSMSMSLGERVTLSCKASEIVGTYVSWFQQKPHQSPKLLIY

[0590] GASNRYTGVPDRFTGSGSATDFTLTISNVQAEDLADYHCGQSYNFPYTFGGGTKLEIK< / INSDSeq_se

[0591] quence>

[0592] < / insdseq>

[0593] < / sequencedata>

[0594] <sequencedata sequenceidnumber="5">

[0595] <insdseq>

[0596] <INSDSeq_length> 8< / INSDSeq_length>

[0597] <INSDSeq_moltype> AA< / INSDSeq_moltype>

[0598] <INSDSeq_division> PAT< / INSDSeq_division>

[0599] <INSDSeq_feature-table>

[0600] <insdfeature>

[0601] <INSDFeature_key>source< / INSDFeature_key>

[0602] <INSDFeature_location>1..8< / INSDFeature_location>

[0603] <INSDFeature_quals>

[0604] <insdqualifier>

[0605] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0606] <INSDQualifier_value>protein< / INSDQualifier_value>

[0607] < / insdqualifier>

[0608] <insdqualifier id="q137">

[0609] <INSDQualifier_name> organism< / INSDQualifier_name>

[0610] <INSDQualifier_value> synthetic construct< / INSDQualifier_value>

[0611] <NonEnglishQualifier_value> synthetic

[0612] design< / NonEnglishQualifier_value>

[0613] < / insdqualifier>

[0614] < / INSDFeature_quals>

[0615] < / insdfeature>

[0616] < / INSDSeq_feature-table>

[0617] <INSDSeq_sequence> GYTFTSYW< / INSDSeq_sequence>

[0618] < / insdseq>

[0619] < / sequencedata>

[0620] <sequencedata sequenceidnumber="6">

[0621] <insdseq>

[0622] <INSDSeq_length>8< / INSDSeq_length>

[0623] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[0624] <INSDSeq_division>PAT< / INSDSeq_division>

[0625] <INSDSeq_feature-table>

[0626] <insdfeature>

[0627] <INSDFeature_key>source< / INSDFeature_key>

[0628] <INSDFeature_location>1..8< / INSDFeature_location>

[0629] <INSDFeature_quals>

[0630] <insdqualifier>

[0631] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0632] <INSDQualifier_value>protein< / INSDQualifier_value>

[0633] < / insdqualifier>

[0634] <insdqualifier id="q138">

[0635] <INSDQualifier_name> organism< / INSDQualifier_name>

[0636] <INSDQualifier_value> synthetic construct< / INSDQualifier_value>

[0637] <NonEnglishQualifier_value> synthetic

[0638] design< / NonEnglishQualifier_value>

[0639] < / insdqualifier>

[0640] < / INSDFeature_quals>

[0641] < / insdfeature>

[0642] < / INSDSeq_feature-table>

[0643] <INSDSeq_sequence>IDPSDSET< / INSDSeq_sequence>

[0644] < / insdseq>

[0645] < / sequencedata>

[0646] <sequencedata sequenceidnumber="7">

[0647] <insdseq>

[0648] <INSDSeq_length> 10< / INSDSeq_length>

[0649] <INSDSeq_moltype> AA< / INSDSeq_moltype>

[0650] <INSDSeq_division> PAT< / INSDSeq_division>

[0651] <INSDSeq_feature-table>

[0652] <insdfeature>

[0653] <INSDFeature_key>source< / INSDFeature_key>

[0654] <INSDFeature_location>1..10< / INSDFeature_location>

[0655] <INSDFeature_quals>

[0656] <insdqualifier>

[0657] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0658] <INSDQualifier_value>protein< / INSDQualifier_value>

[0659] < / insdqualifier>

[0660] <insdqualifier id="q139">

[0661] <INSDQualifier_name> organism< / INSDQualifier_name>

[0662] <INSDQualifier_value> synthetic construct< / INSDQualifier_value>

[0663] <NonEnglishQualifier_value> synthetic

[0664] design< / NonEnglishQualifier_value>

[0665] < / insdqualifier>

[0666] < / INSDFeature_quals>

[0667] < / insdfeature>

[0668] < / INSDSeq_feature-table>

[0669] <INSDSeq_sequence> LIBRARY OF DV< / INSDSeq_sequence>

[0670] < / insdseq>

[0671] < / sequencedata>

[0672] <sequencedata sequenceidnumber="8">

[0673] <insdseq>

[0674] <INSDSeq_length> 6< / INSDSeq_length>

[0675] <INSDSeq_moltype> AA< / INSDSeq_moltype>

[0676] <INSDSeq_division> PAT< / INSDSeq_division>

[0677] <INSDSeq_feature-table>

[0678] <insdfeature>

[0679] <INSDFeature_key>source< / INSDFeature_key>

[0680] <INSDFeature_location>1..6< / INSDFeature_location>

[0681] <INSDFeature_quals>

[0682] <insdqualifier>

[0683] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0684] <INSDQualifier_value>protein< / INSDQualifier_value>

[0685] < / insdqualifier>

[0686] <insdqualifier id="q140">

[0687] <INSDQualifier_name> organism< / INSDQualifier_name>

[0688] <INSDQualifier_value> synthetic construct< / INSDQualifier_value>

[0689] <NonEnglishQualifier_value> synthetic

[0690] design< / NonEnglishQualifier_value>

[0691] < / insdqualifier>

[0692] < / INSDFeature_quals>

[0693] < / insdfeature>

[0694] < / INSDSeq_feature-table>

[0695] <INSDSeq_sequence> EIVGTY< / INSDSeq_sequence>

[0696] < / insdseq>

[0697] < / sequencedata>

[0698] <sequencedata sequenceidnumber="9">

[0699] <insdseq>

[0700] <INSDSeq_length / >

[0701] <INSDSeq_moltype / >

[0702] <INSDSeq_division / >

[0703] <INSDSeq_sequence>000< / INSDSeq_sequence>

[0704] < / insdseq>

[0705] < / sequencedata>

[0706] <sequencedata sequenceidnumber="10">

[0707] <insdseq>

[0708] <INSDSeq_length> 9< / INSDSeq_length>

[0709] <INSDSeq_moltype> AA< / INSDSeq_moltype>

[0710] <INSDSeq_division> PAT< / INSDSeq_division>

[0711] <INSDSeq_feature-table>

[0712] <insdfeature>

[0713] <INSDFeature_key>source< / INSDFeature_key>

[0714] <INSDFeature_location>1..9< / INSDFeature_location>

[0715] <INSDFeature_quals>

[0716] <insdqualifier>

[0717] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0718] <INSDQualifier_value>protein< / INSDQualifier_value>

[0719] < / insdqualifier>

[0720] <insdqualifier id="q142">

[0721] <INSDQualifier_name> organism< / INSDQualifier_name>

[0722] <INSDQualifier_value> synthetic construct< / INSDQualifier_value>

[0723] <NonEnglishQualifier_value> synthetic

[0724] design< / NonEnglishQualifier_value>

[0725] < / insdqualifier>

[0726] < / INSDFeature_quals>

[0727] < / insdfeature>

[0728] < / INSDSeq_feature-table>

[0729] <INSDSeq_sequence> GQSYNFPYT< / INSDSeq_sequence>

[0730] < / insdseq>

[0731] < / sequencedata>

[0732] <sequencedata sequenceidnumber="11">

[0733] <insdseq>

[0734] <INSDSeq_length>351< / INSDSeq_length>

[0735] <INSDSeq_moltype>DNA< / INSDSeq_moltype>

[0736] <INSDSeq_division>PAT< / INSDSeq_division>

[0737] <INSDSeq_feature-table>

[0738] <insdfeature>

[0739] <INSDFeature_key>source< / INSDFeature_key>

[0740] <INSDFeature_location>1..351< / INSDFeature_location>

[0741] <INSDFeature_quals>

[0742] <insdqualifier>

[0743] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0744] <INSDQualifier_value>other DNA< / INSDQualifier_value>

[0745] < / insdqualifier>

[0746] <insdqualifier id="q143">

[0747] <INSDQualifier_name> organism< / INSDQualifier_name>

[0748] <INSDQualifier_value> synthetic construct< / INSDQualifier_value>

[0749] <NonEnglishQualifier_value> synthetic

[0750] design< / NonEnglishQualifier_value>

[0751] < / insdqualifier>

[0752] < / INSDFeature_quals>

[0753] < / insdfeature>

[0754] < / INSDSeq_feature-table>

[0755] <INSDSeq_sequence>caggtgcagctggtgcagagcggagcagaggtggtgaagccaggagcct

[0756] ctgtgaagctgagctgtaaggcctccggctacaccttcacaagctattggatgaactgggtgcggcagag

[0757] accaggacagggactggagtggatcggaatgatcgacccttccgattctgagacccacaatgcccagaag

[0758] tttcagggcaaggccaccctgacagtggacaagagcacctccacagcctacatgcacctgagctccctgc

[0759] ggtccgaggacacagccgtgtactattgcgccaggctgtaccgctggtattttgacgtgtggggagcagg

[0760] aaccacagtgaccgtgtctagc< / INSDSeq_sequence>

[0761] < / insdseq>

[0762] < / sequencedata>

[0763] <sequencedata sequenceidnumber="12">

[0764] <insdseq>

[0765] <INSDSeq_length>117< / INSDSeq_length>

[0766] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[0767] <INSDSeq_division>PAT< / INSDSeq_division>

[0768] <INSDSeq_feature-table>

[0769] <insdfeature>

[0770] <INSDFeature_key>source< / INSDFeature_key>

[0771] <INSDFeature_location>1..117< / INSDFeature_location>

[0772] <INSDFeature_quals>

[0773] <insdqualifier>

[0774] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0775] <INSDQualifier_value>protein< / INSDQualifier_value>

[0776] < / insdqualifier>

[0777] <insdqualifier id="q144">

[0778] <INSDQualifier_name> organism< / INSDQualifier_name>

[0779] <INSDQualifier_value> synthetic construct< / INSDQualifier_value>

[0780] <NonEnglishQualifier_value> synthetic

[0781] design< / NonEnglishQualifier_value>

[0782] < / insdqualifier>

[0783] < / INSDFeature_quals>

[0784] < / insdfeature>

[0785] < / INSDSeq_feature-table>

[0786] <INSDSeq_sequence>QVQLVQSGAEVVKPGASVKLSCKASGYTFTSYWMNWVRQRPGQGLEWIG

[0787] MIDPSDSETHNAQKFQGKATLTVDKSTSTAYMHLSSLRSEDTAVYYCARLYRWYFDVWGAGTTVTVSS< /

[0788] INSDSeq_sequence>

[0789] < / insdseq>

[0790] < / sequencedata>

[0791] <sequencedata sequenceidnumber="13">

[0792] <insdseq>

[0793] <INSDSeq_length>321< / INSDSeq_length>

[0794] <INSDSeq_moltype>DNA< / INSDSeq_moltype>

[0795] <INSDSeq_division>PAT< / INSDSeq_division>

[0796] <INSDSeq_feature-table>

[0797] <insdfeature>

[0798] <INSDFeature_key>source< / INSDFeature_key>

[0799] <INSDFeature_location>1..321< / INSDFeature_location>

[0800] <INSDFeature_quals>

[0801] <insdqualifier>

[0802] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0803] <INSDQualifier_value>other DNA< / INSDQualifier_value>

[0804] < / insdqualifier>

[0805] <insdqualifier id="q145">

[0806] <INSDQualifier_name> organism< / INSDQualifier_name>

[0807] <INSDQualifier_value> synthetic construct< / INSDQualifier_value>

[0808] <NonEnglishQualifier_value> synthetic

[0809] design< / NonEnglishQualifier_value>

[0810] < / insdqualifier>

[0811] < / INSDFeature_quals>

[0812] < / insdfeature>

[0813] < / INSDSeq_feature-table>

[0814] <INSDSeq_sequence>aacatcgtgatgacccagtccccagccacaatgtctatgagcccaggag

[0815] agagggtgaccctgtcctgtagagcctctgagatcgtgggcacatacgtgtcttggtttcagcagaagcc

[0816] aggacaggcacctaggctgctgatctacggagcaagcaacaggtataccggagtgccagcacgcttctcc

[0817] ggctctggcagcggcacagactttaccctgacaatcagctccgtgcagcctgaggacctggccgattatc

[0818] actgcggccagtcttacaatttcccatatacctttggcggcggcacaaagctggagatcaag< / INSDSe

[0819] q_sequence>

[0820] < / insdseq>

[0821] < / sequencedata>

[0822] <sequencedata sequenceidnumber="14">

[0823] <insdseq>

[0824] <INSDSeq_length>107< / INSDSeq_length>

[0825] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[0826] <INSDSeq_division>PAT< / INSDSeq_division>

[0827] <INSDSeq_feature-table>

[0828] <insdfeature>

[0829] <INSDFeature_key>source< / INSDFeature_key>

[0830] <INSDFeature_location>1..107< / INSDFeature_location>

[0831] <INSDFeature_quals>

[0832] <insdqualifier>

[0833] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0834] <INSDQualifier_value>protein< / INSDQualifier_value>

[0835] < / insdqualifier>

[0836] <insdqualifier id="q146">

[0837] <INSDQualifier_name> organism< / INSDQualifier_name>

[0838] <INSDQualifier_value> synthetic construct< / INSDQualifier_value>

[0839] <NonEnglishQualifier_value> synthetic

[0840] design< / NonEnglishQualifier_value>

[0841] < / insdqualifier>

[0842] < / INSDFeature_quals>

[0843] < / insdfeature>

[0844] < / INSDSeq_feature-table>

[0845] <INSDSeq_sequence>NIVMTQSPATMSMSPGERVTLSCRASEIVGTYVSWFQQKPGQAPRLLIY

[0846] GASNRYTGVPARFSGSGSGTDFTLTISSVQPEDLADYHCGQSYNFPYTFGGGTKLEIK< / INSDSeq_se

[0847] quence>

[0848] < / insdseq>

[0849] < / sequencedata>

[0850] <sequencedata sequenceidnumber="15">

[0851] <insdseq>

[0852] <INSDSeq_length>351< / INSDSeq_length>

[0853] <INSDSeq_moltype>DNA< / INSDSeq_moltype>

[0854] <INSDSeq_division>PAT< / INSDSeq_division>

[0855] <INSDSeq_feature-table>

[0856] <insdfeature>

[0857] <INSDFeature_key>source< / INSDFeature_key>

[0858] <INSDFeature_location>1..351< / INSDFeature_location>

[0859] <INSDFeature_quals>

[0860] <insdqualifier>

[0861] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0862] <INSDQualifier_value>other DNA< / INSDQualifier_value>

