Anti-cd47 / anti-pd-l1 antibodies and uses thereof

Abstract

The present application relates to the technical field of antibody drugs, in particular to an anti-CD47 / anti-PD-L1 antibody, a pharmaceutical composition containing the anti-CD47 / anti-PD-L1 antibody and application thereof.The anti-CD47 / anti-PD-L1 antibody of the present application has significant anti-tumor activity and does not have obvious red blood cell toxicity, and can be applied in the preparation of anti-tumor drugs.

Description

Technical Field

[0001] This invention relates to the field of antibody drug technology, and more particularly to anti-CD47 / anti-PD-L1 antibodies, pharmaceutical compositions comprising anti-CD47 / anti-PD-L1 antibodies, and their applications. Background Technology

[0002] CD47, also known as integrin-associated protein (IAP), is a five-transmembrane glycoprotein belonging to the IgG superfamily and is widely expressed in various tissue cells. CD47 can bind to ligands TSP-1 or SIRPα to regulate different cellular functions, including cell migration, adhesion, apoptosis, axonal elongation, cytokine production, and T cell activation. SIRPα is a transmembrane protein containing a typical immune receptor tyrosine inhibitory motif (ITIM) and is mainly expressed on the surface of myeloid hematopoietic cells, such as macrophages and dendritic cells. After CD47 binds to SIRPα, it leads to phosphorylation of ITIMs, thereby recruiting SHP-1 / SHP-2, which in turn inhibits the accumulation of myosin IIA at phagocytic synapses, ultimately inhibiting the phagocytic function of phagocytes.

[0003] Tumor cell "immune escape" is considered a major mechanism in tumor development, progression, and drug resistance. Tumor cells, by highly expressing CD47 molecules, interact with SIRPα on the surface of macrophages, significantly inhibiting macrophage phagocytic activity and avoiding phagocytosis. Blocking the binding of CD47 to SIRPα can eliminate tumor-induced immunosuppression or immune tolerance, effectively killing tumor cells. This provides a very strong theoretical basis for CD47-based tumor immunotherapy.

[0004] In recent years, numerous studies have been conducted both domestically and internationally on various therapeutic approaches targeting the CD47 / SIRPα signaling pathway, among which CD47 blocking antibodies are considered the most promising cancer treatment option. The effectiveness of human CD47 blocking monoclonal antibodies has been demonstrated in multiple preclinical models. However, since erythrocytes and platelets also express CD47 molecules, when antibodies block the interaction between CD47 and SIRPα, these cells may lose the protection of the "don't eat me" signal, thus becoming vulnerable to phagocytosis by macrophages. Therefore, avoiding the side effects of anti-CD47 antibodies, such as platelet degradation, erythrocyte aggregation, erythrocyte depletion, and anemia, is a key consideration when using anti-CD47 antibodies.

[0005] Programmed death-ligand 1 (PD-L1), also known as differentiation cluster 274 (CD274) or B7 homologue 1 (B7-H1), is a 40 kDa type I transmembrane protein that plays a major role in suppressing the immune system during specific events such as pregnancy, tissue allogeneic transplantation, autoimmune diseases, and other disease states such as hepatitis. PD-L1 binds to PD-1 or B7.1 to transmit an inhibitory signal that reduces CD8+. + T cell proliferation in lymph nodes, and supplementation with PD-1 can also control the accumulation of foreign antigen-specific T cells in lymph nodes through apoptosis mediated by further lower regulation of the Bcl-2 gene.

[0006] It has been shown that upregulation of PD-L1 can allow cancer to evade the host's immune system. Analysis of tumor samples from renal cell carcinoma patients has found that high tumor expression of PD-L1 is associated with increased tumor invasiveness and an increased risk of death. Many PD-L1 inhibitors are under development as immuno-oncology therapies and are showing promising results in clinical trials.

[0007] The CD47-SIRPα signaling pathway not only activates the innate immune system, but macrophages also present tumor antigens to CD8+ T cells and CD4+ T cells, further killing tumor cells by promoting T cell activation. Therefore, a bifunctional fusion protein targeting CD47 and PD-L1 could be developed. This fusion protein contains both a CD47-binding portion and a PD-L1-binding portion, blocking both the binding of PD-L1 to PD-1 and the binding of CD47 to SIRPα, thus bridging the innate and adaptive immune signaling pathways and exhibiting better anti-tumor activity, tumor targeting, and lower erythrocyte toxicity. Summary of the Invention

[0008] This invention provides an anti-CD47 / anti-PD-L1 antibody, comprising an anti-CD47 antibody or its antigen-binding fragment and an anti-PD-L1 antibody or its antigen-binding fragment, wherein the anti-CD47 antibody or its antigen-binding fragment comprises a first heavy chain variable region and / or a first light chain variable region, wherein the first heavy chain variable region comprises a complementarity-determining region 1 (H1CDR1), a complementarity-determining region 2 (H1CDR2), and / or a complementarity-determining region 3 (H1CDR3), and the first light chain variable region comprises a complementarity-determining region 1 (L1CDR1), a complementarity-determining region 2 (L1CDR2), and / or a complementarity-determining region 3 (L1CDR3); and the anti-PD-L1 antibody or its antigen-binding fragment is an antibody or its antigen-binding fragment that specifically binds to PD-L1.

[0009] In some embodiments, the present invention provides an anti-CD47 / anti-PD-L1 antibody comprising an anti-CD47 antibody or an antigen-binding fragment thereof and an anti-PD-L1 antibody or an antigen-binding fragment thereof, wherein the anti-CD47 antibody or the antigen-binding fragment thereof comprises a first heavy chain variable region and a first light chain variable region, wherein:

[0010] (1) The first heavy chain variable region includes H1CDR1, H1CDR2 and H1CDR3 selected from the group consisting of:

[0011] (a1) The amino acid sequences shown in SEQ ID NO:1, 2 and 3;

[0012] (a2) The amino acid sequences shown in SEQ ID NO:10, 2 and 11;

[0013] (a3) The amino acid sequences as shown in SEQ ID NO:4, 5 and 6; and

[0014] (a4) The amino acid sequences shown in SEQ ID NO:7, 8 and 9;

[0015] (a5) An amino acid sequence that has at least 85% sequence identity with the amino acid sequences shown in (a1), (a2), (a3), or (a4); and

[0016] (2) The first light chain variable region comprises L1CDR1, L1CDR2 and L1CDR3 selected from the group consisting of:

[0017] (a6) The amino acid sequences shown in SEQ ID NO:12, 13 and 14;

[0018] (a7) The amino acid sequence as shown in SEQ ID NO: 15, 16 and 17; and

[0019] (a8) The amino acid sequences shown in SEQ ID NO:18, 19 and 20;

[0020] (a9) is an amino acid sequence that has at least 85% sequence identity with the amino acid sequences shown in (a6), (a7) or (a8).

[0021] In some embodiments, the anti-CD47 / anti-PD-L1 antibody according to the present invention has the following characteristics:

[0022] H1CDR1, H1CDR2, and H1CDR3 are the first heavy chain variable regions of amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO:1, 2, and 3, respectively; and L1CDR1, L1CDR2, and L1CDR3 are the first light chain variable regions of amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO:12, 13, and 14, respectively.

[0023] H1CDR1, H1CDR2, and H1CDR3 are the first heavy chain variable regions of amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO:10, 2, and 11, respectively; and L1CDR1, L1CDR2, and L1CDR3 are the first light chain variable regions of amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO:12, 13, and 14, respectively.

[0024] H1CDR1, H1CDR2, and H1CDR3 are the first heavy chain variable regions of amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO:4, 5, and 6, respectively; and L1CDR1, L1CDR2, and L1CDR3 are the first light chain variable regions of amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO:15, 16, and 17, respectively; or

[0025] H1CDR1, H1CDR2, and H1CDR3 are the first heavy chain variable regions of amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO:7, 8, and 9, respectively, and L1CDR1, L1CDR2, and L1CDR3 are the first light chain variable regions of amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO:18, 19, and 20, respectively.

[0026] In some embodiments, the anti-CD47 / anti-PD-L1 antibody according to the present invention, wherein the anti-CD47 antibody or its antigen-binding fragment comprises a first heavy chain variable region and a first light chain variable region, wherein:

[0027] (1) The amino acid sequence of the first heavy chain variable region is selected from:

[0028] (b1) Amino acid sequences as shown in SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, and SEQ ID NO:27;

[0029] Amino acid sequences obtained by substituting, deleting, or adding one or more amino acids as shown in (b2)(b1), and which have the same or similar function as the amino acid sequence shown in (b1); and

[0030] (b3) An amino acid sequence that has at least 80% sequence identity with the amino acid sequence shown in (b1); and

[0031] (2) The amino acid sequence of the first light chain variable region is selected from:

[0032] (b4) Amino acid sequences as shown in SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, and SEQ ID NO:28;

[0033] Amino acid sequences obtained by substituting, deleting, or adding one or more amino acids as shown in (b5) and (b4), and which have the same or similar function as the amino acid sequence shown in (b4); and

[0034] (b6) and (b4) show amino acid sequences that have at least 80% sequence identity.

[0035] In some embodiments, the anti-CD47 / anti-PD-L1 antibody according to the present invention, wherein the anti-CD47 antibody or its antigen-binding fragment comprises a first heavy chain variable region and a first light chain variable region, wherein:

[0036] The amino acid sequence of the first heavy chain variable region is SEQ ID NO:21, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to SEQ ID NO:21 and having the same function as SEQ ID NO:21, or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:21; and the amino acid sequence of the first light chain variable region is SEQ ID NO:24, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to SEQ ID NO:24 and having the same function as SEQ ID NO:24, or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:24.

[0037] The amino acid sequence of the first heavy chain variable region is SEQ ID NO:22, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids of SEQ ID NO:22 that has the same function as SEQ ID NO:22 or an amino acid sequence that has at least 85% sequence identity with SEQ ID NO:22, and the amino acid sequence of the first light chain variable region is SEQ ID NO:25, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids of SEQ ID NO:25 that has the same function as SEQ ID NO:25 or an amino acid sequence that has at least 85% sequence identity with SEQ ID NO:25;

[0038] The amino acid sequence of the first heavy chain variable region is SEQ ID NO:23, which is an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to SEQ ID NO:23 and having the same function as SEQ ID NO:23 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:23; and the amino acid sequence of the first light chain variable region is SEQ ID NO:26, which is an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to SEQ ID NO:26 and having the same function as SEQ ID NO:26 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:26.

[0039] The amino acid sequence of the first heavy chain variable region is SEQ ID NO:27, an amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to SEQ ID NO:27 and having the same function as SEQ ID NO:27, or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:27; and the amino acid sequence of the first light chain variable region is SEQ ID NO:24, an amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to SEQ ID NO:24 and having the same function as SEQ ID NO:24, or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:24; or

[0040] The amino acid sequence of the first heavy chain variable region is SEQ ID NO:23, which is an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to SEQ ID NO:23 and having the same function as SEQ ID NO:23 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:23, and the amino acid sequence of the first light chain variable region is SEQ ID NO:28, which is an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to SEQ ID NO:28 and having the same function as SEQ ID NO:28 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:28.

[0041] In some embodiments, the anti-CD47 / anti-PD-L1 antibody according to the present invention, wherein the anti-CD47 antibody or its antigen-binding fragment is a humanized antibody or its antigen-binding fragment, comprising a first heavy chain variable region and a first light chain variable region, wherein:

[0042] (1) The amino acid sequence of the first heavy chain variable region is selected from:

[0043] (c1) The amino acid sequences shown in SEQ ID NO:29, SEQ ID NO:30, and SEQ ID NO:31;

[0044] Amino acid sequences obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequences shown in (c2) and (c1), and which have the same or similar function to the amino acid sequence shown in (c1); and

[0045] (c3) is an amino acid sequence that has at least 80% sequence identity with the amino acid sequence shown in (c1); and

[0046] (2) The amino acid sequence of the first light chain variable region is selected from:

[0047] (c4) Amino acid sequences as shown in SEQ ID NO:32, SEQ ID NO:33, and SEQ ID NO:34;

[0048] Amino acid sequences obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequences shown in (c5) and (c4), and which have the same or similar function to the amino acid sequence shown in (c4); and

[0049] The amino acid sequences shown in (c6) and (c4) have at least 80% sequence identity.

[0050] In some embodiments, the anti-CD47 / anti-PD-L1 antibody according to the present invention, wherein the anti-CD47 antibody or its antigen-binding fragment is a humanized antibody or its antigen-binding fragment, comprising a first heavy chain variable region and a first light chain variable region, wherein

[0051] The amino acid sequence of the first heavy chain variable region is SEQ ID NO:29, which is an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to SEQ ID NO:29 and having the same function as SEQ ID NO:29 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:29; and the amino acid sequence of the first light chain variable region is SEQ ID NO:32, which is an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to SEQ ID NO:32 and having the same function as SEQ ID NO:32 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:32.

[0052] The amino acid sequence of the first heavy chain variable region is SEQ ID NO:30, an amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to SEQ ID NO:30 and having the same function as SEQ ID NO:30, or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:30; and the amino acid sequence of the first light chain variable region is SEQ ID NO:33, an amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to SEQ ID NO:33 and having the same function as SEQ ID NO:33, or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:33; or

[0053] The amino acid sequence of the first heavy chain variable region is SEQ ID NO:31, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to SEQ ID NO:31 that has the same function as SEQ ID NO:31 or an amino acid sequence that has at least 85% sequence identity with SEQ ID NO:31, and the amino acid sequence of the first light chain variable region is SEQ ID NO:34, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to SEQ ID NO:34 that has the same function as SEQ ID NO:34 or an amino acid sequence that has at least 85% sequence identity with SEQ ID NO:34.

[0054] In some embodiments, the present invention provides an anti-CD47 / anti-PD-L1 antibody, comprising an anti-CD47 antibody or an antigen-binding fragment thereof and an anti-PD-L1 antibody or an antigen-binding fragment thereof, wherein:

[0055] The anti-CD47 antibody or its antigen-binding fragment comprises a first heavy chain variable region and a first light chain variable region, wherein:

[0056] (1) The first heavy chain variable region comprises H1CDR1, H1CDR2, and H1CDR3, whose amino acid sequences are SEQ ID NO:4, 5, and 6, respectively, or amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO:4, 5, and 6; and

[0057] (2) The first light chain variable region includes L1CDR1, L1CDR2 and L1CDR3, whose amino acid sequences are SEQ ID NO:15, 16 and 17 or amino acid sequences that have at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO:15, 16 and 17.

[0058] In some preferred embodiments, the present invention provides an anti-CD47 / anti-PD-L1 antibody comprising an anti-CD47 antibody or an antigen-binding fragment thereof and an anti-PD-L1 antibody or an antigen-binding fragment thereof, wherein the anti-CD47 antibody or the antigen-binding fragment thereof is as defined in the above embodiments; and the anti-PD-L1 antibody or the antigen-binding fragment thereof comprises a second heavy chain variable region and / or a second light chain variable region, wherein the second heavy chain variable region comprises a complementarity-determining region 1 (H2CDR1), a complementarity-determining region 2 (H2CDR2), and / or a complementarity-determining region 3 (H2CDR3), and the second light chain variable region comprises a complementarity-determining region 1 (L2CDR1), a complementarity-determining region 2 (L2CDR2), and / or a complementarity-determining region 3 (L2CDR3).

[0059] Further preferably, in some embodiments, the present invention provides an anti-CD47 / anti-PD-L1 antibody, comprising an anti-CD47 antibody or an antigen-binding fragment thereof and an anti-PD-L1 antibody or an antigen-binding fragment thereof, wherein the anti-CD47 antibody or the antigen-binding fragment thereof is as defined in the above embodiments, and the anti-PD-L1 antibody or the antigen-binding fragment thereof comprises a second heavy chain variable region and a second light chain variable region, wherein:

[0060] (1) The second heavy chain variable region comprises H2CDR1, H2CDR2 and H2CDR3 selected from the group consisting of:

[0061] (A1) The amino acid sequences shown in SEQ ID NO:75, 76 and 77;

[0062] (A2) The amino acid sequences shown in SEQ ID NO:81, 82 and 83;

[0063] (A3) The amino acid sequences as shown in SEQ ID NO: 87, 88 and 89; and

[0064] (A4) The amino acid sequences shown in SEQ ID NO: 93, 94 and 95;

[0065] (A5) is an amino acid sequence that has at least 85% sequence identity with the amino acid sequences shown in (A1), (A2), (A3), or (A4); and

[0066] (2) The second light chain variable region comprises L2CDR1, L2CDR2 and L2CDR3 selected from the group consisting of:

[0067] (A6) The amino acid sequences shown in SEQ ID NO:78, 79 and 80;

[0068] (A7) The amino acid sequences shown in SEQ ID NO:84, 85 and 86;

[0069] (A8) The amino acid sequences shown in SEQ ID NO:90, 91 and 92;

[0070] (A9) The amino acid sequences shown in SEQ ID NO:96, 97 and 98;

[0071] (A10) is an amino acid sequence that has at least 85% sequence identity with the amino acid sequences shown in (A6), (A7), (A8) or (A9).

[0072] In some specific embodiments, the present invention provides an anti-CD47 / anti-PD-L1 antibody, comprising an anti-CD47 antibody or an antigen-binding fragment thereof and an anti-PD-L1 antibody or an antigen-binding fragment thereof, wherein:

[0073] The anti-CD47 antibody or its antigen-binding fragment comprises a first heavy chain variable region and a first light chain variable region, wherein:

[0074] (1) The first heavy chain variable region comprises H1CDR1, H1CDR2 and H1CDR3, whose amino acid sequences are SEQ ID NO:4, 5 and 6 or amino acid sequences that have at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO:4, 5 and 6.

[0075] (2) The first light chain variable region comprises L1CDR1, L1CDR2, and L1CDR3, whose amino acid sequences are SEQ ID NO:15, 16, and 17, respectively, or amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO:15, 16, and 17; and

[0076] The anti-PD-L1 antibody or its antigen-binding fragment comprises a second heavy chain variable region and a second light chain variable region, wherein:

[0077] (1) The second heavy chain variable region comprises H2CDR1, H2CDR2 and H2CDR3 selected from the group consisting of:

[0078] (A1) The amino acid sequences shown in SEQ ID NO:75, 76 and 77;

[0079] (A2) The amino acid sequences shown in SEQ ID NO:81, 82 and 83;

[0080] (A3) The amino acid sequences as shown in SEQ ID NO: 87, 88 and 89; and

[0081] (A4) The amino acid sequences shown in SEQ ID NO: 93, 94 and 95;

[0082] (A5) is an amino acid sequence that has at least 85% sequence identity with the amino acid sequences shown in (A1), (A2), (A3), or (A4); and

[0083] (2) The second light chain variable region comprises L2CDR1, L2CDR2 and L2CDR3 selected from the group consisting of:

[0084] (A6) The amino acid sequences shown in SEQ ID NO:78, 79 and 80;

[0085] (A7) The amino acid sequences shown in SEQ ID NO:84, 85 and 86;

[0086] (A8) The amino acid sequences shown in SEQ ID NO:90, 91 and 92;

[0087] (A9) The amino acid sequences shown in SEQ ID NO:96, 97 and 98;

[0088] (A10) is an amino acid sequence that has at least 85% sequence identity with the amino acid sequences shown in (A6), (A7), (A8) or (A9).

[0089] In one specific embodiment, the present invention provides an anti-CD47 / anti-PD-L1 antibody, comprising an anti-CD47 antibody or an antigen-binding fragment thereof and an anti-PD-L1 antibody or an antigen-binding fragment thereof, wherein the anti-CD47 antibody or its antigen-binding fragment comprises a first heavy chain variable region of amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO:4, 5, and 6, respectively, and L1CDR1, L1CDR2, and L1CDR3 having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO:15, 16, and 17, respectively; and a first light chain variable region of amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO:15, 16, and 17, respectively; and the anti-PD-L1 antibody or its antigen-binding fragment comprises H2CDR1, H2CDR2, and H2CDR3 having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO:75, 76, and 77, respectively, and L1CDR3 having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO:15, 16, and 17. The second heavy chain variable regions of amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in NO:75, 76 and 77, and the second light chain variable regions of amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO:78, 79 and 80 or the amino acid sequences shown in SEQ ID NO:78, 79 and 80, respectively.

[0090] In one specific embodiment, the present invention provides an anti-CD47 / anti-PD-L1 antibody, comprising an anti-CD47 antibody or an antigen-binding fragment thereof and an anti-PD-L1 antibody or an antigen-binding fragment thereof, wherein the anti-CD47 antibody or its antigen-binding fragment comprises a first heavy chain variable region of amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO:4, 5, and 6, respectively, and L1CDR1, L1CDR2, and L1CDR3 having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO:15, 16, and 17, respectively; and a first light chain variable region of amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO:15, 16, and 17, respectively; and the anti-PD-L1 antibody or its antigen-binding fragment comprises H2CDR1, H2CDR2, and H2CDR3 having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO:81, 82, and 83, respectively, and L1CDR3 having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO:15, 16, and 17, respectively; and the anti-PD-L1 antibody or its antigen-binding fragment comprises H2CDR1, H2CDR2, and H2CDR3 having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO:15, 16, and 17, respectively. The second heavy chain variable regions of amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in NO:81, 82 and 83, and the second light chain variable regions of amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO:84, 85 and 86 or the amino acid sequences shown in SEQ ID NO:84, 85 and 86, respectively.

[0091] In one specific embodiment, the present invention provides an anti-CD47 / anti-PD-L1 antibody, comprising an anti-CD47 antibody or an antigen-binding fragment thereof and an anti-PD-L1 antibody or an antigen-binding fragment thereof, wherein the anti-CD47 antibody or its antigen-binding fragment comprises a first heavy chain variable region of amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO:4, 5, and 6, respectively, and L1CDR1, L1CDR2, and L1CDR3 having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO:15, 16, and 17, respectively; and a first light chain variable region of amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO:15, 16, and 17, respectively; and the anti-PD-L1 antibody or its antigen-binding fragment comprises H2CDR1, H2CDR2, and H2CDR3 having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO:87, 88, and 89, respectively, and L1CDR3 having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO:15, 16, and 17, respectively; and the anti-PD-L1 antibody or its antigen-binding fragment comprises H2CDR1, H2CDR2, and H2CDR3 having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO:15, 16, and 17, respectively. The second heavy chain variable regions of amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in NO:87, 88 and 89, and the second light chain variable regions of amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO:90, 91 and 92 or the amino acid sequences shown in SEQ ID NO:90, 91 and 92, respectively.

[0092] In one specific embodiment, the present invention provides an anti-CD47 / anti-PD-L1 antibody, comprising an anti-CD47 antibody or an antigen-binding fragment thereof and an anti-PD-L1 antibody or an antigen-binding fragment thereof, wherein the anti-CD47 antibody or its antigen-binding fragment comprises a first heavy chain variable region of amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO:4, 5, and 6, respectively, and L1CDR1, L1CDR2, and L1CDR3 having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO:15, 16, and 17, respectively; and a first light chain variable region of amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO:15, 16, and 17, respectively; and the anti-PD-L1 antibody or its antigen-binding fragment comprises H2CDR1, H2CDR2, and H2CDR3 having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO:93, 94, and 95, respectively, and L1CDR3 having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO:15, 16, and 17, respectively; and the anti-PD-L1 antibody or its antigen-binding fragment comprises H2CDR1, H2CDR2, and H2CDR3 having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO:15, 16, and 17, respectively. The second heavy chain variable regions of amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in NO:93, 94 and 95, and the second light chain variable regions of amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO:96, 97 and 98, respectively, and the second light chain variable regions of amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO:96, 97 and 98.

[0093] In some specific embodiments, the anti-CD47 / anti-PD-L1 antibody according to the present invention, wherein the anti-CD47 antibody or its antigen-binding fragment and the anti-PD-L1 antibody or its antigen-binding fragment are each independently murine antibodies, chimeric antibodies, humanized antibodies or fully human antibodies.

[0094] In some specific embodiments, the present invention provides an anti-CD47 / anti-PD-L1 antibody, wherein the anti-CD47 antibody or its antigen-binding fragment comprises a first heavy chain variable region and a first light chain variable region, wherein:

[0095] (1) The amino acid sequence of the first heavy chain variable region is selected from:

[0096] (b1) The amino acid sequences shown in SEQ ID NO:22 and SEQ ID NO:30;

[0097] Amino acid sequences obtained by substituting, deleting, or adding one or more amino acids as shown in (b2) and (b1) and having the same or similar function as the amino acid sequence shown in (b1); and

[0098] (b3) An amino acid sequence that has at least 80% sequence identity with the amino acid sequence shown in (b1); and

[0099] (2) The amino acid sequence of the first light chain variable region is selected from:

[0100] (b4) The amino acid sequences shown in SEQ ID NO:25 and SEQ ID NO:33;

[0101] Amino acid sequences obtained by substituting, deleting, or adding one or more amino acids as shown in (b5) and (b4) and having the same or similar function to the amino acid sequence shown in (b4); and

[0102] (b6) is an amino acid sequence that has at least 80% sequence identity with the amino acid sequence shown in (b4); and

[0103] The anti-PD-L1 antibody or its antigen-binding fragment comprises a second heavy chain variable region and a second light chain variable region, wherein:

[0104] (1) The amino acid sequence of the second heavy chain variable region is selected from:

[0105] (B1) The amino acid sequences shown in SEQ ID NO:99, 100, 101, 102, 110, 111, 112, 113, 114, 119, 120, 121, 122 and 123;

[0106] Amino acid sequences obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequences shown in (B2) and (B1), and which have the same or similar function to the amino acid sequences shown in (B1); and

[0107] (B3) is an amino acid sequence that has at least 80% sequence identity with the amino acid sequence shown in (B1); and

[0108] (2) The amino acid sequence of the second light chain variable region is selected from:

[0109] (B4) Amino acid sequences as shown in SEQ ID NO: 103, 104, 105, 106, 115, 116, 117, 118, 124, 125, 126;

[0110] Amino acid sequences obtained by substituting, deleting, or adding one or more amino acids as shown in (B5) and (B4), and which have the same or similar function as the amino acid sequence shown in (B4); and

[0111] The amino acid sequences shown in (B6) and (B4) have at least 80% sequence identity.

[0112] In some specific embodiments, the present invention provides an anti-CD47 / anti-PD-L1 antibody, wherein the amino acid sequence of the first heavy chain variable region is SEQ ID NO:22, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to SEQ ID NO:22 and having the same function as SEQ ID NO:22 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:22, and the amino acid sequence of the first light chain variable region is SEQ ID NO:25, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to SEQ ID NO:25 and having the same function as SEQ ID NO:25 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:25.

[0113] In some specific embodiments, the present invention provides an anti-CD47 / anti-PD-L1 antibody, wherein the amino acid sequence of the first heavy chain variable region is SEQ ID NO:22, an amino acid sequence of SEQ ID NO:22 obtained by substitution, deletion or addition of one or more amino acids and functionally identical to SEQ ID NO:22, or an amino acid sequence of SEQ ID NO:22 with at least 85% sequence identity and wherein the H1CDR1, H1CDR2 and H1CDR3 are as shown in SEQ ID NO:4, 5 and 6, and the amino acid sequence of the first light chain variable region is SEQ ID NO:25, an amino acid sequence of SEQ ID NO:25 obtained by substitution, deletion or addition of one or more amino acids and functionally identical to SEQ ID NO:25, or an amino acid sequence of SEQ ID NO:25 with at least 85% sequence identity and wherein the L1CDR1, L1CDR2 and L1CDR3 are as shown in SEQ ID NO:15, 16 and 17.

