Bispecific antibodies targeting pd-l1 and fasl, pharmaceutical compositions and uses thereof

Abstract

The present application provides a bispecific antibody targeting PD-L1 and FasL, which comprises: (a) a PD-L1 antibody capable of specifically binding to PD-L1, and (b) a FasL antibody or a Fas extracellular domain, a truncated or mutant of the Fas extracellular domain, capable of specifically binding to FasL; wherein the heavy chain variable region and the light chain variable region of the FasL antibody are connected to the heavy chain variable region and the light chain variable region of the PD-L1 antibody through a linker sequence, and / or the Fas extracellular domain is connected to the constant region of the PD-L1 antibody through a linker sequence. The present application comprises a preparation method of the molecule and its use as an anticancer drug. The bispecific antibody of the present application comprises a fragment capable of binding to PD-L1 and a fragment specifically binding to FasL, and such bifunctional antibody molecule can produce a synergistic effect and play a high-efficiency antitumor role. Compared with the administration of either of the two antibodies alone, the bispecific antibody of the present application shows a significant synergistic effect in cancer therapy.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a bispecific antibody that simultaneously targets PD-L1 and FasL, a pharmaceutical composition including the bispecific antibody, and its application as an anticancer drug. Background Technology

[0002] Tumor immunotherapy has become a hot topic in cancer treatment research. Under normal circumstances, the human immune system can recognize and eliminate tumor cells in the tumor microenvironment. However, tumor cells can employ a series of strategies to suppress the body's immune system, preventing it from killing tumor cells normally. This allows tumor cells to survive at various stages of the anti-tumor immune response; this process is called immune escape. Different tumors can develop immune tolerance by abnormally inhibiting the immune system's effective recognition and killing of tumor cells at different stages, and may even promote the occurrence and development of tumors. Tumor immunotherapy is a treatment method that controls and eliminates tumors by restarting and maintaining the tumor-immune cycle and restoring the body's normal anti-tumor immune response. This treatment method includes monoclonal antibody immune checkpoint inhibitors, therapeutic antibodies, cancer vaccines, cell therapy, and small molecule inhibitors.

[0003] Currently, the most widespread application in tumor immunotherapy is the development of targeted monoclonal antibodies against regulatory immune checkpoint molecules that inhibit T cell activation, with drugs blocking programmed cell death protein-1 (PD-1) and programmed cell death protein ligand-1 (PD-L1) being the main representatives. PD-1 acts in the effector phase of the immune response, expressed in activated T cells, B cells, and myeloid cells. It has two ligands, the programmed cell death molecule ligands PD-L1 and PD-L2. Both PD-L1 and PD-L2 are expressed in antigen-presenting cells. In addition, PD-L1 is also expressed in various tissues. The binding of PD-1 and PD-L1 mediates co-inhibitory signals for T cell activation, inhibiting the killing function of T cells and playing a negative regulatory role in the human immune response. Chen Lieping's laboratory first discovered that PD-L1 is highly expressed in tumor tissues and inhibits the function of tumor-infiltrating CD8+ T cells, which is an important mechanism for tumor cell immune escape. Therefore, immunomodulation targeting PD-1 / PD-L1 is of great significance in anti-tumor therapy. PD-1 / PD-L1 inhibitors can specifically bind to PD-L1 on tumor cells and inhibit its expression, thereby restoring the tumor cell recognition function of suppressed T cells and achieving an anti-cancer effect through the body's own immune system. Currently, PD-1 / PD-L1-targeted tumor immunotherapy has achieved higher objective response rates while significantly reducing immune-related side effects, and has now been approved by the FDA for more than 10 cancer indications. Furthermore, PD-1 / PD-L1-targeted tumor immunotherapy has also shown significant anti-tumor effects when used in combination with other therapies. While the target mechanism of PD-1 / PD-L1-targeted tumor immunotherapy is well-defined, the efficacy rate is only around 20%, and there is still room for improvement. Therefore, developing more efficient and broader immunotherapies based on this foundation is of great importance. Summary of the Invention

[0004] A first aspect of the present invention provides a bispecific antibody, the bispecific antibody comprising:

[0005] (a) PD-L1 antibodies that can specifically bind to PD-L1, and

[0006] (b) A FasL antibody or Fas extracellular domain, a truncated form or mutant of the Fas extracellular domain that can specifically bind to FasL; wherein the heavy chain variable region and the light chain variable region of the FasL antibody are respectively linked to the heavy chain variable region and the light chain variable region of the PD-L1 antibody via adapter sequences, and / or the Fas extracellular domain is linked to the constant region of the PD-L1 antibody via adapter sequences.