[0863] < / insdqualifier>

[0864] <insdqualifier id="q147">

[0865] <INSDQualifier_name> organism< / INSDQualifier_name>

[0866] <INSDQualifier_value> synthetic construct< / INSDQualifier_value>

[0867] <NonEnglishQualifier_value> synthetic

[0868] design< / NonEnglishQualifier_value>

[0869] < / insdqualifier>

[0870] < / INSDFeature_quals>

[0871] < / insdfeature>

[0872] < / INSDSeq_feature-table>

[0873] <INSDSeq_sequence>caggtgcagctggtgcagagcggagcagaggtggtgaagccaggagcct

[0874] ctgtgaaggtgagctgtaaggcctccggctacaccttcacatcctattggatgaactgggtgcggcagag

[0875] accaggacagggactggagtggatcggaatcatcgacccttccgattctgagacctctaatgcccagaag

[0876] tttcagggccgggtgaccctgacagtggacaagagcacctccacagcctacatgcacctgagctccctga

[0877] ggagcgaggacacagccgtgtactattgcgccaggctgtaccgctggtattttgacgtgtggggagcagg

[0878] aaccacagtgaccgtgtctagc< / INSDSeq_sequence>

[0879] < / insdseq>

[0880] < / sequencedata>

[0881] <sequencedata sequenceidnumber="16">

[0882] <insdseq>

[0883] <INSDSeq_length>117< / INSDSeq_length>

[0884] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[0885] <INSDSeq_division>PAT< / INSDSeq_division>

[0886] <INSDSeq_feature-table>

[0887] <insdfeature>

[0888] <INSDFeature_key>source< / INSDFeature_key>

[0889] <INSDFeature_location>1..117< / INSDFeature_location>

[0890] <INSDFeature_quals>

[0891] <insdqualifier>

[0892] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0893] <INSDQualifier_value>protein< / INSDQualifier_value>

[0894] < / insdqualifier>

[0895] <insdqualifier id="q148">

[0896] <INSDQualifier_name> organism< / INSDQualifier_name>

[0897] <INSDQualifier_value> synthetic construct< / INSDQualifier_value>

[0898] <NonEnglishQualifier_value> synthetic

[0899] design< / NonEnglishQualifier_value>

[0900] < / insdqualifier>

[0901] < / INSDFeature_quals>

[0902] < / insdfeature>

[0903] < / INSDSeq_feature-table>

[0904] <INSDSeq_sequence>QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYWMNWVRQRPGQGLEWIG

[0905] IIDPSDSETSNAQKFQGRVTLTVDKSTSTAYMHLSSLRSEDTAVYYCARLYRWYFDVWGAGTTVTVSS< /

[0906] INSDSeq_sequence>

[0907] < / insdseq>

[0908] < / sequencedata>

[0909] <sequencedata sequenceidnumber="17">

[0910] <insdseq>

[0911] <INSDSeq_length>321< / INSDSeq_length>

[0912] <INSDSeq_moltype>DNA< / INSDSeq_moltype>

[0913] <INSDSeq_division>PAT< / INSDSeq_division>

[0914] <INSDSeq_feature-table>

[0915] <insdfeature>

[0916] <INSDFeature_key>source< / INSDFeature_key>

[0917] <INSDFeature_location>1..321< / INSDFeature_location>

[0918] <INSDFeature_quals>

[0919] <insdqualifier>

[0920] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0921] <INSDQualifier_value>other DNA< / INSDQualifier_value>

[0922] < / insdqualifier>

[0923] <insdqualifier id="q149">

[0924] <INSDQualifier_name> organism< / INSDQualifier_name>

[0925] <INSDQualifier_value> synthetic construct< / INSDQualifier_value>

[0926] <NonEnglishQualifier_value> synthetic

[0927] design< / NonEnglishQualifier_value>

[0928] < / insdqualifier>

[0929] < / INSDFeature_quals>

[0930] < / insdfeature>

[0931] < / INSDSeq_feature-table>

[0932] <INSDSeq_sequence>aacatcgtgatgacccagtccccagccacactgtctctgagcccaggag

[0933] agagggtgaccctgtcctgtagagcctctgagatcgtgggcacatacgtgtcttggtttcagcagaagcc

[0934] aggacaggcacctaggctgctgatctatggcgccagcaacagggcaaccggcatccccgcacgcttctcc

[0935] ggctctggcagcggcacagactttaccctgacaatcagctccctgcagcctgaggacctggccgattact

[0936] attgcggccagtcttacaatttcccatatacctttggcggcggcacaaagctggagatcaag< / INSDSe

[0937] q_sequence>

[0938] < / insdseq>

[0939] < / sequencedata>

[0940] <sequencedata sequenceidnumber="18">

[0941] <insdseq>

[0942] <INSDSeq_length>107< / INSDSeq_length>

[0943] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[0944] <INSDSeq_division>PAT< / INSDSeq_division>

[0945] <INSDSeq_feature-table>

[0946] <insdfeature>

[0947] <INSDFeature_key>source< / INSDFeature_key>

[0948] <INSDFeature_location>1..107< / INSDFeature_location>

[0949] <INSDFeature_quals>

[0950] <insdqualifier>

[0951] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0952] <INSDQualifier_value>protein< / INSDQualifier_value>

[0953] < / insdqualifier>

[0954] <insdqualifier id="q150">

[0955] <INSDQualifier_name> organism< / INSDQualifier_name>

[0956] <INSDQualifier_value> synthetic construct< / INSDQualifier_value>

[0957] <NonEnglishQualifier_value> synthetic

[0958] design< / NonEnglishQualifier_value>

[0959] < / insdqualifier>

[0960] < / INSDFeature_quals>

[0961] < / insdfeature>

[0962] < / INSDSeq_feature-table>

[0963] <INSDSeq_sequence>NIVMTQSPATLSLSPGERVTLSCRASEIVGTYVSWFQQKPGQAPRLLIY

[0964] GASNRATGIPARFSGSGSGTDFTLTISSLQPEDLADYYCGQSYNFPYTFGGGTKLEIK< / INSDSeq_se

[0965] quence>

[0966] < / insdseq>

[0967] < / sequencedata>

[0968] <sequencedata sequenceidnumber="19">

[0969] <insdseq>

[0970] <INSDSeq_length>351< / INSDSeq_length>

[0971] <INSDSeq_moltype>DNA< / INSDSeq_moltype>

[0972] <INSDSeq_division>PAT< / INSDSeq_division>

[0973] <INSDSeq_feature-table>

[0974] <insdfeature>

[0975] <INSDFeature_key>source< / INSDFeature_key>

[0976] <INSDFeature_location>1..351< / INSDFeature_location>

[0977] <INSDFeature_quals>

[0978] <insdqualifier>

[0979] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[0980] <INSDQualifier_value>other DNA< / INSDQualifier_value>

[0981] < / insdqualifier>

[0982] <insdqualifier id="q151">

[0983] <INSDQualifier_name> organism< / INSDQualifier_name>

[0984] <INSDQualifier_value> synthetic construct< / INSDQualifier_value>

[0985] <NonEnglishQualifier_value> synthetic

[0986] design< / NonEnglishQualifier_value>

[0987] < / insdqualifier>

[0988] < / INSDFeature_quals>

[0989] < / insdfeature>

[0990] < / INSDSeq_feature-table>

[0991] <INSDSeq_sequence>caggtgcagctggtgcagagcggagcagaggtggtgaagccaggagcct

[0992] ctgtgaaggtgagctgtaaggcctccggctacaccttcacatcctattggatgaactgggtgcggcaggc

[0993] accaggacagggactggagtggatcggcatcatcgacccttccgattctgagacctcttacgcccagaag

[0994] tttcagggcagggtgaccctgacagtggacaagagcacctccacagcctatatggagctgagctccctgc

[0995] gcagcgaggacacagccgtgtactattgcgcccggctgtacagatggtattttgacgtgtggggagcagg

[0996] aaccacagtgaccgtgtctagc< / INSDSeq_sequence>

[0997] < / insdseq>

[0998] < / sequencedata>

[0999] <sequencedata sequenceidnumber="20">

[1000] <insdseq>

[1001] <INSDSeq_length>117< / INSDSeq_length>

[1002] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[1003] <INSDSeq_division>PAT< / INSDSeq_division>

[1004] <INSDSeq_feature-table>

[1005] <insdfeature>

[1006] <INSDFeature_key>source< / INSDFeature_key>

[1007] <INSDFeature_location>1..117< / INSDFeature_location>

[1008] <INSDFeature_quals>

[1009] <insdqualifier>

[1010] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[1011] <INSDQualifier_value>protein< / INSDQualifier_value>

[1012] < / insdqualifier>

[1013] <insdqualifier id="q152">

[1014] <INSDQualifier_name> organism< / INSDQualifier_name>

[1015] <INSDQualifier_value> synthetic construct< / INSDQualifier_value>

[1016] <NonEnglishQualifier_value> synthetic

[1017] design< / NonEnglishQualifier_value>

[1018] < / insdqualifier>

[1019] < / INSDFeature_quals>

[1020] < / insdfeature>

[1021] < / INSDSeq_feature-table>

[1022] <INSDSeq_sequence>QVQLVQSGAEVVKPGASVKVSCKASGYTFTSYWMNWVRQAPGQGLEWIG

[1023] IIDPSDSETSYAQKFQGRVTLTVDKSTSTAYMELSSLRSEDTAVYYCARLYRWYFDVWGAGTTVTVSS< /

[1024] INSDSeq_sequence>

[1025] < / insdseq>

[1026] < / sequencedata>

[1027] <sequencedata sequenceidnumber="21">

[1028] <insdseq>

[1029] <INSDSeq_length>321< / INSDSeq_length>

[1030] <INSDSeq_moltype>DNA< / INSDSeq_moltype>

[1031] <INSDSeq_division>PAT< / INSDSeq_division>

[1032] <INSDSeq_feature-table>

[1033] <insdfeature>

[1034] <INSDFeature_key>source< / INSDFeature_key>

[1035] <INSDFeature_location>1..321< / INSDFeature_location>

[1036] <INSDFeature_quals>

[1037] <insdqualifier>

[1038] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[1039] <INSDQualifier_value>other DNA< / INSDQualifier_value>

[1040] < / insdqualifier>

[1041] <insdqualifier id="q153">

[1042] <INSDQualifier_name> organism< / INSDQualifier_name>

[1043] <INSDQualifier_value> synthetic construct< / INSDQualifier_value>

[1044] <NonEnglishQualifier_value> synthetic

[1045] design< / NonEnglishQualifier_value>

[1046] < / insdqualifier>

[1047] < / INSDFeature_quals>

[1048] < / insdfeature>

[1049] < / INSDSeq_feature-table>

[1050] <INSDSeq_sequence>aacatcgtgatgacccagtccccagccacactgtctctgagcccaggag

[1051] agagggtgaccctgtcctgtagagcctctgagatcgtgggcacatacctgtcttggtatcagcagaagcc

[1052] aggacaggcacctaggctgctgatctacggagccagcaccagggcaacaggcatccccgcacgcttctcc

[1053] ggctctggcagcggcaccgactttaccctgacaatcagctccctgcagcctgaggattttgccgtgtact

[1054] attgcggccagtcttacaatttcccatatacctttggcggcggcacaaagctggagatcaag< / INSDSe

[1055] q_sequence>

[1056] < / insdseq>

[1057] < / sequencedata>

[1058] <sequencedata sequenceidnumber="22">

[1059] <insdseq>

[1060] <INSDSeq_length>107< / INSDSeq_length>

[1061] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[1062] <INSDSeq_division>PAT< / INSDSeq_division>

[1063] <INSDSeq_feature-table>

[1064] <insdfeature>

[1065] <INSDFeature_key>source< / INSDFeature_key>

[1066] <INSDFeature_location>1..107< / INSDFeature_location>

[1067] <INSDFeature_quals>

[1068] <insdqualifier>

[1069] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[1070] <INSDQualifier_value>protein< / INSDQualifier_value>

[1071] < / insdqualifier>

[1072] <insdqualifier id="q154">

[1073] <INSDQualifier_name> organism< / INSDQualifier_name>

[1074] <INSDQualifier_value> synthetic construct< / INSDQualifier_value>

[1075] <NonEnglishQualifier_value> synthetic

[1076] design< / NonEnglishQualifier_value>

[1077] < / insdqualifier>

[1078] < / INSDFeature_quals>

[1079] < / insdfeature>

[1080] < / INSDSeq_feature-table>

[1081] <INSDSeq_sequence>NIVMTQSPATLSLSPGERVTLSCRASEIVGTYLSWYQQKPGQAPRLLIY

[1082] GASTRATGIPARFSGSGSGTDFTLTISSLQPEDFAVYYCGQSYNFPYTFGGGTKLEIK< / INSDSeq_se

[1083] quence>

[1084] < / insdseq>

[1085] < / sequencedata>

[1086] <sequencedata sequenceidnumber="23">

[1087] <insdseq>

[1088] <INSDSeq_length>351< / INSDSeq_length>

[1089] <INSDSeq_moltype>DNA< / INSDSeq_moltype>

[1090] <INSDSeq_division>PAT< / INSDSeq_division>

[1091] <INSDSeq_feature-table>

[1092] <insdfeature>

[1093] <INSDFeature_key>source< / INSDFeature_key>

[1094] <INSDFeature_location>1..351< / INSDFeature_location>

[1095] <INSDFeature_quals>

[1096] <insdqualifier>

[1097] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[1098] <INSDQualifier_value>other DNA< / INSDQualifier_value>