[0114] In some specific embodiments, the anti-CD47 / anti-PD-L1 antibody according to the present invention may be a murine antibody, which further contains a heavy chain constant region of murine IgG1, IgG2, IgG3 or IgG4 or variants thereof, and a light chain constant region of murine κ chain or variants thereof.

[0115] In some preferred embodiments, the anti-CD47 / anti-PD-L1 antibody according to the present invention further comprises a heavy chain constant region of mouse IgG1 or IgG2 or a variant thereof, and a light chain constant region of mouse κ chain or a variant thereof.

[0116] In some specific embodiments, the present invention provides an anti-CD47 / anti-PD-L1 antibody, wherein the anti-CD47 antibody or its antigen-binding fragment comprises a first heavy chain variable region and a first light chain variable region, wherein:

[0117] (1) The amino acid sequence of the first heavy chain variable region is selected from:

[0118] (c1) The amino acid sequence as shown in SEQ ID NO:30;

[0119] Amino acid sequences obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequences shown in (c2) and (c1) and having the same or similar function to the amino acid sequence shown in (c1); and

[0120] (c3) is an amino acid sequence that has at least 80% sequence identity with the amino acid sequence shown in (c1); and

[0121] (2) The amino acid sequence of the first light chain variable region is selected from:

[0122] (c4) The amino acid sequence as shown in SEQ ID NO:33;

[0123] Amino acid sequences obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequences shown in (c5) and (c4) and having the same or similar function to the amino acid sequence shown in (c4); and

[0124] (c6) and (c4) show amino acid sequences with at least 80% sequence identity; and

[0125] The anti-PD-L1 antibody or its antigen-binding fragment comprises a second heavy chain variable region and a second light chain variable region, wherein:

[0126] (1) The amino acid sequence of the second heavy chain variable region is selected from:

[0127] (C1) The amino acid sequences shown in SEQ ID NO: 110, 111, 112, 113, 114, 119, 120, 121, 122 and 123;

[0128] Amino acid sequences obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequences shown in (C2) and (C1), and which have the same or similar function to the amino acid sequence shown in (C1); and

[0129] (C3) and (C1) show amino acid sequences with at least 80% sequence identity; and

[0130] (2) The amino acid sequence of the second light chain variable region is selected from:

[0131] (C4) The amino acid sequences shown in SEQ ID NO: 115, 116, 117, 118, 124, 125, 126;

[0132] Amino acid sequences obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequences shown in (C5) and (C4), and which have the same or similar function to the amino acid sequence shown in (C4); and

[0133] The amino acid sequences shown in (C6) and (C4) have at least 80% sequence identity.

[0134] In some specific embodiments, the present invention provides an anti-CD47 / anti-PD-L1 antibody, wherein the amino acid sequence of the first heavy chain variable region is SEQ ID NO:30, an amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to SEQ ID NO:30 and functionally identical to SEQ ID NO:30, or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:30; and the amino acid sequence of the first light chain variable region is SEQ ID NO:33, an amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to SEQ ID NO:33 and functionally identical to SEQ ID NO:33, or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:33; and the amino acid sequence of the second heavy chain variable region is the amino acid sequence shown in SEQ ID NO:110, 111, 112, 113, or 114, an amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to SEQ ID NO:110, 111, 112, 113, or 114 and functionally identical to SEQ ID NO:110, 111, 112, 113, or 114, or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:110, 111, 112, 113, or 114. The amino acid sequences NO:110, 111, 112, 113 or 114 have at least 85% sequence identity, and the amino acid sequence of the second light chain variable region is SEQ ID NO:115, 116, 117 or 118, which are amino acid sequences obtained by substitution, deletion or addition of one or more amino acids of SEQ ID NO:115, 116, 117 or 118 and have the same function as SEQ ID NO:115, 116, 117 or 118 or amino acid sequences with at least 85% sequence identity.

[0135] In some specific embodiments, the present invention provides an anti-CD47 / anti-PD-L1 antibody, wherein the amino acid sequence of the first heavy chain variable region is SEQ ID NO:30, an amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to SEQ ID NO:30 and functionally identical to SEQ ID NO:30, or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:30; and the amino acid sequence of the first light chain variable region is SEQ ID NO:33, an amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to SEQ ID NO:33 and functionally identical to SEQ ID NO:33, or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:33; and the amino acid sequence of the second heavy chain variable region is the amino acid sequence shown in SEQ ID NO:119, 120, 121, 122, or 123, an amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to SEQ ID NO:119, 120, 121, 122, or 123 and functionally identical to SEQ ID NO:119, 120, 121, 122, or 123, or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:30; and the amino acid sequence of the second heavy chain variable region is the amino acid sequence shown in SEQ ID NO:119, 120, 121, 122, or 123, an amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to SEQ ID NO:119, 120, 121, 122, or 123, or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:30; The amino acid sequences NO:119, 120, 121, 122 or 123 have at least 85% sequence identity, and the amino acid sequence of the second light chain variable region is SEQ ID NO:124, 125 or 126, which is an amino acid sequence of SEQ ID NO:124, 125 or 126 obtained by substitution, deletion or addition of one or more amino acids and has the same function as SEQ ID NO:124, 125 or 126 or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:124, 125 or 126.

[0136] In some specific embodiments, the present invention provides an anti-CD47 / anti-PD-L1 antibody, wherein the amino acid sequence of the first heavy chain variable region is SEQ ID NO:30, an amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to SEQ ID NO:30 and functionally identical to SEQ ID NO:30, or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:30, and wherein the H1CDR1, H1CDR2, and H1CDR3 are as shown in SEQ ID NO:4, 5, and 6; and the amino acid sequence of the first light chain variable region is SEQ ID NO:33, an amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to SEQ ID NO:33 and functionally identical to SEQ ID NO:33, or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:33, and wherein the L1CDR1, L1CDR2, and L1CDR3 are as shown in SEQ ID NO:15, 16, and 17; and the amino acid sequence of the second heavy chain variable region is the amino acid sequence shown in SEQ ID NO:110, 111, 112, 113, or 114, SEQ ID NO:30 ... The amino acid sequences of SEQ ID NO:110, 111, 112, 113, or 114 obtained by substitution, deletion, or addition of one or more amino acids and functionally identical to SEQ ID NO:110, 111, 112, 113, or 114, or having at least 85% sequence identity with SEQ ID NO:110, 111, 112, 113, or 114, and the H2CDR1, H2CDR2, and H2CDR3 as shown in SEQ ID NO:75, 76, and 77, and the amino acid sequence of the second light chain variable region is SEQ ID NO:115, 116, 117, or 118, the amino acid sequences of SEQ ID NO:115, 116, 117, or 118 obtained by substitution, deletion, or addition of one or more amino acids and functionally identical to SEQ ID NO:115, 116, 117, or 118, or the amino acid sequences of SEQ ID NO:115, 116, 117, or 118, obtained by substitution, deletion, or addition of one or more amino acids and functionally identical to SEQ ID NO:115, 116, 117, or 118, or the amino acid sequences of SEQ ID NO:110, 111, 112, 113, or 114, are shown in SEQ ID NO:75, 76, and 77. NO:115, 116, 117 or 118 have at least 85% sequence identity and the L2CDR1, L2CDR2 and L2CDR3 have the amino acid sequences shown in SEQ ID NO:78, 79 and 80.

[0137] In some specific embodiments, the present invention provides an anti-CD47 / anti-PD-L1 antibody, wherein the amino acid sequence of the first heavy chain variable region is SEQ ID NO:30, an amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to SEQ ID NO:30 and functionally identical to SEQ ID NO:30, or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:30, and wherein the H1CDR1, H1CDR2, and H1CDR3 are as shown in SEQ ID NO:4, 5, and 6; and the amino acid sequence of the first light chain variable region is SEQ ID NO:33, an amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to SEQ ID NO:33 and functionally identical to SEQ ID NO:33, or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:33, and wherein the L1CDR1, L1CDR2, and L1CDR3 are as shown in SEQ ID NO:15, 16, and 17; and the amino acid sequence of the second heavy chain variable region is the amino acid sequence shown in SEQ ID NO:110, 111, 112, 113, or 114, SEQ ID NO:30 ... The amino acid sequences of SEQ ID NO:110, 111, 112, 113, or 114 obtained by substitution, deletion, or addition of one or more amino acids and functionally identical to SEQ ID NO:110, 111, 112, 113, or 114, or having at least 85% sequence identity with SEQ ID NO:110, 111, 112, 113, or 114, and the H2CDR1, H2CDR2, and H2CDR3 as shown in SEQ ID NO:87, 88, and 89, and the amino acid sequence of the second light chain variable region is SEQ ID NO:115, 116, 117, or 118, the amino acid sequences of SEQ ID NO:115, 116, 117, or 118 obtained by substitution, deletion, or addition of one or more amino acids and functionally identical to SEQ ID NO:115, 116, 117, or 118, or the amino acid sequences of SEQ ID NO:115, 116, 117, or 118, obtained by substitution, deletion, or addition of one or more amino acids and functionally identical to SEQ ID NO:115, 116, 117, or 118, or the amino acid sequences of SEQ ID NO:110, 111, 112, 113, or 114, are shown in SEQ ID NO:87, 88, and 89. NO:115, 116, 117 or 118 have at least 85% sequence identity and the L2CDR1, L2CDR2 and L2CDR3 have amino acid sequences as shown in SEQ ID NO:90, 91 and 92.

[0138] In some preferred embodiments, the present invention provides an anti-CD47 / anti-PD-L1 antibody, wherein the amino acid sequence of the first heavy chain variable region is SEQ ID NO:30, an amino acid sequence of SEQ ID NO:30 obtained by substitution, deletion or addition of one or more amino acids and functionally identical to SEQ ID NO:30, or having at least 85%, or at least 90%, or at least 95%, or at least 98% sequence identity with SEQ ID NO:30, and the amino acid sequences of H1CDR1, H1CDR2 and H1CDR3 as shown in SEQ ID NO:4, 5 and 6; and the amino acid sequence of the first light chain variable region is SEQ ID NO:33, an amino acid sequence of SEQ ID NO:33 obtained by substitution, deletion or addition of one or more amino acids and functionally identical to SEQ ID NO:33, or having at least 85%, or at least 90%, or at least 95%, or at least 98% sequence identity with SEQ ID NO:33, and the amino acid sequences of L1CDR1, L1CDR2 and L1CDR3 as shown in SEQ ID NO:15, 16 and 17; and the amino acid sequence of the second heavy chain variable region is SEQ ID NO:30. The amino acid sequence shown in SEQ ID NO:112, the amino acid sequence obtained by substituting, deleting or adding one or more amino acids to SEQ ID NO:112 and having the same function as SEQ ID NO:112, or having at least 85%, or at least 90%, or at least 95%, or at least 98% sequence identity with SEQ ID NO:112 and the amino acid sequences of H2CDR1, H2CDR2 and H2CDR3 as shown in SEQ ID NO:75, 76 and 77, and the amino acid sequence of the second light chain variable region is SEQ ID NO:116, the amino acid sequence obtained by substituting, deleting or adding one or more amino acids to SEQ ID NO:116 and having the same function as SEQ ID NO:116, or having at least 85%, or at least 90%, or at least 95%, or at least 98% sequence identity with SEQ ID NO:116 and the amino acid sequences of L2CDR1, L2CDR2 and L2CDR3 as shown in SEQ ID NO:78, 79 and 80.

[0139] In some other preferred embodiments, the present invention provides an anti-CD47 / anti-PD-L1 antibody, wherein the amino acid sequence of the first heavy chain variable region is SEQ ID NO:30, an amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to SEQ ID NO:30 and functionally identical to SEQ ID NO:30, or having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with SEQ ID NO:30, and the H1CDR1, H1CDR2, and H1CDR3 are as shown in SEQ ID NO:4, 5, and 6; and the amino acid sequence of the first light chain variable region is SEQ ID NO:33, an amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to SEQ ID NO:33 and functionally identical to SEQ ID NO:33, or having at least 85%, at least 90%, at least 95%, or at least 98% sequence identity with SEQ ID NO:33, and the L1CDR1, L1CDR2, and L1CDR3 are as shown in SEQ ID NO:30. The amino acid sequences shown in NO:15, 16, and 17; and the amino acid sequence of the second heavy chain variable region is the amino acid sequence shown in SEQ ID NO:123, which is an amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to SEQ ID NO:123 and has the same function as SEQ ID NO:123, or has at least 85%, or at least 90%, or at least 95%, or at least 98% sequence identity with SEQ ID NO:123, and the H2CDR1, H2CDR2, and H2CDR3 are as shown in SEQ ID NO:87, 88, and 89; and the amino acid sequence of the second light chain variable region is SEQ ID NO:126, which is an amino acid sequence obtained by substituting, deleting, or adding one or more amino acids to SEQ ID NO:126 and has the same function as SEQ ID NO:126, or has at least 85%, or at least 90%, or at least 95%, or at least 98% sequence identity with SEQ ID NO:126, and the L2CDR1, L2CDR2, and L2CDR3 are as shown in SEQ ID NO:90, 91, and 92.

[0140] In some embodiments, the present invention provides an anti-CD47 / anti-PD-L1 humanized antibody, wherein the heavy chain comprises a heavy chain constant region of human IgG1, IgG2, IgG3, IgG4 or variants thereof, and the light chain comprises a light chain constant region of human κ, λ chains or variants thereof.

[0141] In a preferred embodiment of the present invention, the murine anti-CD47 / anti-PD-L1 antibody may further comprise a light chain constant region of a murine κ, λ chain or a variant thereof, and / or further comprise a heavy chain constant region of a murine IgG1, IgG2, IgG3 or IgG4 or a variant thereof.

[0142] In a preferred embodiment of the present invention, the anti-CD47 / anti-PD-L1 antibody according to the present invention further comprises a light chain constant region of a murine κ, λ chain or a mutant sequence thereof. The heavy chain of the anti-CD47 antibody or a murine antigen-binding fragment thereof further comprises a heavy chain constant region of a murine IgG1, IgG2, IgG3, IgG4 or a mutant sequence thereof, preferably comprising a human IgG1, IgG2, IgG4 heavy chain constant region.

[0143] In some specific embodiments, the anti-CD47 / anti-PD-L1 antibody according to the present invention further comprises a heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4 or variants thereof, and a light chain constant region of human κ, λ chains or variants thereof. In some preferred embodiments, the anti-CD47 humanized antibody or antigen-binding fragment of the present invention further comprises a heavy chain constant region of human IgG1, IgG2, IgG4 or variants thereof, and a light chain constant region of human κ chain or variants thereof.

[0144] In a preferred embodiment of the present invention, the anti-CD47 / anti-PD-L1 antibody according to the present invention further comprises a heavy chain constant region of murine IgG1, IgG2, IgG3, IgG4 or their mutant sequences, preferably comprising a heavy chain constant region of human IgG or their mutant sequences; the antibody light chain of the anti-PD-L1 antibody or its antigen-binding fragment further comprises a light chain constant region of murine κ, λ chains or their mutant sequences.

[0145] In some specific embodiments, the anti-CD47 / anti-PD-L1 antibody according to the present invention further comprises a heavy chain constant region of human IgG1, IgG2, IgG3, or IgG4 or variants thereof, and a light chain constant region of human κ, λ chains or variants thereof. In some preferred embodiments, the anti-CD47 humanized antibody or antigen-binding fragment of the present invention further comprises a heavy chain constant region of human IgG4 or variants thereof, and a light chain constant region of human κ chain or variants thereof.

[0146] In some embodiments, the present invention provides an anti-CD47 / anti-PD-L1 antibody, wherein the anti-CD47 antibody or its antigen-binding fragment and the anti-PD-L1 antibody or its antigen-binding fragment are Fab, Fv, sFv, or F(ab)2, respectively. In a specific embodiment, the anti-CD47 / anti-PD-L1 antibody provided by the present invention is scF(ab)2.

[0147] Preferably, the anti-CD47 / anti-PD-L1 antibody in the above embodiments of the present invention is an anti-CD47 / anti-PD-L1 bispecific antibody. In some embodiments, the bispecific antibody is a human antibody or a humanized antibody. In some embodiments, one of the binding specificities is against CD47, while the other binding specificity is against any other antigen. In some embodiments, one of the binding specificities is against CD47, while the other binding specificity is against PD-L1. In some embodiments, the bispecific antibody can bind to two different epitopes of CD47. The bispecific antibody can also be used to target cytotoxic agents to cells expressing CD47. These antibodies have a CD47 binding arm and a cytotoxic agent binding arm, such as saponin, interferon-α, vinca alkaloids, ricin A chain, methotrexate, or a radioactive isotope hapten. The bispecific antibodies of the present invention can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies).

[0148] Methods for preparing bispecific antibodies are known in the art. Traditionally, the recombinant preparation of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-light chain pairs, where the two heavy chains have different specificities (Millstein and Cuello, Nature 305:537 (1983)). Due to the random allocation of the immunoglobulin heavy and light chains, these quadromas can produce a mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. Purification of the correct molecule is typically performed via affinity chromatography, which is cumbersome and yields low product. Similar methods are disclosed in WO93 / 08829 and Traunecker et al., EMBO J. 10:3655 (1991).

[0149] According to a different method, an antibody variable region having desired binding specificity (antibody-antigen binding site) is fused to an immunoglobulin constant region sequence. In some embodiments, fusion is performed with an immunoglobulin heavy chain constant region comprising at least a portion of the hinge, CH2, and CH3 regions. In some embodiments, a first heavy chain constant region (CH1) containing the site necessary for binding to the light chain is present in at least a portion of the fusion. DNA encoding the immunoglobulin heavy chain fusion fragment and, if desired, the immunoglobulin light chain is inserted into separate expression vectors and co-transfected into a suitable host organism. In embodiments where optimal yield is provided when the proportions of the three polypeptide chains used for construction are unequal, this provides great flexibility in adjusting the relative proportions of the three polypeptide fragments. However, when high yields are produced by expressing at least two polypeptide chains in equal proportions or when the proportions are not particularly significant, it is possible to insert the coding sequences of two or all three polypeptide chains into a single expression vector.

[0150] In one embodiment of the method, the bispecific antibody comprises a hybrid immunoglobulin heavy chain having a first binding specificity in one arm and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. This asymmetric structure is found to facilitate the separation of the desired bispecific substance from the unwanted immunoglobulin chain composition because the presence of the immunoglobulin light chain in only half of the bispecific molecule provides a convenient separation pathway. This method is disclosed in WO 94 / 04690. Further information on the generation of bispecific antibodies can be found, for example, Sureshetal., Methods in Enzymology 121:210 (1986).

[0151] According to another method, the interface between a pair of antibody molecules can be modified to maximize the percentage of heterodimers recovered from recombinant cell cultures. This interface contains at least a portion of the antibody's constant region CH3 domain. In this method, one or more small amino acid side chains at the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). By replacing the large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine), a compensatory "cavity" of the same or similar size as the large side chain is created at the interface of the second antibody molecule. This provides a mechanism to increase the yield of heterodimers compared to other unwanted end products such as homodimers.

[0152] Bispecific antibodies include cross-linked or “heteroconjugated” antibodies. For example, one heteroconjugated antibody may be conjugated with avidin, and another heteroconjugated antibody may be conjugated with biotin. Heteroconjugated antibodies can be prepared using any convenient cross-linking method. Suitable cross-linking agents are well known in the art and, along with many cross-linking techniques, are disclosed in U.S. Patent No. 4,676,980.

[0153] The bispecific antibody of the present invention can be generated from antibody fragments. For example, chemical linking techniques can be used to prepare bispecific antibodies. Brennane et al., Science 229:81 (1985) describes a method for generating F(ab')2 fragments by cleaving intact antibodies through proteolytic hydrolysis. These fragments are decomposed in the presence of sodium arsenite, a dithiol complexing agent (used to stabilize neighboring dithiols and prevent the formation of intermolecular disulfide bonds). The resulting Fab' fragment is then converted into a thionitrobenzoate (TNB) derivative. One of the Fab'-TNB derivatives is then reduced back to Fab'-thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of another Fab'-TNB derivative to form a bispecific antibody.

[0154] Fab'-SH fragments can be directly recovered from *E. coli*, and these fragments can be chemically conjugated to form bispecific antibodies. Shalaby et al., *J. Exp. Med.* 175:217-225 (1992) described the generation of a fully humanized bispecific antibody molecule, F(ab')2. Each Fab' fragment was secreted separately by *E. coli* and chemically conjugated in vitro to form bispecific antibodies.

[0155] In some embodiments, the bispecific antibody fragments of the present invention can be generated and isolated directly from recombinant cell cultures. For example, bispecific antibodies can be generated using leucine zippers (Kostelny et al., J. Immunol. 148(5):1547-1553(1992)). Leucine zipper peptides from Fos and Jun proteins are linked to the Fab' portions of two different antibodies via gene fusion. The antibody homodimer is broken down in the hinge region to form monomers, which are then re-oxidized to form antibody heterodimers. This method can also be used to generate antibody homodimers. Biantibody technology provides other mechanisms for preparing bispecific antibody fragments. The bispecific antibody fragments contain a heavy chain variable region (VH) and a light chain variable region (VL) linked by a linker that is too short for the two domains on the same chain to pair. Thus, the VH and VL domains on one fragment are forced to pair with complementary VL and VH domains on another fragment, thereby forming two antigen-binding sites. In another implementation, a bispecific antibody fragment can be constructed using a single-chain Fv(sFv) dimer.

[0156] This invention covers multivalent antibodies having more than two valences, for example, trispecific antibodies can be prepared. Multivalent antibodies can be internalized (and / or dissimilarized) by cells expressing antigens that the antibody binds more quickly than bivalent antibodies. The antibodies of this invention can be multivalent antibodies having three or more antigen-binding sites (e.g., tetravalent antibodies) that can be readily generated by recombinant expression of nucleic acids encoding antibody polypeptide chains. Multivalent antibodies may comprise a dimerizing domain and three or more antigen-binding sites. In some embodiments, the dimerizing domain comprises (or consists of) an Fc region or a hinge region. In this case, the antibody will comprise an Fc region and three or more antigen-binding sites at the N-terminus of the Fc region. In some embodiments, the multivalent antibody comprises (or consists of) three to about eight antigen-binding sites. In some embodiments, the multivalent antibody comprises four antigen-binding sites. The multivalent antibody comprises at least one polypeptide chain (e.g., two polypeptide chains), wherein said polypeptide chain comprises two or more variable regions. The multivalent antibodies of this invention may further comprise at least two (e.g., four) light chain variable region polypeptides. The multivalent antibody of the present invention may comprise, for example, about two to about eight light chain variable region polypeptides. The light chain variable region polypeptides of the present invention comprise light chain variable regions and optionally further comprise CL domains.

[0157] In bispecific antibodies containing both a CD47-targeting moiety and a PD-L1-targeting moiety, one of the CD47-targeting moiety and the PD-L1-targeting moiety can be a full-length antibody, and the other can be an antigen-binding fragment (e.g., scFv) containing a heavy chain CDR, a light chain CDR, or a combination thereof. The full-length antibody targeting one of the CD47 and PD-L1 proteins and the antigen-binding fragment targeting the other protein can be chemically linked (e.g., covalently linked) directly or via a peptide linker. The antigen-binding fragment (e.g., scFv) can be directly or via a peptide linker to the N-terminus of the full-length antibody (e.g., the N-terminus of the light or heavy chain of the full-length antibody), the C-terminus of the full-length antibody (e.g., the C-terminus of the heavy chain (or the Fc or CH3 domain) of the full-length antibody), or both.

[0158] In one embodiment, the bispecific antibody may comprise a full-length anti-CD47 antibody, an antigen-binding fragment of an anti-PD-L1 antibody (e.g., scFab, scFv), and a peptide linker between them. In other embodiments, the bispecific antibody may comprise a full-length anti-CD47 antibody, an antigen-binding fragment of an anti-PD-L1 antibody (e.g., scFab, scFv), and a peptide linker between them.

[0159] In one embodiment, the scFv contained in the bispecific antibody may contain heavy chain variable regions and light chain variable regions in any order. For example, the scFv contained in the bispecific antibody may contain heavy chain variable regions and light chain variable regions, as well as peptide linkers optionally located therebetween, in a direction from the N-terminus to the C-terminus. Alternatively, the scFv contained in the bispecific antibody may contain light chain variable regions and heavy chain variable regions, as well as peptide linkers optionally located therebetween, in a direction from the N-terminus to the C-terminus.

[0160] In some embodiments, the peptide linker may include, for example, Gly, Asn, and / or Ser residues, and may also include neutral amino acids such as Thr and / or Ala. The amino acid sequence suitable for the peptide linker may be those known in the relevant art. Meanwhile, the length of the peptide linker may be determined differently within such limits as to ensure that the function of the fusion protein is not affected. For example, the peptide linker may be formed by including a total of about 1 to about 100, about 2 to about 50, or about 5 to about 25 residues selected from the group consisting of Gly, Asn, Ser, Thr, and Ala. In one embodiment, the peptide linker may be represented as (GmSl)n (m, l, and n are independently integers from about 1 to about 10, particularly from about 2 to about 5).

[0161] In another embodiment, the PD-L1 targeting portion and the CD47 targeting portion can both be full-length antibodies or antigen-binding fragments containing heavy chain CDRs, light chain CDRs, or combinations thereof.

[0162] In another embodiment, the bispecific antibody may be in the form of a heterodimer comprising a first arm and a second arm, the first arm comprising a pair of first heavy chains and a first light chain targeting one of CD47 and PD-L1, and the second arm comprising a pair of second heavy chains and a second light chain targeting the other.

[0163] In one embodiment, the full-length antibody may be in the form of a full-length immunoglobulin (e.g., IgG, IgM, IgA, IgE, or IgD, such as human IgG, human IgM, human IgA, human IgE, or human IgD), and the antigen-binding fragment may be selected from the group consisting of Fab, Fab', F(ab')2, Fd, Fv, scFv, scFab, single-chain antibody, sdFv, etc. For example, the full-length antibody may be in the form of full-length human IgG (human IgG1, human IgG2, human IgG3, or human IgG4), and the antigen-binding fragment may be scFv.

[0164] For example, the antibodies described herein may contain flexible linker sequences or may be modified to add functional parts (e.g., PEG, drugs, toxins, or labels).