[0007] Apoptosis is the autonomous, orderly cell death controlled by genes in order to maintain homeostasis. The Fas / FasL signaling pathway is an important signaling pathway for apoptosis. Fas, also known as CD95, is a transmembrane protein belonging to the tumor necrosis factor receptor superfamily. Its binding to its ligand FasL initiates apoptotic signal transduction, leading to apoptosis. Fas is a universally expressed receptor molecule that can be expressed on the surface of various cells, but FasL is typically expressed only on activated T cells and NK cells. Further research has revealed a close relationship between FasL and tumor development. FasL can inhibit the activity of CD8+ T cells in the immune microenvironment; tumor expression of FasL can induce apoptosis of tumor-infiltrating T lymphocytes; high FasL expression at tumor sites leads to a decrease in the number of T cells, thus making the tumor more prone to becoming a "cold" tumor; and studies have shown that overexpression of FasL at tumor sites promotes tumor metastasis and development.

[0008] This invention designs bispecific antibodies targeting PD-L1 and FasL to achieve dual anti-tumor effects from the two antibodies, which not only improves the effectiveness of treatment but also reduces the dosage, lowers side effects, and enhances safety.

[0009] In one embodiment of the present invention, the PD-L1 antibody can be linked to a complete FasL antibody or to the Fas extracellular domain (Fas-ECD). Inhibition of Fas / FasL proteins can be achieved through the binding of the FasL antibody to a FasL ligand or through the binding of the Fas-ECD to a FasL ligand. During their research, the inventors discovered that FasL includes soluble FasL (sFasL) and membrane-expressed FasL (mFasL), both of which can exert an immune effect. Therefore, in designing the FasL protein targeting mechanism, both FasL antibodies and Fas-ECD were selected, as both can target FasL and exert an immune effect.

[0010] In one embodiment of the present invention, the bispecific antibody includes a PD-L1 antibody and one or two Fas extracellular domains (Fas-ECDs) connected by adapter sequences on both sides of the heavy chain constant region at the C-terminus of the PD-L1 antibody.

[0011] In another embodiment of the present invention, the bispecific antibody comprises a PD-L1 antibody and a FasL antibody, wherein the N-terminal heavy chain variable region and light chain variable region of the PD-L1 antibody are respectively linked to the N-terminal heavy chain variable region and light chain variable region of the FasL antibody via adapter sequences. In another embodiment, the N-terminal heavy chain variable region and light chain variable region of the FasL antibody are respectively linked to the N-terminal heavy chain variable region and light chain variable region of the PD-L1 antibody via adapter sequences.

[0012] In one embodiment of the present invention, the bispecific antibody includes a PD-L1 antibody, a FasL antibody, and a Fas extracellular domain. The N-terminal heavy chain variable region and light chain variable region of the PD-L1 antibody are respectively connected to the N-terminal heavy chain variable region and light chain variable region of the FasL antibody via adapter sequences. Furthermore, one or two Fas extracellular domains are connected to both sides of the C-terminal heavy chain constant region of the PD-L1 antibody or FasL antibody formed by linking the PD-L1 antibody and FasL antibody to form the bispecific antibody via adapter sequences. In another embodiment, the N-terminal heavy chain variable region and light chain variable region of the FasL antibody are respectively connected to the N-terminal heavy chain variable region and light chain variable region of the PD-L1 antibody via adapter sequences. Furthermore, one or two Fas extracellular domains are connected to both sides of the C-terminal heavy chain constant region of the bispecific antibody formed by linking the FasL antibody and PD-L1 antibody to form the bispecific antibody via adapter sequences.