[1099] < / insdqualifier>

[1100] <insdqualifier id="q155">

[1101] <INSDQualifier_name> organism< / INSDQualifier_name>

[1102] <INSDQualifier_value> synthetic construct< / INSDQualifier_value>

[1103] <NonEnglishQualifier_value> synthetic

[1104] design< / NonEnglishQualifier_value>

[1105] < / insdqualifier>

[1106] < / INSDFeature_quals>

[1107] < / insdfeature>

[1108] < / INSDSeq_feature-table>

[1109] <INSDSeq_sequence>caggtgcagctggtgcagagcggagccgaagtggtgaagcctggcgcct

[1110] ctgtgaagctgtcctgtaaggcctctggctacacctttacatcctactggatgaactgggtgcggcagag

[1111] accaggccagtgcctggagtggatcggcatgatcgacccatctgattccgagacccacaatgcccagaag

[1112] tttcagggcaaggccacactgaccgtggacaagagcacctccacagcctatatgcacctgtcctctctga

[1113] gaagcgaggatacagccgtgtattactgtgccagactgtatcggtggtactttgacgtgtggggcgccgg

[1114] caccacagtgaccgtgtctagc< / INSDSeq_sequence>

[1115] < / insdseq>

[1116] < / sequencedata>

[1117] <sequencedata sequenceidnumber="24">

[1118] <insdseq>

[1119] <INSDSeq_length>117< / INSDSeq_length>

[1120] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[1121] <INSDSeq_division>PAT< / INSDSeq_division>

[1122] <INSDSeq_feature-table>

[1123] <insdfeature>

[1124] <INSDFeature_key>source< / INSDFeature_key>

[1125] <INSDFeature_location>1..117< / INSDFeature_location>

[1126] <INSDFeature_quals>

[1127] <insdqualifier>

[1128] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[1129] <INSDQualifier_value>protein< / INSDQualifier_value>

[1130] < / insdqualifier>

[1131] <insdqualifier id="q156">

[1132] <INSDQualifier_name> organism< / INSDQualifier_name>

[1133] <INSDQualifier_value> synthetic construct< / INSDQualifier_value>

[1134] <NonEnglishQualifier_value> synthetic

[1135] design< / NonEnglishQualifier_value>

[1136] < / insdqualifier>

[1137] < / INSDFeature_quals>

[1138] < / insdfeature>

[1139] < / INSDSeq_feature-table>

[1140] <INSDSeq_sequence>QVQLVQSGAEVVKPGASVKLSCKASGYTFTSYWMNWVRQRPGQCLEWIG

[1141] MIDPSDSETHNAQKFQGKATLTVDKSTSTAYMHLSSLRSEDTAVYYCARLYRWYFDVWGAGTTVTVSS< /

[1142] INSDSeq_sequence>

[1143] < / insdseq>

[1144] < / sequencedata>

[1145] <sequencedata sequenceidnumber="25">

[1146] <insdseq>

[1147] <INSDSeq_length>321< / INSDSeq_length>

[1148] <INSDSeq_moltype>DNA< / INSDSeq_moltype>

[1149] <INSDSeq_division>PAT< / INSDSeq_division>

[1150] <INSDSeq_feature-table>

[1151] <insdfeature>

[1152] <INSDFeature_key>source< / INSDFeature_key>

[1153] <INSDFeature_location>1..321< / INSDFeature_location>

[1154] <INSDFeature_quals>

[1155] <insdqualifier>

[1156] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[1157] <INSDQualifier_value>other DNA< / INSDQualifier_value>

[1158] < / insdqualifier>

[1159] <insdqualifier id="q157">

[1160] <INSDQualifier_name> organism< / INSDQualifier_name>

[1161] <INSDQualifier_value> synthetic construct< / INSDQualifier_value>

[1162] <NonEnglishQualifier_value> synthetic

[1163] design< / NonEnglishQualifier_value>

[1164] < / insdqualifier>

[1165] < / INSDFeature_quals>

[1166] < / insdfeature>

[1167] < / INSDSeq_feature-table>

[1168] <INSDSeq_sequence>aatatcgtgatgacccagtctcctgccacaatgtctatgagccctggcg

[1169] agagagtgaccctgtcttgtagggccagcgagatcgtgggcacctacgtgagctggttccagcagaagcc

[1170] aggccaggccccaagactgctgatctatggcgccagcaaccggtatacaggcgtgcctgccagattttct

[1171] ggctccggctctggcaccgatttcacactgaccatctctagcgtgcagcccgaggatctggccgattacc

[1172] actgtggccagtcctacaacttcccctatacattcgggtgcggcactaaactcgagatcaaa< / INSDSe

[1173] q_sequence>

[1174] < / insdseq>

[1175] < / sequencedata>

[1176] <sequencedata sequenceidnumber="26">

[1177] <insdseq>

[1178] <INSDSeq_length>107< / INSDSeq_length>

[1179] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[1180] <INSDSeq_division>PAT< / INSDSeq_division>

[1181] <INSDSeq_feature-table>

[1182] <insdfeature>

[1183] <INSDFeature_key>source< / INSDFeature_key>

[1184] <INSDFeature_location>1..107< / INSDFeature_location>

[1185] <INSDFeature_quals>

[1186] <insdqualifier>

[1187] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[1188] <INSDQualifier_value>protein< / INSDQualifier_value>

[1189] < / insdqualifier>

[1190] <insdqualifier id="q158">

[1191] <INSDQualifier_name> organism< / INSDQualifier_name>

[1192] <INSDQualifier_value> synthetic construct< / INSDQualifier_value>

[1193] <NonEnglishQualifier_value> synthetic

[1194] design< / NonEnglishQualifier_value>

[1195] < / insdqualifier>

[1196] < / INSDFeature_quals>

[1197] < / insdfeature>

[1198] < / INSDSeq_feature-table>

[1199] <INSDSeq_sequence>NIVMTQSPATMSMSPGERVTLSCRASEIVGTYVSWFQQKPGQAPRLLIY

[1200] GASNRYTGVPARFSGSGSGTDFTLTISSVQPEDLADYHCGQSYNFPYTFGCGTKLEIK< / INSDSeq_se

[1201] quence>

[1202] < / insdseq>

[1203] < / sequencedata>

[1204] <sequencedata sequenceidnumber="27">

[1205] <insdseq>

[1206] <INSDSeq_length>354< / INSDSeq_length>

[1207] <INSDSeq_moltype>DNA< / INSDSeq_moltype>

[1208] <INSDSeq_division>PAT< / INSDSeq_division>

[1209] <INSDSeq_feature-table>

[1210] <insdfeature>

[1211] <INSDFeature_key>source< / INSDFeature_key>

[1212] <INSDFeature_location>1..354< / INSDFeature_location>

[1213] <INSDFeature_quals>

[1214] <insdqualifier>

[1215] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[1216] <INSDQualifier_value>other DNA< / INSDQualifier_value>

[1217] < / insdqualifier>

[1218] <insdqualifier id="q159">

[1219] <INSDQualifier_name> organism< / INSDQualifier_name>

[1220] <INSDQualifier_value> synthetic construct< / INSDQualifier_value>

[1221] <NonEnglishQualifier_value> synthetic

[1222] design< / NonEnglishQualifier_value>

[1223] < / insdqualifier>

[1224] < / INSDFeature_quals>

[1225] < / insdfeature>

[1226] < / INSDSeq_feature-table>

[1227] <INSDSeq_sequence>gaagtgcagctggttgagagcggcggaggactggtgaaacctggaggat

[1228] ctctgaagctgagctgcgctgcttccggattcacttttagcaactccgccgtgagctgggtgagacaaac

[1229] acctgagaagaggctggagtgggtggctacaatcacaagcggcgtgagctacacctactacctggactcc

[1230] gtgaggggcagattcacaatcagcagggacaacgccaagaacaccctgtacctgcagatgagcagcctga

[1231] ggagcgaggataccgctatgtactactgtaccaggcagttcaagggcaacgccctggattactggggcca

[1232] aggaacctctgtgacagtgagctcc< / INSDSeq_sequence>

[1233] < / insdseq>

[1234] < / sequencedata>

[1235] <sequencedata sequenceidnumber="28">

[1236] <insdseq>

[1237] <INSDSeq_length>118< / INSDSeq_length>

[1238] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[1239] <INSDSeq_division>PAT< / INSDSeq_division>

[1240] <INSDSeq_feature-table>

[1241] <insdfeature>

[1242] <INSDFeature_key>source< / INSDFeature_key>

[1243] <INSDFeature_location>1..118< / INSDFeature_location>

[1244] <INSDFeature_quals>

[1245] <insdqualifier>

[1246] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[1247] <INSDQualifier_value>protein< / INSDQualifier_value>

[1248] < / insdqualifier>

[1249] <insdqualifier id="q160">

[1250] <INSDQualifier_name> organism< / INSDQualifier_name>

[1251] <INSDQualifier_value> synthetic construct< / INSDQualifier_value>

[1252] <NonEnglishQualifier_value> synthetic

[1253] design< / NonEnglishQualifier_value>

[1254] < / insdqualifier>

[1255] < / INSDFeature_quals>

[1256] < / insdfeature>

[1257] < / INSDSeq_feature-table>

[1258] <INSDSeq_sequence>EVQLVESGGGLVKPGGSLKLSCAASGFTFSNSAVSWVRQTPEKRLEWVA

[1259] TITSGVSYTYYLDSVRGRFTISRDNAKNTLYLQMSSLRSEDTAMYYCTRQFKGNALDYWGQGTSVTVSS<

[1260] / INSDSeq_sequence>

[1261] < / insdseq>

[1262] < / sequencedata>

[1263] <sequencedata sequenceidnumber="29">

[1264] <insdseq>

[1265] <INSDSeq_length>339< / INSDSeq_length>

[1266] <INSDSeq_moltype>DNA< / INSDSeq_moltype>

[1267] <INSDSeq_division>PAT< / INSDSeq_division>

[1268] <INSDSeq_feature-table>

[1269] <insdfeature>

[1270] <INSDFeature_key>source< / INSDFeature_key>

[1271] <INSDFeature_location>1..339< / INSDFeature_location>

[1272] <INSDFeature_quals>

[1273] <insdqualifier>

[1274] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[1275] <INSDQualifier_value>other DNA< / INSDQualifier_value>

[1276] < / insdqualifier>

[1277] <insdqualifier id="q161">

[1278] <INSDQualifier_name> organism< / INSDQualifier_name>

[1279] <INSDQualifier_value> synthetic construct< / INSDQualifier_value>

[1280] <NonEnglishQualifier_value> synthetic

[1281] design< / NonEnglishQualifier_value>

[1282] < / insdqualifier>

[1283] < / INSDFeature_quals>

[1284] < / insdfeature>

[1285] < / INSDSeq_feature-table>

[1286] <INSDSeq_sequence>gacattgtgatgacccagtccccttcctccctgacagtgacagctggag

[1287] agaaggtgacaatgtcctgcaagagcagccagagcctgctgaactccggaaatcagaagaactacctgac

[1288] ctggtaccagcagaagcccggacaaccacctaagctgctgatctactgggcctccaccagggaaagcgga

[1289] gttcctgatagattcaccggcagcggctccggaacagatttcacactgacaatcaacaacttccaggctg

[1290] aggacctggccgtgtactactgtcagaacgactacttctaccctctgaccttcggcgccggaacaaagct

[1291] ggaactgaag< / INSDSeq_sequence>

[1292] < / insdseq>

[1293] < / sequencedata>

[1294] <sequencedata sequenceidnumber="30">

[1295] <insdseq>

[1296] <INSDSeq_length>113< / INSDSeq_length>

[1297] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[1298] <INSDSeq_division>PAT< / INSDSeq_division>

[1299] <INSDSeq_feature-table>

[1300] <insdfeature>

[1301] <INSDFeature_key>source< / INSDFeature_key>

[1302] <INSDFeature_location>1..113< / INSDFeature_location>

[1303] <INSDFeature_quals>

[1304] <insdqualifier>

[1305] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[1306] <INSDQualifier_value>protein< / INSDQualifier_value>

[1307] < / insdqualifier>

[1308] <insdqualifier id="q162">

[1309] <INSDQualifier_name> organism< / INSDQualifier_name>

[1310] <INSDQualifier_value> synthetic construct< / INSDQualifier_value>

[1311] <NonEnglishQualifier_value> synthetic

[1312] design< / NonEnglishQualifier_value>

[1313] < / insdqualifier>

[1314] < / INSDFeature_quals>

[1315] < / insdfeature>

[1316] < / INSDSeq_feature-table>

[1317] <INSDSeq_sequence>DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQP

[1318] PKLLIYWASTRESGVPDRFTGSGSGTDFTLTINNFQAEDLAVYYCQNDYFYPLTFGAGTKLELK< / INSD

[1319] Seq_sequence>

[1320] < / insdseq>

[1321] < / sequencedata>

[1322] <sequencedata sequenceidnumber="31">

[1323] <insdseq>

[1324] <INSDSeq_length> 8< / INSDSeq_length>

[1325] <INSDSeq_moltype> AA< / INSDSeq_moltype>

[1326] <INSDSeq_division> PAT< / INSDSeq_division>

[1327] <INSDSeq_feature-table>

[1328] <insdfeature>

[1329] <INSDFeature_key>source< / INSDFeature_key>

[1330] <INSDFeature_location>1..8< / INSDFeature_location>

[1331] <INSDFeature_quals>

[1332] <insdqualifier>

[1333] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[1334] <INSDQualifier_value>protein< / INSDQualifier_value>

[1335] < / insdqualifier>

[1336] <insdqualifier id="q163">

[1337] <INSDQualifier_name> organism< / INSDQualifier_name>

[1338] <INSDQualifier_value> synthetic construct< / INSDQualifier_value>

[1339] <NonEnglishQualifier_value> synthetic

[1340] design< / NonEnglishQualifier_value>

[1341] < / insdqualifier>

[1342] < / INSDFeature_quals>

[1343] < / insdfeature>

[1344] < / INSDSeq_feature-table>

[1345] <INSDSeq_sequence> GFTFSNSA< / INSDSeq_sequence>

[1346] < / insdseq>

[1347] < / sequencedata>

[1348] <sequencedata sequenceidnumber="32">

[1349] <insdseq>

[1350] <INSDSeq_length>8< / INSDSeq_length>

[1351] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[1352] <INSDSeq_division>PAT< / INSDSeq_division>

[1353] <INSDSeq_feature-table>

[1354] <insdfeature>

[1355] <INSDFeature_key>source< / INSDFeature_key>

[1356] <INSDFeature_location>1..8< / INSDFeature_location>

[1357] <INSDFeature_quals>

[1358] <insdqualifier>

[1359] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[1360] <INSDQualifier_value>protein< / INSDQualifier_value>