[0165] In some specific embodiments, the anti-CD47 / anti-PD-L1 bispecific antibody according to the present invention has the structure of (VL-CL)-peptide linker-(VH)-IgG4CH for the anti-CD47 antibody or its antigen-binding fragment, and the structure of (VL-CL)-peptide linker-(VH)-IgG4CH for the anti-PD-L1 antibody or its antigen-binding fragment. In some specific embodiments, the peptide linker is in the form of (GGGGS)n, where n is 1-12, preferably 3-10, more preferably 6-8, for example, 6, 7, or 8 GGGGS repeat sequences. In some other specific implementations, the IgG4CH in (VL-CL)-peptide linker-(VH)-IgG4CH targeting the CD47 region is an IgG4CH segment containing mutations of S228P, L235E, Y349C, T366S, L368A, and Y407V to form a "Hole" structure, and the IgG4CH in (VL-CL)-peptide linker-(VH)-IgG4CH targeting the PD-L1 region is an IgG4CH segment containing mutations of S228P, L235E, T366W, and S354C to form a "Knob" structure. In some specific embodiments, the amino acid sequence of VL in (VL-CL)-peptide linker-(VH)-IgG4CH targeting the CD47 region is SEQ ID NO: 33, the amino acid sequence of CL is SEQ ID NO: 131, the amino acid sequence of VH is SEQ ID NO: 30, and the amino acid sequence of IgG4CH is SEQ ID NO: 133; and / or the amino acid sequence of VL in (VL-CL)-peptide linker-(VH)-IgG4CH targeting the PD-L1 region is SEQ ID NO: 116, the amino acid sequence of CL is SEQ ID NO: 131, the amino acid sequence of VH is SEQ ID NO: 112, and the amino acid sequence of IgG4CH is SEQ ID NO: 132. In some other specific embodiments, the amino acid sequence of VL in (VL-CL)-peptide linker-(VH)-IgG4CH targeting the CD47 region is SEQ ID NO: 33, the amino acid sequence of CL is SEQ ID NO: 131, the amino acid sequence of VH is SEQ ID NO: 30, and the amino acid sequence of IgG4CH is SEQ ID NO: 133; and / or the amino acid sequence of VL in (VL-CL)-peptide linker-(VH)-IgG4CH targeting the PD-L1 region is SEQ ID NO: 126, the amino acid sequence of CL is SEQ ID NO: 131, the amino acid sequence of VH is SEQ ID NO: 123, and the amino acid sequence of IgG4CH is SEQ ID NO: 132.

[0166] Another aspect of the present invention provides an anti-PD-L1 antibody or an antigen-binding fragment thereof, comprising a heavy chain variable region and a light chain variable region, wherein:

[0167] (1) The heavy chain variable region comprises CDR1, CDR2 and CDR3 selected from the group consisting of:

[0168] (A1) The amino acid sequences shown in SEQ ID NO:75, 76 and 77;

[0169] (A2) The amino acid sequences shown in SEQ ID NO:81, 82 and 83;

[0170] (A3) The amino acid sequences as shown in SEQ ID NO: 87, 88 and 89; and

[0171] (A4) The amino acid sequences shown in SEQ ID NO: 93, 94 and 95;

[0172] (A5) is an amino acid sequence that has at least 85% sequence identity with the amino acid sequences shown in (A1), (A2), (A3), or (A4); and

[0173] (2) The light chain variable region comprises CDR1, CDR2 and CDR3 selected from the group consisting of:

[0174] (A6) The amino acid sequences shown in SEQ ID NO:78, 79 and 80;

[0175] (A7) The amino acid sequences shown in SEQ ID NO:84, 85 and 86;

[0176] (A8) The amino acid sequences shown in SEQ ID NO:90, 91 and 92;

[0177] (A9) The amino acid sequences shown in SEQ ID NO:96, 97 and 98;

[0178] (A10) is an amino acid sequence that has at least 85% sequence identity with the amino acid sequences shown in (A6), (A7), (A8) or (A9).

[0179] In some embodiments, the anti-PD-L1 antibody or its antigen-binding fragment according to the present invention comprises:

[0180] The CDR1, CDR2, and CDR3 are heavy chain variable regions of amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO:75, 76, and 77, respectively, and the CDR1, CDR2, and CDR3 are light chain variable regions of amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO:78, 79, and 80, respectively;

[0181] The CDR1, CDR2, and CDR3 are heavy chain variable regions of amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO:87, 88, and 89, respectively; and the CDR1, CDR2, and CDR3 are light chain variable regions of amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO:90, 91, and 92, respectively; or

[0182] The CDR1, CDR2, and CDR3 are heavy chain variable regions of amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO:93, 94, and 95, respectively, and light chain variable regions of amino acid sequences having at least 85% sequence identity with the amino acid sequences shown in SEQ ID NO:96, 97, and 98, respectively.

[0183] In some embodiments, the anti-PD-L1 antibody or its antigen-binding fragment according to the present invention, wherein:

[0184] (1) The amino acid sequence of the heavy chain variable region is selected from:

[0185] (B1) The amino acid sequences shown in SEQ ID NO:99, 100, 101, 102, 110, 111, 112, 113, 114, 119, 120, 121, 122 and 123;

[0186] Amino acid sequences obtained by substituting, deleting, or adding one or more amino acids to the amino acid sequences shown in (B2) and (B1), and which have the same or similar function to the amino acid sequences shown in (B1); and

[0187] (B3) is an amino acid sequence that has at least 80% sequence identity with the amino acid sequence shown in (B1); and

[0188] (2) The amino acid sequence of the light chain variable region is selected from:

[0189] (B4) Amino acid sequences as shown in SEQ ID NO: 103, 104, 105, 106, 115, 116, 117, 118, 124, 125, 126;

[0190] Amino acid sequences obtained by substituting, deleting, or adding one or more amino acids as shown in (B5) and (B4), and which have the same or similar function as the amino acid sequence shown in (B4); and

[0191] The amino acid sequences shown in (B6) and (B4) have at least 80% sequence identity.

[0192] In some embodiments, the anti-PD-L1 antibody or its antigen-binding fragment according to the present invention, wherein:

[0193] The amino acid sequence of the heavy chain variable region is the amino acid sequence shown in SEQ ID NO: 110, 111, 112, 113 or 114, or an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to SEQ ID NO: 110, 111, 112, 113 or 114 that is functionally identical to SEQ ID NO: 110, 111, 112, 113 or 114, or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 110, 111, 112, 113 or 114. The amino acid sequence of the light chain variable region is SEQ ID NO: 115, 116, 117 or 118, or an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to SEQ ID NO: 115, 116, 117 or 118 that is functionally identical to SEQ ID NO: 115, 116, 117 or 118, or an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 115, 116, 117 or 118.

[0194] In some embodiments, the anti-PD-L1 antibody or its antigen-binding fragment according to the present invention, wherein:

[0195] The amino acid sequence of the heavy chain variable region is the amino acid sequence shown in SEQ ID NO:119, 120, 121, 122 or 123, which is an amino acid sequence obtained by substituting, deleting or adding one or more amino acids of SEQ ID NO:119, 120, 121, 122 or 123 and has the same function as SEQ ID NO:119, 120, 121, 122 or 123, or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:119, 120, 121, 122 or 123. The amino acid sequence of the light chain variable region is SEQ ID NO:124, 125 or 126, which is an amino acid sequence obtained by substituting, deleting or adding one or more amino acids of SEQ ID NO:124, 125 or 126 and has the same function as SEQ ID NO:124, 125 or 126, or an amino acid sequence having at least 85% sequence identity with SEQ ID NO:124, 125 or 126.

[0196] In some embodiments, the anti-PD-L1 antibody or its antigen-binding fragment according to the present invention, wherein the amino acid sequence of the heavy chain variable region is the amino acid sequence shown in SEQ ID NO:112, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to SEQ ID NO:112 and functionally identical to SEQ ID NO:112, or having at least 85%, or at least 90%, or at least 95%, or at least 98% sequence identity with SEQ ID NO:112, and the H2CDR1, H2CDR2 and H2CDR3 are as shown in SEQ ID NO:75, 76 and 77, and the amino acid sequence of the light chain variable region is SEQ ID NO:116, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to SEQ ID NO:116 and functionally identical to SEQ ID NO:116, or having at least 85%, or at least 90%, or at least 95%, or at least 98% sequence identity with SEQ ID NO:116, and the L2CDR1, L2CDR2 and L2CDR3 are as shown in SEQ ID NO:78, 79 and 80.

[0197] In some embodiments, the anti-PD-L1 antibody or its antigen-binding fragment according to the present invention, wherein the amino acid sequence of the heavy chain variable region is the amino acid sequence shown in SEQ ID NO:123, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to SEQ ID NO:123 and having the same function as SEQ ID NO:123, or having at least 85%, or at least 90%, or at least 95%, or at least 98% sequence identity with SEQ ID NO:123, and the amino acid sequences of H2CDR1, H2CDR2 and H2CDR3 are as shown in SEQ ID NO:87, 88 and 89, and the amino acid sequence of the light chain variable region is SEQ ID NO:126, an amino acid sequence obtained by substituting, deleting or adding one or more amino acids to SEQ ID NO:126 and having the same function as SEQ ID NO:126, or having at least 85%, or at least 90%, or at least 95%, or at least 98% sequence identity with SEQ ID NO:126, and the amino acid sequences of L2CDR1, L2CDR2 and L2CDR3 are as shown in SEQ ID NO:90, 91 and 92.

[0198] In some embodiments, the anti-PD-L1 antibody or its antigen-binding fragment according to the present invention is a humanized antibody or a fully human antibody.

[0199] Another aspect of the invention provides isolated nucleic acids. In some embodiments, the isolated nucleic acids according to the invention encode the anti-CD47 / anti-PD-L1 antibody or anti-PD-L1 antibody of the invention. In some embodiments, the isolated nucleic acids according to the invention encode the anti-CD47 antibody of the invention or its antigen-binding fragment. In other embodiments, the isolated nucleic acids according to the invention encode the anti-PD-L1 antibody of the invention or its antigen-binding fragment.

[0200] In one specific embodiment, the nucleic acid isolated according to the present invention encodes the first heavy chain variable region SEQ ID NO:30 as shown in SEQ ID NO:36, and the nucleotide sequence encoding the first light chain variable region SEQ ID NO:33 as shown in SEQ ID NO:39. In another specific embodiment, the nucleic acid isolated according to the present invention encodes the second heavy chain variable region SEQ ID NO:112 as shown in SEQ ID NO:127, and the nucleotide sequence encoding the second light chain variable region SEQ ID NO:116 as shown in SEQ ID NO:129. In yet another specific embodiment, the nucleic acid isolated according to the present invention encodes the second heavy chain variable region SEQ ID NO:123 as shown in SEQ ID NO:128, and the nucleotide sequence encoding the second light chain variable region SEQ ID NO:126 as shown in SEQ ID NO:130.

[0201] Another aspect of the present invention provides expression vectors. In some embodiments, the expression vectors of the present invention express the anti-CD47 / anti-PD-L1 bispecific antibody or the anti-PD-L1 antibody of the present invention. In some embodiments, the expression vectors of the present invention express the anti-CD47 antibody of the present invention or its antigen-binding fragment. In other embodiments, the expression vectors of the present invention express the anti-PD-L1 antibody of the present invention or its antigen-binding fragment. In some embodiments, according to the expression vectors of the present invention, the vectors expressing the anti-CD47 antibody of the present invention or its antigen-binding fragment and the vectors expressing the anti-PD-L1 antibody of the present invention or its antigen-binding fragment are the same expression vector. The expression vectors of the present invention contain the isolated nucleic acid molecules of the present invention.

[0202] Another aspect of the present invention provides a host cell transformed with the expression vector as described above.

[0203] In some embodiments, the host cell according to the invention is selected from prokaryotic and eukaryotic cells. In some embodiments, the host cell is a bacterium, preferably *Escherichia coli*. In another preferred embodiment, the host cell is a mammalian cell.

[0204] Another aspect of the present invention provides a method for preparing the anti-CD47 / anti-PD-L1 bispecific antibody or anti-PD-L1 antibody of the present invention, comprising the steps of expressing the antibody in the host cells and isolating the antibody from the host cells.

[0205] Another aspect of the present invention provides a pharmaceutical composition comprising the anti-CD47 / anti-PD-L1 bispecific antibody of the present invention and a pharmaceutically acceptable carrier. In some embodiments, the present invention provides a pharmaceutical composition comprising the anti-CD47 / anti-PD-L1 bispecific antibody of the present invention, and further comprising other active ingredients, such as other antibodies, targeted drugs, etc. In some embodiments, the pharmaceutically acceptable carrier is selected from antioxidants, peptides, proteins, hydrophilic polymers, amino acids, sugars, chelating agents, sugar alcohols, ions, and surfactants. In one specific embodiment, the pharmaceutically acceptable carrier is a buffered aqueous solution. In another specific embodiment, the pharmaceutically acceptable carrier is in the form of liposomes.

[0206] Another aspect of the present invention provides a chimeric antigen receptor (CAR) fusion protein comprising the anti-CD47 antibody or its antigen-binding fragment of the present invention and / or an anti-PD-L1 antibody or its antigen-binding fragment. In some embodiments, the chimeric antigen receptor fusion protein comprises the anti-CD47 antibody or its antigen-binding fragment of the present invention, which is a V-type antigen targeting the CD47 antigen. H and V L The single-chain variable fragment (scFv). In other embodiments, the chimeric antigen receptor fusion protein comprises the anti-PD-L1 antibody of the present invention or its antigen-binding fragment, which is a V-type antigen targeting the PD-L1 antigen. H and V L The single-chain variable fragment (scFv). In other embodiments, the chimeric antigen receptor fusion protein comprises a V targeting the CD47 antigen. H and V L The first single-chain variable fragment (scFv) and V targeting the PD-L1 antigen H and V L The second single-stranded variable fragment (scFv). The V targeting the CD47 antigen. H and V L The first scFv has the first heavy chain variable regions H1CDR1, H1CDR2, and H1CDR3 and the first light chain variable regions L1CDR1, L1CDR2, and L1CDR3 described in the above embodiments. The V targeting the PD-L1 antigen... H and V L The second scFv has the second heavy chain variable regions H2CDR1, H2CDR2 and H2CDR3 and the second light chain variable regions L2CDR1, L2CDR2 and L2CDR3 as described in the above embodiments.

[0207] The anti-CD47 / anti-PD-L1 bispecific antibody of the present invention can be mixed with pharmaceutically acceptable carriers, diluents, or excipients to prepare a pharmaceutical formulation suitable for oral or parenteral administration. Administration methods include, but are not limited to, oral, intradermal, intramuscular, intraperitoneal, intravenous, intracerebral, intraocular, intratracheal, subcutaneous, and intranasal routes. The formulation can be administered via any route, such as by infusion or bolus, or by absorption through the epithelium or mucous membranes (e.g., oral mucosa or rectum). Administration can be systemic or local. The formulation can be prepared by methods known in the art and contains carriers, diluents, or excipients conventionally used in the field of pharmaceutical formulations.

[0208] Another aspect of the invention provides a method for treating and / or preventing diseases associated with CD47, PD-L1, or both, the method comprising administering to an individual in need an anti-CD47 / anti-PD-L1 bispecific antibody of the invention or a pharmaceutical composition of the invention.

[0209] Another aspect of the present invention provides the use of the anti-CD47 / anti-PD-L1 bispecific antibody or anti-PD-L1 antibody or pharmaceutical composition of the present invention in the preparation of medicaments for treating and / or preventing diseases associated with CD47, PD-L1, or both. In some embodiments, the diseases associated with CD47, PD-L1, or both include hematologic malignancies, lymphoma, breast cancer, lung cancer, gastric cancer, colorectal cancer, esophageal cancer, ovarian cancer, cervical cancer, kidney cancer, bladder cancer, pancreatic cancer, glioma, and / or melanoma. The tumor can be any tumor expressing the PD-L1 protein, such as bladder cancer, liver cancer, colon cancer, rectal cancer, endometrial cancer, leukemia, lymphoma, pancreatic cancer, lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, etc.), breast cancer, urethral cancer, head and neck cancer, gastrointestinal cancer, stomach cancer, esophageal cancer, ovarian cancer, kidney cancer, melanoma, prostate cancer, thyroid cancer, etc. The tumor can be primary or metastatic. In some embodiments, the present invention provides the use of the above-described anti-CD47 / anti-PD-L1 bispecific antibody or the pharmaceutical composition of the present invention in the preparation of an antitumor medicament, for example, the tumor being selected from hematologic malignancies, lymphoma, breast cancer, lung cancer, gastric cancer, colorectal cancer, esophageal cancer, ovarian cancer, cervical cancer, kidney cancer, bladder cancer, pancreatic cancer, glioma, and melanoma.

[0210] The anti-CD47 / anti-PD-L1 bispecific antibody provided by this invention has significant anti-tumor effects, can significantly inhibit tumor growth, and does not have obvious erythrocyte toxicity. The immunogenicity of the humanized antibody is greatly reduced, effectively eliminating the rejection reaction of the human immune system to exogenous monoclonal antibodies. It can be used in the preparation of drugs for the treatment of various tumor diseases and has broad market prospects.

[0211] definition

[0212] Unless otherwise defined, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. The nomenclature and techniques used in cell and tissue culture, molecular biology, and protein and oligo or polynucleotide chemistry and hybridization described herein are well-known and commonly used in the art. Standard techniques were used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipid transfection). Enzymatic reactions and purification techniques were performed according to the manufacturer's instructions or those commonly used in the art or described herein. The foregoing techniques and methods are generally used as described in several comprehensive and more specific references well-known in the art and cited and discussed herein. See, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (2nd ed., Cold Spring Harbor Laboratory Press, New York (1989)). The nomenclature and laboratory methods and techniques used in analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein are well-known and commonly used in the art.

[0213] In this invention, the term "at least 80% sequence identity" refers to at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In this invention, the term "at least 85% sequence identity" refers to at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some preferred embodiments, the sequence identity described in this invention may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. Sequence comparison and identity percentage determination between two sequences can be performed using the BLASTN / BLASTP algorithm on the National Center for Biotechnology Institute website.

[0214] In antibody molecules, three hypervariable regions of the light chain and three hypervariable regions of the heavy chain are arranged in three-dimensional space relative to each other to form an antigen-binding surface. The antigen-binding surface is complementary to the three-dimensional surface of the antigen to which it is bound, and each of the three hypervariable regions of the heavy and light chains is called a "complementarity-determining region" or "CDR". The allocation of amino acids to each domain is defined according to Kabat, Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, MD (1987 and 1991)) or Chothia and Lesk, J. Mol. Biol. 196:901-917 (1987), Chothia et al., Nature 342:878-883 (1989).

[0215] The "antibody" of this invention refers to a polypeptide or polypeptide complex that specifically recognizes and binds to an antigen. An antibody can be a complete antibody and any antigen-binding fragment or single chain thereof. The "antibody" of this invention includes any protein or peptide containing at least a portion of an Ig molecule that has biological activity in binding to an antigen. Examples of the "antibody" of this invention include, but are not limited to, the CDR of the heavy or light chain or its ligand-binding portion, the variable region of the heavy or light chain, the constant region of the heavy or light chain, the frame region, or any portion thereof.

[0216] The "antigen-binding fragments" described in this invention include Fab fragments, Fab' fragments, F(ab')2 fragments, and Fv fragments and scFv fragments that bind to human CD47 or PD-L1, all possessing antigen-binding activity. The Fv fragment contains a variable region of the first heavy chain and a variable region of the first light chain of the antibody, but no constant region, and is the smallest antibody fragment with all antigen-binding sites. Generally, Fv antibodies also contain a polypeptide linker between the VH and VL domains and are capable of forming the structure required for antigen binding. Two antibody variable regions can also be linked into a single polypeptide chain using different linkers, termed a single-chain antibody or single-chain Fv (scFv). The anti-CD47 or anti-PD-L1 antibody of this invention can be a single-chain variable region fragment (scFv), derived from a single-chain polypeptide of the antibody, retaining the ability to bind antigens. Examples of scFv include antibody polypeptides formed by recombinant DNA technology, wherein the Fv regions of the immunoglobulin heavy chain (H chain) and light chain (L chain) fragments are linked via spacer sequences. Various methods for preparing scFv are well known to those skilled in the art.

[0217] The antibody described in this invention refers to an immunoglobulin molecule or its immunoactive portion, that is, a molecule containing an antigen-binding site that specifically binds to an antigen (and with which it reacts immunely). "Specific binding" means that the antibody reacts with one or more antigenic determinants of the antigen without reacting with other peptides or with very low affinity (Kd > 10). -6Antibodies can bind to other peptides. Antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, dAb (domain antibody), single-chain, Fab, Fab' and F(ab')2 fragments, Fv, scFv, and Fab expression libraries. Monoclonal antibodies (mAbs) are antibodies obtained from a single clonal cell line, which is not limited to eukaryotic, prokaryotic, or phage clonal cell lines. Monoclonal antibodies or antigen-binding fragments can be obtained through recombinant techniques such as hybridoma technology, recombinant technology, phage display technology, and synthetic techniques such as CDR grafting or other existing technologies.

[0218] The "mouse-derived antibody" described in this invention is a monoclonal antibody against human CD47 prepared based on the knowledge and skills in the art. During preparation, the test subject is injected with CD47 antigen, and then hybridomas expressing antibodies with the desired sequence or functional characteristics are isolated.

[0219] The "chimeric antibody" described in this invention is an antibody formed by fusing the variable region of a murine antibody with the constant region of a human antibody, which can reduce the immune response induced by murine antibodies. To establish a chimeric antibody, a hybridoma secreting a murine-specific monoclonal antibody is first established. Then, the variable region gene is cloned from mouse hybridoma cells, and the constant region gene of the human antibody is cloned as needed. The mouse variable region gene and the human constant region gene are then linked to form a chimeric gene, which is inserted into a human vector. Finally, the chimeric antibody molecule is expressed in a eukaryotic or prokaryotic industrial system.

[0220] The "humanized antibody" described in this invention, also known as a CDR transplantation antibody, is an antibody produced by transplanting a mouse CDR sequence into a human antibody variable region framework (FR). Such variable region framework sequences can be obtained from public DNA databases or publicly available references, such as from the ImMunoGeneTics (IMGT) website http: / / imgt.cines.fr or from the journal Immunoglobulins, 2001 ISBN012441351.

[0221] The "bispecific antibody" described in this invention refers to a monoclonal antibody that has binding specificity to at least two different antigens.

[0222] The “peptide linker” described in this invention may be those comprising 1 to 10, particularly 2 to 50, any amino acids, and may include any kind of amino acids without any limitation. Attached Figure Description

[0223] Figure 1 The results are from an ELISA assay to determine the binding activity of anti-CD47 humanized antibody to monkey CD47.

[0224] Figure 2This is the result of an ELISA assay to determine the binding activity of anti-CD47 humanized antibody to human CD47.

[0225] Figure 3 The results are from an ELISA assay to measure the binding activity of anti-CD47 humanized antibody to CD47 on the cell surface.

[0226] Figure 4 These are the results of a red blood cell agglutination test, where RBC is the positive control and PBS is the blank control.

[0227] Figure 5 This is the result of the FACS assay for the blocking activity of anti-CD47 humanized antibody.

[0228] Figure 6 This is the result of an anti-tumor trial using the anti-CD47 humanized antibody Hu34-39-PE in a human gastric cancer NUGC-4 xenograft model.

[0229] Figure 7 This is the result of an anti-tumor trial using the anti-CD47 humanized antibody Hu26T-31-PE in a human gastric cancer NUGC-4 xenograft model.

[0230] Figure 8 This is a schematic diagram of the structure of the dual-antibody ScFab (HuPL7-21Ks / Hu34-39Hs).

[0231] Figure 9 This is a schematic diagram of the ScFabHuPL7-21Ks sequence.

[0232] Figure 10 This is a schematic diagram of the ScFabHu34-39Hs sequence.

[0233] Figure 11 These are the experimental results of the bispecific antibodies ScFab (HuPL7-21Ks / Hu34-39Hs) and ScFab (HuPL16-42Ks / Hu34-39Hs) binding to PD-L1 (A), competing with PD-1 (B), binding with CD47 (C), competing with SIRPα (D), and competing with CD80 (E).

[0234] Figure 12 The results show the experimental findings at the cellular level of the bispecific antibodies ScFab (HuPL7-21Ks / Hu34-39Hs) and ScFab (HuPL16-42Ks / Hu34-39Hs) binding to PD-L1 on the cell surface (A), binding to CD47 on the cell surface (B), blocking the binding of PD-1 to PD-L1 (C), and blocking the binding of SIRPα to CD47 (D).

[0235] Figure 13These are the experimental results of double binding (A, B) and double blocking (B) of CD47 / PD-L1 by bispecific antibodies ScFab (HuPL7-21Ks / Hu34-39Hs) and ScFab (HuPL16-42Ks / Hu34-39Hs) on Raji-hPD-L1 cells.

[0236] Figure 14 The results show the inhibitory effect of the bispecific antibody ScFab (HuPL7-21Ks / Hu34-39Hs) on the growth of xenografts in mice. Detailed Implementation

[0237] The following representative embodiments are provided to better illustrate the invention and are not intended to limit the scope of protection of the invention. Experimental methods not specifically described in the following embodiments are generally performed under conventional conditions, such as those described in Cold Spring Harbor's Antibody Technology Manual, Molecular Cloning Manual, etc., or as recommended by the raw material or commercial manufacturer. Unless otherwise specified, all materials and reagents used in the embodiments are commercially available.

[0238] Example 1: Preparation of CD47 antigen protein and anti-CD47 positive control antibody

[0239] 1. Construction of expression vectors for antigen protein and positive control antibody

[0240] (1) Construction of expression vector for antigen protein

[0241] A gene fragment encoding the full length of CD47 protein was synthesized, and its amino acid sequence was designed as shown in SEQ ID NO:41. It was then cloned into the eukaryotic expression plasmid pTargeT to obtain its expression plasmid pTargeT-CD47.

[0242] The amino acid sequence of the extracellular region of human CD47 protein was fused with the amino acid sequence of hIgG1-Fc or his tag, as shown in SEQ ID NO:42 and SEQ ID NO:43, respectively. After codon optimization of the above amino acid sequences, the tagged extracellular region gene fragments CD47-hFc and CD47-his were synthesized and cloned into the eukaryotic expression plasmid pHR, respectively, to obtain the expression plasmids pHR-CD47-hFc and pHR-CD47-his.

[0243] The amino acid sequence of the extracellular region of human CD47 protein was fused with the amino acid sequence of mIgG1-Fc, and the amino acid sequence design is shown in SEQ ID NO:44. After codon optimization of this amino acid sequence, the complete expression plasmid pcDNA3.1(+)-TPA-CD47-mIgG1-Fc was synthesized.

[0244] The sequence of SIRPα is shown in SEQ ID NO:45. After codon optimization of the sequence, the complete expression plasmid pcDNA3.1(+)-SIRPα-myc-His was synthesized.

[0245] (2) Construction of expression vector for positive control antibody

[0246] The antibody AB6.12-IgG4P (hereinafter referred to as AB06.12-4P) disclosed in patent application WO2013 / 119714 was used as a positive control antibody. The amino acid sequence of AB06.12-4P is shown below:

[0247] AB06.12-4P heavy chain amino acid sequence: SEQ ID NO:46;

[0248] AB06.12-4P light chain amino acid sequence: SEQ ID NO:47.

[0249] The amino acid sequences corresponding to the above antibody sequences were codon-optimized to obtain the heavy and light chain expression plasmids pcDNA3.1(+)-SHC025-hG4 and pcDNA3.1(+)-SHC025-hk for the positive control antibody AB06.12-4P. The heavy chain gene fragment was then cloned into the eukaryotic expression plasmid pHR containing the constant region of the IgG4 light chain, resulting in the eukaryotic expression plasmid pHR-SHC025-hG4-4PE for the heavy chain of AB06.12-4P, and the light chain expression plasmid pcDNA3.1(+)-SHC025-hk.