[0013] In this invention, the PD-L1 antibody can be a complete PD-L1 antibody capable of specifically binding to PD-L1, or a mutant or truncated version of a PD-L1 antibody capable of specifically binding to PD-L1. Similarly, the FasL antibody can be a complete FasL antibody, a mutant or truncated version of a FasL antibody. Likewise, the Fas extracellular domain can also be a mutant or truncated version.

[0014] In this invention, both the PD-L1 antibody and the FasL antibody comprise two light chains and two heavy chains, each light chain comprising a light chain variable region and a light chain constant region, and each heavy chain comprising a heavy chain variable region and a heavy chain constant region; wherein, the amino acid sequence of the light chain variable region of the PD-L1 antibody is shown in SEQ ID NO:1, and is encoded by the nucleotide sequence shown in SEQ ID NO:2; the amino acid sequence of the heavy chain variable region of the PD-L1 antibody is shown in SEQ ID NO:3, and is encoded by the nucleotide sequence shown in SEQ ID NO:4;

[0015] The amino acid sequence of the light chain variable region of the FasL antibody is shown in SEQ ID NO:5, which is encoded by the nucleotide sequence shown in SEQ ID NO:6; the amino acid sequence of the heavy chain variable region of the PD-L1 antibody is shown in SEQ ID NO:7, which is encoded by the nucleotide sequence shown in SEQ ID NO:8.

[0016] In this invention, the adapter sequence can be a commonly used (G4S)3 adapter sequence. Specifically, the nucleotide sequence of the (G4S)3 adapter sequence is shown in SEQ ID NO:17, and the amino acid sequence is shown in SEQ ID NO:18. The adapter sequence connects two antibodies or corresponding portions of antibodies without affecting the function of the bispecific antibody.

[0017] A first aspect of the present invention provides a pharmaceutical composition comprising the bispecific antibody described herein and optionally a pharmaceutically acceptable carrier or excipient.

[0018] Another aspect of the present invention provides the use of the bispecific antibody described herein in the preparation of a medicament for treating PD-L1 and FasL-related cancers.

[0019] In the application of this invention, the bispecific antibody can treat one or more of the following cancers: urothelial carcinoma, small cell lung cancer, triple-negative breast cancer, liver cancer, melanoma, pancreatic cancer, gastric cancer, ovarian cancer, kidney cancer, colorectal cancer, breast cancer, prostate cancer, and bladder cancer.

[0020] This invention is based on the discovery that the bispecific antibody of this invention comprises a fragment capable of binding to PD-L1 and a fragment specifically binding to FasL. This bifunctional antibody molecule can produce a synergistic effect, simultaneously targeting PD-L1 and FasL, thereby exerting a highly effective anti-tumor effect. Compared to the efficacy of administering these two antibodies alone, the bispecific antibody of this invention shows a significant synergistic effect in cancer treatment. Attached Figure Description

[0021] Figure 1 This is a schematic diagram of the structure of a monoclonal antibody (PD-L1 monoclonal antibody or FasL monoclonal antibody).

[0022] Figure 2 This is a schematic diagram of a bispecific antibody that uses PD-L1 monoclonal antibody as the main component, with two heavy chains on both sides of the C-terminus of the PD-L1 monoclonal antibody each expressing a Fas-ECD.

[0023] Figure 3This is a schematic diagram of a bispecific antibody based on a PD-L1 monoclonal antibody, with two Fas-ECD antibodies fused to the two heavy chains on either side of the C-terminus of the PD-L1 monoclonal antibody.

[0024] Figure 4 This is a schematic diagram showing a PD-L1 monoclonal antibody as the main body, with the light and heavy chains of the corresponding FasL antibody fused to the N-terminus of the light and heavy chains on both sides of the N-terminus of the PD-L1 monoclonal antibody, respectively; or a FasL monoclonal antibody as the main body, with the light and heavy chains of the corresponding PD-L1 antibody fused to the N-terminus of the light and heavy chains on both sides of the N-terminus of the FasL monoclonal antibody, respectively. Figure 4 In this study, VL1 and VH1 are the light chain variable region and heavy chain variable region of one of the PD-L1 monoclonal antibodies and the FasL monoclonal antibody, while VL2 and VH2 are the light chain variable region and heavy chain variable region of the other of the PD-L1 monoclonal antibody and the FasL monoclonal antibody. The two antibodies are linked end-to-end through an adapter sequence.