[1361] < / insdqualifier>

[1362] <insdqualifier id="q164">

[1363] <INSDQualifier_name> organism< / INSDQualifier_name>

[1364] <INSDQualifier_value> synthetic construct< / INSDQualifier_value>

[1365] <NonEnglishQualifier_value> synthetic

[1366] design< / NonEnglishQualifier_value>

[1367] < / insdqualifier>

[1368] < / INSDFeature_quals>

[1369] < / insdfeature>

[1370] < / INSDSeq_feature-table>

[1371] <INSDSeq_sequence>ITSGVSYT< / INSDSeq_sequence>

[1372] < / insdseq>

[1373] < / sequencedata>

[1374] <sequencedata sequenceidnumber="33">

[1375] <insdseq>

[1376] <INSDSeq_length> 11< / INSDSeq_length>

[1377] <INSDSeq_moltype> AA< / INSDSeq_moltype>

[1378] <INSDSeq_division> PAT< / INSDSeq_division>

[1379] <INSDSeq_feature-table>

[1380] <insdfeature>

[1381] <INSDFeature_key>source< / INSDFeature_key>

[1382] <INSDFeature_location>1..11< / INSDFeature_location>

[1383] <INSDFeature_quals>

[1384] <insdqualifier>

[1385] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[1386] <INSDQualifier_value>protein< / INSDQualifier_value>

[1387] < / insdqualifier>

[1388] <insdqualifier id="q165">

[1389] <INSDQualifier_name> organism< / INSDQualifier_name>

[1390] <INSDQualifier_value> synthetic construct< / INSDQualifier_value>

[1391] <NonEnglishQualifier_value> synthetic

[1392] design< / NonEnglishQualifier_value>

[1393] < / insdqualifier>

[1394] < / INSDFeature_quals>

[1395] < / insdfeature>

[1396] < / INSDSeq_feature-table>

[1397] <INSDSeq_sequence> TRQFKGNALDY< / INSDSeq_sequence>

[1398] < / insdseq>

[1399] < / sequencedata>

[1400] <sequencedata sequenceidnumber="34">

[1401] <insdseq>

[1402] <INSDSeq_length> 12< / INSDSeq_length>

[1403] <INSDSeq_moltype> AA< / INSDSeq_moltype>

[1404] <INSDSeq_division> PAT< / INSDSeq_division>

[1405] <INSDSeq_feature-table>

[1406] <insdfeature>

[1407] <INSDFeature_key>source< / INSDFeature_key>

[1408] <INSDFeature_location>1..12< / INSDFeature_location>

[1409] <INSDFeature_quals>

[1410] <insdqualifier>

[1411] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[1412] <INSDQualifier_value>protein< / INSDQualifier_value>

[1413] < / insdqualifier>

[1414] <insdqualifier id="q166">

[1415] <INSDQualifier_name> organism< / INSDQualifier_name>

[1416] <INSDQualifier_value> synthetic construct< / INSDQualifier_value>

[1417] <NonEnglishQualifier_value> synthetic

[1418] design< / NonEnglishQualifier_value>

[1419] < / insdqualifier>

[1420] < / INSDFeature_quals>

[1421] < / insdfeature>

[1422] < / INSDSeq_feature-table>

[1423] <INSDSeq_sequence> QSLLNSGNQKNY< / INSDSeq_sequence>

[1424] < / insdseq>

[1425] < / sequencedata>

[1426] <sequencedata sequenceidnumber="35">

[1427] <insdseq>

[1428] <INSDSeq_length / >

[1429] <INSDSeq_moltype / >

[1430] <INSDSeq_division / >

[1431] <INSDSeq_sequence>000< / INSDSeq_sequence>

[1432] < / insdseq>

[1433] < / sequencedata>

[1434] <sequencedata sequenceidnumber="36">

[1435] <insdseq>

[1436] <INSDSeq_length> 9< / INSDSeq_length>

[1437] <INSDSeq_moltype> AA< / INSDSeq_moltype>

[1438] <INSDSeq_division> PAT< / INSDSeq_division>

[1439] <INSDSeq_feature-table>

[1440] <insdfeature>

[1441] <INSDFeature_key>source< / INSDFeature_key>

[1442] <INSDFeature_location>1..9< / INSDFeature_location>

[1443] <INSDFeature_quals>

[1444] <insdqualifier>

[1445] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[1446] <INSDQualifier_value>protein< / INSDQualifier_value>

[1447] < / insdqualifier>

[1448] <insdqualifier id="q168">

[1449] <INSDQualifier_name> organism< / INSDQualifier_name>

[1450] <INSDQualifier_value> synthetic construct< / INSDQualifier_value>

[1451] <NonEnglishQualifier_value> synthetic

[1452] design< / NonEnglishQualifier_value>

[1453] < / insdqualifier>

[1454] < / INSDFeature_quals>

[1455] < / insdfeature>

[1456] < / INSDSeq_feature-table>

[1457] <INSDSeq_sequence> QNDYFYPLT< / INSDSeq_sequence>

[1458] < / insdseq>

[1459] < / sequencedata>

[1460] <sequencedata sequenceidnumber="37">

[1461] <insdseq>

[1462] <INSDSeq_length>118< / INSDSeq_length>

[1463] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[1464] <INSDSeq_division>PAT< / INSDSeq_division>

[1465] <INSDSeq_feature-table>

[1466] <insdfeature>

[1467] <INSDFeature_key>source< / INSDFeature_key>

[1468] <INSDFeature_location>1..118< / INSDFeature_location>

[1469] <INSDFeature_quals>

[1470] <insdqualifier>

[1471] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[1472] <INSDQualifier_value>protein< / INSDQualifier_value>

[1473] < / insdqualifier>

[1474] <insdqualifier id="q169">

[1475] <INSDQualifier_name> organism< / INSDQualifier_name>

[1476] <INSDQualifier_value> synthetic construct< / INSDQualifier_value>

[1477] <NonEnglishQualifier_value> synthetic

[1478] design< / NonEnglishQualifier_value>

[1479] < / insdqualifier>

[1480] < / INSDFeature_quals>

[1481] < / insdfeature>

[1482] < / INSDSeq_feature-table>

[1483] <INSDSeq_sequence>EVQLVESGGGLVKPGGSLRLSCAASGFTFSNSAVSWVRQAPGKRLEWVA

[1484] TITSGVSYTYYLDSVRGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCTRQFKGNALDYWGQGTSVTVSS<

[1485] / INSDSeq_sequence>

[1486] < / insdseq>

[1487] < / sequencedata>

[1488] <sequencedata sequenceidnumber="38">

[1489] <insdseq>

[1490] <INSDSeq_length>354< / INSDSeq_length>

[1491] <INSDSeq_moltype>DNA< / INSDSeq_moltype>

[1492] <INSDSeq_division>PAT< / INSDSeq_division>

[1493] <INSDSeq_feature-table>

[1494] <insdfeature>

[1495] <INSDFeature_key>source< / INSDFeature_key>

[1496] <INSDFeature_location>1..354< / INSDFeature_location>

[1497] <INSDFeature_quals>

[1498] <insdqualifier>

[1499] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[1500] <INSDQualifier_value>other DNA< / INSDQualifier_value>

[1501] < / insdqualifier>

[1502] <insdqualifier id="q170">

[1503] <INSDQualifier_name> organism< / INSDQualifier_name>

[1504] <INSDQualifier_value> synthetic construct< / INSDQualifier_value>

[1505] <NonEnglishQualifier_value> synthetic

[1506] design< / NonEnglishQualifier_value>

[1507] < / insdqualifier>

[1508] < / INSDFeature_quals>

[1509] < / insdfeature>

[1510] < / INSDSeq_feature-table>

[1511] <INSDSeq_sequence>gaggtgcagctggtggagagcggcggcggcctggtgaagcccggcggca

[1512] gcctgaggctgtcctgcgccgcctctggcttcaccttttctaacagcgccgtgtcttgggtgaggcaggc

[1513] acctggcaagcgcctggagtgggtggccaccatcacatccggcgtgtcttacacctactatctggacagc

[1514] gtgcggggcagattcacaatcagcagagataacgccaagaacagcctgtatctgcagatgaactctctgc

[1515] gggccgaggacaccgccgtgtactattgtacaagacagtttaagggcaatgccctggattactggggcca

[1516] gggcaccagcgtgacagtgagctcc< / INSDSeq_sequence>

[1517] < / insdseq>

[1518] < / sequencedata>

[1519] <sequencedata sequenceidnumber="39">

[1520] <insdseq>

[1521] <INSDSeq_length>118< / INSDSeq_length>

[1522] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[1523] <INSDSeq_division>PAT< / INSDSeq_division>

[1524] <INSDSeq_feature-table>

[1525] <insdfeature>

[1526] <INSDFeature_key>source< / INSDFeature_key>

[1527] <INSDFeature_location>1..118< / INSDFeature_location>

[1528] <INSDFeature_quals>

[1529] <insdqualifier>

[1530] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[1531] <INSDQualifier_value>protein< / INSDQualifier_value>

[1532] < / insdqualifier>

[1533] <insdqualifier id="q171">

[1534] <INSDQualifier_name> organism< / INSDQualifier_name>

[1535] <INSDQualifier_value> synthetic construct< / INSDQualifier_value>

[1536] <NonEnglishQualifier_value> synthetic

[1537] design< / NonEnglishQualifier_value>

[1538] < / insdqualifier>

[1539] < / INSDFeature_quals>

[1540] < / insdfeature>

[1541] < / INSDSeq_feature-table>

[1542] <INSDSeq_sequence>EVQLVESGGGLVKPGGSLRLSCAASGFTFSNSAVSWVRQAPGKGLEWVS

[1543] TITSGVSYTYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCTRQFKGNALDYWGQGTSVTVSS<

[1544] / INSDSeq_sequence>

[1545] < / insdseq>

[1546] < / sequencedata>

[1547] <sequencedata sequenceidnumber="40">

[1548] <insdseq>

[1549] <INSDSeq_length>354< / INSDSeq_length>

[1550] <INSDSeq_moltype>DNA< / INSDSeq_moltype>

[1551] <INSDSeq_division>PAT< / INSDSeq_division>

[1552] <INSDSeq_feature-table>

[1553] <insdfeature>

[1554] <INSDFeature_key>source< / INSDFeature_key>

[1555] <INSDFeature_location>1..354< / INSDFeature_location>

[1556] <INSDFeature_quals>

[1557] <insdqualifier>

[1558] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[1559] <INSDQualifier_value>other DNA< / INSDQualifier_value>

[1560] < / insdqualifier>

[1561] <insdqualifier id="q172">

[1562] <INSDQualifier_name> organism< / INSDQualifier_name>

[1563] <INSDQualifier_value> synthetic construct< / INSDQualifier_value>

[1564] <NonEnglishQualifier_value> synthetic

[1565] design< / NonEnglishQualifier_value>

[1566] < / insdqualifier>

[1567] < / INSDFeature_quals>

[1568] < / insdfeature>

[1569] < / INSDSeq_feature-table>

[1570] <INSDSeq_sequence>gaggtgcagctggtggagagcggcggcggcctggtgaagcccggcggca

[1571] gcctgaggctgtcctgcgcagcatctggcttcaccttttctaacagcgccgtgtcttgggtgaggcaggc

[1572] acctggcaagggactggagtgggtgagcaccatcacatccggcgtgtcttacacctactatgccgactcc

[1573] gtgaagggccggttcacaatcagcagagataacgccaagaacagcctgtatctgcagatgaactctctga

[1574] gggccgaggacaccgccgtgtactattgtacacgccagtttaagggcaatgccctggattactggggcca

[1575] gggcaccagcgtgacagtgagctcc< / INSDSeq_sequence>

[1576] < / insdseq>

[1577] < / sequencedata>

[1578] <sequencedata sequenceidnumber="41">

[1579] <insdseq>

[1580] <INSDSeq_length>118< / INSDSeq_length>

[1581] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[1582] <INSDSeq_division>PAT< / INSDSeq_division>

[1583] <INSDSeq_feature-table>

[1584] <insdfeature>

[1585] <INSDFeature_key>source< / INSDFeature_key>

[1586] <INSDFeature_location>1..118< / INSDFeature_location>

[1587] <INSDFeature_quals>

[1588] <insdqualifier>

[1589] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[1590] <INSDQualifier_value>protein< / INSDQualifier_value>

[1591] < / insdqualifier>

[1592] <insdqualifier id="q173">

[1593] <INSDQualifier_name> organism< / INSDQualifier_name>

[1594] <INSDQualifier_value> synthetic construct< / INSDQualifier_value>

[1595] <NonEnglishQualifier_value> synthetic

[1596] design< / NonEnglishQualifier_value>

[1597] < / insdqualifier>

[1598] < / INSDFeature_quals>

[1599] < / insdfeature>

[1600] < / INSDSeq_feature-table>

[1601] <INSDSeq_sequence>EVQLVESGGGLVKPGGSLRLSCAASGFTFSNSAVSWVRQAPGKGLEWVS

[1602] SITSGVSYTYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCTRQFKGNALDYWGQGTSVTVSS<

[1603] / INSDSeq_sequence>

[1604] < / insdseq>

[1605] < / sequencedata>

[1606] <sequencedata sequenceidnumber="42">

[1607] <insdseq>

[1608] <INSDSeq_length>354< / INSDSeq_length>

[1609] <INSDSeq_moltype>DNA< / INSDSeq_moltype>

[1610] <INSDSeq_division>PAT< / INSDSeq_division>

[1611] <INSDSeq_feature-table>

[1612] <insdfeature>

[1613] <INSDFeature_key>source< / INSDFeature_key>

[1614] <INSDFeature_location>1..354< / INSDFeature_location>

[1615] <INSDFeature_quals>

[1616] <insdqualifier>

[1617] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[1618] <INSDQualifier_value>other DNA< / INSDQualifier_value>