[0250] 2. Expression and purification of antigen protein and positive control antibody

[0251] (1) Construction of stable cell lines for antigen proteins

[0252] The eukaryotic expression plasmid pTargeT-CD47 was electroporated into CHO-K1 cells (Shanghai Institute of Cell Biology, Chinese Academy of Sciences) at 160V for 15msec using a square pulse. Cells were then cultured at 37℃ in a 5% CO2 incubator. After 24 hours, the cells were cultured under pressure with medium containing 500ug / ml G418. After 16 days, the pooled positivity rate was detected using FACS. Cells seeded with the electroporated plasmid were plated (1x10⁶ cells / mL). 6Cells were incubated at a density of 100 μL / well (cells / ml). The cells were incubated with PE mouse anti-human CD47 antibody (BD, 556046). Mean values ​​at 585 nm were read using a flow cytometer (BD, FACSJazz), and data were analyzed using GraphPad. Positive cell lines were subcloned, and the cloned CHO-K1 cell line, which highly expresses CD47, was selected and named CHO-K1-E5.

[0253] (2) Expression of tag antigen protein and positive control antibody

[0254] The seeding density in a 1L cell culture flask is 0.5 x 10⁶ cells / mL. 6 293F cells per ml were added to fresh, preheated FreeStyle 293 expression medium to achieve a total volume of 250 mL after inoculation. The cells were then incubated overnight in a humidified CO2 incubator at 37°C with 8% CO2. Take 8.5 mL of FreeStyle 293 expression medium, add 500 μL of 1 mg / mL PEI solution, mix well, and add 250 μg of the plasmid to be transfected into 8.5 mL of FreeStyle 293 expression medium, mix well, and transfect the tag antigen protein plasmids pHR-CD47-hFc, pHR-CD47-his, pcDNA3.1(+)-TPA-CD47-mIgG1-Fc, and pcDNA3.1(+)-SIRPα-myc-His separately; co-transfect the positive control antibody AB06.12-4P heavy chain plasmid pHR-SHC025-hG4-4PE and the light chain plasmid pcDNA3.1(+)-SHC025-hk. Add the mixture of PEI and FreeStyle 293 expression medium to the plasmid, mix well, and then add to the cell culture. Incubate at 37°C in an 8% CO2 humidified CO2 incubator. On days 1 and 3 post-transfection, cells were fed with 2.5 ml of glutamine (200 mM stock solution) and 5 ml of glucose (180 g / L stock solution) per flask. When cell viability decreased to 65%–75%, the cell supernatant was collected. The cell culture was centrifuged at 1500 rpm for 5 min, and the supernatant was collected. Then, the culture was centrifuged again at 8000 rpm for 20 min, and the supernatant was collected once more.

[0255] (3) Affinity chromatography column purification

[0256] Purification was performed using AKTA (GE, AKTA pure-150) with different affinity chromatography columns based on the protein properties (see Table 1 for affinity chromatography columns suitable for different proteins). The specific purification steps are as follows:

[0257] Table 1. Affinity chromatography columns adapted to different proteins

[0258]

[0259] Cleaning: Clean the equipment and pipelines with ultrapure water for 2 minutes at a flow rate of 10 mL / min, and then clean the chromatography system with 0.1 M NaOH;

[0260] Column connection: Connect the chromatography column to the chromatography equipment and rinse with ultrapure water for 5 min; then rinse with 0.1M NaOH for 30 min, and retain for 5 min;

[0261] Equilibration: 20mM PB + 0.15M NaCl, pH 7.2, equilibrated for 5 column volumes (CVs);

[0262] Loading: Load the cell expression supernatant and retain for 5 minutes;

[0263] Post-equilibration: 20mM PB + 0.15M NaCl, pH 7.2, equilibrated for 5 CVs;

[0264] Elution: Elute with 50 mM acetic acid, pH 3.4, retention time 5 min. Start collecting when UV280 reaches approximately 50 mAu, and stop collecting when it drops to approximately 50 mAu. Adjust the sample pH to 7.0 with 1 M Tris-HCl, pH 9.0.

[0265] Reequilibration: 20mM PB + 0.15M NaCl, pH 7.2, equilibrate for 3 CVs, retention time 5 min;

[0266] Online cleaning: Clean with 0.1M NaOH for 30 minutes, retain for 5 minutes;

[0267] Cleaning and storage: Wash with purified water for 10 min, then 20% ethanol for 2 CVs.

[0268] Example 2: Preparation of anti-CD47 monoclonal antibody

[0269] 1. Preparation of hybridoma monoclonal antibodies

[0270] (1) Animal Immunization

[0271] SJL strain mice were immunized using a method of co-immunization with anti-CD47 antigen proteins of different labels and adjuvants. The initial antigen used was 50 μg, and the subsequent antigen used was 25 μg.

[0272] The adjuvant can be Quick Antibody-Mouse 5W (Beijing Bio-Long Immunotherapy Co., Ltd.) or Titer Max (Sigma) with CpG (synthesized by Genscript Biotech Co., Ltd.) / Alum (thermo) adjuvant as a separator. CD47 antigen protein samples with different labels are added dropwise to the adjuvant solution, vortexing continuously to ensure thorough mixing. Refer to the manufacturer's instructions for adjuvant dosage. After mixing thoroughly to form a water-in-oil emulsion, SJL mice are immunized.

[0273] Cell lines expressing high levels of CD47 molecules, such as CCRF-CEM and CHO-K1-E5, were also used to immunize mice to induce antibody production. Cultured human acute lymphoblastic leukemia cells (CCRF-CEM) and CHO-K1-E5-positive single cells obtained in Example 1 were digested with trypsin, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cell pellet was resuspended in PBS. Samples were taken and counted using a cell counter. The remaining sample was centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cell pellet was resuspended in PBS. An appropriate amount of PBS was added to obtain 1x10⁻⁶ cells / mL. 8 Cell suspension of 10 cells / ml. Each mouse in the experimental group was immunized with 1x10 cells / ml. 7 Each cell.

[0274] The immunization schedule is shown in Table 2:

[0275] Table 2. Immunization regimens for mice

[0276]

[0277]

[0278] *im (intramuscular injection); sc (subcutaneous injection); ip (intraperitoneal injection).

[0279] (2) Hybridoma fusion

[0280] Spleen cell acquisition and preparation: After booster immunization, mice were sacrificed and immersed in 75% alcohol. The spleen was dissected, ground with a grinding rod, and filtered through a cell sieve to prepare a single-cell suspension. The spleen cell suspension was centrifuged at 2000 rpm for 5 min, and the supernatant was discarded. 2 mL of erythrocyte lysis buffer was added, and the erythrocytes were lysed at room temperature for 2 min. PBS was added to 20 mL, and the suspension was centrifuged at 1500 rpm for 7 min, the supernatant was discarded, and the cells were resuspended for viable cell counting. Sp2 / 0 cells were collected from the culture flask, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, and the cells were resuspended for viable cell counting. A 1:1 ratio of spleen cells to Sp2 / 0 cells was mixed, centrifuged at 1500 rpm for 7 min, and the supernatant was discarded. The cells were resuspended in 20 mL of electroporation buffer and centrifuged at 1500 rpm for 7 min. The supernatant was discarded, and the process was repeated once. Cells were resuspended in appropriate amounts of electroporation buffer to maintain a cell concentration of 2 × 10⁶ cells / mL. 7 Approximately 100 cells / mL. Add the cell suspension to a 9mL electroporation fusion bath for fusion. After fusion, transfer the cell suspension to 15mL RPMI 1640 complete medium containing 20% ​​FBS and incubate at room temperature for 20 min. Resuspend the fused cells in RPMI 1640 medium containing 1×HAT, 1×BIOMYC3, and 20% FBS. Add 100μl of the cell suspension to several 96-well cell culture plates, ensuring approximately 4×10⁶ cells per well. 4 Cells per well were cultured in a 37°C cell culture incubator. After 5 days, 100 μL of RPMI 1640 complete medium (containing 20% ​​FBS, 1×HAT, and 1×BIOMYC-3) was added per well.

[0281] (3) Screening of hybridomas and subclonal supernatants

[0282] One week after fusion, cell supernatants were collected, and hybridoma supernatants capable of binding CD47-his protein or CD47 on the cell surface were screened using ELISA. Antibodies targeting CD47 but not hFc or mFc were then screened using CD47-his. The ability of the hybridoma supernatants to block CD47-SIRPα interaction was then analyzed using ELISA. SIRPα-myc-his was coated onto an ELISA plate, and a mixture of recombinant human protein CD47-hFc and hybridoma supernatant was added and incubated for 2 hours. Then, HRP-labeled anti-human IgG Fc-specific antibody (Jackson Immuno Research) was added and incubated for 1 hour. The absorbance at 450 nm was measured using an ELISA reader. The hybridoma parent clones with binding and blocking abilities were expanded and cultured, and their binding and blocking activities were retested. Positive hybridoma clones with both binding and blocking abilities were obtained through further screening.

[0283] Positive cell lines were subcloned using the limiting dilution method. After one week of culture, the binding activity of the subclone supernatant to CD47 molecules and the activity of blocking CD47-SIRPα interaction were detected by ELISA. Three double-positive cell lines were obtained and labeled as SHC025-26, SHC025-34, and SHC025-58, respectively.

[0284] 2. Subtype identification

[0285] Antibody subtypes were identified according to the instructions of the SBA Clonotyping Systerm-C57BL / 6-HRP (SouthernBiotech, catalog number: 5300-05B) mouse antibody subtype identification kit. The results are shown in Table 3.

[0286] Table 3. Results of Antibody Subtype Identification

[0287] name Antibody subtypes SHC025-26 IgG1 / k SHC025-34 IgG2c / k SHC025-58 IgG2b / k

[0288] 3. Preparation of monoclonal antibodies

[0289] Based on the subclonal supernatant activity analysis, the parent monoclonal antibody clones SHC025-26, SHC025-34, and SHC025-58 were identified and expanded. The culture conditions were 1640 medium containing 10% fetal bovine serum, 1x NAEE, 1x sodium pyruvate, and 1% penicillin-streptomycin. When cell confluence was >80%, the cells were passaged and expanded. When the culture volume reached approximately 50 ml, the supernatant was collected, and the antibodies were purified. The obtained antibodies were confirmed to have good purity by SDS-PAGE gel electrophoresis.

[0290] 4. Monoclonal antibody sequencing

[0291] The subcloned positive hybridoma cells were expanded and cultured. An appropriate amount of cells were taken and total RNA was extracted according to the instructions of the RNeasy Plus Mini Kit (Qiagen, 74134). The first strand of cDNA was synthesized using the Prime Script 1st strand cDNA Synthesis Kit (Takara, 6110A) reverse transcription kit.

[0292] Based on the variable region of mouse antibody subtypes, specific primers were designed (containing homologous arm sequences at the 5' end for homologous recombination with eukaryotic expression vectors). PCR amplification of the antibody variable region gene was performed using cDNA as a template, thereby obtaining gene fragments of the mouse antibody light and heavy chain variable regions, respectively. Primers were designed (References: 1. Anke Krebber, Susanne Bornhauser, Jorg Burmester et al. Reliable cloning of functional antibody variable domains from hybridomas and spleen cell repertoires employing areengineered phage display system. Journal of Immunological Methods, 1997, 201: 35–55; 2. Simon Koren Miha...). Colja Venturini et al. Antibody variable-region sequencing as a method for hybridoma cell-line authentication, 2008, 78:1071–1078), DNA sequencing was performed to obtain the sequence, and the sequencing results are shown in Table 4.

[0293] Table 4 Sequence List of Anti-CD47 Murine Monoclonal Antibodies

[0294] Antibody Heavy chain variable region amino acid sequence Light chain variable region amino acid sequence SHC025-26 SEQ ID NO:21 SEQ ID NO:24 SHC025-34 SEQ ID NO:22 SEQ ID NO:25 SHC025-58 SEQ ID NO:23 SEQ ID NO:26

[0295] The CDR sequences of antibody SHC025-34 for VH are SEQ ID NO: 4, 5, and 6, respectively; the CDR sequences of VL are SEQ ID NO: 15, 16, and 17, respectively. Example 3: Construction of anti-CD47 chimeric antibody.

[0296] The purified mouse antibody light chain and heavy chain variable region gene fragments (purification steps are described in Example 1) were co-transformed with linearized eukaryotic expression plasmids containing human antibody light chain or heavy chain constant regions into E. coli DH5α competent cells. The mixture was evenly spread on the surface of agar plates containing the corresponding antibiotics and incubated overnight at 37°C. Several single colonies were then picked for DNA sequencing. The chimeric antibodies that were correctly sequenced were labeled as SHC025-26CHI, SHC025-34CHI, and SHC025-58CHI, respectively.

[0297] Positive clones with correct sequencing were inoculated into 2×YT liquid medium containing the corresponding antibiotics and cultured at 37°C with shaking for more than 12 hours. Then, the bacterial cells were collected for plasmid extraction to obtain chimeric antibody light chain and heavy chain expression plasmids. The concentration and purity of the plasmids were detected using a nucleic acid quantification analyzer.

[0298] The chimeric antibody was transfected into HEK293E cells, expressed and purified to obtain a large amount of antibody, and then subjected to purity testing, activity analysis and affinity testing.

[0299] Sequencing revealed that each of the heavy chain CDRs in SHC025-26 contains a cysteine ​​residue at position 118, and the light chain CDR in SHC025-58 contains a cysteine ​​residue at position 56. During expression, these cysteine ​​residues in the CDR region randomly pair with other cysteine ​​residues on the antibody molecule, forming disulfide bridges and significantly affecting antibody purity. To address this issue, the amino acid sequences of SHC025-26CHI and SHC025-58CHI were modified as follows: C118 of the heavy chain in SHC025-26CHI was mutated to T, labeled as SHC025-26CHI-T; C56 of the light chain in SHC025-58CHI was mutated to A, labeled as SHC025-58CHI-A. The mutant genes were constructed using site-directed mutagenesis. The sequencing results of the chimeric antibody are shown in Table 5.

[0300] Table 5. Sequence listing of anti-CD47 chimeric antibodies

[0301]

[0302]

[0303] The CDR sequences of VH of antibody SHC025-34CHI are SEQ ID NO:4, 5, and 6, respectively, and the CDR sequences of VL are SEQ ID NO:15, 16, and 17, respectively.

[0304] Example 4: Construction and Production of Anti-CD47 Humanized Antibody

[0305] Based on the results of chimeric antibody activity analysis and affinity KD value, SHC025-34CHI, SHC025-58CHI-A, and SHC025-26CHI-T were selected for humanized antibody modification.

[0306] The humanization of antibodies began with comparison with mouse antibody sequences in the Immunogenetic Database (IMGT). Mouse lineages for the variable regions of the SHC025-34CHI, SHC025-58CHI-A, and SHC025-26CHI-T antibodies were identified. Homology comparison revealed that the FR regions of the heavy chain variable regions of these antibodies were most similar to human antibody germline genes IGHV1-8*01, IGHV3-21*04, and IGHV1-2*02, respectively. The FR sequences of the light chain variable regions were most similar to human antibody germline genes IGKV3-11*01, IGKV1-5*01, and IGKV4-1*01, respectively. Using the FR1-FR3 frames of the SHC025-34CHI / SHC025-58CHI-A antibody as templates, a fully human frame region with similar 3D structure but lower immunogenicity was searched in the human frame region library to replace the FR1-FR3 sequences of SHC025-34CHI / SHC025-58CHI-A. 3D modeling of the full-length heavy / light chain sequences was performed, and structural alignment analysis was conducted with the original antibody heavy / light chain sequences. Considering both antigenicity and 3D structural similarity, six humanized heavy chain variable regions (see SEQ ID NO: 48, 49, 50, 51, 52, 53) and four humanized light chain variable regions (see SEQ ID NO: 54, 55, 56, 57) of SHC025-34CHI and six humanized heavy chain variable regions (see SEQ ID NO: 54, 55, 56, 57) of SHC025-58CHI-A were finally selected. Further optimization was performed on the variable regions of the heavy and light chains (SEQ ID NOs: 58, 59, 60, 61, 62, 63) and 5 humanized light chain variable regions (SEQ ID NOs: 64, 65, 66, 67, 68). The non-CDR regions of the SHC025-34CHI / SHC025-58CHI-A humanized antibody achieved over 95% humanization. The variable region sequences of the heavy and light chains of SHC025-26CHI-T were used for structural alignment analysis in Protein Data Bank. The closest FR1-FR3 sequences were selected to replace the murine sequences, and amino acid sites that played a crucial role in antibody structural stability in structural simulations were mutated to murine amino acid residues. Finally, 4 humanized heavy chain variable regions (SEQ ID NOs: 69, 70, 71, 72) and 2 humanized light chain variable regions (SEQ ID NOs: 73, 74) of SHC025-26CHI-T were obtained.

[0307] The designed humanized antibody light chain and heavy chain variable region amino acid sequences were reverse transcribed into corresponding nucleotide sequences, generating oligonucleotide fragments with complementary sequences between adjacent fragments. These oligonucleotide fragments were annealed and ligated using overlap PCR, and then the complete light chain and heavy chain variable region nucleotide fragments were amplified using specific primers (containing homologous arm sequences at the 5' end for homologous recombination with the eukaryotic expression vector). The purified light chain variable region nucleotide fragments were co-transformed into *E. coli* DH5α competent cells with a linearized eukaryotic expression plasmid containing the IgG4 light chain constant region. Similarly, the purified heavy chain variable region nucleotide fragments were co-transformed into *E. coli* DH5α competent cells with a eukaryotic expression plasmid containing the S228P / L235E mutant IgG4 heavy chain constant region. The transformed competent cells were evenly spread on agar plates containing the corresponding antibiotics and incubated overnight at 37°C. Single colonies were then picked for DNA sequencing.

[0308] Positive clones with correct sequencing were inoculated into 2×YT liquid medium containing the corresponding antibiotics and cultured at 37°C with shaking for more than 12 hours. Then, the bacterial cells were collected for plasmid extraction to obtain humanized antibody light chain and heavy chain expression plasmids. The concentration and purity of the plasmids were detected using a nucleic acid quantification analyzer.

[0309] The plasmid was transfected into HEK293E cells, and a large amount of antibody was obtained through expression and purification. The purity, activity and affinity were then tested.

[0310] Humanized antibodies with good purity, activity, and affinity were selected and labeled as Hu26T-31-PE, Hu34-39-PE, and Hu58A-14-PE, with sequences shown in Table 6. The sources of the humanized antibodies are shown in Table 7.

[0311] Table 6 Sequence List of Anti-CD47 Humanized Antibodies

[0312]

[0313] The CDR sequences of VH of antibody Hu34-39-PE are SEQ ID NO:4, 5, and 6, respectively, and the CDR sequences of VL are SEQ ID NO:15, 16, and 17, respectively.

[0314] Table 7 Humanized Sequence Design Information

[0315]

[0316]

[0317] Example 5: Assay of anti-CD47 antibody binding activity to monkey CD47 (ELISA)

[0318] The binding activity of the antibodies was analyzed using protein-based ELISA. Cynomolgus monkey CD47-His (0.1 μg / well, ACRO Biosystems, Cat. No. CD7-C52H1-50ug) was coated onto 96-well microplates. The anti-CD47 antibody provided in this invention was added to the microplate as a primary antibody starting at 2 μg / mL, with 5-fold serial dilutions, resulting in 8 concentrations: 2000 ng / mL, 400 ng / mL, 80 ng / mL, 16 ng / mL, 3.2 ng / mL, 0.64 ng / mL, 0.128 ng / mL, and 0 ng / mL. The plates were incubated at 37°C for 1.5 h. The positive control antibody was AB06.12-4P. The secondary antibody used was Anti-Human IgG HRP (Jackson, 109-035-003, 1:10000), with the addition of chromogenic buffer TMB (3,3',5,5'-tetramethylbenzidine). After termination, the OD450 value was read using a microplate reader (Thermo, Multiskan FC). EC was generated using GraphPad. 50 The result is as follows Figure 1 As shown.

[0319] Experimental results show that the humanized anti-CD47 antibodies Hu26T-31-PE, Hu34-39-PE, and Hu58A-14-PE provided by this invention all have the ability to bind to CD47 in cynomolgus monkeys, and the binding ability is comparable to that of the positive control antibody AB06.12-4P.

[0320] Example 6: Assay of anti-CD47 antibody binding activity to human CD47 (ELISA)

[0321] The binding activity of the antibody was analyzed by ELISA. Human CD47-His protein (0.1 μg / well, prepared in Examples 1 and 2) was coated onto a 96-well microplate and incubated at 37°C for 2 h. After washing three times with 1xPBST, the plate was blocked overnight at 4°C with 5% skim milk. After washing three times with 1xPBST, the anti-CD47 antibody provided in this invention was added to the ELISA plate as the primary antibody, starting at 2 μg / mL and serially diluted 5-fold, for a total of 8 concentrations: 2000 ng / mL, 400 ng / mL, 80 ng / mL, 16 ng / mL, 3.2 ng / mL, 0.64 ng / mL, 0.128 ng / mL, and 0 ng / mL. The plate was incubated at 37°C for 1.5 h. The positive control antibody was AB06.12-4P. After washing five times with 1xPBST, the secondary antibody was Anti-Human IgG HRP (Jackson, 109-035-003, 1:10000), and incubated at 37°C for 40 min. After washing five times with 1xPBST, the chromogenic solution TMB was added, and the OD450 value was read using a ELISA reader (Thermo, Multiskan FC). EC was generated using GraphPad. 50 The result is as follows Figure 2 As shown.

[0322] Experimental results show that the humanized anti-CD47 antibodies Hu26T-31-PE, Hu34-39-PE, and Hu58A-14-PE provided by this invention all have the ability to bind to human CD47, and the binding ability is comparable to that of the positive control antibody AB06.12-4P.

[0323] Example 7: Assay of anti-CD47 antibody binding to CD47 on cell surface (ELISA)

[0324] Antibody binding activity was analyzed using cell-based ELISA. CHO-K1-E5 cells were used at 1 x 10⁶ cells per well. 5Cells were seeded in a single-cell culture plate and incubated overnight at 37°C with 5% CO2. The next day, cells were fixed with 4% paraformaldehyde and blocked with skim milk for 1 hour. The cells were then gently washed with 1xPBS. The anti-CD47 antibody provided in this invention was added to the cells as the primary antibody in 5-fold serial dilutions, starting at 2 μg / mL, for a total of 8 concentrations: 2000 ng / mL, 400 ng / mL, 80 ng / mL, 16 ng / mL, 3.2 ng / mL, 0.64 ng / mL, 0.128 ng / mL, and 0 ng / mL. Cells were incubated at 37°C for 1.5 hours. The positive control antibody was AB06.12-4P. The secondary antibody was Anti-Human IgG HRP (Jackson, 109-035-003, 1:10000), with TMB chromogenic buffer added. After termination, the OD450 value was read using a microplate reader (Thermo, Multiskan FC). EC was generated using GraphPad. 50 The result is as follows Figure 3 As shown.

[0325] Experimental results show that the humanized anti-CD47 antibodies Hu26T-31-PE, Hu34-39-PE, and Hu58A-14-PE provided by this invention can all bind to CD47 on the cell surface, and their binding ability is comparable to that of the positive control antibody AB06.12-4P.

[0326] Example 8: Determination of the affinity between anti-CD47 antibody and human CD47 protein

[0327] The affinity of the humanized anti-CD47 antibody prepared in Examples 1 and 2 for the antigen CD47(19-136)-hFC was determined using the Fortebio Octet. First, the antigen CD47(19-136)-hFc was biotinylated and then desalted 3-4 times by centrifugation with PBS using a 10kD ultrafiltration tube. The actual concentration of the biotinylated antigen CD47-hFc-Biotin was determined using Nanodrop. CD47-hFc-Biotin was diluted to 5ug / ml with SD buffer (0.02% Tween 20 + 0.1% BSA solution). The humanized anti-CD47 antibody was serially diluted 4-fold with SD buffer to concentrations of 10ug / ml, 2.5ug / ml, 0.625ug / ml, and 0ug / ml. The antigen was immobilized using an SA sensor, and affinity was determined according to the operating procedure of the Fortebio Octet RED96. Specific parameters and experimental results are shown in Table 8.

[0328] Table 8. Affinity determination with human CD47 protein

[0329]

[0330] Experimental results showed that, compared with the positive control antibody, the humanized anti-CD47 antibody Hu26T-31-PE had a higher affinity for binding to human CD47 protein.

[0331] Example 9: Erythrocyte agglutination assay using anti-CD47 antibody

[0332] Add 5 mL of blood to 40 mL of PBS, centrifuge gently at 2000 rpm for 5 min, discard the supernatant, wash three times with PBS, and then resuspend the red blood cells in PBS. Prepare a 2% red blood cell suspension according to the hematocrit. The initial antibody concentration to be analyzed is between 1-20 μM, serially diluted 2-fold, for a total of 24 concentration gradients. Add 50 μL of the above different concentrations of antibody to a round-bottom 96-well plate, then add 50 μL of the above 2% red blood cell suspension, mix well, and incubate at room temperature. Observe for agglutination after 2 hours. Rabbit polyclonal antibody RBC antibody (Rockland, 109-4139) was used as a positive control for red blood cell agglutination. The results are as follows. Figure 4 As shown. Figure 4 As shown, the antibodies (rabbit polyclonal antibody RBC, AB06.12-4P antibody, and the antibody to be tested in this invention) added to the 96-well plate from left to right were serially diluted 2-fold starting from 20 μM. RBC represents the positive control group (using rabbit polyclonal antibody RBC, which significantly induced erythrocyte agglutination), and PBS represents the blank control group. Small, round dots with neat edges indicate no cell agglutination; slightly irregular edges indicate a small amount of erythrocyte agglutination; and sheets covering the bottom of the wells indicate most erythrocyte agglutination.

[0333] Testing revealed that the anti-CD47 antibody Hu5F9-G4 disclosed in patent application WO2011 / 143624 caused significant agglutination of most erythrocytes within the same concentration range, a common adverse phenomenon of anti-CD47 antibodies. However, under the same conditions, as experimental results show, Hu26T-31-PE, Hu34-39-PE, and Hu58A-14-PE of this invention did not cause erythrocyte agglutination. Therefore, the antibodies of this invention are significantly superior to antibody Hu5F9-G4 in this respect.

[0334] Example 10: Determination of the CD47 blocking activity of anti-CD47 antibody

[0335] The ability of the anti-CD47 antibody provided in this invention to block SIRPa from binding to CD47 on the cell surface was detected using FACS.

[0336] CHO-K1-E5 positive cell lines were used as CD47 providers. The binding ability of CD47 to SIRPa was observed in the presence of serially diluted anti-CD47 antibody. PE Streptavidin (Biolegend, 405203, 1:200) was used as a secondary antibody to monitor changes in SIRPa-Biotin levels. AB06.12-4P served as a positive control to block SIRPa binding to CD47 on the cell surface. Flow cytometry (BD, FACSJazz) was used to read the mean value at 585 nm, and IC50 was generated using GraphPad. The results are shown below. Figure 5 As shown.

[0337] Experimental results showed that the blocking activities were ordered as follows: Hu26T-31-PE≧Hu34-39-PE≧AB06.12-P>Hu58A-14-PE.

[0338] Example 11: Anti-tumor assay of anti-CD47 antibody in a human gastric cancer NUGC-4 xenograft model

[0339] 1. Experimental Materials

[0340] (1) Experimental cells and animals

[0341] The NUGC-4 human gastric cancer cells were purchased from the American Type Culture Collection (ATCC).