[0025] Figure 5 Therefore Figure 4 This diagram illustrates the expression of a Fas-ECD by fusing a PD-L1 monoclonal antibody or a FasL antibody to both sides of the C-terminus of a bispecific antibody. Figure 5 In this study, VL1 and VH1 are the light chain variable region and heavy chain variable region of one of the PD-L1 monoclonal antibodies and the FasL monoclonal antibody, while VL2 and VH2 are the light chain variable region and heavy chain variable region of the other of the PD-L1 monoclonal antibody and the FasL monoclonal antibody. The two antibodies are linked end-to-end through an adapter sequence.

[0026] Figure 6 Therefore Figure 4 Based on bispecific antibodies, two Fas-ECDs are fused to both sides of the C-terminus of either a PD-L1 monoclonal antibody or a FasL antibody. Figure 6 In this study, VL1 and VH1 are the light chain variable region and heavy chain variable region of one of the PD-L1 monoclonal antibodies and the FasL monoclonal antibody, while VL2 and VH2 are the light chain variable region and heavy chain variable region of the other of the PD-L1 monoclonal antibody and the FasL monoclonal antibody. The two antibodies are linked end-to-end through an adapter sequence.

[0027] Figure 7 The results of non-reducing SDS-PAGE in Example 2 of this invention show the purity of the purified PD-L1 / FasL protein. Most proteins are stable, while proteins containing Fas ECD in their conformation are slightly less stable, with some aggregation and degradation.

[0028] Figure 8The results of the reducing SDS-PAGE in Example 2 of this invention show the purity of the purified PD-L1 / FasL protein. Most proteins are stable, while proteins containing Fas ECD in their conformation are slightly less stable, with some aggregation and degradation.

[0029] Figures 9 to 19 The results of SEC-HPLC in Example 2 of this invention show the aggregates and degradation of the purified PD-L1 / FasL protein. Most proteins showed good stability, while a few proteins exhibited partial aggregates and degradation; among them,

[0030] Figure 9 These are the SEC-HPLC results for proteins numbered 60 / 61;

[0031] Figure 10 These are the SEC-HPLC results for proteins numbered 141 / 142;

[0032] Figure 11 These are the SEC-HPLC results for proteins numbered 101 / 102;

[0033] Figure 12 This is the SEC-HPLC result of protein number 101 / 167;

[0034] Figure 13 This is the SEC-HPLC result of protein number 101 / 168;

[0035] Figure 14 This is the SEC-HPLC result of protein number 161 / 157;

[0036] Figure 15 These are the SEC-HPLC results for proteins numbered 161 / 164;

[0037] Figure 16 These are the SEC-HPLC results for proteins numbered 161 / 166;

[0038] Figure 17 This is the SEC-HPLC result of protein number 162 / 159;

[0039] Figure 18 These are the SEC-HPLC results for proteins numbered 162 / 163;

[0040] Figure 19 These are the SEC-HPLC results for proteins numbered 162 / 165.

[0041] Figure 20This is the experimental result of the PD-L1 / FasL bispecific antibody blocking the PD-1 / PD-L1 signaling pathway at the molecular level in Example 3 of the present invention.

[0042] Figure 21 This is the target binding activity curve of the PD-L1 / FasL bispecific antibody at the cellular level in Example 4 of the present invention. (A) Binding activity curve of PD-L1 / FasL bispecific antibody with CHO-K1-PD-L1 stable cell line. (B) Binding activity curve of PD-L1 / FasL bispecific antibody with CHO-K1-FasL stable cell line.

[0043] Figure 22 This is the inhibition curve of PD-L1 / FasL bispecific antibody against recombinant human free FasL-induced apoptosis in Jurkat cells in Example 5 of the present invention.

[0044] Figure 23 The curve showing the inhibitory effect of the PD-L1 / FasL bispecific antibody on Jurkat cell apoptosis induced by the CHO-K1-FasL stable cell line was determined by the Annexin-V method in Example 6 of this invention.