[1619] < / insdqualifier>

[1620] <insdqualifier id="q174">

[1621] <INSDQualifier_name> organism< / INSDQualifier_name>

[1622] <INSDQualifier_value> synthetic construct< / INSDQualifier_value>

[1623] <NonEnglishQualifier_value> synthetic

[1624] design< / NonEnglishQualifier_value>

[1625] < / insdqualifier>

[1626] < / INSDFeature_quals>

[1627] < / insdfeature>

[1628] < / INSDSeq_feature-table>

[1629] <INSDSeq_sequence>gaggtgcagctggtggagagcggcggcggcctggtgaagcccggcggca

[1630] gcctgaggctgtcctgcgcagcatctggcttcaccttttctaacagcgccgtgtcttgggtgaggcaggc

[1631] acctggcaagggactggagtgggtgagctccatcacatccggcgtgtcttacacctactatgccgactcc

[1632] gtgaagggccggttcacaatcagcagagataacgccaagaacagcctgtatctgcagatgaactctctga

[1633] gggccgaggacaccgccgtgtactattgtacacgccagtttaagggcaatgccctggattactggggcca

[1634] gggcaccagcgtgacagtgagctcc< / INSDSeq_sequence>

[1635] < / insdseq>

[1636] < / sequencedata>

[1637] <sequencedata sequenceidnumber="43">

[1638] <insdseq>

[1639] <INSDSeq_length>113< / INSDSeq_length>

[1640] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[1641] <INSDSeq_division>PAT< / INSDSeq_division>

[1642] <INSDSeq_feature-table>

[1643] <insdfeature>

[1644] <INSDFeature_key>source< / INSDFeature_key>

[1645] <INSDFeature_location>1..113< / INSDFeature_location>

[1646] <INSDFeature_quals>

[1647] <insdqualifier>

[1648] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[1649] <INSDQualifier_value>protein< / INSDQualifier_value>

[1650] < / insdqualifier>

[1651] <insdqualifier id="q175">

[1652] <INSDQualifier_name> organism< / INSDQualifier_name>

[1653] <INSDQualifier_value> synthetic construct< / INSDQualifier_value>

[1654] <NonEnglishQualifier_value> synthetic

[1655] design< / NonEnglishQualifier_value>

[1656] < / insdqualifier>

[1657] < / INSDFeature_quals>

[1658] < / insdfeature>

[1659] < / INSDSeq_feature-table>

[1660] <INSDSeq_sequence>DIVMTQSPSSLSVSPGERVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQP

[1661] PKLLIYWASTRESGVPDRFSGSGSGTDFTLTINSFQAEDVAVYYCQNDYFYPLTFGAGTKLELK< / INSD

[1662] Seq_sequence>

[1663] < / insdseq>

[1664] < / sequencedata>

[1665] <sequencedata sequenceidnumber="44">

[1666] <insdseq>

[1667] <INSDSeq_length> 339< / INSDSeq_length>

[1668] <INSDSeq_moltype> DNA< / INSDSeq_moltype>

[1669] <INSDSeq_division> PAT< / INSDSeq_division>

[1670] <INSDSeq_feature-table>

[1671] <insdfeature>

[1672] <INSDFeature_key>source< / INSDFeature_key>

[1673] <INSDFeature_location>1..339< / INSDFeature_location>

[1674] <INSDFeature_quals>

[1675] <insdqualifier>

[1676] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[1677] <INSDQualifier_value>other DNA< / INSDQualifier_value>

[1678] < / insdqualifier>

[1679] <insdqualifier id="q176">

[1680] <INSDQualifier_name> organism< / INSDQualifier_name>

[1681] <INSDQualifier_value> synthetic construct< / INSDQualifier_value>

[1682] <NonEnglishQualifier_value> synthetic

[1683] design< / NonEnglishQualifier_value>

[1684] < / insdqualifier>

[1685] < / INSDFeature_quals>

[1686] < / insdfeature>

[1687] < / INSDSeq_feature-table>

[1688] <INSDSeq_sequence> gatatcgtgatgacccagagcccatctagcctgtccgtgtctccaggcg

[1689] agagagtgaccatgagctgcaagtcttcccagagcctgctgaatagcggcaaccagaagaactacctgac

[1690] atggtaccagcagaagccaggccagcctccaaagctgctgatctactgggccagcacaagagagagcggc

[1691] gtgccagacagattctctggcagcggctctggcaccgacttcacactgaccatcaactccttccaggccg

[1692] aggatgtggccgtgtattactgccagaacgattacttctatcccctgacattcggcgccggcacaaagct

[1693] gagctgaag< / INSDSeq_sequence>

[1694] < / insdseq>

[1695] < / sequencedata>

[1696] <sequencedata sequenceidnumber="45">

[1697] <insdseq>

[1698] <INSDSeq_length>113< / INSDSeq_length>

[1699] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[1700] <INSDSeq_division>PAT< / INSDSeq_division>

[1701] <INSDSeq_feature-table>

[1702] <insdfeature>

[1703] <INSDFeature_key>source< / INSDFeature_key>

[1704] <INSDFeature_location>1..113< / INSDFeature_location>

[1705] <INSDFeature_quals>

[1706] <insdqualifier>

[1707] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[1708] <INSDQualifier_value>protein< / INSDQualifier_value>

[1709] < / insdqualifier>

[1710] <insdqualifier id="q177">

[1711] <INSDQualifier_name> organism< / INSDQualifier_name>

[1712] <INSDQualifier_value> synthetic construct< / INSDQualifier_value>

[1713] <NonEnglishQualifier_value> synthetic

[1714] design< / NonEnglishQualifier_value>

[1715] < / insdqualifier>

[1716] < / INSDFeature_quals>

[1717] < / insdfeature>

[1718] < / INSDSeq_feature-table>

[1719] <INSDSeq_sequence>DIVMTQSPSSLSVSPGERVTISCKSSQSLLNSGNQKNYLTWYQQKPGQP

[1720] PKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSFQAEDVAVYYCQNDYFYPLTFGAGTKLELK< / INSD

[1721] Seq_sequence>

[1722] < / insdseq>

[1723] < / sequencedata>

[1724] <sequencedata sequenceidnumber="46">

[1725] <insdseq>

[1726] <INSDSeq_length>339< / INSDSeq_length>

[1727] <INSDSeq_moltype>DNA< / INSDSeq_moltype>

[1728] <INSDSeq_division>PAT< / INSDSeq_division>

[1729] <INSDSeq_feature-table>

[1730] <insdfeature>

[1731] <INSDFeature_key>source< / INSDFeature_key>

[1732] <INSDFeature_location>1..339< / INSDFeature_location>

[1733] <INSDFeature_quals>

[1734] <insdqualifier>

[1735] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[1736] <INSDQualifier_value>other DNA< / INSDQualifier_value>

[1737] < / insdqualifier>

[1738] <insdqualifier id="q178">

[1739] <INSDQualifier_name> organism< / INSDQualifier_name>

[1740] <INSDQualifier_value> synthetic construct< / INSDQualifier_value>

[1741] <NonEnglishQualifier_value> synthetic

[1742] design< / NonEnglishQualifier_value>

[1743] < / insdqualifier>

[1744] < / INSDFeature_quals>

[1745] < / insdfeature>

[1746] < / INSDSeq_feature-table>

[1747] <INSDSeq_sequence>gacatcgtgatgacccagtctccaagctccctgagcgtgtccccaggag

[1748] agagggtgacaatctcctgcaagtctagccagtctctgctgaacagcggcaatcagaagaactacctgac

[1749] ctggtatcagcagaagcctggccagccccctaagctgctgatctactgggcctccaccagggagtctgga

[1750] gtgccagacagattttctggcagcggctccggcacagatttcaccctgacaatctcctcttttcaggccg

[1751] aggacgtggccgtgtactattgtcagaatgattacttctatcccctgacctttggcgccggcacaaagct

[1752] ggagctgaag< / INSDSeq_sequence>

[1753] < / insdseq>

[1754] < / sequencedata>

[1755] <sequencedata sequenceidnumber="47">

[1756] <insdseq>

[1757] <INSDSeq_length>113< / INSDSeq_length>

[1758] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[1759] <INSDSeq_division>PAT< / INSDSeq_division>

[1760] <INSDSeq_feature-table>

[1761] <insdfeature>

[1762] <INSDFeature_key>source< / INSDFeature_key>

[1763] <INSDFeature_location>1..113< / INSDFeature_location>

[1764] <INSDFeature_quals>

[1765] <insdqualifier>

[1766] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[1767] <INSDQualifier_value>protein< / INSDQualifier_value>

[1768] < / insdqualifier>

[1769] <insdqualifier id="q179">

[1770] <INSDQualifier_name> organism< / INSDQualifier_name>

[1771] <INSDQualifier_value> synthetic construct< / INSDQualifier_value>

[1772] <NonEnglishQualifier_value> synthetic

[1773] design< / NonEnglishQualifier_value>

[1774] < / insdqualifier>

[1775] < / INSDFeature_quals>

[1776] < / insdfeature>

[1777] < / INSDSeq_feature-table>

[1778] <INSDSeq_sequence>DIVMTQSPSSLSVSPGERVTISCKSSQSLLNSGNQKNYLAWYQQKPGQP

[1779] PKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYFYPLTFGAGTKLELK< / INSD

[1780] Seq_sequence>

[1781] < / insdseq>

[1782] < / sequencedata>

[1783] <sequencedata sequenceidnumber="48">

[1784] <insdseq>

[1785] <INSDSeq_length>339< / INSDSeq_length>

[1786] <INSDSeq_moltype>DNA< / INSDSeq_moltype>

[1787] <INSDSeq_division>PAT< / INSDSeq_division>

[1788] <INSDSeq_feature-table>

[1789] <insdfeature>

[1790] <INSDFeature_key>source< / INSDFeature_key>

[1791] <INSDFeature_location>1..339< / INSDFeature_location>

[1792] <INSDFeature_quals>

[1793] <insdqualifier>

[1794] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[1795] <INSDQualifier_value>other DNA< / INSDQualifier_value>

[1796] < / insdqualifier>

[1797] <insdqualifier id="q180">

[1798] <INSDQualifier_name> organism< / INSDQualifier_name>

[1799] <INSDQualifier_value> synthetic construct< / INSDQualifier_value>

[1800] <NonEnglishQualifier_value> synthetic

[1801] design< / NonEnglishQualifier_value>

[1802] < / insdqualifier>

[1803] < / INSDFeature_quals>

[1804] < / insdfeature>

[1805] < / INSDSeq_feature-table>

[1806] <INSDSeq_sequence>gacatcgtgatgacccagtctccaagctccctgagcgtgtccccaggag

[1807] agagggtgacaatctcctgcaagtctagccagtctctgctgaacagcggcaatcagaagaactacctggc

[1808] ctggtatcagcagaagcctggccagccccctaagctgctgatctactgggcctccaccagggagtctgga

[1809] gtgccagacagattctctggcagcggctccggcacagacttcaccctgacaatctcctctctgcaggccg

[1810] aggacgtggccgtgtactattgtcagaatgattacttctatcccctgacctttggcgccggcacaaagct

[1811] ggagctgaag< / INSDSeq_sequence>

[1812] < / insdseq>

[1813] < / sequencedata>

[1814] <sequencedata sequenceidnumber="49">

[1815] <insdseq>

[1816] <INSDSeq_length>7< / INSDSeq_length>

[1817] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[1818] <INSDSeq_division>PAT< / INSDSeq_division>

[1819] <INSDSeq_feature-table>

[1820] <insdfeature>

[1821] <INSDFeature_key>source< / INSDFeature_key>

[1822] <INSDFeature_location>1..7< / INSDFeature_location>

[1823] <INSDFeature_quals>

[1824] <insdqualifier>

[1825] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[1826] <INSDQualifier_value>protein< / INSDQualifier_value>

[1827] < / insdqualifier>

[1828] <insdqualifier id="q181">

[1829] <INSDQualifier_name> organism< / INSDQualifier_name>

[1830] <INSDQualifier_value> synthetic construct< / INSDQualifier_value>

[1831] <NonEnglishQualifier_value> synthetic

[1832] design< / NonEnglishQualifier_value>

[1833] < / insdqualifier>

[1834] < / INSDFeature_quals>

[1835] < / insdfeature>

[1836] < / INSDSeq_feature-table>

[1837] <INSDSeq_sequence>ENLYFQG< / INSDSeq_sequence>

[1838] < / insdseq>

[1839] < / sequencedata>

[1840] <sequencedata sequenceidnumber="50">

[1841] <insdseq>

[1842] <INSDSeq_length>20< / INSDSeq_length>

[1843] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[1844] <INSDSeq_division>PAT< / INSDSeq_division>

[1845] <INSDSeq_feature-table>

[1846] <insdfeature>

[1847] <INSDFeature_key>source< / INSDFeature_key>

[1848] <INSDFeature_location>1..20< / INSDFeature_location>

[1849] <INSDFeature_quals>

[1850] <insdqualifier>

[1851] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[1852] <INSDQualifier_value>protein< / INSDQualifier_value>

[1853] < / insdqualifier>

[1854] <insdqualifier id="q182">

[1855] <INSDQualifier_name> organism< / INSDQualifier_name>

[1856] <INSDQualifier_value> synthetic construct< / INSDQualifier_value>

[1857] <NonEnglishQualifier_value> synthetic

[1858] design< / NonEnglishQualifier_value>

[1859] < / insdqualifier>

[1860] < / INSDFeature_quals>

[1861] < / insdfeature>

[1862] < / INSDSeq_feature-table>

[1863] <INSDSeq_sequence>GGGGSGGGGSGGGGSGGGGS< / INSDSeq_sequence>

[1864] < / insdseq>

[1865] < / sequencedata>

[1866] <sequencedata sequenceidnumber="51">

[1867] <insdseq>

[1868] <INSDSeq_length>244< / INSDSeq_length>

[1869] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[1870] <INSDSeq_division>PAT< / INSDSeq_division>

[1871] <INSDSeq_feature-table>

[1872] <insdfeature>

[1873] <INSDFeature_key>source< / INSDFeature_key>

[1874] <INSDFeature_location>1..244< / INSDFeature_location>

[1875] <INSDFeature_quals>

[1876] <insdqualifier>

[1877] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[1878] <INSDQualifier_value>protein< / INSDQualifier_value>