[0342] NOD-Scid mice, female, 5-8 weeks old, weighing 18-20 grams, were purchased from Shanghai Lingchang Biotechnology Co., Ltd.

[0343] (2) Test sample and reference sample

[0344] The control standard Isotype IgG4 (catalog number AB170091) was purchased from Crown Bioscience Inc. and used as a negative control.

[0345] Before the experiment, the humanized anti-CD47 antibody of the present invention was prepared with PBS to two concentrations of 0.6 mg / mL and 0.3 mg / mL, and Isotype IgG4 and AB 06.12-4P were prepared to 0.6 mg / mL.

[0346] (3) Experimental methods

[0347] NUGC-4 human gastric cancer cells were cultured in RMPI1640 medium containing 10% fetal bovine serum, 100 U / mL penicillin, and 100 μg / mL streptomycin in an incubator at 37°C and 5% CO2. Cells were passaged weekly with 2 mL of 1×EDTA solution. When cell saturation reached 80%-90%, cells were harvested, counted, and seeded. Cells containing 5×10⁶ cells were then introduced into a culture medium. 6Cells were mixed with 100 μL of Matrigel in PBS (final volume 200 μL) and inoculated into the right posterior side of mice, with a cell number of 5 × 10⁶ cells. 6 / each. Wait until the tumor grows to a size of 150-200 mm. 3 Grouping began at that time. Intraperitoneal administration was administered three times a week. Tumor diameter was measured three times a week using calipers, and tumor volume was calculated using the formula: V = 0.5a × b 2 , where a and b represent the long and short diameters of the tumor, respectively. The antitumor efficacy of the antibody was evaluated using the relative tumor proliferation rate (T / C%). The relative tumor proliferation rate (T / C%) was calculated as follows: T / C% = TRTV / CRTV × 100% (TRTV: RTV of the treatment group; CRTV: RTV of the negative control group). RTV = V21 / V0, where V0 is the tumor volume measured at the time of administration (d0), and V21 is the tumor volume measured on day 21 after administration. The tumor volumes of the treatment group and the solvent group on the last day (day 21) were analyzed using a T-test, performed with GraphPad Prism. The results are shown in Table 9.

[0348] Table 9 Results of anti-tumor trials using the NUGC-4 human gastric cancer xenograft model

[0349]

[0350] ***p<0.001 vs.Isotype IgG4; **p<0.005 vs.Isotype IgG4

[0351] Experimental results showed that the antibodies provided by this invention had significant antitumor effects in a NOD-SCID mouse xenograft model of human gastric cancer NUGC-4 cells. The tumor-suppressing effects of Hu26T-31-PE and Hu34-39-PE at a dose of 3 mg / kg were comparable to those of the reference antibody AB06.12-4P at a dose of 6 mg / kg, while the tumor-suppressing effects of Hu26T-31-PE and Hu34-39-PE at a dose of 6 mg / kg were superior to those of the reference antibody AB06.12-4P at a dose of 6 mg / kg. One week after drug withdrawal, tumor recurrence occurred in the 6 mg / kg reference antibody group, while no recurrence was observed in the Hu26T-31-PE and Hu34-39-PE groups. Figure 6 , Figure 7 The invention suggests that the anti-CD47 antibody provided by this invention unexpectedly has a more significant effect in inhibiting tumor growth.

[0352] Example 12: Obtaining Anti-human PD-L1 Antibody

[0353] 1. Animal immunization

[0354] BALB / c and SJL strain mice and SD strain rats were immunized with mFc-tagged PD-L1 antigen protein (purchased from Beijing Baipusaisi Biotechnology Co., Ltd.) and adjuvant.

[0355] The initial adjuvant was Freund's Adjuvant, Complete (SIGMA, F5881-10ML), followed by Freund's Adjuvant, Incomplete (SIGMA, F5506-10ML). PD-L1 antigen protein samples with different labels were added dropwise to the adjuvant solution, vortexing constantly to ensure thorough mixing. The dosage of the adjuvant was followed according to the manufacturer's instructions. After mixing thoroughly to form a water-in-oil emulsion, mice or rats were immunized. The immunization protocol is shown in Table 10.

[0356] Table 10 Immunization Regimens

[0357]

[0358] *intramuscular injection

[0359] 2. Cell fusion

[0360] Mouse spleens were aseptically harvested and prepared into cell suspensions. Cell fusion was performed at a spleen cell to Sp2 / 0 cell ratio of 1:1. The fused cell suspension was transferred to 15 mL of RPMI 1640 complete medium containing 20% ​​FBS and incubated at room temperature for 20 min. The fused cells were resuspended in RPMI 1640 medium containing 1×HAT, 1×BIOMYC3, and 20% FBS. The cell suspension was added at 100 μL / well to several 96-well cell culture plates, ensuring approximately 4 × 10⁶ cells per well. 4 Cells per well were cultured in a 37°C cell culture incubator. After 5 days, 100 μL of RPMI 1640 complete medium (containing 20% ​​FBS, 1×HAT, and 1×BIOMYC-3) was added per well.

[0361] 3. Screening for positive clones

[0362] One week after fusion, cell supernatants were collected, and hybridoma mother clones with binding and blocking abilities were screened by ELISA and expanded for culture. Binding and blocking activities were retested, and hybridoma-positive cell lines with binding and blocking abilities were obtained through further screening. The positive cell lines were subcloned using limiting dilution. After one week of culture, the binding activity of the subclone supernatant to PD-L1 molecules and the activity blocking PD-L1 / PD-1 interaction were detected by ELISA, yielding four preferred double-positive cell lines: PL-7, PL-15, PL-16, and PL-18.

[0363] 4. Obtaining the variable region sequence of the anti-PD-L1 antibody

[0364] The subcloned positive hybridoma cells were expanded and cultured. An appropriate amount of cells were taken and total RNA was extracted according to the instructions of the RNeasy Plus Mini Kit (Qiagen, 74134). The first strand of cDNA was synthesized using the Prime Script 1st strand cDNA Synthesis Kit (Takara, 6110A) reverse transcription kit.

[0365] Based on the mouse antibody subtype variable region, specific primers were designed (containing a homologous arm sequence at the 5' end for homologous recombination with the eukaryotic expression vector). PCR amplification of the antibody variable region gene was performed using cDNA as a template, thereby obtaining gene fragments of the mouse antibody light and heavy chain variable regions, named SHS009PL-7, SHS009PL-15, SHS009PL-16, and SHS009PL-18, respectively. Primers were designed (References: 1. Anke Krebber, Susanne Bornhauser, Jorg Burmester et al. Reliable cloning of functional antibody variable domains from hybridas and spleen cell repertoires employing a reengineered phage display system. Journal of Immunological Methods, 1997, 201:35–55; 2. Simon Koren Miha...). Colja Venturini et al. Antibody variable-region sequencing as a method for hybridoma cell-line authentication, 2008, 78:1071–1078), DNA sequencing was performed to obtain the sequence, and the sequencing results are shown in Table 11.

[0366] Table 11 Sequence List of Anti-PD-L1 Murine Monoclonal Antibodies

[0367]

[0368] 5. Construction of chimeric antibodies

[0369] The purified mouse antibody light chain and heavy chain variable region gene fragments were co-transformed into E. coli DH5α competent cells with linearized eukaryotic expression plasmids containing human antibody light chain or heavy chain constant region gene fragments, respectively. The chimeric antibodies with correct sequencing were selected and labeled as PL-7CHI, PL-15CHI, PL-16CHI, and PL-18CHI, respectively. The sequencing results of the chimeric antibodies are shown in Table 12.

[0370] The amino acid sequences of the light and heavy chain variable regions of PL-7CHI, PL-15CHI, PL-16CHI, and PL-18CHI are identical to the amino acid sequences of the light and heavy chain variable regions of the murine antibodies SHS009PL-7, SHS009PL-15, SHS009PL-16, and SHS009PL-18, respectively.

[0371] Chimeric antibody light chain plasmids and heavy chain plasmids were extracted and transfected into HEK 293F cells. After expression and purification, a large number of antibodies were obtained and subjected to purity detection, activity analysis and affinity detection.

[0372] Table 12 Sequence Listing of Anti-PD-L1 Chimeric Antibodies

[0373] chimeric antibodies Heavy chain variable region amino acid sequence Light chain variable region amino acid sequence PL-7CHI SEQ ID NO: 99 SEQ ID NO: 103 PL-15CHI SEQ ID NO: 100 SEQ ID NO: 104 PL-16CHI SEQ ID NO: 101 SEQ ID NO: 105 PL-18CHI SEQ ID NO: 102 SEQ ID NO: 106

[0374] 6. Humanization of anti-PD-L1 antibodies

[0375] Based on the results of chimeric antibody activity analysis and affinity KD value, PL-7CHI and PL-16CHI were selected for humanized antibody modification.

[0376] Humanization of the murine monoclonal chimeric antibodies PL-7CHI and PL-16CHI was performed using the classic CDR transplantation strategy. The FR1-FR3 frames of the PL-7CHI and PL-16CHI antibodies were used as templates. Fully human frames with similar 3D structures but lower immunogenicity were searched in the human frame region library to replace the FR1-FR3 sequences of PL-7CHI / PL-16CHI. Homology analysis showed that the FR regions of the heavy chain variable regions of the PL-7CHI and PL-16CHI antibodies were most similar to the human antibody germline genes M99683|IGHV4-31*02 (SEQ ID NO:107) and X62109|IGHV1-3*01 (SEQ ID NO:108), respectively. The FR regions of the light chain variable regions of the PL-7CHI and PL-16CHI antibodies were most similar to the human antibody germline gene Z00023|IGKV4-1*01 (SEQ ID NO:109). The full-length humanized heavy / light chain sequences were 3D modeled and structurally compared with the original antibody heavy / light chain sequences. Taking into account antigenicity and 3D structural similarity, amino acid sites that play a key role in antibody structural stability were mutated to murine amino acid residues in the structural simulation. Finally, five humanized heavy chains of PL-7CHI (PL-7CHI humanized heavy chain variable region sequences: VH1-0 (SEQ ID NO:110), VH1-1 (SEQ ID NO:111), VH1-2 (SEQ ID NO:112), VH1-3 (SEQ ID NO:113) or VH1-4 (SEQ ID NO:114)) and four humanized light chains (PL-7CHI humanized light chain variable region sequences: VL1-0 (SEQ ID NO:115), VL1-1 (SEQ ID NO:116), VL1-2 (SEQ ID NO:117) or VL1-3 (SEQ ID NO:118)) and five humanized heavy chains of PL-16CHI (PL-16CHI humanized heavy chain variable region sequences: VH1-0 (SEQ ID NO:119), VH1-1 (SEQ ID NO:120), VH1-2 (SEQ ID NO:119), VH1-1 (SEQ ID NO:111), VH1-2 (SEQ ID NO:112), VH1-3 (SEQ ID NO:113), VH1-4 (SEQ ID NO:114)) were obtained. Based on this, we obtained multiple humanized antibodies through different combinations of light and heavy chains. After activity testing, the highest-scoring anti-PD-L1 humanized antibody sequences were identified as HuPL7-21 and HuPL16-42. The sequences included SEQ ID NO:121, VH1-3 (SEQ ID NO:122) or VH1-4 (SEQ ID NO:123), and three humanized light chains (PL-16CHI humanized light chain variable region sequences: VL1-0 (SEQ ID NO:124), VL1-1 (SEQ ID NO:125), or VL1-2 (SEQ ID NO:126)).

[0377] Example 13 Production of Humanized Anti-PD-L1 Antibody

[0378] The designed humanized antibody light chain and heavy chain variable region amino acid sequences were synthesized into corresponding nucleotide coding sequences, and oligonucleotide fragments containing complementary sequences between adjacent fragments were generated. The oligonucleotide fragments were annealed and ligated by overlap PCR, and then the complete light chain and heavy chain variable region nucleotide fragments were amplified using specific primers (containing homologous arm sequences at the 5' end for homologous recombination with eukaryotic expression vectors). The purified light chain variable region nucleotide fragments were co-transformed into E. coli DH5α competent cells with a linearized eukaryotic expression plasmid containing the IgG4 light chain constant region, and the purified heavy chain variable region nucleotide fragments were co-transformed into E. coli DH5α competent cells with a eukaryotic expression plasmid containing the S228P / L235E mutant IgG4 heavy chain constant region. The competent cells transformed with the plasmids were evenly spread on the surface of agar plates containing the corresponding antibiotics and incubated overnight at 37°C. Several single colonies were then picked for DNA sequencing.

[0379] Plasmids were extracted from the correctly sequenced positive clones to obtain humanized antibody light and heavy chain expression plasmids. The concentration and purity of the plasmids were then detected using a nucleic acid quantification analyzer.

[0380] The plasmid was transfected into HEK293 F cells, and a large amount of antibody was obtained through expression and purification. Purity, activity, and affinity were then analyzed. The sequence is shown in Table 13.

[0381] Table 13 Sequence List of Anti-PD-L1 Humanized Antibodies

[0382]

[0383] The CDR sequences of the VH of antibody HuPL7-21 are SEQ ID NO: 75, 76, and 77 for CDR1, CDR2, and CDR3, respectively, and the CDR sequences of the VL are SEQ ID NO: 78, 79, and 80, respectively. The CDR sequences of the VH of antibody HuPL16-42 are SEQ ID NO: 87, 88, and 89 for CDR1, CDR2, and CDR3, respectively, and the CDR sequences of the VL are SEQ ID NO: 90, 91, and 92, respectively.

[0384] Example 14 Construction, expression and purification of anti-CD47 / PD-L1 bispecific antibody

[0385] 1. Construction of a bispecific anti-CD47 / anti-PD-L1 antibody expression vector

[0386] Bispecific antibodies against CD47 and PD-L1 were constructed using genetic engineering techniques, and their structural forms are as follows: Figure 8 As shown: This bispecific antibody is formed by heterodimerization of two single-chain antibodies, one anti-PD-L1 and the other anti-CD47. Unlike natural IgG antibodies, in this bispecific antibody, the light chains of both the anti-PD-L1 and anti-CD47 antibodies are linked to the N-terminus of the antibody heavy chain by additional flexible linker peptides. These linker peptides are GGGGS repeat sequences containing glycine (G) and serine (S) residues, preferably containing 8 GGGGS repeat sequences. In addition, to promote the formation of heterodimers, based on the above-mentioned S228P / L235E mutation, the S354C / T366W mutation is added to the CH3 domain of the anti-PD-L1 antibody single chain, and the Y349C / T366S / L368A / Y407V mutation is added to the CH3 domain of the anti-CD47 antibody single chain. Based on this structural form, using the above-mentioned anti-PD-L1 antibody HuPL7-21 (light chain variable region SEQ ID NO: 116 and constant region CL SEQ ID NO: 131) or HuPL16-42 (light chain variable region SEQ ID NO: 126 and constant region CL SEQ ID NO: 131) sequence, single-chain nucleotide fragments of anti-PD-L1 antibody, namely ScFabHuPL7-21Ks or ScFabHuPL16-42Ks, are obtained through gene synthesis; using the above-mentioned anti-CD47 antibody Hu34-39 (light chain variable region SEQ ID NO: 33 and constant region CL SEQ ID NO: 131) sequence, single-chain nucleotide fragments of anti-CD47 antibody, namely ScFabHu34-39Hs, are obtained through gene synthesis. The nucleotide fragments ScFabHuPL7-21Ks (or ScFabHuPL16-42Ks) and ScFabHu34-39Hs (containing homologous arms of appropriate length both upstream and downstream) were co-transformed into E. coli DH5α competent cells with the linearized eukaryotic expression plasmid pHR. The transformed competent cells were evenly spread on agar plates containing the corresponding antibiotics and incubated overnight at 37°C. Single colonies were then picked for DNA sequencing. Positive clones with correct sequencing results were subjected to plasmid extraction to obtain the ScFabHuPL7-21Ks (or ScFabHuPL16-42Ks) and ScFabHu34-39Hs expression vectors.

[0387] A schematic diagram of the ScFabHuPL7-21Ks sequence is shown below. Figure 9 As shown,

[0388] Among them: HuPL7-21VL-CL: humanized PD-L1 monoclonal antibody HuPL7-21 light chain;

[0389] (GGGGS)8: A flexible linker peptide consisting of 8 GGGGS repeat sequences;

[0390] HuPL7-21VH: The heavy chain variable region of the humanized PD-L1 monoclonal antibody HuPL7-21;

[0391] IgG4CH / Ks: Contains the IgG4 heavy chain constant region with a "Knobs" structure formed by the S228P / L235E / S354C / T366W mutation (specific sequence example SEQ ID NO:132);

[0392] A schematic diagram of the ScFabHu34-39Hs sequence is shown below. Figure 10 As shown,

[0393] Among them: Hu34-39VL-CL: humanized CD47 monoclonal antibody Hu34-39 light chain;

[0394] (GGGGS)6: A flexible linker peptide consisting of six GGGGS repeat sequences;

[0395] Hu34-39VH: Humanized CD47 monoclonal antibody Hu34-39 heavy chain variable region;

[0396] IgG4CH / Hs: Contains the IgG4 heavy chain constant region with a “Hole” structure formed by the S228P / L235E / Y349C / T366S / L368A / Y407V mutation (specific sequence as shown in SEQ ID NO:133).

[0397] The CL sequence in the above structure is shown in SEQ ID NO:131.

[0398] 2. Transient expression of anti-CD47 / anti-PD-L1 bispecific antibody in Expi-CHO cells

[0399] Expi-CHO cells were transfected with the co-recombinant plasmids of the expression vectors ScFabHuPL7-21Ks (or ScFabHuPL16-42Ks) and ScFabHu34-39Hs using the Expi-Fectamine CHO Transfection Kit. After culturing in serum-free medium for 14 days, the supernatant of Expi-CHO cells was collected, and the expression of bispecific antibodies was detected by Western blotting. The bispecific antibody formed by the dimerization of the two single-chain molecules ScFabHuPL7-21Ks and ScFabHu34-39Hs was named ScFab(HuPL7-21Ks / Hu34-39Hs), and the bispecific antibody formed by the dimerization of the two single-chain molecules ScFabHuPL16-42Ks and ScFabHu34-39Hs was named ScFab(HuPL16-42Ks / Hu34-39Hs).

[0400] 3. Purification of bispecific anti-CD47 / anti-PD-L1 antibody

[0401] After the bispecific antibody of this invention is expressed and secreted in Expi-CHO cells, it is purified using Protein A affinity chromatography. The specific method is as follows: After equilibrating the Protein A affinity chromatography column with buffer, the supernatant of Expi-CHO cell culture medium concentrated by ultrafiltration is injected, and the concentration is monitored at A280 (nm). Washing is performed with washing buffer until all unbound proteins are eluted, followed by elution with elution buffer to obtain the corresponding bispecific antibody. The purity of the purified bispecific antibody is determined by SEC-HPLC, and the molecular weight is determined by LC-MS. After quality identification, it is used for subsequent pharmaceutical research. SEC-HPLC and LC-MS identification results show that the purity of both ScFab (HuPL7-21Ks / Hu34-39Hs) and ScFab (HuPL16-42Ks / Hu34-39Hs) reaches over 95%, and the determined molecular weight matches the theoretical value.

[0402] Example 15 Construction of a stable cell line expressing high levels of hPD-L1

[0403] 1. Construction of stable cell lines CHO-K1-hPD-L1 and Raji-hPD-L1 with high expression of hPD-L1:

[0404] The eukaryotic expression plasmid pTargeT-hPD-L1, containing the extracellular region sequence of hPD-L1 (human PD-L1) (UniProtKB-Q9NZQ7(PD-L1Human)>sp|Q9NZQ7|19-238, SEQ ID NO:134), was electroporated into CHO-K1 cells (from the Shanghai Institute of Cell Biology, Chinese Academy of Sciences) and Raji cells (from Yiming Angke Biotechnology Co., Ltd.) and cultured at 37°C in a 5% CO2 incubator. After 24 h, the cells were cultured under pressure in medium containing 500 μg / ml G418. After 12 days, the pool positivity rate was detected by FACS. The electroporated cells were plated into 1x10⁻¹ plates. 6 Cells were incubated at a density of 100 μL / well with FITC anti-human PD-L1 antibody (SINO BIOLOGICAL, 10084-MMB6-F) at 4°C for 60 min. The mean value of the FITC channel was read by flow cytometry. After data analysis, positive cell lines were selected for subcloning. The cloned CHO-K1 / Raji cell lines were selected. These cell lines expressed PD-L1 molecules at high levels and were named CHO-K1-hPD-L1 and Raji-hPD-L1.

[0405] Example 16: In vitro binding and blocking experiment of anti-CD47 / anti-PD-L1 bispecific antibody ELISA

[0406] 1. Anti-CD47 / anti-PD-L1 bispecific antibody PD-L1 binding ELISA assay

[0407] Human PD-L1-His protein (0.5 μg / mL, 100 μL / well) was coated onto a 96-well microplate and incubated at 37°C for 2 h. After washing three times with 1xPBST, the plate was blocked overnight at 4°C with 5% skim milk, followed by three washes with 1xPBST. The secondary antibody concentration was serially diluted 5-fold starting at 10 μg / mL and added to the microplate. The plate was incubated at 37°C for 1.5 h. The control antibody was Atezolizumab (Sino Biological, Cat: 68049-H001, abbreviated Ate). After washing five times with 1xPBST, HRP-Anti-Human IgG secondary antibody (Jackson, 109-035-003, 1:10000) was added and incubated at 37°C for 40 min. After washing five times with 1xPBST, TMB chromogenic buffer was added. After termination, the OD450 value and EC50 were read using a Thermo, Multiskan FC microplate reader. 50 The results are as follows Figure 11 As shown.

[0408] Experimental results showed that the bispecific antibodies ScFab (HuPL7-21Ks / Hu34-39Hs) and ScFab (HuPL16-42Ks / Hu34-39Hs) both possessed the ability to bind to human PD-L1. Their binding affinity to human PD-L1 was comparable to that of Atezolizumab.

[0409] 2. Competitive ELISA assay between anti-CD47 / anti-PD-L1 bispecific antibody and PD-1

[0410] The activity of the antibody in blocking the binding of PD1 to PD-L1 was analyzed using ELISA. Human PD-L1-hFC protein (2 μg / ml, 100 μl / well) was coated onto a 96-well microplate and incubated at 37°C for 2 h. After washing three times with 1xPBST, the plate was blocked overnight at 4°C with 5% skim milk. After washing three times with 1xPBST, bispecific antibodies ScFab (HuPL7-21Ks / Hu34-39Hs) or ScFab (HuPL16-42Ks / Hu34-39Hs) were used as primary antibodies, starting at 10 μg / mL and serially diluted 3-fold to obtain 8 concentrations. In the presence of these serially diluted anti-PD-L1 antibodies, they were added to the ELISA plate along with 1 μg / mL of PD-1-mFc and incubated at 37°C for 1.5 h. The control antibody was Atezolizumab. After washing five times with 1xPBST, the secondary antibody, Anti-Mouse IgG HRP (Jackson, 109-035-003, 1:10000), was used for incubation at 37°C for 1 h. After washing five times with 1xPBST, TMB chromogenic buffer was added, and the OD450 value was read using a microplate reader after the incubation period. The results are as follows: Figure 11 As shown.

[0411] Experimental results showed that the bispecific antibodies ScFab (HuPL7-21Ks / Hu34-39Hs) and ScFab (HuPL16-42Ks / Hu34-39Hs) both had the ability to block the binding of PD-L1 and PD-1, and their blocking ability was comparable to that of Atezolizumab.

[0412] 3. Anti-CD47 / anti-PD-L1 bispecific antibody CD47 binding ELISA assay

[0413] The binding activity of the antibody to CD47 was analyzed using ELISA. Human CD47-His protein (0.5 μg / mL, 100 μL / well) was coated onto a 96-well microplate and incubated at 37°C for 2 h. After washing three times with 1xPBST, the plate was blocked overnight at 4°C with 5% skim milk. After washing three times with 1xPBST, the anti-CD47 / anti-PD-L1 bispecific antibody provided in this invention was serially diluted 5-fold starting at 50 μg / mL, for a total of 8 concentrations, and incubated at 37°C for 1.5 h. The control antibody was Hu34-39-PE. After washing five times with 1xPBST, HRP-Anti-Human IgG (1:10000) was used as the secondary antibody and incubated at 37°C for 40 min. After washing five times with 1xPBST, TMB chromogenic buffer was added, and the OD450 value was read using a Thermo Multiskan FC microplate reader. EC 50 The results are as follows Figure 11 As shown.

[0414] Experimental results showed that both bispecific antibodies ScFab (HuPL7-21Ks / Hu34-39Hs) and ScFab (HuPL16-42Ks / Hu34-39Hs) had the ability to bind to human CD47, but their binding activity was reduced to varying degrees. ScFab (HuPL7-21Ks / Hu34-39Hs) bound EC47 to EC47. 50 EC value compared to Hu34-39-PE 50 The value is 40 times higher.

[0415] 4. Competitive ELISA assay between anti-CD47 / anti-PD-L1 bispecific antibody and SIRPα

[0416] The blocking activity of the antibody was analyzed by ELISA. Human CD47-His protein (0.4 μg / ml, 100 μl / well) was coated onto a 96-well microplate and incubated at 37°C for 2 h. After washing three times with 1xPBST, the plate was blocked overnight at 4°C with 5% skim milk. After washing three times with 1xPBST, bispecific antibodies ScFab (HuPL7-21Ks / Hu34-39Hs) or ScFab (HuPL16-42Ks / Hu34-39Hs) were used as primary antibodies, starting at 10 μg / mL and serially diluted 3-fold to a total of 8 concentrations. In the presence of serially diluted anti-CD47 antibody, these were added to the ELISA plate along with 2 μg / mL SIRPα-biotin and incubated at 37°C for 1.5 h. The control antibody was Hu34-39-PE. After washing five times with 1xPBST, secondary antibody SA-HRP (Jackson, 109-035-003, 1:10000) was used for incubation at 37°C for 1 h. After washing five times with 1xPBST, chromogenic buffer TMB was added, and the OD450 value was read using an ELISA reader. The results are as follows: Figure 11 As shown.

[0417] Experimental results showed that the bispecific antibodies ScFab (HuPL7-21Ks / Hu34-39Hs) and ScFab (HuPL16-42Ks / Hu34-39Hs) both had the ability to block the binding of CD47 to SIRPα, and their blocking ability was lower than that of Hu34-39-PE, with an IC50 value of 1.5%. 50 The value increased by about 40 times.

[0418] 5. Competition ELISA assay between anti-CD47 / anti-PD-L1 bispecific antibody and CD80

[0419] The activity of the antibody blocking PD-L1 binding to CD80 was analyzed using ELISA. CD80-hFc protein (8 μg / mL, 100 μL / well) was coated onto 96-well microplates and incubated overnight at 4°C. After washing three times with 1xPBST, the plates were blocked with 5% skim milk at 37°C for 2 h. After washing three times with 1xPBST, bispecific antibodies ScFab (HuPL7-21Ks / Hu34-39Hs) or ScFab (HuPL16-42Ks / Hu34-39Hs) were used as primary antibodies, starting at 30 μg / mL and serially diluted 3-fold to a total of 8 concentrations. These were added to the microplate along with PD-L1-mFc in the presence of the serially diluted anti-PD-L1 antibody and incubated at 37°C for 1.5 h. Atezolizumab was used as the control antibody. After washing five times with 1xPBST, Anti-Mouse IgG HRP was used as the secondary antibody and incubated at 37°C for 1 h. After washing five times with 1xPBST, TMB chromogenic solution was added, and the reaction was stopped. The OD450 value was then read using a microplate reader. The results are as follows: Figure 11 As shown.

[0420] Experimental results showed that the bispecific antibodies ScFab (HuPL7-21Ks / Hu34-39Hs) and ScFab (HuPL16-42Ks / Hu34-39Hs) both had the ability to block the binding of PD-L1 to CD80, and their blocking ability was comparable to that of Atezolizumab.

[0421] The above experimental results show that the bispecific antibody of the present invention can differentially bind to PD-L1 / CD47, thereby ensuring anti-tumor activity while potentially reducing antibody toxicity, such as hematologic toxicity.

[0422] Example 17: Cellular Binding / Blocking Assay of Anti-CD47 / Anti-PD-L1 Bispecific Antibody

[0423] 1. The ability of bispecific antibodies ScFab (HuPL7-21Ks / Hu34-39Hs) and ScFab (HuPL16-42Ks / Hu34-39Hs) to bind to PD-L1 / CD47 on the cell surface was detected by FACS.

[0424] The CHO-K1-hPD-L1 / CHO-K1-hCD47 stable cell line was used as the PD-L1 / CD47 provider. Serially diluted anti-PD-L1 / anti-CD47 bispecific antibodies were added to the cell plate as primary antibodies and incubated at 4°C for 1.5 h. PE Anti-Human IgG was used as the secondary antibody and incubated at 4°C for 1 h. Atezolizumab and Hu34-39-PE were used as positive controls. Flow cytometry was used to read the product of the mean and parent values ​​at 585 nm. The results are shown below. Figure 12 As shown.

[0425] Experimental results showed that the bispecific antibodies ScFab (HuPL7-21Ks / Hu34-39Hs) and ScFab (HuPL16-42Ks / Hu34-39Hs) both possessed the ability to bind to human PD-L1 on the cell surface, and their binding ability was comparable to that of Atezolizumab. The binding activity of the bispecific antibodies ScFab (HuPL7-21Ks / Hu34-39Hs) and ScFab (HuPL16-42Ks / Hu34-39Hs) to CD47 on the cell surface was lower than that of the Hu34-39-PE monoclonal antibody, EC... 50 It increases by about 4 times, and Emax decreases by about 2 times.

[0426] 2. The ability of bispecific antibodies ScFab (HuPL7-21Ks / Hu34-39Hs) and ScFab (HuPL16-42Ks / Hu34-39Hs) to block PD-1 / SIRPα binding to the cell surface was detected by FACS.

[0427] The CHO-K1-hPD-L1 / CHO-K1-hCD47 stable cell line was used as a PD-L1 / CD47 provider. The binding abilities of PD-L1 to PD-1 and CD47 to SIRPα were observed in the presence of serially diluted anti-PD-L1 / anti-CD47 antibodies. Bispecific antibodies ScFab (HuPL7-21Ks / Hu34-39Hs) or ScFab (HuPL16-42Ks / Hu34-39Hs) were used as primary antibodies. After serial dilution, they were added to cell plates with 1 μg / mL PD-1-mFc and SIRPα-biotin, respectively, and incubated at 37°C for 1.5 h. PE-Anti-Mouse IgG / PE-SA was used as the secondary antibody. Atezolizumab was used as a positive control to block PD-1-mFc binding to PD-L1 on the cell surface, and Hu34-39-PE was used as a positive reference to block SIRPα binding to CD47 on the cell surface. The results are as follows: Figure 12 As shown.

[0428] Experimental results showed that the bispecific antibodies ScFab (HuPL7-21Ks / Hu34-39Hs) and ScFab (HuPL16-42Ks / Hu34-39Hs) both possessed the ability to block the binding of human PD-1 to CHO-K1-PD-L1 and SIRPα to CHO-K1-CD47, and their ability to block the binding of PD-1 to CHO-K1-PD-L1 was comparable to that of Atezolizumab. The bispecific antibodies showed a lower ability to block the binding of SIRPα to CHO-K1-CD47 compared to Hu34-39-PE, with an IC50 value lower than that of Hu34-39-PE. 50 The value increased by 3 times.

[0429] According to ELISA and FACS results, the binding and blocking abilities of the bispecific antibody molecules ScFab (HuPL7-21Ks / Hu34-39Hs) and ScFab (HuPL16-42Ks / Hu34-39Hs) with PD-L1 were consistent with those of Atezolizumab. However, the binding ability of the bispecific antibody molecules to CD47 was reduced. ELISA detection of EC 50 Elevated by approximately 40 times, FACS detects EC 50 The value increased by approximately 4-fold, while Emax decreased by 2-fold. This indicates that the differential binding of the bispecific antibody molecules ScFab (HuPL7-21Ks / Hu34-39Hs) and ScFab (HuPL16-42Ks / Hu34-39Hs) to PD-L1 / CD47 helps enhance the tumor targeting of the bispecific antibody and reduce its adverse reactions, especially those against erythrocytes.

[0430] Example 18: FACS Detection of Dual Binding and Dual Blocking of Anti-CD47 / Anti-PD-L1 Bispecific Antibody with Raji-hPD-L1 Cells

[0431] Raji-hPD-L1 tumor cells were purchased from Yiming Angke Biomedical Technology Co., Ltd. In addition to high expression of hPD-L1, Raji-hPD-L1 cells also highly express hCD47. The bispecific antibodies ScFab (HuPL7-21Ks / Hu34-39Hs) and ScFab (HuPL16-42Ks / Hu34-39Hs) bound to both hPD-L1 and hCD47 on the surface of Raji-hPD-L1 cells, and simultaneously blocked the binding of CD47 / SIRPα and PD-1 / PD-L1, demonstrating bi-arm binding and bi-arm blocking activity. Bi-arm blocking: 2.4 × 10⁶ cells were seeded per well at the bottom of a U-shaped plate. 5 Raji-hPD-L1 cells were prepared by adding primary antibody at a working concentration starting from 2.5 μg / mL, with 9 serial dilutions (2-fold). SIRPα-mFc and PD-1-mFc were mixed evenly at a final concentration of 1 μg / mL. 50 μl of antibody and 50 μl of antigen were premixed per well, for a total of 100 μl, and added to each well. The cells were incubated at 4°C for 1.5 h. Secondary antibody was added, and the cells were washed three times with 200 μl of cell stain buffer. Then, 0.8 μl of PE-anti-mouse-IgG-Fc was added to each well, and the cells were incubated at 4°C for 1 h. After washing three times with 200 μl of cell stain buffer and resuspending the cells in 100 μl of cell stain buffer, flow cytometry was used for analysis. For double-arm cytometry, 1.5 x 10-1 cells were seeded per well on the bottom of a U-shaped plate. 6 Raji-hPD-L1 cells were incubated with primary antibody at a working concentration of 10 μg / mL, serially diluted 5-fold for a total of 8 concentrations, and incubated at 4°C for 1.5 h. Secondary antibody was then added, followed by washing three times with 200 μl of cell stain buffer. 0.8 μl of PE-anti-human-IgG-Fc was added to each well, and the cells were incubated at 4°C for 1 h. After washing three times with 200 μl of cell stain buffer and resuspending the cells in 100 μl of cell stain buffer, the cells were analyzed by flow cytometry.

[0432] Experimental results are as follows Figure 13 As shown, the bispecific antibody of the present invention binds to both target cells and can simultaneously block the binding of CD47 / SIRPα and PD-1 / PD-L1 on the surface of both target cells.

[0433] Example 19: Detection of Anti-CD47 / Anti-PD-L1 Bispecific Antibody's Inhibition of Xenograft Growth in Mice. NSG mice exhibit NOD, Prkdcscid, and IL2rgnull deletion / mutation characteristics, making them the most immunodeficient and suitable tool mice for human cell transplantation, showing almost no rejection response to human cells and tissues. This invention utilizes NSG mice (purchased from Beijing Biocytogen Gene Biotechnology Co., Ltd.) and Raji-hPD-L1 tumor cells to establish a tumor transplantation model—the Raji-PBMC-NSG model—to study the anti-tumor effect of the anti-CD47 / anti-PD-L1 bispecific antibody ScFab (HuPL7-21Ks / Hu34-39Hs) in the Raji-hPD-L1 lymphoma subcutaneous transplantation model. CD47 monoclonal antibody 5F9 was purchased from Sino Biological Inc. (Cat: 68063-H001). Table 14 shows the experimental design scheme for the anti-tumor effect of the test drugs in the Raji-PBMC-NSG tumor model.

[0434] Table 14 Experimental Design Scheme for Raji-PBMC-NSG Model Drug Testing

[0435] Group N Antibody mouse strains Dosage (mg / kg) Dosage volume (ul / g) route of administration Frequency of administration 1 6 PBS NSG --- 10 iv qw×3 2 6 Atezolizumab NSG 10 10 iv qw×3 3 6 Atezolizumab+5F9 NSG 10+10 10 iv qw×3 4 6 BiAb NSG 10 10 iv qw×3 5 6 BiAb NSG 20 10 iv qw×3

[0436] a: N refers to the number of mice in each group; b: BiAb refers to the bispecific antibody ScFab (HuPL7-21Ks / Hu34-39Hs).

[0437] The changes in tumor volume over time in each group are as follows: Figure 14 As shown, the dual antibody of this invention exhibits significantly better tumor-suppressing effects than Atezolizumab, and is superior to the combination of monoclonal antibodies (Atezolizumab + 5F9).

[0438] Example 20: Acute toxicity test of anti-CD47 / anti-PD-L1 bispecific antibody in cynomolgus monkeys.

[0439] This embodiment is a toxicity test of a single intravenous infusion of the anti-CD47 / anti-PD-L1 bispecific antibody ScFab (HuPL7-21Ks / Hu34-39Hs) (BiAb) of the present invention into cynomolgus monkeys. The bispecific antibody was administered at doses of 10, 30, and 100 mg / kg, with one male and one female cynomolgus monkey in each group. The anti-CD47 monoclonal antibody Hu34-39-PE was administered at a dose of 30 mg / kg, and Hu5F9 was administered at a dose of 20 mg / kg, with two cynomolgus monkeys in each group.

[0440] A single intravenous infusion was administered, with an observation period of 21 days. Blood samples were collected via the femoral vein at different time points for the detection of blood cell counts, coagulation function indicators, and blood biochemical indicators.

[0441] The drug safety evaluation results showed that, as of day 21, no monkeys died in any group, and there were no abnormalities in general condition, food intake, weight, etc. in any group.

[0442] Animals administered the bispecific antibody ScFab (HuPL7-21Ks / Hu34-39Hs) showed no changes in RBC count, HGB content, or RET%. At doses of 10, 30, and 100 mg / kg, the bispecific antibody ScFab (HuPL7-21Ks / Hu34-39Hs) did not exhibit erythrocytic toxicity or other hematologic toxicity.

[0443] Animals administered the monoclonal antibody Hu5F9 (20 mg / kg) showed a significant decrease in RBC count and HGB content, and a significant increase in RET%. Animals administered the monoclonal antibody Hu34-39-PE (30 mg / kg) showed a certain degree of decrease in RBC count and HGB content, and an increase in RET%, but less pronounced than with Hu5F9. Hu5F9 exhibited significant erythrocytotoxicity, while Hu34-39-PE showed less erythrocytotoxicity than Hu5F9. The bispecific antibody ScFab (HuPL7-21Ks / Hu34-39Hs) demonstrated significantly better erythrocyte safety than both Hu34-39-PE and Hu5F9.

[0444] The above embodiments demonstrate that the anti-CD47 / anti-PD-L1 bispecific antibody molecule ScFab (HuPL7-21Ks / Hu34-39Hs) of the present invention differentially binds to CD47 and PD-L1, effectively retaining its binding and blocking activity to PD-L1, while exhibiting no erythrocyte toxicity or other hematologic toxicity, thus demonstrating excellent safety.

[0445] Example 21 Detection of the inhibitory effect of anti-PD-L1 antibody on xenograft growth in mice

[0446] This invention utilizes NSG mice (purchased from Beijing Biocytogen Biotechnology Co., Ltd., China) and Raji-PD-L1 tumor cells (purchased from Yiming Angke Biomedical Technology Co., Ltd., China) to establish a tumor transplantation model—the Raji-PBMC-NSG model—to investigate the anti-tumor effect of the antibody of this invention in a subcutaneous transplantation model of Raji-hPD-L1 lymphoma. The anti-PD-L1 positive control antibody was Atezolizumab (Sino Biological, Cat:68049-H001). Six mice were used in each group. The negative control group received physiological saline (PBS). The dosage of the anti-PD-L1 antibody HuPL7-21 and Atezolizumab of this invention was 10 mg / kg, respectively. Administration was via intraperitoneal injection, twice weekly for three consecutive weeks.

[0447] At the end of the experiment, the tumor inhibition rate (TGI) of each group was calculated.TV )%, as shown in Table 15.

[0448] Table 15 Effects of various antibodies on tumor volume in Raji-PBMC-NSG model mice

[0449]

[0450] The anti-PD-L1 antibody HuPL7-21 of this invention has a significantly better in vivo tumor-suppressing effect than Atezolizumab.