[1879] < / insdqualifier>

[1880] <insdqualifier id="q183">

[1881] <INSDQualifier_name> organism< / INSDQualifier_name>

[1882] <INSDQualifier_value> synthetic construct< / INSDQualifier_value>

[1883] <NonEnglishQualifier_value> synthetic

[1884] design< / NonEnglishQualifier_value>

[1885] < / insdqualifier>

[1886] < / INSDFeature_quals>

[1887] < / insdfeature>

[1888] < / INSDSeq_feature-table>

[1889] <INSDSeq_sequence>QVQLVQSGAEVVKPGASVKLSCKASGYTFTSYWMNWVRQRPGQCLEWIG

[1890] MIDPSDSETHNAQKFQGKATLTVDKSTSTAYMHLSSLRSEDTAVYYCARLYRWYFDVWGAGTTVTVSSGG

[1891] GGSGGGGSGGGGSGGGGSNIVMTQSPATMSMSPGERVTLSCRASEIVGTYVSWFQQKPGQAPRLLIYGAS

[1892] NRYTGVPARFSGSGSGTDFTLTISSVQPEDLADYHCGQSYNFPYTFGCGTKLEIK< / INSDSeq_seque

[1893] nce>

[1894] < / insdseq>

[1895] < / sequencedata>

[1896] <sequencedata sequenceidnumber="52">

[1897] <insdseq>

[1898] <INSDSeq_length> 232< / INSDSeq_length>

[1899] <INSDSeq_moltype> AA< / INSDSeq_moltype>

[1900] <INSDSeq_division> PAT< / INSDSeq_division>

[1901] <INSDSeq_feature-table>

[1902] <insdfeature>

[1903] <INSDFeature_key>source< / INSDFeature_key>

[1904] <INSDFeature_location>1..232< / INSDFeature_location>

[1905] <INSDFeature_quals>

[1906] <insdqualifier>

[1907] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[1908] <INSDQualifier_value>protein< / INSDQualifier_value>

[1909] < / insdqualifier>

[1910] <insdqualifier id="q184">

[1911] <INSDQualifier_name> organism< / INSDQualifier_name>

[1912] <INSDQualifier_value> synthetic construct< / INSDQualifier_value>

[1913] <NonEnglishQualifier_value> synthetic

[1914] design< / NonEnglishQualifier_value>

[1915] < / insdqualifier>

[1916] < / INSDFeature_quals>

[1917] < / insdfeature>

[1918] < / INSDSeq_feature-table>

[1919] <INSDSeq_sequence> EPKSSDKTHTCPPCPAPELLGGPSVFLFPKPKDTLMISRTPEVTCVVV

[1920] DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK

[1921] TISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF

[1922] FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK< / INSDSeq_sequence>

[1923] < / insdseq>

[1924] < / sequencedata>

[1925] <sequencedata sequenceidnumber="53">

[1926] <insdseq>

[1927] <INSDSeq_length>476< / INSDSeq_length>

[1928] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[1929] <INSDSeq_division>PAT< / INSDSeq_division>

[1930] <INSDSeq_feature-table>

[1931] <insdfeature>

[1932] <INSDFeature_key>source< / INSDFeature_key>

[1933] <INSDFeature_location>1..476< / INSDFeature_location>

[1934] <INSDFeature_quals>

[1935] <insdqualifier>

[1936] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[1937] <INSDQualifier_value>protein< / INSDQualifier_value>

[1938] < / insdqualifier>

[1939] <insdqualifier id="q185">

[1940] <INSDQualifier_name> organism< / INSDQualifier_name>

[1941] <INSDQualifier_value> synthetic construct< / INSDQualifier_value>

[1942] <NonEnglishQualifier_value> synthetic

[1943] design< / NonEnglishQualifier_value>

[1944] < / insdqualifier>

[1945] < / INSDFeature_quals>

[1946] < / insdfeature>

[1947] < / INSDSeq_feature-table>

[1948] <INSDSeq_sequence>QVQLVQSGAEVVKPGASVKLSCKASGYTFTSYWMNWVRQRPGQCLEWIG

[1949] MIDPSDSETHNAQKFQGKATLTVDKSTSTAYMHLSSLRSEDTAVYYCARLYRWYFDVWGAGTTVTVSSGG

[1950] GGSGGGGSGGGGSGGGGSNIVMTQSPATMSMSPGERVTLSCRASEIVGTYVSWFQQKPGQAPRLLIYGAS

[1951] NRYTGVPARFSGSGSGTDFTLTISSVQPEDLADYHCGQSYNFPYTFGCGTKLEIKEPKSSDKTHTCPPCP

[1952] APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTY

[1953] RVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPCRDELTKNQVSLWCLVK

[1954] GFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKS

[1955] LSLSPGK< / INSDSeq_sequence>

[1956] < / insdseq>

[1957] < / sequencedata>

[1958] <sequencedata sequenceidnumber="54">

[1959] <insdseq>

[1960] <INSDSeq_length>1428< / INSDSeq_length>

[1961] <INSDSeq_moltype>DNA< / INSDSeq_moltype>

[1962] <INSDSeq_division>PAT< / INSDSeq_division>

[1963] <INSDSeq_feature-table>

[1964] <insdfeature>

[1965] <INSDFeature_key>source< / INSDFeature_key>

[1966] <INSDFeature_location>1..1428< / INSDFeature_location>

[1967] <INSDFeature_quals>

[1968] <insdqualifier>

[1969] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[1970] <INSDQualifier_value>other DNA< / INSDQualifier_value>

[1971] < / insdqualifier>

[1972] <insdqualifier id="q186">

[1973] <INSDQualifier_name> organism< / INSDQualifier_name>

[1974] <INSDQualifier_value> synthetic construct< / INSDQualifier_value>

[1975] <NonEnglishQualifier_value> synthetic

[1976] design< / NonEnglishQualifier_value>

[1977] < / insdqualifier>

[1978] < / INSDFeature_quals>

[1979] < / insdfeature>

[1980] < / INSDSeq_feature-table>

[1981] <INSDSeq_sequence>caggtgcagctggtgcagagcggagccgaagtggtgaagcctggcgcct

[1982] ctgtgaagctgtcctgtaaggcctctggctacacctttacatcctactggatgaactgggtgcggcagag

[1983] accaggccagtgcctggagtggatcggcatgatcgacccatctgattccgagacccacaatgcccagaag

[1984] tttcagggcaaggccacactgaccgtggacaagagcacctccacagcctatatgcacctgtcctctctga

[1985] gaagcgaggatacagccgtgtattactgtgccagactgtatcggtggtactttgacgtgtggggcgccgg

[1986] caccacagtgaccgtgtctagcggaggcggaggatctggaggaggaggcagtggcggagggggaagcggc

[1987] ggaggaggatcaaatatcgtgatgacccagtctcctgccacaatgtctatgagccctggcgagagagtga

[1988] ccctgtcttgtagggccagcgagatcgtgggcacctacgtgagctggttccagcagaagccaggccaggc

[1989] cccaagactgctgatctatggcgccagcaaccggtatacaggcgtgcctgccagattttctggctccggc

[1990] tctggcaccgatttcacactgaccatctctagcgtgcagcccgaggatctggccgattaccactgtggcc

[1991] agtcctacaacttcccctatacattcgggtgcggcactaaactcgagatcaaagagcccaaatcctccga

[1992] caagacacacacctgtcccccctgtcctgctcccgaactcctcggagggccctccgtgtttctgttccct

[1993] ccaaagcctaaagatactctgatgattagtaggactcccgaagtcacctgtgtggtcgtggacgtgtcac

[1994] acgaagatcctgaggtcaaattcaactggtacgtggacggcgtcgaggtgcataatgccaagaccaagcc

[1995] ccgcgaggaacagtacaacagcacctatcgagtcgtgtccgtcctgacagtgctgcaccaggattggctg

[1996] aacgggaaggagtataagtgcaaagtgagcaataaggctctgcccgcacctatcgagaagaccatttcca

[1997] aggctaaaggacagccccgcgagcctcaggtgtacacactgcccccttgccgggacgaactgaccaagaa

[1998] ccaggtctccctgtggtgtctggtgaagggcttctacccaagcgatatcgccgtggagtgggaatccaat

[1999] ggacagcccgagaacaattacaagaccacaccacccgtgctggactccgatggcagcttcttcctgtatt

[2000] ccaagctgacagtggacaagagccggtggcagcaggggaacgtcttcagttgctcagtgatgcacgaggc

[2001] cctgcacaatcattacactcagaagagcctgtccctgtctccaggcaaa< / INSDSeq_sequence>

[2002] < / insdseq>

[2003] < / sequencedata>

[2004] <sequencedata sequenceidnumber="55">

[2005] <insdseq>

[2006] <INSDSeq_length> 329< / INSDSeq_length>

[2007] <INSDSeq_moltype> AA< / INSDSeq_moltype>

[2008] <INSDSeq_division> PAT< / INSDSeq_division>

[2009] <INSDSeq_feature-table>

[2010] <insdfeature>

[2011] <INSDFeature_key>source< / INSDFeature_key>

[2012] <INSDFeature_location>1..329< / INSDFeature_location>

[2013] <INSDFeature_quals>

[2014] <insdqualifier>

[2015] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[2016] <INSDQualifier_value>protein< / INSDQualifier_value>

[2017] < / insdqualifier>

[2018] <insdqualifier id="q187">

[2019] <INSDQualifier_name> organism< / INSDQualifier_name>

[2020] <INSDQualifier_value> synthetic construct< / INSDQualifier_value>

[2021] <NonEnglishQualifier_value> synthetic

[2022] design< / NonEnglishQualifier_value>

[2023] < / insdqualifier>

[2024] < / INSDFeature_quals>

[2025] < / insdfeature>

[2026] < / INSDSeq_feature-table>

[2027] <INSDSeq_sequence> ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG

[2028] VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLG

[2029] GPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL

[2030] TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSD

[2031] IAVEWESNGQPENNYKTTPPVLDSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[2032] < / INSDSeq_sequence>

[2033] < / insdseq>

[2034] < / sequencedata>

[2035] <sequencedata sequenceidnumber="56">

[2036] <insdseq>

[2037] <INSDSeq_length>447< / INSDSeq_length>

[2038] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[2039] <INSDSeq_division>PAT< / INSDSeq_division>

[2040] <INSDSeq_feature-table>

[2041] <insdfeature>

[2042] <INSDFeature_key>source< / INSDFeature_key>

[2043] <INSDFeature_location>1..447< / INSDFeature_location>

[2044] <INSDFeature_quals>

[2045] <insdqualifier>

[2046] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[2047] <INSDQualifier_value>protein< / INSDQualifier_value>

[2048] < / insdqualifier>

[2049] <insdqualifier id="q188">

[2050] <INSDQualifier_name> organism< / INSDQualifier_name>

[2051] <INSDQualifier_value> synthetic construct< / INSDQualifier_value>

[2052] <NonEnglishQualifier_value> synthetic

[2053] design< / NonEnglishQualifier_value>

[2054] < / insdqualifier>

[2055] < / INSDFeature_quals>

[2056] < / insdfeature>

[2057] < / INSDSeq_feature-table>

[2058] <INSDSeq_sequence>EVQLVESGGGLVKPGGSLRLSCAASGFTFSNSAVSWVRQAPGKGLEWVS

[2059] SITSGVSYTYYADSVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYYCTRQFKGNALDYWGQGTSVTVSSA

[2060] STKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTV

[2061] PSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPE

[2062] VTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL

[2063] PAPIEKTISKAKGQPREPQVCTLPPSRDELTKNQVSLSCAVKGFYPSDIAVEWESNGQPENNYKTTPPVL

[2064] DSDGSFFLVSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG< / INSDSeq_sequence>

[2065] < / insdseq>

[2066] < / sequencedata>

[2067] <sequencedata sequenceidnumber="57">

[2068] <insdseq>

[2069] <INSDSeq_length>1341< / INSDSeq_length>

[2070] <INSDSeq_moltype>DNA< / INSDSeq_moltype>

[2071] <INSDSeq_division>PAT< / INSDSeq_division>

[2072] <INSDSeq_feature-table>

[2073] <insdfeature>

[2074] <INSDFeature_key>source< / INSDFeature_key>

[2075] <INSDFeature_location>1..1341< / INSDFeature_location>

[2076] <INSDFeature_quals>

[2077] <insdqualifier>

[2078] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[2079] <INSDQualifier_value>other DNA< / INSDQualifier_value>

[2080] < / insdqualifier>

[2081] <insdqualifier id="q189">

[2082] <INSDQualifier_name> organism< / INSDQualifier_name>

[2083] <INSDQualifier_value> synthetic construct< / INSDQualifier_value>

[2084] <NonEnglishQualifier_value> synthetic

[2085] design< / NonEnglishQualifier_value>

[2086] < / insdqualifier>

[2087] < / INSDFeature_quals>

[2088] < / insdfeature>

[2089] < / INSDSeq_feature-table>

[2090] <INSDSeq_sequence>gaggtgcagctggtggagagcggcggcggcctggtgaagcccggcggca

[2091] gcctgaggctgtcctgcgcagcatctggcttcaccttttctaacagcgccgtgtcttgggtgaggcaggc

[2092] acctggcaagggactggagtgggtgagctccatcacatccggcgtgtcttacacctactatgccgactcc

[2093] gtgaagggccggttcacaatcagcagagataacgccaagaacagcctgtatctgcagatgaactctctga

[2094] gggccgaggacaccgccgtgtactattgtacacgccagtttaagggcaatgccctggattactggggcca

[2095] gggcaccagcgtgacagtgagctccgcctccacaaaggggcccagcgtgtttcctctcgccccctcctcc

[2096] aaaagcaccagcggaggaaccgctgctctcggatgtctggtgaaggactacttccctgaacccgtcaccg

[2097] tgagctggaatagcggcgctctgacaagcggagtccatacattccctgctgtgctgcaaagcagcggact

[2098] ctattccctgtccagcgtcgtcacagtgcccagcagcagcctgggcacccagacctacatctgtaacgtc

[2099] aaccacaagccctccaacaccaaggtggacaagaaagtggagcccaaatcctgcgacaagacccatacct

[2100] gcccaccttgtccagctccagagctgctgggaggaccaagcgtgttcctgtttccacccaagcctaaaga

[2101] caccctgatgatcagcagaaccccagaggtcacatgtgtggtcgtggacgtgtcccacgaggaccccgaa

[2102] gtcaagtttaactggtacgtggatggcgtggaggtccataatgccaagaccaagccccgcgaagagcagt

[2103] acaactcaacttatcgagtcgtgagcgtgctgaccgtcctgcaccaggactggctgaacggaaaggagta

[2104] taagtgcaaagtgtctaacaaggccctgcctgctccaatcgagaagaccattagcaaggccaaaggccag

[2105] cccagagaacctcaggtgtgcaccctgcctccaagcagggatgagctgacaaagaaccaggtcagcctgt

[2106] cctgtgctgtgaaaggattctacccctccgacatcgcagtggagtgggaatctaatggccagcctgagaa

[2107] caattataagaccacaccccctgtgctggattcagacggcagcttcttcctggtgagcaagctgactgtg

[2108] gataagtcaaggtggcagcagggcaacgtgttcagctgtagtgtgatgcacgaggccctgcacaatcatt

[2109] acacccagaaatcactgagcctgtcccccggg< / INSDSeq_sequence>

[2110] < / insdseq>

[2111] < / sequencedata>

[2112] <sequencedata sequenceidnumber="58">

[2113] <insdseq>

[2114] <INSDSeq_length>107< / INSDSeq_length>

[2115] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[2116] <INSDSeq_division>PAT< / INSDSeq_division>