[0451] Although the present invention has been described in detail above, those skilled in the art will understand that various modifications and changes can be made to the invention without departing from its spirit and scope. The scope of the invention is not limited to the detailed description above, but should be attributed to the claims. sequence list <110> Nanjing Shenghe Pharmaceutical Co., Ltd. <120> Anti-CD47 / anti-PD-L1 antibodies and their applications <150> 202010128900X <151> 2020-02-28 <160> 134 <170> SIPOSequenceListing 1.0 <210> 1 <211> 8 <212> PRT <213> Mouse source (Mus musculus) <400> 1 Gly Tyr Ile Phe Thr Asp Tyr Tyr 1 5 <210> 2 <211> 8 <212> PRT <213> Mouse source (Mus musculus) <400> 2 Ile Thr Pro Gly Ser Gly Leu Thr 1 5 <210> 3 <211> 12 <212> PRT <213> Mouse source (Mus musculus) <400> 3 Ala Arg Cys Ser Tyr Gly Ser Ser Phe Pro His Val 1 5 10 <210> 4 <211> 8 <212> PRT <213> Mouse (Mus musculus) <400> 4 Gly Tyr Thr Phe Thr Asn Tyr Trp 1 5 <210> 5 <211> 8 <212> PRT <213> Mouse (Mus musculus) <400> 5 Ile Thr Pro Gly Arg Gly Glu Thr 1 5 <210> 6 <211> 10 <212> PRT <213> Mouse (Mus musculus) <400> 6 Ser Arg Trp Gly Leu Arg Arg Gly Asp Tyr 1 5 10 <210> 7 <211> 8 <212> PRT <213> Mouse (Mus musculus) <400> 7 Gly Phe Ile Phe Ser Arg Phe Gly 1 5 <210> 8 <211> 8 <212> PRT <213> Mouse (Mus musculus) <400> 8 Ile Ser Ser Gly Gly Ser Tyr Thr 1 5 <210> 9 <211> 14 <212> PRT <213> Mouse (Mus musculus) <400> 9 Ala Arg Gln Gly Leu Leu Asp Tyr Leu Tyr Ala Leu Asp Tyr 1 5 10 <210> 10 <211> 8 <212> PRT <213> Mouse (Mus musculus) <400> 10 Gly Tyr Thr Phe Thr Asp Tyr Tyr 1 5 <210> 11 <211> 12 <212> PRT <213> Mouse (Mus musculus) <400> 11 Ala Arg Thr Ser Tyr Gly Ser Ser Phe Pro His Val 1 5 10 <210> 12 <211> 12 <212> PRT <213> Mouse (Mus musculus) <400> 12 Gln Ser Leu Leu Asn Val Asn Asp Gln Lys Asn Tyr 1 5 10 <210> 13 <211> 3 <212> PRT <213> Mouse (Mus musculus) <400> 13 Phe Ala Ser 1 <210> 14 <211> 9 <212> PRT <213> Mouse origin (Mus musculus) <400> 14 Gln Gln His Tyr Ser Thr Pro Leu Thr 1 5 <210> 15 <211> 7 <212> PRT <213> Mouse origin (Mus musculus) <400> 15 Ser Ser Val Ser Ser Ser Tyr 1 5 <210> 16 <211> 3 <212> PRT <213> Mouse origin (Mus musculus) <400> 16 Ser Thr Ser 1 <210> 17 <211> 9 <212> PRT <213> Mouse origin (Mus musculus) <400> 17 Gln Gln Tyr Ser Gly Tyr Pro Leu Thr 1 5 <210> 18 <211> 6 <212> PRT <213> Mouse origin (Mus musculus) <400> 18 Gln Asp Ile Asn Ser Phe 1 5 <210> 19 <211> 3 <212> PRT <213> Mouse origin (Mus musculus) <400> 19 Arg Ala Tyr 1 <210> 20 <211> 9 <212> PRT <213> Mouse (Mus musculus) <400> 20 Gln Gln Tyr Val Glu Phe Pro Pro Thr 1 5 <210> 21 <211> 119 <212> PRT <213> Mouse (Mus musculus) <400> 21 Gln Val Gln Leu Lys Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Asp Tyr 20 25 30 Tyr Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Ala Arg Ile Tyr Pro Gly Ser Gly Leu Thr Tyr Tyr Asn Ala Lys Phe 50 55 60 Lys Gly Lys Ala Thr Val Thr Ala Glu Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Cys Ser Tyr Gly Ser Ser Phe Pro His Val Trp Gly Thr Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115 <210> 22 <211> 117 <212> PRT <213> Mouse (Mus musculus) <400> 22 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Trp Ile Thr Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asp Ile Thr Pro Gly Arg Gly Glu Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Ser Lys Ala Thr Leu Thr Val Asp Thr Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Thr Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Leu Arg Arg Gly Asp Tyr Trp Gly Gln Gly Thr Ser 100 105 110 Val Thr Val Ser Ser 115 <210> 23 <211> 121 <212> PRT <213> Mouse (Mus musculus) <400> 23 Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Glu Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Ser Arg Phe 20 25 30 Gly Met Ala Trp Val Arg Gln Thr Pro Asp Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Leu Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr 65 70 75 80 Leu Gln Met Arg Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Gln Gly Leu Leu Asp Tyr Leu Tyr Ala Leu Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Ala Val Thr Val Ser Ser 115 120 <210> 24 <211> 113 <212> PRT <213> Mouse (Mus musculus) <400> 24 Asp Ile Val Met Thr Gln Ser Pro Phe Ser Leu Ala Met Ser Val Gly 1 5 10 15 Gln Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Val 20 25 30 Asn Asp Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Ser Pro Lys Leu Leu Val Tyr Phe Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ile Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Asp Tyr Phe Cys Gln Gln 85 90 95 His Tyr Ser Thr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu 100 105 110 Lys <210> 25 <211> 108 <212> PRT <213> Mouse (Mus musculus) <400> 25 Glu Asn Val Leu Thr Gln Ser Pro Ala Ile Met Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Ser Ser 20 25 30 Tyr Leu Asn Trp Tyr Gln Gln Lys Ser Gly Ala Ser Pro Lys Leu Trp 35 40 45 Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Gly Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Val Glu 65 70 75 80 Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Gly Tyr Pro 85 90 95 Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Val Lys 100 105 <210> 26 <211> 107 <212> PRT <213> Mouse (Mus musculus) <400> 26 Asp Ile Lys Met Ile Gln Ser Pro Ser Ser Met Tyr Ala Gly Leu Gly 1 5 10 15 Glu Arg Val Thr Phe Asn Cys Lys Ala Ser Gln Asp Ile Asn Ser Phe 20 25 30 Leu Cys Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Arg Pro Leu Ile 35 40 45 Tyr Arg Ala Tyr Arg Leu Val Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Val Gly Ser Gly Gln Asp Tyr Ser Leu Thr Ile Ser Ser Leu Asp Tyr 65 70 75 80 Glu Asp Leu Gly Ile Tyr Tyr Cys Gln Gln Tyr Val Glu Phe Pro Pro 85 90 95 Thr Phe Gly Ala Gly Thr Met Leu Glu Leu Lys 100 105 <210> 27 <211> 119 <212> PRT <213> Mouse (Mus musculus) <400> 27 Gln Val Gln Leu Lys Gln Ser Gly Ala Glu Leu Val Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Leu Ser Cys Lys Ala Ser Gly Tyr Ile Phe Thr Asp Tyr 20 25 30 Tyr Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Ala Arg Ile Tyr Pro Gly Ser Gly Leu Thr Tyr Tyr Asn Ala Lys Phe 50 55 60 Lys Gly Lys Ala Thr Val Thr Ala Glu Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Phe Cys 85 90 95 Ala Arg Thr Ser Tyr Gly Ser Ser Phe Pro His Val Trp Gly Thr Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115 <210> 28 <211> 107 <212> PRT <213> Mouse (Mus musculus) <400> 28 Asp Ile Lys Met Ile Gln Ser Pro Ser Ser Met Tyr Ala Gly Leu Gly 1 5 10 15 Glu Arg Val Thr Phe Asn Cys Lys Ala Ser Gln Asp Ile Asn Ser Phe 20 25 30 Leu Ala Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Arg Pro Leu Ile 35 40 45 Tyr Arg Ala Tyr Arg Leu Val Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Val Gly Ser Gly Gln Asp Tyr Ser Leu Thr Ile Ser Ser Leu Asp Tyr 65 70 75 80 Glu Asp Leu Gly Ile Tyr Tyr Cys Gln Gln Tyr Val Glu Phe Pro Pro 85 90 95 Thr Phe Gly Ala Gly Thr Met Leu Glu Leu Lys 100 105 <210> 29 <211> 119 <212> PRT <213> Artificial Sequence <400> 29 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Tyr Pro Gly Ser Gly Leu Thr Tyr Tyr Asn Ala Lys Phe 50 55 60 Lys Gly Arg Ala Thr Val Thr Ala Asp Lys Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Ser Tyr Gly Ser Ser Phe Pro His Val Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115 <210> 30 <211> 117 <212> PRT <213> Artificial Sequence <400> 30 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Trp Ile Thr Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asp Ile Thr Pro Gly Arg Gly Glu Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Val Asp Ile Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Leu Arg Arg Gly Asp Tyr Trp Gly Gln Gly Thr Ser 100 105 110 Val Thr Val Ser Ser 115 <210> 31 <211> 121 <212> PRT <213> Artificial Sequence <400> 31 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Ser Arg Phe 20 25 30 Gly Met Ala Trp Val Arg Gln Thr Pro Asp Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Leu Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr 65 70 75 80 Leu Gln Met Arg Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Gln Gly Leu Leu Asp Tyr Leu Tyr Ala Leu Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Ala Val Thr Val Ser Ser 115 120 <210> 32 <211> 113 <212> PRT <213> Artificial Sequence(Artificial Sequence) <400> 32 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Leu Asn Val 20 25 30 Asn Asp Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45 Ala Pro Lys Leu Leu Ile Tyr Phe Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln 85 90 95 His Tyr Ser Thr Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 33 <211> 108 <212> PRT <213> Artificial Sequence <400> 33 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Ser Ser Ser 20 25 30 Tyr Leu Asn Trp Tyr Gln Gln Lys Ser Gly Ala Ser Pro Lys Leu Trp 35 40 45 Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Gly Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Val Glu 65 70 75 80 Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Gly Tyr Pro 85 90 95 Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Val Lys 100 105 <210> 34 <211> 107 <212> PRT <213> Artificial Sequence <400> 34 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Ile Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Ser Phe 20 25 30 Leu Ala Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Arg Pro Leu Ile 35 40 45 Tyr Arg Ala Tyr Arg Leu Val Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Val Gly Ser Gly Gln Asp Tyr Ser Leu Thr Ile Ser Ser Leu Asp Tyr 65 70 75 80 Glu Asp Leu Gly Ile Tyr Tyr Cys Gln Gln Tyr Val Glu Phe Pro Pro 85 90 95 Thr Phe Gly Ala Gly Thr Met Leu Glu Leu Lys 100 105 <210> 35 <211> 357 <212> DNA <213> Artificial Sequence <400> 35 caggtgcagc tggtgcagag cggagccgaa gtgaagaagc ccggtgccag cgtgaaagta 60 agctgcaagg ccagcggcta caccttcacc gactactaca tgaactgggt gaggcaggcc 120 cctggacaag gcctggagtg gatcggcagg atctaccccg gcagcggcct gacctactat 180 aacgccaagt tcaagggcag ggccaccgtg accgccgaca agtccaccag caccgtgtac 240 atggagctga gcagcctgag gagcgaggac accgccgtgt attactgcgc caggaccagc 300 tacggcagca gcttccccca cgtgtggggc cagggcacca ccgtgaccgt gagctct 357 <210> 36 <211> 351 <212> DNA <213> Artificial Sequence <400> 36 caggttcagc tgcaagagag cggtcccggc ctggtgaaac ccagccagac cctgagcctg 60 acctgcaagg ccagcggcta cactttcact aactactgga tcacctgggt gaagcagcga 120 cccggccagg gcctggagtg gatcggcgac atcacccctg ggaggggaga gaccaactac 180 aaccagaagt tcaagggcag ggtgaccctg accgtggaca tcagcgcctc cactgcctat 240 atggagctga gcagcctgag gagcgaggac accgccgtgt actactgcag caggtggggc 300 ctgaggaggg gcgattactg gggccaaggg accagcgtga ccgtgagctc t 351 <210> 37 <211> 363 <212> DNA <213> Artificial Sequence <400> 37 gaggtccagc tgctggagag cggcggtggc ctcgtgcagc ccggaggcag cctgaggctg 60 agctgcgcgg caagcggctt catcttcagc aggttcggca tggcctgggt gaggcagacc 120 ccagacaaga ggctggagtg ggtggcaact atcagcagcg gaggaagtta cacctactac 180 cccgacagcg tgaagggcag gctgaccatc agtagggaca acgccaagac caccctgtac 240 ctgcagatga ggagcctgaa gagcgaggac accgccatgt actactgcgc caggcagggc 300 ctgctcgact atctgtacgc cctggactat tggggccagg gcactgccgt gaccgtgagc 360 agc 363 <210> 38 <211> 339 <212> DNA <213> Artificial Sequence <400> 38 gacatccaga tgacccagag cccaagcagc ctgagcgcca gcgtgggcga cagggtgacc 60 atcacctgca agagcagcca aagcctgctg aacgtgaacg accagaagaa ctacctggcc 120 tggtatcagc agaagccggg caaggccccc aagctgctca tctactttgc cagcaccagg 180 gagagcggcg tgcccagcag gttcagcggc agcggaagtg gcaccgactt caccctcacc 240 atcagctccc tgcaacccga ggacttcgcc acctactact gtcaacagca ctacagcaca 300 cccctgacct tcggccaggg caccaagttg gagatcaag 339 <210> 39 <211> 324 <212> DNA <213> Artificial Sequence <400> 39 gagatcgtgc tgacccagag ccctggcacc ttgagcctga gtcccggaga gagggccacc 60 ctgagctgca gggcctcaag ctccgtgagc agcagctatc tgaattggta tcagcagaag 120 agcggagcca gccccaagct gtggatctac agcaccagca acctggcaag cggcgtgccc 180 ggcaggttca gcggcagtgg cagcgggacc agctacagcc tgaccatcag tagcgtggag 240 gccgaggacg ccgccaccta ctactgccag cagtacagcg gataccctct gaccttcgga 300 gccggaacca agttggaggt gaag 324 <210> 40 <211> 321 <212> DNA <213> Artificial Sequence <400> 40 gacatccaga tgacccagag cccaagcagc gtgagcgcca gcatcggcga cagggtgacc 60 ataacctgca aggccagcca agacatcaac agcttcctgg catggttcca gcagaagccc 120 ggcaagagcc ccaggcccct gatctacagg gcctacaggc tggtagacgg ggtgcccagc 180 aggttcagcg gcgtgggcag cggccaggac tacagcctga ccatcagcag cctggactac 240 gaggacctgg gcatctacta ctgccagcag tacgtggagt tccccccgac cttcggtgca 300 gggaccatgc tggagctgaa g 321 <210> 41 <211> 323 <212> PRT <213> Artificial Sequence <400> 41 Met Trp Pro Leu Val Ala Ala Leu Leu Leu Gly Ser Ala Cys Cys Gly 1 5 10 15 Ser Ala Gln Leu Leu Phe Asn Lys Thr Lys Ser Val Glu Phe Thr Phe 20 25 30 Cys Asn Asp Thr Val Val Ile Pro Cys Phe Val Thr Asn Met Glu Ala 35 40 45 Gln Asn Thr Thr Glu Val Tyr Val Lys Trp Lys Phe Lys Gly Arg Asp 50 55 60 Ile Tyr Thr Phe Asp Gly Ala Leu Asn Lys Ser Thr Val Pro Thr Asp 65 70 75 80 Phe Ser Ser Ala Lys Ile Glu Val Ser Gln Leu Leu Lys Gly Asp Ala 85 90 95 Ser Leu Lys Met Asp Lys Ser Asp Ala Val Ser His Thr Gly Asn Tyr 100 105 110 Thr Cys Glu Val Thr Glu Leu Thr Arg Glu Gly Glu Thr Ile Ile Glu 115 120 125 Leu Lys Tyr Arg Val Val Ser Trp Phe Ser Pro Asn Glu Asn Ile Leu 130 135 140 Ile Val Ile Phe Pro Ile Phe Ala Ile Leu Leu Phe Trp Gly Gln Phe 145 150 155 160 Gly Ile Lys Thr Leu Lys Tyr Arg Ser Gly Gly Met Asp Glu Lys Thr 165 170 175 Ile Ala Leu Leu Val Ala Gly Leu Val Ile Thr Val Ile Val Ile Val 180 185 190 Gly Ala Ile Leu Phe Val Pro Gly Glu Tyr Ser Leu Lys Asn Ala Thr 195 200 205 Gly Leu Gly Leu Ile Val Thr Ser Thr Gly Ile Leu Ile Leu Leu His 210 215 220 Tyr Tyr Val Phe Ser Thr Ala Ile Gly Leu Thr Ser Phe Val Ile Ala 225 230 235 240 Ile Leu Val Ile Gln Val Ile Ala Tyr Ile Leu Ala Val Val Gly Leu 245 250 255 Ser Leu Cys Ile Ala Ala Cys Ile Pro Met His Gly Pro Leu Leu Ile 260 265 270 Ser Gly Leu Ser Ile Leu Ala Leu Ala Gln Leu Leu Gly Leu Val Tyr 275 280 285 Met Lys Phe Val Ala Ser Asn Gln Lys Thr Ile Gln Pro Pro Arg Lys 290 295 300 Ala Val Glu Glu Pro Leu Asn Ala Phe Lys Glu Ser Lys Gly Met Met 305 310 315 320 Asn Asp Glu <210> 42 <211> 350 <212> PRT <213> Artificial Sequence <400> 42 Gln Leu Leu Phe Asn Lys Thr Lys Ser Val Glu Phe Thr Phe Cys Asn 1 5 10 15 Asp Thr Val Val Ile Pro Cys Phe Val Thr Asn Met Glu Ala Gln Asn 20 25 30 Thr Thr Glu Val Tyr Val Lys Trp Lys Phe Lys Gly Arg Asp Ile Tyr 35 40 45 Thr Phe Asp Gly Ala Leu Asn Lys Ser Thr Val Pro Thr Asp Phe Ser 50 55 60 Ser Ala Lys Ile Glu Val Ser Gln Leu Leu Lys Gly Asp Ala Ser Leu 65 70 75 80 Lys Met Asp Lys Ser Asp Ala Val Ser His Thr Gly Asn Tyr Thr Cys 85 90 95 Glu Val Thr Glu Leu Thr Arg Glu Gly Glu Thr Ile Ile Glu Leu Lys 100 105 110 Tyr Arg Val Val Ser Trp Glu Pro Lys Ser Cys Asp Lys Thr His Thr 115 120 125 Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe 130 135 140 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 145 150 155 160 Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val 165 170 175 Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 180 185 190 Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val 195 200 205 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 210 215 220 Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser 225 230 235 240 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 245 250 255 Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 260 265 270 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 275 280 285 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 290 295 300 Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp 305 310 315 320 Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 325 330 335 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 340 345 350 <210> 43 <211> 124 <212> PRT <213> Artificial Sequence <400> 43 Gln Leu Leu Phe Asn Lys Thr Lys Ser Val Glu Phe Thr Phe Cys Asn 1 5 10 15 Asp Thr Val Val Ile Pro Cys Phe Val Thr Asn Met Glu Ala Gln Asn 20 25 30 Thr Thr Glu Val Tyr Val Lys Trp Lys Phe Lys Gly Arg Asp Ile Tyr 35 40 45 Thr Phe Asp Gly Ala Leu Asn Lys Ser Thr Val Pro Thr Asp Phe Ser 50 55 60 Ser Ala Lys Ile Glu Val Ser Gln Leu Leu Lys Gly Asp Ala Ser Leu 65 70 75 80 Lys Met Asp Lys Ser Asp Ala Val Ser His Thr Gly Asn Tyr Thr Cys 85 90 95 Glu Val Thr Glu Leu Thr Arg Glu Gly Glu Thr Ile Ile Glu Leu Lys 100 105 110 Tyr Arg Val Val Ser Trp His His His His His His 115 120 <210> 44 <211> 354 <212> PRT <213> Artificial Sequence <400> 44 Gln Leu Leu Phe Asn Lys Thr Lys Ser Val Glu Phe Thr Phe Cys Asn 1 5 10 15 Asp Thr Val Val Ile Pro Cys Phe Val Thr Asn Met Glu Ala Gln Asn 20 25 30 Thr Thr Glu Val Tyr Val Lys Trp Lys Phe Lys Gly Arg Asp Ile Tyr 35 40 45 Thr Phe Asp Gly Ala Leu Asn Lys Ser Thr Val Pro Thr Asp Phe Ser 50 55 60 Ser Ala Lys Ile Glu Val Ser Gln Leu Leu Lys Gly Asp Ala Ser Leu 65 70 75 80 Lys Met Asp Lys Ser Asp Ala Val Ser His Thr Gly Asn Tyr Thr Cys 85 90 95 Glu Val Thr Glu Leu Thr Arg Glu Gly Glu Thr Ile Ile Glu Leu Lys 100 105 110 Tyr Arg Val Val Ser Trp Phe Ser Pro Glu Pro Arg Gly Pro Thr Ile 115 120 125 Lys Pro Cys Pro Pro Cys Lys Cys Pro Ala Pro Asn Leu Leu Gly Gly 130 135 140 Pro Ser Val Phe Ile Phe Pro Pro Lys Ile Lys Asp Val Leu Met Ile 145 150 155 160 Ser Leu Ser Pro Ile Val Thr Cys Val Val Val Asp Val Ser Glu Asp 165 170 175 Asp Pro Asp Val Gln Ile Ser Trp Phe Val Asn Asn Val Glu Val His 180 185 190 Thr Ala Gln Thr Gln Thr His Arg Glu Asp Tyr Asn Ser Thr Leu Arg 195 200 205 Val Val Ser Ala Leu Pro Ile Gln His Gln Asp Trp Met Ser Gly Lys 210 215 220 Glu Phe Lys Cys Lys Val Asn Asn Lys Asp Leu Pro Ala Pro Ile Glu 225 230 235 240 Arg Thr Ile Ser Lys Pro Lys Gly Ser Val Arg Ala Pro Gln Val Tyr 245 250 255 Val Leu Pro Pro Pro Glu Glu Glu Met Thr Lys Lys Gln Val Thr Leu 260 265 270 Thr Cys Met Val Thr Asp Phe Met Pro Glu Asp Ile Tyr Val Glu Trp 275 280 285 Thr Asn Asn Gly Lys Thr Glu Leu Asn Tyr Lys Asn Thr Glu Pro Val 290 295 300 Leu Asp Ser Asp Gly Ser Tyr Phe Met Tyr Ser Lys Leu Arg Val Glu 305 310 315 320 Lys Lys Asn Trp Val Glu Arg Asn Ser Tyr Ser Cys Ser Val Val His 325 330 335 Glu Gly Leu His Asn His His Thr Thr Lys Ser Phe Ser Arg Thr Pro 340 345 350 Gly Lys <210> 45 <211> 338 <212> PRT <213> Artificial Sequence <400> 45 Glu Leu Gln Val Ile Gln Pro Asp Lys Ser Val Leu Val Ala Ala Gly 1 5 10 15 Glu Thr Ala Thr Leu Arg Cys Thr Ala Thr Ser Leu Ile Pro Val Gly 20 25 30 Pro Ile Gln Trp Phe Arg Gly Ala Gly Pro Gly Arg Glu Leu Ile Tyr 35 40 45 Asn Gln Lys Glu Gly His Phe Pro Arg Val Thr Thr Val Ser Asp Leu 50 55 60 Thr Lys Arg Asn Asn Met Asp Phe Ser Ile Arg Ile Gly Asn Ile Thr 65 70 75 80 Pro Ala Asp Ala Gly Thr Tyr Tyr Cys Val Lys Phe Arg Lys Gly Ser 85 90 95 Pro Asp Asp Val Glu Phe Lys Ser Gly Ala Gly Thr Glu Leu Ser Val 100 105 110 Arg Ala Lys Pro Ser Ala Pro Val Val Ser Gly Pro Ala Ala Arg Ala 115 120 125 Thr Pro Gln His Thr Val Ser Phe Thr Cys Glu Ser His Gly Phe Ser 130 135 140 Pro Arg Asp Ile Thr Leu Lys Trp Phe Lys Asn Gly Asn Glu Leu Ser 145 150 155 160 Asp Phe Gln Thr Asn Val Asp Pro Val Gly Glu Ser Val Ser Tyr Ser 165 170 175 Ile His Ser Thr Ala Lys Val Val Leu Thr Arg Glu Asp Val His Ser 180 185 190 Gln Val Ile Cys Glu Val Ala His Val Thr Leu Gln Gly Asp Pro Leu 195 200 205 Arg Gly Thr Ala Asn Leu Ser Glu Thr Ile Arg Val Pro Pro Thr Leu 210 215 220 Glu Val Thr Gln Gln Pro Val Arg Ala Glu Asn Gln Val Asn Val Thr 225 230 235 240 Cys Gln Val Arg Lys Phe Tyr Pro Gln Arg Leu Gln Leu Thr Trp Leu 245 250 255 Glu Asn Gly Asn Val Ser Arg Thr Glu Thr Ala Ser Thr Val Thr Glu 260 265 270 Asn Lys Asp Gly Thr Tyr Asn Trp Met Ser Trp Leu Leu Val Asn Val 275 280 285 Ser Ala His Arg Asp Asp Val Lys Leu Thr Cys Gln Val Glu His Asp 290 295 300 Gly Gln Pro Ala Val Ser Lys Ser His Asp Leu Lys Val Ser Ala His 305 310 315 320 Pro Lys Glu Gln Gly Ser Asn Thr Ala Ala Glu Asn Thr Gly Ser Asn 325 330 335 Glu Arg <210> 46 <211> 466 <212> PRT <213> Artificial Sequence <400> 46 Met Glu Phe Gly Leu Ser Trp Leu Phe Leu Val Ala Ile Leu Lys Gly 1 5 10 15 Val Gln Cys Gln Met Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys 20 25 30 Thr Gly Ser Ser Val Lys Val Ser Cys Lys Ala Ser Gly Phe Asn Ile 35 40 45 Lys Asp Tyr Tyr Leu His Trp Val Arg Gln Ala Pro Gly Gln Ala Leu 50 55 60 Glu Trp Met Gly Trp Ile Asp Pro Asp Gln Gly Asp Thr Glu Tyr Ala 65 70 75 80 Gln Lys Phe Gln Asp Arg Val Thr Ile Thr Arg Asp Arg Ser Met Ser 85 90 95 Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Met 100 105 110 Tyr Tyr Cys Asn Ala Ala Tyr Gly Ser Ser Ser Tyr Pro Met Asp Tyr 115 120 125 Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser Ala Ser Thr Lys Gly 130 135 140 Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser 145 150 155 160 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val 165 170 175 Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe 180 185 190 Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 195 200 205 Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val 210 215 220 Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys 225 230 235 240 Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly 245 250 255 Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 260 265 270 Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu 275 280 285 Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 290 295 300 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg 305 310 315 320 Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 325 330 335 Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu 340 345 350 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr 355 360 365 Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu 370 375 380 Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 385 390 395 400 Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 405 410 415 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp 420 425 430 Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His 435 440 445 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu 450 455 460 Gly Lys 465 <210> 47 <211> 236 <212> PRT <213> Artificial Sequence <400> 47 Met Asp Met Arg Val Pro Ala Gln Leu Leu Gly Leu Leu Leu Leu Trp 1 5 10 15 Phe Pro Gly Ser Arg Cys Asn Ile Gln Met Thr Gln Ser Pro Ser Ala 20 25 30 Met Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser 35 40 45 Gln Asp Ile His Arg Tyr Leu Ser Trp Phe Gln Gln Lys Pro Gly Lys 50 55 60 Val Pro Lys His Leu Ile Tyr Arg Ala Asn Arg Leu Val Ser Gly Val 65 70 75 80 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr 85 90 95 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln 100 105 110 Tyr Asp Glu Phe Pro Tyr Thr Phe Gly Gly Gly Thr Lys Val Glu Ile 115 120 125 Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp 130 135 140 Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn 145 150 155 160 Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu 165 170 175 Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp 180 185 190 Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr 195 200 205 Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser 210 215 220 Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 225 230 235 <210> 48 <211> 117 <212> PRT <213> Artificial Sequence <400> 48 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Trp Ile Thr Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asp Ile Thr Pro Gly Arg Gly Glu Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Leu Thr Ile Ser Ile Asp Thr Ser Lys Thr Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Leu Arg Arg Gly Asp Tyr Trp Gly Gln Gly Thr Ser 100 105 110 Val Thr Val Ser Ser 115 <210> 49 <211> 117 <212> PRT <213> Artificial Sequence <400> 49 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Trp Ile Thr Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asp Ile Thr Pro Gly Arg Gly Glu Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Leu Thr Ile Ser Ile Asp Thr Ser Lys Thr Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Leu Arg Arg Gly Asp Tyr Trp Gly Gln Gly Thr Ser 100 105 110 Val Thr Val Ser Ser 115 <210> 50 <211> 117 <212> PRT <213> Artificial Sequence <400> 50 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Trp Ile Thr Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asp Ile Thr Pro Gly Arg Gly Glu Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Ser Lys Ala Thr Leu Thr Val Asp Thr Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Thr Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Leu Arg Arg Gly Asp Tyr Trp Gly Gln Gly Thr Ser 100 105 110 Val Thr Val Ser Ser 115 <210> 51 <211> 118 <212> PRT <213> Artificial Sequence <400> 51 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Arg Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Trp Ile Thr Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asp Ile Thr Pro Gly Arg Gly Glu Thr Asn Tyr Asn Pro Ser Leu 50 55 60 Lys Ser Arg Leu Thr Ile Ser Ile Asp Thr Ser Lys Thr Gln Phe Ser 65 70 75 80 Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Ile Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Leu Arg Arg Gly Asp Tyr Trp Gly Gln Gly Thr Ser 100 105 110 Val Thr Val Ser Ser His 115 <210> 52 <211> 117 <212> PRT <213> Artificial Sequence <400> 52 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Trp Ile Thr Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asp Ile Thr Pro Gly Arg Gly Glu Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Val Asp Ile Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Leu Arg Arg Gly Asp Tyr Trp Gly Gln Gly Thr Ser 100 105 110 Val Thr Val Ser Ser 115 <210> 53 <211> 117 <212> PRT <213> Artificial Sequence <400> 53 Gln Val Gln Leu Gln Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Trp Ile Thr Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Asp Ile Thr Pro Gly Arg Gly Glu Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Val Asp Ile Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Leu Arg Arg Gly Asp Tyr Trp Gly Gln Gly Thr Ser 100 105 110 Val Thr Val Ser Ser 115 <210> 54 <211> 108 <212> PRT <213> Artificial Sequence <400> 54 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Ser Ser Ser 20 25 30 Tyr Leu Asn Trp Tyr Gln Gln Lys Ser Gly Ala Ser Pro Lys Leu Trp 35 40 45 Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Gly Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Val Glu 65 70 75 80 Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Gly Tyr Pro 85 90 95 Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Val Lys 100 105 <210> 55 <211> 108 <212> PRT <213> Artificial Sequence(Artificial Sequence) <400> 55 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Ser Ser Ser 20 25 30 Tyr Leu Asn Trp Tyr Gln Gln Lys Ser Gly Ala Ser Pro Lys Leu Trp 35 40 45 Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Ser Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Gly Tyr Pro 85 90 95 Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Val Lys 100 105 <210> 56 <211> 108 <212> PRT <213> Artificial Sequence(Artificial Sequence) <400> 56 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Ser Ser Ser 20 25 30 Tyr Leu Asn Trp Tyr Gln Gln Lys Ser Gly Ala Ser Pro Lys Leu Trp 35 40 45 Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Gly Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Val Glu 65 70 75 80 Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Gly Tyr Pro 85 90 95 Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Val Lys 100 105 <210> 57 <211> 108 <212> PRT <213> Artificial Sequence(Artificial Sequence) <400> 57 Glu Ile Val Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Ser Ser Val Ser Ser Ser 20 25 30 Tyr Leu Asn Trp Tyr Gln Gln Lys Ser Gly Ala Ser Pro Lys Leu Trp 35 40 45 Ile Tyr Ser Thr Ser Asn Leu Ala Ser Gly Val Pro Gly Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Ser Val Glu 65 70 75 80 Ala Glu Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Gly Tyr Pro 85 90 95 Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Val Lys 100 105 <210> 58 <211> 121 <212> PRT <213> Artificial Sequence <400> 58 Glu Val Gln Leu Val Glu Ser Gly Gly Asp Leu Val Glu Pro Gly Gly 1 5 10 15 Ser Leu Lys Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Ser Arg Phe 20 25 30 Gly Met Ala Trp Val Arg Gln Thr Pro Asp Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Ser Gly Gly Ser Tyr Thr Arg Tyr Ser Pro Ser Phe 50 55 60 Gln Gly Gln Val Thr Met Ser Val Asp Lys Ser Ile Thr Thr Ala Tyr 65 70 75 80 Leu Gln Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Gln Gly Leu Leu Asp Tyr Leu Tyr Ala Leu Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Ala Val Thr Val Ser Ser 115 120 <210> 59 <211> 121 <212> PRT <213> Artificial Sequence <400> 59 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Ser Arg Phe 20 25 30 Gly Met Ala Trp Val Arg Gln Thr Pro Asp Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Leu Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr 65 70 75 80 Leu Gln Met Arg Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Gln Gly Leu Leu Asp Tyr Leu Tyr Ala Leu Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Ala Val Thr Val Ser Ser 115 120 <210> 60 <211> 121 <212> PRT <213> Artificial Sequence <400> 60 Leu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Ser Arg Phe 20 25 30 Gly Met Ala Trp Val Arg Gln Thr Pro Asp Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Ser Gly Gly Ser Tyr Thr Asn Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Arg Val Thr Leu Thr Val Asp Ile Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gln Gly Leu Leu Asp Tyr Leu Tyr Ala Leu Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Ala Val Thr Val Ser Ser 115 120 <210> 61 <211> 121 <212> PRT <213> Artificial Sequence <400> 61 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Ser Arg Phe 20 25 30 Gly Met Ala Trp Val Arg Gln Thr Pro Asp Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Ser Gly Gly Ser Tyr Thr Tyr Tyr Pro Asp Ser Val 50 55 60 Lys Gly Arg Leu Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr Leu Tyr 65 70 75 80 Leu Gln Met Arg Ser Leu Lys Ser Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Gln Gly Leu Leu Asp Tyr Leu Tyr Ala Leu Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Ala Val Thr Val Ser Ser 115 120 <210> 62 <211> 121 <212> PRT <213> Artificial Sequence <400> 62 Leu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Ile Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Ser Arg Phe 20 25 30 Gly Met Ala Trp Val Arg Gln Thr Pro Asp Lys Arg Leu Glu Trp Val 35 40 45 Note: There seems to be a typo in the original text where " " should probably be " " and " " should probably be " ". The translation has been done as accurately as possible based on the provided text.Ala Thr Ile Ser Ser Gly Gly Ser Tyr Thr Arg Tyr Ser Pro Ser Phe 50 55 60 Gln Gly Gln Val Thr Met Ser Val Asp Lys Ser Ile Thr Thr Ala Tyr 65 70 75 80 Leu Gln Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Gln Gly Leu Leu Asp Tyr Leu Tyr Ala Leu Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Ala Val Thr Val Ser Ser 115 120 <210> 63 <211> 121 <212> PRT <213> Artificial Sequence <400> 63 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Ser Arg Phe 20 25 30 Gly Met Ala Trp Val Arg Gln Thr Pro Asp Lys Arg Leu Glu Trp Val 35 40 45 Ala Thr Ile Ser Ser Gly Gly Ser Tyr Thr Arg Tyr Ser Pro Ser Phe 50 55 60 Gln Gly Gln Val Thr Met Ser Val Asp Lys Ser Ile Thr Thr Ala Tyr 65 70 75 80 Leu Gln Trp Asn Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Gln Gly Leu Leu Asp Tyr Leu Tyr Ala Leu Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Ala Val Thr Val Ser Ser 115 120 <210> 64 <211> 107 <212> PRT <213> Artificial Sequence(Artificial Sequence) <400> 64 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Ile Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Ser Phe 20 25 30 Leu Thr Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Arg Pro Leu Ile 35 40 45 Tyr Arg Ala Tyr Arg Leu Val Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Val Gly Ser Gly Gln Asp Tyr Ser Leu Thr Ile Ser Ser Leu Asp Tyr 65 70 75 80 Glu Asp Leu Gly Ile Tyr Tyr Cys Gln Gln Tyr Val Glu Phe Pro Pro 85 90 95 Thr Phe Gly Ala Gly Thr Met Leu Glu Leu Lys 100 105 <210> 65 <211> 107 <212> PRT <213> Artificial Sequence <400> 65 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Ile Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Ser Phe 20 25 30 Leu Thr Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Arg Pro Leu Ile 35 40 45 Tyr Arg Ala Tyr Arg Leu Val Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Tyr Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Val Glu Phe Pro Pro 85 90 95 Thr Phe Gly Ala Gly Thr Met Leu Glu Leu Lys 100 105 <210> 66 <211> 107 <212> PRT <213> Artificial Sequence <400> 66 Asp Ile Lys Met Ile Gln Ser Pro Ser Ser Met Tyr Ala Gly Leu Gly 1 5 10 15 Glu Arg Val Thr Phe Asn Cys Lys Ala Ser Gln Asp Ile Asn Ser Phe 20 25 30 Leu Thr Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Arg Pro Leu Ile 35 40 45 Tyr Arg Ala Tyr Arg Leu Val Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Tyr Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Val Glu Phe Pro Pro 85 90 95 Thr Phe Gly Ala Gly Thr Met Leu Glu Leu Lys 100 105 <210> 67 <211> 107 <212> PRT <213> Artificial Sequence <400> 67 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Ile Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Ile Asn Ser Phe 20 25 30 Leu Thr Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Arg Pro Leu Ile 35 40 45 Tyr Arg Ala Tyr Arg Leu Val Asp Gly Val Pro Asp Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Ala 65 70 75 80 Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln Tyr Val Glu Phe Pro Pro 85 90 95 Thr Phe Gly Ala Gly Thr Met Leu Glu Leu Lys 100 105 <210> 68 <211> 107 <212> PRT <213> Artificial Sequence <400> 68 Asp Ile Lys Met Ile Gln Ser Pro Ser Ser Met Tyr Ala Gly Leu Gly 1 5 10 15 Glu Arg Val Thr Phe Asn Cys Lys Ala Ser Gln Asp Ile Asn Ser Phe 20 25 30 Leu Thr Trp Phe Gln Gln Lys Pro Gly Lys Ser Pro Arg Pro Leu Ile 35 40 45 Tyr Arg Ala Tyr Arg Leu Val Asp Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Val Gly Ser Gly Gln Asp Tyr Ser Leu Thr Ile Ser Ser Leu Asp Tyr 65 70 75 80 Glu Asp Leu Gly Ile Tyr Tyr Cys Gln Gln Tyr Val Glu Phe Pro Pro 85 90 95 Thr Phe Gly Ala Gly Thr Met Leu Glu Leu Lys 100 105 <210> 69 <211> 119 <212> PRT <213> Artificial Sequence <400> 69 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Tyr Pro Gly Ser Gly Leu Thr Tyr Tyr Asn Ala Lys Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Arg Asp Thr Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Ser Tyr Gly Ser Ser Phe Pro His Val Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115 <210> 70 <211> 119 <212> PRT <213> Artificial Sequence(Artificial Sequence) <400> 70 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Tyr Pro Gly Ser Gly Leu Thr Tyr Tyr Asn Ala Lys Phe 50 55 60 Lys Gly Arg Val Thr Met Thr Ala Asp Lys Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Ser Tyr Gly Ser Ser Phe Pro His Val Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115 <210> 71 <211> 119 <212> PRT <213> Artificial Sequence <400> 71 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Tyr Pro Gly Ser Gly Leu Thr Tyr Tyr Asn Ala Lys Phe 50 55 60 Lys Gly Arg Ala Thr Val Thr Ala Asp Lys Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Ser Tyr Gly Ser Ser Phe Pro His Val Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115 <210> 72 <211> 119 <212> PRT <213> Artificial Sequence <400> 72 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Asp Tyr 20 25 30 Tyr Met Asn Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Arg Ile Tyr Pro Gly Ser Gly Leu Thr Tyr Tyr Asn Ala Lys Phe 50 55 60 Lys Gly Arg Ala Thr Val Thr Ala Asp Lys Ser Thr Ser Thr Val Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Arg Thr Ser Tyr Gly Ser Ser Phe Pro His Val Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115 <210> 73 <211> 113 <212> PRT <213> Artificial Sequence <400> 73 天冬氨酸-异亮氨酸-谷氨酰胺-甲硫氨酸-苏氨酸-谷氨酰胺-丝氨酸-脯氨酸-丝氨酸-丝氨酸-亮氨酸-丝氨酸-丙氨酸-丝氨酸-缬氨酸-甘氨酸 1 5 10 15 天冬氨酸-精氨酸-缬氨酸-苏氨酸-异亮氨酸-苏氨酸-半胱氨酸-赖氨酸-丝氨酸-丝氨酸-谷氨酰胺-丝氨酸-亮氨酸-亮氨酸-天冬酰胺-缬氨酸 20 25 30 天冬酰胺-天冬氨酸-谷氨酰胺-赖氨酸-天冬酰胺-酪氨酸-亮氨酸-丙氨酸-色氨酸-酪氨酸-谷氨酰胺-谷氨酰胺-赖氨酸-脯氨酸-甘氨酸-赖氨酸 35 40 45 丙氨酸-脯氨酸-赖氨酸-亮氨酸-亮氨酸-异亮氨酸-酪氨酸-苯丙氨酸-丙氨酸-丝氨酸-苏氨酸-精氨酸-谷氨酸-丝氨酸-甘氨酸-缬氨酸 50 55 60 脯氨酸-丝氨酸-精氨酸-苯丙氨酸-丝氨酸-甘氨酸-丝氨酸-甘氨酸-丝氨酸-甘氨酸-苏氨酸-天冬氨酸-苯丙氨酸-苏氨酸-亮氨酸-苏氨酸 65 70 75 80 异亮氨酸-丝氨酸-丝氨酸-亮氨酸-谷氨酰胺-脯氨酸-谷氨酸-天冬氨酸-苯丙氨酸-丙氨酸-苏氨酸-酪氨酸-酪氨酸-半胱氨酸-谷氨酰胺-谷氨酰胺 85 90 95 组氨酸-酪氨酸-丝氨酸-苏氨酸-脯氨酸-亮氨酸-苏氨酸-苯丙氨酸-甘氨酸-谷氨酰胺-甘氨酸-苏氨酸-赖氨酸-亮氨酸-谷氨酸-异亮氨酸 100 105 110 赖氨酸 <210> 74 <211> 113 <212> PRT <213> Artificial Sequence <400> 74 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ser Ser Gln Ser Leu Leu Asn Val 20 25 30 Asn Asp Gln Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys 35 40 45 Ala Pro Lys Leu Leu Val Tyr Phe Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Pro Glu Asp Phe Ala Thr Tyr Phe Cys Gln Gln 85 90 95 His Tyr Ser Thr Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 75 <211> CDS 10 <212> PRT <213> Mouse (Mus musculus) <400> 75 Gly Asp Ser Ile Thr Ser Gly Tyr Trp Asn 1 5 10 <210> 76 <211> CDS 16 <212> PRT <213> Mouse (Mus musculus) <400> 76 Tyr Ile Ser Tyr Thr Gly Ser Thr Tyr Tyr Asn Leu Ser Leu Lys Ser 1 5 10 15 It should be noted that in the original text, <211> only has a number, which is not very clear in the context. I have added "CDS" according to common sense in the translation to make it more complete and understandable. If this is not in line with your requirements, please adjust it according to the actual situation. <210> 77 <211> 10 <212> PRT <213> Mouse origin (Mus musculus) <400> 77 Tyr Ile Leu Trp Leu Arg His Phe Asp Tyr 1 5 10 <210> 78 <211> 17 <212> PRT <213> Mouse origin (Mus musculus) <400> 78 Lys Ser Ser Gln Ser Leu Leu Tyr Ser Ser Asn Gln Lys Asn Ser Leu 1 5 10 15 Ala <210> 79 <211> 7 <212> PRT <213> Mouse origin (Mus musculus) <400> 79 Trp Ala Ser Thr Arg Glu Ser 1 5 <210> 80 <211> 9 <212> PRT <213> Mouse origin (Mus musculus) <400> 80 Gln Gln Tyr Tyr Gly Tyr Pro Leu Thr 1 5 <210> 81 <211> 5 <212> PRT <213> Mouse origin (Mus musculus) <400> 81 Ser Asp Tyr Trp Asn 1 5 <210> 82 <211> 16 <212> PRT <213> From Mus musculus <400> 82 Tyr Ile Ser Tyr Thr Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys Arg 1 5 10 15 <210> 83 <211> 10 <212> PRT <213> From Mus musculus <400> 83 Tyr Arg Asp Trp Pro Gly Tyr Phe Asp Val 1 5 10 <210> 84 <211> 11 <212> PRT <213> From Mus musculus <400> 84 Arg Ala Ser Lys Ser Ile Arg Lys Phe Leu Ala 1 5 10 <210> 85 <211> 7 <212> PRT <213> From Mus musculus <400> 85 Ser Gly Ser Thr Leu Gln Ser 1 5 <210> 86 <211> 9 <212> PRT <213> From Mus musculus <400> 86 Gln His Tyr Asn Glu Tyr Pro Phe Thr 1 5 <210> 87 <211> 10 [[ID=7 0]]<212> PRT <213> Mouse origin (Mus musculus) <400> 87 Gly Tyr Thr Phe Thr Arg Asn Thr Met His 1 5 10 <210> 88 <211> 17 <212> PRT <213> Mouse origin (Mus musculus) <400> 88 Phe Ile Asp Pro His Asn Thr Tyr Thr Arg Tyr Asn Gln Lys Phe Lys 1 5 10 15 Asp <210> 89 <211> 5 <212> PRT <213> Mouse origin (Mus musculus) <400> 89 Trp Pro Met Asp Tyr 1 5 <210> 90 <211> 17 <212> PRT <213> Mouse origin (Mus musculus) <400> 90 Lys Ser Ser Gln Ser Leu Ser Trp Ser Glu Asn Gln Asn Asn Tyr Leu 1 5 10 15 Ser <210> 91 <211> 7 <212> PRT <213> Mouse origin (Mus musculus) <400> 91 Gly Ala Ser Ile Arg Glu Ser 1 5 <210> 92 <211> 11 <212> PRT <213> From Mus musculus <400> 92 Gln His Asn His Gly Ser Phe Leu Pro Tyr Thr 1 5 10 <210> 93 <211> 5 <212> PRT <213> From Mus musculus <400> 93 Ser Asn Trp Met Asn 1 5 <210> 94 <211> 17 <212> PRT <213> From Mus musculus ...<400> 94 Met Ile His Leu Tyr Asp Ser Glu Thr Lys Leu Asn Pro Asn Phe Gln 1 5 10 15 Asp <210> 95 <211> 9 ...<212> PRT <213> From Mus musculus <400> 95 Ser Ala Gly Asn Tyr Arg Phe Ala Tyr 1 5 <210> 96 <211> 11 <212> PRT <213> From Mus musculus <400> 96 Lys Ala Ser Gln Ser Val Ser Asn Glu Val Ala 1 5 10 <210> 97 <211> 7 <212> PRT <213> From Mus musculus <400> 97 Ser Ala Ser Ser Arg Tyr Thr 1 5 <210> 98 <211> 9 <212> PRT <213> Mouse (Mus musculus) <400> 98 Gln Gln His Tyr Ser Ser Pro Leu Thr 1 5 <210> 99 <211> 118 <212> PRT <213> Mouse (Mus musculus) <400> 99 Glu Val Gln Leu Gln Glu Ser Gly Pro Ser Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ser Val Thr Gly Asp Ser Ile Thr Ser Gly 20 25 30 Tyr Trp Asn Trp Ile Arg Lys Phe Pro Gly Asn Lys Leu Glu Tyr Met 35 40 45 Gly Tyr Ile Ser Tyr Thr Gly Ser Thr Tyr Tyr Asn Leu Ser Leu Lys 50 55 60 Ser Arg Ile Ser Ile Thr Arg Asp Thr Ser Lys Asn Gln Tyr Tyr Leu 65 70 75 80 Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95 Arg Tyr Ile Leu Trp Leu Arg His Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Thr Leu Thr Val Ser Ser 115 <210> 100 <211> 118 <212> PRT <213> Mouse (Mus musculus) <400> 100 Glu Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Ala Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ser Val Thr Gly Tyr Ser Ile Thr Ser Asp 20 25 30 Tyr Trp Asn Trp Ile Arg Lys Phe Pro Gly Asn Lys Leu Glu Tyr Met 35 40 45 Gly Tyr Ile Ser Tyr Thr Gly Ser Thr Tyr Tyr Asn Pro Ser Leu Lys 50 55 60 Arg Arg Val Ser Ile Thr Arg Asp Thr Ser Lys Asn Asn Tyr Tyr Leu 65 70 75 80 Gln Leu Asn Ser Val Thr Thr Glu Asp Thr Ala Thr Tyr Tyr Cys Ala 85 90 95 Arg Tyr Arg Asp Trp Pro Gly Tyr Phe Asp Val Trp Gly Ala Gly Thr 100 105 110 Thr Val Thr Val Ser Ser 115 <210> 101 <211> 114 <212> PRT <213> Mouse origin (Mus musculus) <400> 101 Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ala Arg Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Asn 20 25 30<e Thr Met His Trp Ile Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Phe Ile Asp Pro His Asn Thr Tyr Thr Arg Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Ser Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Trp Pro Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val 100 105 110 Ser Ser <210> 102 <211> 118 <212> PRT <213> Mouse origin (Mus musculus) <400> 102 Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Arg Pro Gly Thr 1 5 10 15 Ser Val Asn Leu Ser Cys Lys Ala Ser Gly Tyr Ser Phe Thr Ser Asn 20 25 30 Trp Met Asn Trp Val Lys Gln Arg Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Met Ile His Leu Tyr Asp Ser Glu Thr Lys Leu Asn Pro Asn Phe 50 55 60 Gln Asp Lys Ala Thr Leu Thr Val Asp Lys Ser Ser Ser Ile Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Pro Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Ala Gly Asn Tyr Arg Phe Ala Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ala 115 <210> 103 <211> 113 <212> PRT <213> Mouse (Mus musculus) <400> 103 Asp Ile Val Met Ser Gln Ser Pro Ser Ser Leu Ala Val Ser Val Gly 1 5 10 15 Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Ser Asn Gln Lys Asn Ser Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Ser Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Lys Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Gly Tyr Pro Leu Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu 100 105 110 Lys <210> 104 <211> 107 <212> PRT <213> Mouse (Mus musculus) <400> 104 Asp Val Gln Ile Thr Gln Ser Pro Ser Tyr Leu Ala Ala Ser Pro Gly 1 5 10 15 Glu Thr Ile Thr Ile Asn Cys Arg Ala Ser Lys Ser Ile Arg Lys Phe 20 25 30 Leu Ala Trp Tyr Gln Glu Lys Pro Gly Lys Thr Asn Lys Leu Leu Ile[[ID=Z5]] 35 40 45 Tyr Ser Gly Ser Thr Leu Gln Ser Gly Ile Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Thr Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln His Tyr Asn Glu Tyr Pro Phe 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 105 <211> 115 <212> PRT <213> Mouse (Mus musculus) <400> 105 Asp Ile Val Met Thr Gln Phe Pro Ser Ser Leu Ala Val Thr Ala Gly 1 5 10 15 Glu Lys Val Thr Met Arg Cys Lys Ser Ser Gln Ser Leu Ser Trp Ser 20 25 30 Glu Asn Gln Asn Asn Tyr Leu Ser Trp Tyr Gln Gln Lys Gln Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Ile Arg Glu Ser Trp Val 50 55 60 Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Asn Val His Ala Glu Asp Leu Gly Val Tyr Phe Cys Gln His 85 90 95 Asn His Gly Ser Phe Leu Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu 100 105 110 Glu Ile Lys 115 <210> 106 <211> 107 <212> PRT <213> Mouse (Mus musculus) <400> 106 Ser Ile Val Met Thr Gln Thr Pro Lys Phe Leu Pro Val Thr Ala Glu 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Ser Val Ser Asn Glu 20 25 30 Val Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Lys Leu Leu Ile 35 40 45 Tyr Ser Ala Ser Ser Arg Tyr Thr Gly Val Pro Asp Arg Phe Ile Gly 50 55 60 Gly Gly Ser Ala Thr Asp Phe Thr Phe Thr Ile Ser Ser Val Gln Val 65 70 75 80 Glu Asp Leu Ala Val Tyr Phe Cys Gln Gln His Tyr Ser Ser Pro Leu 85 90 95 Thr Phe Gly Ala Gly Thr Lys Leu Glu Leu Lys 100 105 <210> 107 <211> 99 <212> PRT <213> Artificial Sequence <400> 107 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Gly Ser Ile Ser Ser Gly 20 25 30 Gly Tyr Tyr Trp Ser Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu 35 40 45 Trp Ile Gly Tyr Ile Tyr Tyr Ser Gly Ser Thr Tyr Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe 65 70 75 80 Ser Leu Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr 85 90 95 Cys Ala Arg <210> 108 <211> 98 <212> PRT <213> Artificial Sequence <400> 108 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Trp Ile Asn Ala Gly Asn Gly Asn Thr Lys Tyr Ser Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg <210> 109 <211> 101 <212> PRT <213> Artificial Sequence <400> 109 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly<{} 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr Ser 20 25 30 Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Ser Thr Pro 100 <210> 110 <211> 118 <212> PRT <213> Artificial Sequence <400> 110 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Ser Ile Thr Ser Gly 20 25 30 Tyr Trp Asn Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Ile Ser Tyr Thr Gly Ser Thr Tyr Tyr Asn Leu Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 7� 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Tyr Ile Leu Trp Leu Arg His Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 111 <211> 118 <212> PRT <213> Artificial Sequence <400> 111 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Ser Ile Thr Ser Gly 20 25 30 Tyr Trp Asn Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Tyr Ile 35 40 45 Gly Tyr Ile Ser Tyr Thr Gly Ser Thr Tyr Tyr Asn Leu Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Val Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Tyr Ile Leu Trp Leu Arg His Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 112 <211> 118 <212> PRT <213> Artificial Sequence <400> 112 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Ser Ile Thr Ser Gly 20 25 30 Tyr Trp Asn Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Tyr Ile 35 40 45 Gly Tyr Ile Ser Tyr Thr Gly Ser Thr Tyr Tyr Asn Leu Ser Leu Lys 50 55 60 Ser Arg Val Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Phe Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Tyr Ile Leu Trp Leu Arg His Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 113 <211> 118 <212> PRT <213> Artificial Sequence <400> 113 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Ser Ile Thr Ser Gly 20 25 30 Tyr Trp Asn Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Tyr Ile 35 40 45 Gly Tyr Ile Ser Tyr Thr Gly Ser Thr Tyr Tyr Asn Leu Ser Leu Lys 50 55 60 Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Tyr Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Tyr Ile Leu Trp Leu Arg His Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 114 <211> 118 <212> PRT <213> Artificial Sequence <400> 114 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Asp Ser Ile Thr Ser Gly 20 25 30 Tyr Trp Asn Trp Ile Arg Gln His Pro Gly Lys Gly Leu Glu Tyr Met 35 40 45 Gly Tyr Ile Ser Tyr Thr Gly Ser Thr Tyr Tyr Asn Leu Ser Leu Lys 50 55 60 Ser Arg Ile Thr Ile Ser Arg Asp Thr Ser Lys Asn Gln Tyr Ser Leu 65 70 75 80 Lys Leu Ser Ser Val Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Tyr Ile Leu Trp Leu Arg His Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Leu Thr Val Ser Ser 115 <210> 115 <211> 113 <212> PRT <213> Artificial Sequence(Artificial Sequence) <400> 115 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Ser Asn Gln Lys Asn Ser Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Gly Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 116 <211> 113 <212> PRT <213> Artificial Sequence <400> 116 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Ser Asn Gln Lys Asn Ser Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Gly Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 117 <211> 113 <212> PRT <213> Artificial Sequence <400> 117 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Val Thr Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Ser Asn Gln Lys Asn Ser Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Gly Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 118 <211> 113 <212> PRT <213> Artificial Sequence <400> 118 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Tyr Ser 20 25 30 Ser Asn Gln Lys Asn Ser Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Gln 85 90 95 Tyr Tyr Gly Tyr Pro Leu Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile 100 105 110 Lys <210> 119 <211> 114 <212> PRT <213> Artificial Sequence <400> 119 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Asn 20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Phe Ile Asp Pro His Asn Thr Tyr Thr Arg Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Ile Thr Arg Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Trp Pro Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val 100 105 110 Ser Ser <210> 120 <211> 114 <212> PRT <213> Artificial Sequence <400> 120 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Asn 20 25 30 Thr Met His Trp Val Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Phe Ile Asp Pro His Asn Thr Tyr Thr Arg Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Ile Thr Ala Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Trp Pro Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val 100 105 110 Ser Ser <210> 121 <211> 114 <212> PRT <213> Artificial Sequence <400> 121 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Asn 20 25 30 Thr Met His Trp Ile Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Met 35 40 45 Gly Phe Ile Asp Pro His Asn Thr Tyr Thr Arg Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Val Thr Ile Thr Ala Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Trp Pro Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val 100 105 110 Ser Ser <210> 122 <211> 114 <212> PRT <213> Artificial Sequence <400> 122 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Ala Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Asn 20 25 30 Thr Met His Trp Ile Arg Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile 35 40 45 Gly Phe Ile Asp Pro His Asn Thr Tyr Thr Arg Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Ala Thr Ile Thr Ala Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Trp Pro Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val 100 105 110 Ser Ser <210> 123 <211> 114 <212> PRT <213> Artificial Sequence <400> 123 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Ala Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Arg Asn 20 25 30 Thr Met His Trp Ile Lys Gln Ala Pro Gly Gln Arg Leu Glu Trp Ile 35 40 45 Gly Phe Ile Asp Pro His Asn Thr Tyr Thr Arg Tyr Asn Gln Lys Phe 50 55 60 Lys Asp Arg Ala Thr Leu Thr Ala Asp Thr Ser Ala Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Trp Pro Met Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val 100 105 110 Ser Ser <210> 124 <211> 115 <212> PRT <213> Artificial Sequence <400> 124 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Ser Trp Ser 20 25 30 Glu Asn Gln Asn Asn Tyr Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Ile Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln His 85 90 95 Asn His Gly Ser Phe Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu 100 105 110 Glu Ile Lys 115 <210> 125 <211> 115 <212> PRT <213> Artificial Sequence <400> 125 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Val Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Ser Trp Ser 20 25 30 Glu Asn Gln Asn Asn Tyr Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Ile Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln His 85 90 95 Asn His Gly Ser Phe Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu 100 105 110 Glu Ile Lys 115 <210> 126 <211> 115 <212> PRT <213> Artificial Sequence <400> 126 Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly 1 5 10 15 Glu Arg Val Thr Met Arg Cys Lys Ser Ser Gln Ser Leu Ser Trp Ser 20 25 30 Glu Asn Gln Asn Asn Tyr Leu Ser Trp Tyr Gln Gln Lys Pro Gly Gln 35 40 45 Pro Pro Lys Leu Leu Ile Tyr Gly Ala Ser Ile Arg Glu Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr 65 70 75 80 Ile Ser Ser Val Gln Ala Glu Asp Val Gly Val Tyr Tyr Cys Gln His 85 90 95 Asn His Gly Ser Phe Leu Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu 100 105 110 Glu Ile Lys 115 <210> 127 <211> 354 <212> DNA <213> Artificial Sequence <400> 127 caggttcagc tgcaggagtc cggccctggc ctggtgaagc ccagccagac cctgagcctg 60 This region contains the sequence: caggttcagc etc., up to cctgagcctg at 60 bases. acctgtaccg tgagcggcga cagcatcact agcggctact ggaactggat caggcaacac 120 acctgtaccg etc., up to caggcaacac at 120 bases. cccggcaagg gcctggagta catcggctac atcagctaca ccggcagcac ctactacaac 180 cccggcaagg etc., up to ctactacaac at 180 bases. ctgagtctga agagcagggt gaccatcagc cgggacacca gcaagaacca gttctctctg 240 ctgagtctga etc., up to gttctctctg at 240 bases. aagctgagca gcgtgactgc ggcggatacc gccgtgtact actgcgccag gtacatcctg 300 aagctgagca etc., up to gtacatcctg at 300 bases. tggttgaggc acttcgacta ctggggccag ggcaccctgg tgaccgttag ctct 354 tggttgaggc etc., up to ctct at 354 bases. <210> 128 <211> 342 <212> DNA <213> Artificial Sequence <400> 128 caggtgcagc tggtgcagag cggagctgag gtggccaagc caggcgccag cgtgaagatg 60 caggtgcagc etc., up to cgtgaagatg at 60 bases. tcctgcaagg ccagcggcta cacctttacc cggaacacaa tgcactggat taagcaggcc 120 tcctgcaagg etc., up to taagcaggcc at 120 bases. cccggccagc ggctggagtg gatcggcttc atcgacccac acaataccta caccagatac 180 cccggccagc etc., up to caccagatac at 180 bases. [[ID=:30]]aaccagaagt ttaaggaccg ggccaccctg accgccgata catcagcatc aacagcttac 240 aaccagaagt etc., up to aacagcttac at 240 bases. It should be noted that this translation is for the purpose of presenting the patent text in English while maintaining the original format and content as accurately as possible. The actual understanding and interpretation of such biological sequence data require in-depth knowledge of relevant biological and patent concepts.atggagctga gtagtctgag atctgaggat acagccgtgt actattgcgc tagatggcct 300 atggattact ggggccaggg aaccctggtg accgtgtcta gc 342 <210> 129 <211> 339 <212> DNA <213> Artificial Sequence <400> 129 gacatcgtga tgacccagag ccccgactcc ctggccgtga gcctgggcga gagggccacc 60 atctcctgca agagcagtca gagccttctg tatagctcca accagaagaa cagcctggcc 120 tggtatcaac agaagcccgg tcaacccccc aagctgctga tctactgggc ctcaaccagg 180 gaaagcggcg tgcccgacag gttcagcggc agtggcagcg gcaccgactt caccctgacc 240 atcagctcct tgcaggccga ggacgtggca gtgtactact gccagcagta ctacggctac 300 cccctgacat tcggaggggg aaccaagttg gagatcaag 339 <210> 130 <211> 345 <212> DNA <213> Artificial Sequence <400> 130 gatatcgtga tgacccagag cccagatagc ctggccgtga gcctgggaga gagagtgacc 60 atgagatgta agtctagtca gagtctgagt tggagcgaga atcagaacaa ttacctgagc 120 tggtatcagc agaagcctgg ccagcctcca aagctgctga tctacggcgc ctccatccgg 180 gagagcggag tgcccgaccg cttcagcggc agcggcagcg gcacagactt caccctgacc 240 atcagcagcg tgcaggccga ggatgtgggc gtgtactact gtcagcacaa ccacggcagc 300 ttcctgccct acaccttcgg gcagggcacc aagctggaga tcaag 345 <210> 131 <211> 107 <212> PRT <213> Artificial Sequence <400> 131 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20 25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 100 105 <210> 132 <211> 327 <212> PRT <213> Artificial Sequence <400> 132 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100 105 110 Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly Lys 325 <210> 133 <211> 327 <212> PRT <213> Artificial Sequence <400> 133 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100 105 110 Glu Phe Glu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220 Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu Ser Leu Gly Lys 325 <210> 134 <211> 220 <212> PRT <213> Artificial Sequence <400> 134 Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser 1 5 10 15 Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu 20 25 30 Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile Ile Gln 35 40 45 Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg 50 55 60 Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala 65 70 75 80 Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Arg Cys 85 90 95 Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val 100 105 110 Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro 115 120 125 Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys 130 135 140 Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys 145 150 155 160 Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn Val Thr 165 170 175 Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr 180 185 190 Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile 195 200 205 Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg 210 215 220