[2117] <INSDSeq_feature-table>

[2118] <insdfeature>

[2119] <INSDFeature_key>source< / INSDFeature_key>

[2120] <INSDFeature_location>1..107< / INSDFeature_location>

[2121] <INSDFeature_quals>

[2122] <insdqualifier>

[2123] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[2124] <INSDQualifier_value>protein< / INSDQualifier_value>

[2125] < / insdqualifier>

[2126] <insdqualifier id="q190">

[2127] <INSDQualifier_name> organism< / INSDQualifier_name>

[2128] <INSDQualifier_value> synthetic construct< / INSDQualifier_value>

[2129] <NonEnglishQualifier_value> synthetic

[2130] design< / NonEnglishQualifier_value>

[2131] < / insdqualifier>

[2132] < / INSDFeature_quals>

[2133] < / insdfeature>

[2134] < / INSDSeq_feature-table>

[2135] <INSDSeq_sequence>RTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQS

[2136] GNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC< / INSDSeq_se

[2137] quence>

[2138] < / insdseq>

[2139] < / sequencedata>

[2140] <sequencedata sequenceidnumber="59">

[2141] <insdseq>

[2142] <INSDSeq_length>220< / INSDSeq_length>

[2143] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[2144] <INSDSeq_division>PAT< / INSDSeq_division>

[2145] <INSDSeq_feature-table>

[2146] <insdfeature>

[2147] <INSDFeature_key>source< / INSDFeature_key>

[2148] <INSDFeature_location>1..220< / INSDFeature_location>

[2149] <INSDFeature_quals>

[2150] <insdqualifier>

[2151] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[2152] <INSDQualifier_value>protein< / INSDQualifier_value>

[2153] < / insdqualifier>

[2154] <insdqualifier id="q191">

[2155] <INSDQualifier_name> organism< / INSDQualifier_name>

[2156] <INSDQualifier_value> synthetic construct< / INSDQualifier_value>

[2157] <NonEnglishQualifier_value> synthetic

[2158] design< / NonEnglishQualifier_value>

[2159] < / insdqualifier>

[2160] < / INSDFeature_quals>

[2161] < / insdfeature>

[2162] < / INSDSeq_feature-table>

[2163] <INSDSeq_sequence>DIVMTQSPSSLSVSPGERVTISCKSSQSLLNSGNQKNYLAWYQQKPGQP

[2164] PKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYFYPLTFGAGTKLELKRTVAAP

[2165] SVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSK

[2166] ADYEKHKVYACEVTHQGLSSPVTKSFNRGEC< / INSDSeq_sequence>

[2167] < / insdseq>

[2168] < / sequencedata>

[2169] <sequencedata sequenceidnumber="60">

[2170] <insdseq>

[2171] <INSDSeq_length>660< / INSDSeq_length>

[2172] <INSDSeq_moltype>DNA< / INSDSeq_moltype>

[2173] <INSDSeq_division>PAT< / INSDSeq_division>

[2174] <INSDSeq_feature-table>

[2175] <insdfeature>

[2176] <INSDFeature_key>source< / INSDFeature_key>

[2177] <INSDFeature_location>1..660< / INSDFeature_location>

[2178] <INSDFeature_quals>

[2179] <insdqualifier>

[2180] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[2181] <INSDQualifier_value>other DNA< / INSDQualifier_value>

[2182] < / insdqualifier>

[2183] <insdqualifier id="q192">

[2184] <INSDQualifier_name> organism< / INSDQualifier_name>

[2185] <INSDQualifier_value> synthetic construct< / INSDQualifier_value>

[2186] <NonEnglishQualifier_value> synthetic

[2187] design< / NonEnglishQualifier_value>

[2188] < / insdqualifier>

[2189] < / INSDFeature_quals>

[2190] < / insdfeature>

[2191] < / INSDSeq_feature-table>

[2192] <INSDSeq_sequence>gacatcgtgatgacccagtctccaagctccctgagcgtgtccccaggag

[2193] agagggtgacaatctcctgcaagtctagccagtctctgctgaacagcggcaatcagaagaactacctggc

[2194] ctggtatcagcagaagcctggccagccccctaagctgctgatctactgggcctccaccagggagtctgga

[2195] gtgccagacagattctctggcagcggctccggcacagacttcaccctgacaatctcctctctgcaggccg

[2196] aggacgtggccgtgtactattgtcagaatgattacttctatcccctgacctttggcgccggcacaaagct

[2197] ggagctgaagcgtacggtggcagccccatctgtcttcatttttccccctagtgacgagcagctgaaatcc

[2198] ggaacagcctctgtggtctgtctgctgaacaatttctaccctcgcgaagccaaggtgcagtggaaagtcg

[2199] ataacgctctgcagagtggcaattcacaggagagcgtgactgaacaggactccaaggattctacctatag

[2200] tctgagctccactctgaccctgtccaaagcagattacgaaaagcacaaagtgtatgcctgtgaggtcacc

[2201] caccaggggctgagttctccagtcaccaaatccttcaacagaggcgaatgt< / INSDSeq_sequence>

[2202] < / insdseq>

[2203] < / sequencedata>

[2204] <sequencedata sequenceidnumber="61">

[2205] <insdseq>

[2206] <INSDSeq_length> 330< / INSDSeq_length>

[2207] <INSDSeq_moltype> AA< / INSDSeq_moltype>

[2208] <INSDSeq_division> PAT< / INSDSeq_division>

[2209] <INSDSeq_feature-table>

[2210] <insdfeature>

[2211] <INSDFeature_key>source< / INSDFeature_key>

[2212] <INSDFeature_location>1..330< / INSDFeature_location>

[2213] <INSDFeature_quals>

[2214] <insdqualifier>

[2215] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[2216] <INSDQualifier_value>protein< / INSDQualifier_value>

[2217] < / insdqualifier>

[2218] <insdqualifier id="q193">

[2219] <INSDQualifier_name> organism< / INSDQualifier_name>

[2220] <INSDQualifier_value> synthetic construct< / INSDQualifier_value>

[2221] <NonEnglishQualifier_value> synthetic

[2222] design< / NonEnglishQualifier_value>

[2223] < / insdqualifier>

[2224] < / INSDFeature_quals>

[2225] < / insdfeature>

[2226] < / INSDSeq_feature-table>

[2227] <INSDSeq_sequence> ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG

[2228] VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLG

[2229] GPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL

[2230] TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSD

[2231] IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[2232] K< / INSDSeq_sequence>

[2233] < / insdseq>

[2234] < / sequencedata>

[2235] <sequencedata sequenceidnumber="62">

[2236] <insdseq>

[2237] <INSDSeq_length>232< / INSDSeq_length>

[2238] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[2239] <INSDSeq_division>PAT< / INSDSeq_division>

[2240] <INSDSeq_feature-table>

[2241] <insdfeature>

[2242] <INSDFeature_key>source< / INSDFeature_key>

[2243] <INSDFeature_location>1..232< / INSDFeature_location>

[2244] <INSDFeature_quals>

[2245] <insdqualifier>

[2246] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[2247] <INSDQualifier_value>protein< / INSDQualifier_value>

[2248] < / insdqualifier>

[2249] <insdqualifier id="q194">

[2250] <INSDQualifier_name> organism< / INSDQualifier_name>

[2251] <INSDQualifier_value> synthetic construct< / INSDQualifier_value>

[2252] <NonEnglishQualifier_value> synthetic

[2253] design< / NonEnglishQualifier_value>

[2254] < / insdqualifier>

[2255] < / INSDFeature_quals>

[2256] < / insdfeature>

[2257] < / INSDSeq_feature-table>

[2258] <INSDSeq_sequence>EPKSSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVV

[2259] DVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK

[2260] TISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSF

[2261] FLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK< / INSDSeq_sequence>

[2262] < / insdseq>

[2263] < / sequencedata>

[2264] <sequencedata sequenceidnumber="63">

[2265] <insdseq>

[2266] <INSDSeq_length> 329< / INSDSeq_length>

[2267] <INSDSeq_moltype> AA< / INSDSeq_moltype>

[2268] <INSDSeq_division> PAT< / INSDSeq_division>

[2269] <INSDSeq_feature-table>

[2270] <insdfeature>

[2271] <INSDFeature_key>source< / INSDFeature_key>

[2272] <INSDFeature_location>1..329< / INSDFeature_location>

[2273] <INSDFeature_quals>

[2274] <insdqualifier>

[2275] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[2276] <INSDQualifier_value>protein< / INSDQualifier_value>

[2277] < / insdqualifier>

[2278] <insdqualifier id="q195">

[2279] <INSDQualifier_name> organism< / INSDQualifier_name>

[2280] <INSDQualifier_value> synthetic construct< / INSDQualifier_value>

[2281] <NonEnglishQualifier_value> synthetic

[2282] design< / NonEnglishQualifier_value>

[2283] < / insdqualifier>

[2284] < / INSDFeature_quals>

[2285] < / insdfeature>

[2286] < / INSDSeq_feature-table>

[2287] <INSDSeq_sequence> ASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSG

[2288] VHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLG

[2289] GPSVFLFPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVL

[2290] TVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSD

[2291] IAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

[2292] < / INSDSeq_sequence>

[2293] < / insdseq>

[2294] < / sequencedata>

[2295] <sequencedata sequenceidnumber="64">

[2296] <insdseq>

[2297] <INSDSeq_length>444< / INSDSeq_length>

[2298] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[2299] <INSDSeq_division>PAT< / INSDSeq_division>

[2300] <INSDSeq_feature-table>

[2301] <insdfeature>

[2302] <INSDFeature_key>source< / INSDFeature_key>

[2303] <INSDFeature_location>1..444< / INSDFeature_location>

[2304] <INSDFeature_quals>

[2305] <insdqualifier>

[2306] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[2307] <INSDQualifier_value>protein< / INSDQualifier_value>

[2308] < / insdqualifier>

[2309] <insdqualifier id="q196">

[2310] <INSDQualifier_name> organism< / INSDQualifier_name>

[2311] <INSDQualifier_value> synthetic construct< / INSDQualifier_value>

[2312] <NonEnglishQualifier_value> synthetic

[2313] design< / NonEnglishQualifier_value>

[2314] < / insdqualifier>

[2315] < / INSDFeature_quals>

[2316] < / insdfeature>

[2317] < / INSDSeq_feature-table>

[2318] <INSDSeq_sequence>QVQLVQSGAEVKKPGASVKVSCKASGYTFTNYNMHWVRQAPGQRLEWMG

[2319] TIYPGNDDTSYNQKFKDRVTITADTSASTAYMELSSLRSEDTAVYYCARGGYRAMDYWGQGTLVTVSSAS

[2320] TKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVP

[2321] SSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCV

[2322] VVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSI

[2323] EKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDG

[2324] SFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK< / INSDSeq_sequence>

[2325] < / insdseq>

[2326] < / sequencedata>

[2327] <sequencedata sequenceidnumber="65">

[2328] <insdseq>

[2329] <INSDSeq_length>219< / INSDSeq_length>

[2330] <INSDSeq_moltype>AA< / INSDSeq_moltype>

[2331] <INSDSeq_division>PAT< / INSDSeq_division>

[2332] <INSDSeq_feature-table>

[2333] <insdfeature>

[2334] <INSDFeature_key>source< / INSDFeature_key>

[2335] <INSDFeature_location>1..219< / INSDFeature_location>

[2336] <INSDFeature_quals>

[2337] <insdqualifier>

[2338] <INSDQualifier_name>mol_type< / INSDQualifier_name>

[2339] <INSDQualifier_value>protein< / INSDQualifier_value>

[2340] < / insdqualifier>

[2341] <insdqualifier id="q197">

[2342] <INSDQualifier_name> organism< / INSDQualifier_name>

[2343] <INSDQualifier_value> synthetic construct< / INSDQualifier_value>

[2344] <NonEnglishQualifier_value> synthetic

[2345] design< / NonEnglishQualifier_value>

[2346] < / insdqualifier>

[2347] < / INSDFeature_quals>

[2348] < / insdfeature>

[2349] < / INSDSeq_feature-table>

[2350] <INSDSeq_sequence>DIVMTQSPLSLPVTPGEPASISCRSSQSIVYSNGNTYLGWYLQKPGQSP

[2351] QLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDVGVYYCFQGSHVPYTFGQGTKLEIKRTVAAPS

[2352] VFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKA

[2353] DYEKHKVYACEVTHQGLSSPVTKSFNRGEC< / INSDSeq_sequence>

[2354] < / insdseq>

[2355] < / sequencedata>

[2356]

[2357] <---

Claims

1. An anti-CLDN18.2 / anti-CD47 bispecific antibody comprising a first protein functional region and a second protein functional region, wherein: the first protein functional region is an antibody against CLDN18.2 (claudin 18.2) or an antigen-binding fragment thereof; and the second protein functional region is an anti-CD47 (integrin-associated protein) antibody or its antigen-binding fragment; wherein the antibody against CLDN18.2 comprises a heavy chain variable region comprising HCDR1-HCDR3 and a light chain variable region comprising LCDR1-LCDR3, wherein: the amino acid sequence of HCDR1 is set forth in SEQ ID NO:31, the amino acid sequence of HCDR2 is set forth in SEQ ID NO:32, and the amino acid sequence of HCDR3 is set forth in SEQ ID NO:33; and the amino acid sequence of LCDR1 is set forth in SEQ ID NO:34, the amino acid sequence of LCDR2 is WAS, and the amino acid sequence of LCDR3 is set forth in SEQ ID NO:36; the anti-CD47 antibody comprises a heavy chain variable region comprising HCDR1-HCDR3 and a light chain variable region comprising LCDR1-LCDR3, where: the amino acid sequence of HCDR1 is set forth in SEQ ID NO:5, the amino acid sequence of HCDR2 is set forth in SEQ ID NO:6, and the amino acid sequence of HCDR3 is set forth in SEQ ID NO:7; and the amino acid sequence of LCDR1 is set forth in SEQ ID NO:8, the amino acid sequence of LCDR2 is GAS, and the amino acid sequence of LCDR3 is set forth in SEQ ID NO:10; and the first protein functional region is a single-chain variable fragment fusion protein (scFv), and the second protein functional region is a semi-molecular monovalent antibody (half of an IgG molecule, IgG-HM); or the first protein functional region is a semi-molecular monovalent antibody (half of an IgG molecule, IgG-HM), and the second protein functional region is a single-chain variable fragment fusion protein (scFv); wherein the single-chain variable fragment fusion protein consists of a single-chain variable fragment, a hinge region and an Fc fragment, or consists of a single-chain variable fragment and a heavy chain constant region.