Claims

1. An anti-CD47 / anti-PD-Ll antibody comprising an anti-CD47 antibody or antigen-binding fragment thereof and an anti-PD-Ll antibody or antigen-binding fragment thereof, wherein: the anti-CD47 antibody or antigen-binding fragment thereof comprises a first heavy chain variable region and a first light chain variable region, wherein: (1) the first heavy chain variable region comprises H1CDR1, H1CDR2, and H1CDR3, whose amino acid sequences are SEQ ID NOs: 4, 5, and 6, respectively; (2) the first light chain variable region comprises L1CDR1, L1CDR2, and L1CDR3, whose amino acid sequences are SEQ ID NOs: 15, 16, and 17, respectively; and the anti-PD-Ll antibody or antigen-binding fragment thereof comprises a second heavy chain variable region and a second light chain variable region, wherein: (1) the second heavy chain variable region comprises H2CDR1, H2CDR2, and H2CDR3, whose amino acid sequences are SEQ ID NOs: 75, 76, and 77, respectively; and (2) the second light chain variable region comprises L2CDR1, L2CDR2, and L2CDR3, whose amino acid sequences are SEQ ID NOs: 78, 79, and 80, respectively.

2. The anti-CD47 / anti-PD-Ll antibody of claim 1, wherein: (1) the amino acid sequence of the first heavy chain variable region is the amino acid sequence set forth in SEQ ID NO: 30; and (2) the amino acid sequence of the first light chain variable region is the amino acid sequence set forth in SEQ ID NO:

33.

3. The anti-CD47 / anti-PD-Ll antibody of claim 1, comprising an anti-PD-Ll antibody or antigen-binding fragment thereof, wherein the anti-PD-Ll antibody or antigen-binding fragment thereof comprises a second heavy chain variable region and a second light chain variable region, wherein: (1) the amino acid sequence of the second heavy chain variable region is the amino acid sequence set forth in SEQ ID NO: 112; and (2) the amino acid sequence of the second light chain variable region is the amino acid sequence set forth in SEQ ID NO:

116.

4. The anti-CD47 / anti-PD-Ll antibody of claim 2, comprising an anti-PD-Ll antibody or antigen-binding fragment thereof, wherein the anti-PD-Ll antibody or antigen-binding fragment thereof comprises a second heavy chain variable region and a second light chain variable region, wherein: (1) the amino acid sequence of the second heavy chain variable region is the amino acid sequence set forth in SEQ ID NO: 112; and (2) the amino acid sequence of the second light chain variable region is the amino acid sequence set forth in SEQ ID NO:

116.

5. The anti-CD47 / anti-PD-Ll antibody of any one of claims 1-4, wherein the antibody is a humanized antibody or a fully human antibody.

6. The anti-CD47 / anti-PD-Ll antibody of any one of claims 1-4, wherein the antibody is a bispecific antibody.

7. An isolated nucleic acid encoding the anti-CD47 / anti-PD-Ll antibody of any one of claims 1-6.

8. The nucleic acid of claim 7, wherein: (1) the nucleotide sequence encoding the first heavy chain variable region amino acid sequence is set forth in SEQ ID NO: 36; (2) the nucleotide sequence encoding the first light chain variable region amino acid sequence is set forth in SEQ ID NO: 39; (3) the nucleotide sequence encoding the second heavy chain variable region amino acid sequence is set forth in SEQ ID NO: 127; and (4) the nucleotide sequence encoding the second light chain variable region amino acid sequence is set forth in SEQ ID NO:

129.

9. An expression vector comprising the nucleic acid of claim 7 or 8.

10. A host cell transformed with the expression vector of claim 9, said host cell being selected from the group consisting of prokaryotic and eukaryotic cells.

11. The host cell of claim 10, which is a mammalian cell.

12. A method of producing the anti-CD47 / anti-PD-Ll antibody of any one of claims 1-6, comprising the steps of expressing the antibody in the host cell of claim 10 or 11, and isolating the antibody from the host cell.

13. A pharmaceutical composition comprising the anti-CD47 / anti-PD-Ll antibody of any one of claims 1-6 and a pharmaceutically acceptable carrier.

14. Use of the anti-CD47 / anti-PD-Ll antibody of any one of claims 1-6 or the pharmaceutical composition of claim 13 for the manufacture of a medicament for the treatment of a tumor, said tumor being lymphoma, breast cancer, lung cancer, gastric cancer, intestinal cancer, esophageal cancer, ovarian cancer, cervical cancer, kidney cancer, bladder cancer, pancreatic cancer, glioma, and / or melanoma.

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