2. Anti-CLDN18.2 / anti-CD47 bispecific antibody according to claim 1, where the amino acid sequence of the heavy chain variable region of the antibody against CLDN18.2 is selected from SEQ ID NO:28, SEQ ID NO:37, SEQ ID NO:39 and SEQ ID NO:41; and the amino acid sequence of the variable region of the light chain of the antibody against CLDN18.2 is selected from SEQ ID NO:30, SEQ ID NO:43, SEQ ID NO:45 and SEQ ID NO:

47.

3. The anti-CLDN18.2 / anti-CD47 bispecific antibody of claim 1, wherein the anti-CLDN18.2 antibody: the amino acid sequence of the variable region of the heavy chain is set forth in SEQ ID NO:28, and the amino acid sequence of the variable region of the light chain is set forth in SEQ ID NO:30; the amino acid sequence of the variable region of the heavy chain is set forth in SEQ ID NO:37, and the amino acid sequence of the variable region of the light chain is set forth in SEQ ID NO:43; the amino acid sequence of the variable region of the heavy chain is set forth in SEQ ID NO:37, and the amino acid sequence of the variable region of the light chain is set forth in SEQ ID NO:45; the amino acid sequence of the variable region of the heavy chain is set forth in SEQ ID NO:37, and the amino acid sequence of the variable region of the light chain is set forth in SEQ ID NO:47; the amino acid sequence of the variable region of the heavy chain is set forth in SEQ ID NO:39, and the amino acid sequence of the variable region of the light chain is set forth in SEQ ID NO:43; the amino acid sequence of the variable region of the heavy chain is set forth in SEQ ID NO:39, and the amino acid sequence of the variable region of the light chain is set forth in SEQ ID NO:45; the amino acid sequence of the variable region of the heavy chain is set forth in SEQ ID NO:39, and the amino acid sequence of the variable region of the light chain is set forth in SEQ ID NO:47; the amino acid sequence of the variable region of the heavy chain is set forth in SEQ ID NO:41, and the amino acid sequence of the variable region of the light chain is set forth in SEQ ID NO:43; the amino acid sequence of the variable region of the heavy chain is set forth in SEQ ID NO:41, and the amino acid sequence of the variable region of the light chain is set forth in SEQ ID NO:45; or the amino acid sequence of the variable region of the heavy chain is set forth in SEQ ID NO:41, and the amino acid sequence of the variable region of the light chain is set forth in SEQ ID NO:

47.

4. The anti-CLDN18.2 / anti-CD47 bispecific antibody of claim 1, wherein the heavy chain constant region of the anti-CLDN18.2 antibody is the C region of the Ig gamma-1 chain or the C region of the Ig gamma-4 chain; the light chain constant region of the anti-CLDN18.2 antibody is the C region of the Ig kappa chain.

5. The anti-CLDN18.2 / anti-CD47 bispecific antibody of claim 1, wherein the anti-CLDN18.2 antibody or antigen-binding fragment thereof is selected from Fab, Fab', F(ab')2, Fv, single-chain variable fragment (scFv), or humanized antibody.

6. Anti-CLDN18.2 / anti-CD47 bispecific antibody according to claim 1, where The anti-CLDN18.2 antibody contains a non-CDR region derived from a human antibody.

7. Anti-CLDN18.2 / anti-CD47 bispecific antibody according to claim 1, where: EC 50 the anti-CLDN18.2 antibody, with respect to binding to a cell expressing CLDN18.2, is less than or equal to 15 nM, less than or equal to 10 nM, or less than or equal to 5 nM; and / or EC 50 the anti-CLDN18.2 antibody, with respect to binding to a cell expressing both CLDN18.2 and CD47, is less than or equal to 10 nM, less than or equal to 5 nM, or less than or equal to 2 nM; where each EC 50 determined by FACS.

8. Anti-CLDN18.2 / anti-CD47 bispecific antibody according to claim 1, where: the amino acid sequence of the heavy chain variable region of the anti-CD47 antibody is selected from SEQ ID NO:2, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:20 and SEQ ID NO:24; and the amino acid sequence of the variable region of the light chain of the antibody against CD47 is selected from SEQ ID NO:4, SEQ ID NO:14, SEQ ID NO:18, SEQ ID NO:22 and SEQ ID NO:

26.

9. The anti-CLDN18.2 / anti-CD47 bispecific antibody of claim 1, wherein the anti-CD47 antibody: the amino acid sequence of the variable region of the heavy chain is set forth in SEQ ID NO:2, and the amino acid sequence of the variable region of the light chain is set forth in SEQ ID NO:4; the amino acid sequence of the variable region of the heavy chain is set forth in SEQ ID NO:12, and the amino acid sequence of the variable region of the light chain is set forth in SEQ ID NO:14; the amino acid sequence of the variable region of the heavy chain is set forth in SEQ ID NO:12, and the amino acid sequence of the variable region of the light chain is set forth in SEQ ID NO:18; the amino acid sequence of the variable region of the heavy chain is set forth in SEQ ID NO:12, and the amino acid sequence of the variable region of the light chain is set forth in SEQ ID NO:22; the amino acid sequence of the variable region of the heavy chain is set forth in SEQ ID NO:12, and the amino acid sequence of the variable region of the light chain is set forth in SEQ ID NO:26; the amino acid sequence of the variable region of the heavy chain is set forth in SEQ ID NO:16, and the amino acid sequence of the variable region of the light chain is set forth in SEQ ID NO:14; the amino acid sequence of the variable region of the heavy chain is set forth in SEQ ID NO:16, and the amino acid sequence of the variable region of the light chain is set forth in SEQ ID NO:18; the amino acid sequence of the variable region of the heavy chain is set forth in SEQ ID NO:16, and the amino acid sequence of the variable region of the light chain is set forth in SEQ ID NO:22; the amino acid sequence of the variable region of the heavy chain is set forth in SEQ ID NO:16, and the amino acid sequence of the variable region of the light chain is set forth in SEQ ID NO:26; the amino acid sequence of the variable region of the heavy chain is set forth in SEQ ID NO:20, and the amino acid sequence of the variable region of the light chain is set forth in SEQ ID NO:14; the amino acid sequence of the variable region of the heavy chain is set forth in SEQ ID NO:20, and the amino acid sequence of the variable region of the light chain is set forth in SEQ ID NO:18; the amino acid sequence of the variable region of the heavy chain is set forth in SEQ ID NO:20, and the amino acid sequence of the variable region of the light chain is set forth in SEQ ID NO:22; the amino acid sequence of the variable region of the heavy chain is set forth in SEQ ID NO:20, and the amino acid sequence of the variable region of the light chain is set forth in SEQ ID NO:26; the amino acid sequence of the variable region of the heavy chain is set forth in SEQ ID NO:24, and the amino acid sequence of the variable region of the light chain is set forth in SEQ ID NO:14; the amino acid sequence of the variable region of the heavy chain is set forth in SEQ ID NO:24, and the amino acid sequence of the variable region of the light chain is set forth in SEQ ID NO:18; the amino acid sequence of the variable region of the heavy chain is set forth in SEQ ID NO:24, and the amino acid sequence of the variable region of the light chain is set forth in SEQ ID NO:22; or the amino acid sequence of the variable region of the heavy chain is set forth in SEQ ID NO:24, and the amino acid sequence of the variable region of the light chain is set forth in SEQ ID NO:

26.

10. The anti-CLDN18.2 / anti-CD47 bispecific antibody of claim 1, wherein the heavy chain constant region of the anti-CD47 antibody is the C region of the Ig gamma-1 chain or the C region of the Ig gamma-4 chain; the light chain constant region of the anti-CD47 antibody is the C region of the Ig kappa chain.

11. Anti-CLDN18.2 / anti-CD47 bispecific antibody according to claim 1, where the first protein functional region is a single-chain variable fragment fusion protein directed to CLDN18.2, and the second protein functional region is a semi-molecular monovalent antibody directed to CD47; or the first protein functional region is a semi-molecular monovalent antibody directed against CLDN18.2, and the second protein functional region is a single-chain variable fragment fusion protein directed against CD47.

12. Anti-CLDN18.2 / anti-CD47 bispecific antibody according to claim 1, where: the hinge region and Fc region are the hinge region and Fc region of human IgG1; the hinge region and the Fc portion have the amino acid sequence set forth in SEQ ID NO:62; the heavy chain constant region is the heavy chain constant region of human IgG1; or the heavy chain constant region has the amino acid sequence set forth in SEQ ID NO:

63.

13. The anti-CLDN18.2 / anti-CD47 bispecific antibody of claim 1, wherein the Fc region of the fusion protein or the heavy chain constant region of the single-chain variable region has a Knob mutation (S354C and T366W mutations); and The constant region of the heavy chain of the semi-molecular monovalent antibody is the constant region of the heavy chain of human IgG1 and has a "hole" mutation (Hole mutation) (mutations Y349C, T366S, L368A and Y407V).

14. The anti-CLDN18.2 / anti-CD47 bispecific antibody of claim 1, comprising the peptide chain set forth in SEQ ID NO:53, the peptide chain set forth in SEQ ID NO:56, and the peptide chain set forth in SEQ ID NO:

59.

15. Anti-CLDN18.2 / anti-CD47 bispecific antibody according to claim 14, where: the peptide chain set forth in SEQ ID NO:53 and the peptide chain set forth in SEQ ID NO:56 are linked by one or more hinge region disulfide bonds, and the peptide chain set forth in SEQ ID NO:56 and the peptide chain set forth in SEQ ID NO:59 are linked by one or more disulfide bonds; or the peptide chain set forth in SEQ ID NO:53 and the peptide chain set forth in SEQ ID NO:56 are linked by two hinge region disulfide bonds, and the peptide chain set forth in SEQ ID NO:56 and the peptide chain set forth in SEQ ID NO:59 are linked by one disulfide bond.

16. Anti-CLDN18.2 / anti-CD47 bispecific antibody according to claim 1, where: EC 50 the bispecific antibody, with respect to binding to a cell expressing CLDN18.2, is less than or equal to 20 nM, less than or equal to 15 nM, or less than or equal to 12 nM; EC 50 the bispecific antibody, with respect to binding to CD47 on the surface of the erythrocyte membrane, is greater than or equal to 20 nM, greater than or equal to 40 nM, or greater than or equal to 50 nM; and / or EC 50 the bispecific antibody, with respect to binding to a cell expressing both CLDN18.2 and CD47, is less than or equal to 10 nM, less than or equal to 5 nM, or less than or equal to 2 nM; where each EC 50determined by FACS.

17. The anti-CLDN18.2 / anti-CD47 bispecific antibody of claim 1 that does not induce erythrocyte agglutination at a concentration of 3000 nM or less.

18. The anti-CLDN18.2 / anti-CD47 bispecific antibody of claim 1, having ADCP activity, ADCC activity, and CDC activity.

19. An isolated nucleic acid molecule encoding the anti-CLDN18.2 / anti-CD47 bispecific antibody of any one of claims 1 to 18.

20. An expression vector containing the isolated nucleic acid molecule according to claim 19.

21. A host cell for expressing an anti-CLDN18.2 / anti-CD47 bispecific antibody according to any one of claims 1-18, comprising the expression vector according to claim 20.

22. A pharmaceutical composition for the treatment or prevention of a tumor, comprising an effective amount of an anti-CLDN18.2 / anti-CD47 bispecific antibody according to any one of claims 1-18 and one or more pharmaceutically acceptable excipients, wherein the tumor is a CD47- and / or CLDN18.2-positive tumor.

23. The pharmaceutical composition of claim 22, wherein the tumor is one or more selected from biliary tract cancer, bronchogenic carcinoma, lymphoma, ovarian cancer, esophageal cancer, melanoma, hematological malignancy, glioblastoma, lung cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, liver cancer, stomach cancer, breast cancer, brain cancer, pancreatic cancer, thyroid cancer, head and neck cancer, and kidney cancer.

24. Use of an anti-CLDN18.2 / anti-CD47 bispecific antibody according to any one of claims 1-18 in the manufacture of a medicament for the treatment or prevention of a tumor, wherein the tumor is a CD47- and / or CLDN18.2-positive tumor.

25. The use according to claim 24, wherein the tumor is one or more selected from biliary tract cancer, bronchogenic carcinoma, lymphoma, ovarian cancer, esophageal cancer, melanoma, hematological malignancy, glioblastoma, lung cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, liver cancer, stomach cancer, breast cancer, brain cancer, pancreatic cancer, thyroid cancer, head and neck cancer, and kidney cancer.

26. A method for treating or preventing a tumor, comprising the step of administering to a subject in need thereof an effective amount of an anti-CLDN18.2 / anti-CD47 bispecific antibody according to any one of claims 1-18, wherein the tumor is a CD47- and / or CLDN18.2-positive tumor.

27. The method of treating or preventing a tumor according to claim 26, characterized by one or more of the following: (1) the tumor is one or more selected from biliary tract cancer, bronchogenic carcinoma, lymphoma, ovarian cancer, esophageal cancer, melanoma, hematological malignancy, glioblastoma, lung cancer, prostate cancer, bladder cancer, colon cancer, rectal cancer, liver cancer, stomach cancer, breast cancer, brain cancer, pancreatic cancer, thyroid cancer, head and neck cancer, and kidney cancer; (2) the drug is administered before or after surgery and / or before or after radiation therapy; (3) the bispecific antibody is administered at a standard dose of 0.1-100 mg / kg body weight, 5-50 mg or 5-15 mg / kg body weight; (4) the drug is administered once every 3 days, 4 days, 5 days, 6 days, 10 days, 1 week, 2 weeks, or 3 weeks; (5) The route of administration is intravenous drip infusion or intravenous injection.

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