[0045] Figure 24 The curve showing the inhibitory effect of the PD-L1 / FasL bispecific antibody on Jurkat cell apoptosis induced by the CHO-K1-FasL stable cell line, as determined by the lactate dehydrogenase release method in Example 7 of this invention.

[0046] Figure 25 This is a curve showing the neutralization effect of the PD-L1 / FasL bispecific antibody on free FasL expressed in the CHO-K1-FasL cell line in Example 8 of the present invention.

[0047] Figure 26 This is Example 9 of the present invention, illustrating the inhibitory effect of the PD-L1 / FasL bispecific antibody on CD8+ T cell apoptosis induced by the CHO-K1-FasL-PD-L1 cell line. (A) shows the proportion curve of CD8+ T cells in the co-culture system after the addition of the PD-L1 / FasL bispecific antibody. (B) shows a comparison of the inhibitory effects of the PD-L1 / FasL bispecific antibody on CD8+ T cell apoptosis induced by the CHO-K1-FasL-PD-L1 stable cell line. (C) shows the expression level of IFN-γ when CD8+ T cells and the CHO-K1-FasL-PD-L1 stable cell line are co-cultured.

[0048] Figure 27The figures show the effects of adding PD-L1 / FasL bispecific antibody to the mixed lymphocyte experimental system in Example 10 of this invention on the expression levels of cytokines IFN-γ and IL-2. (A) shows the effect of PD-L1 / FasL bispecific antibody on the expression of cytokine IFN-γ in CD8+ T cells. (B) shows the effect of PD-L1 / FasL bispecific antibody on the expression of cytokine IL-2 in CD8+ T cells. Detailed Implementation

[0049] The present invention will now be described in detail through specific embodiments.

[0050] In this invention, antibody refers to antibody (immunoglobulin, Ig) as generally understood by those skilled in the art.

[0051] Antibodies are composed of four polypeptide chains, which are linked by a varying number of interchain disulfide bonds, forming a "Y"-shaped structure that constitutes the basic unit of an antibody.

[0052] Antibody molecules contain four polypeptide chains. The two chains with the larger molecular weight are called heavy chains (H), while the two chains with the smaller molecular weight are called light chains (L). The two H chains and the two L chains in the same antibody molecule have the same amino acid composition.

[0053] The N-terminus of both the heavy and light chains contains approximately 110 amino acids with highly variable sequences, while the remaining amino acid sequences are relatively constant. Therefore, the region of the light and heavy chains with highly variable amino acid sequences near the N-terminus is called the variable region (V), and the region of the light and heavy chains with relatively stable amino acid sequences near the C-terminus is called the constant region (C).

[0054] In this application, the variable region of the heavy chain is represented by VH, and the variable region of the light chain is represented by VL; the constant region of the heavy chain is represented by CH, and the constant region of the light chain is represented by CL.

[0055] VH and VL each contain three regions with highly variable amino acid composition and sequence, called hypervariable regions (HVR) or complementarity determining regions (CDR), including CDR1, CDR2, and CDR3.

[0056] The lengths of the constant light chain regions of different types (κ or λ) antibodies are basically the same, but the CH lengths of different classes of Ig are different. For example, IgG, IgA and IgD include CH1, CH2 and CH3, while IgM and IgE include CH1, CH2, CH3 and CH4.

[0057] Papain can cleave the IgG heavy chain into three fragments near the amino terminus at the interchain disulfide bond in the hinge region: two identical antigen-binding fragments (Fab) and one crystallizable fragment (Fc). Each Fab fragment consists of a complete light chain and a portion of the heavy chain (VH and CH1). This fragment has monovalent antibody activity and can only bind to one corresponding antigen epitope; therefore, it cannot form a large immune complex after binding to the corresponding antigen. The Fc fragment consists of the CH2 and CH3 functional regions of the two heavy chains connected by interchain disulfide bonds in the hinge region and has no antigen-binding activity.

[0058] In this invention, the amino acid sequences of all light chain variable regions of the PD-L1 antibody are identical, as shown in SEQ ID NO:1, which is encoded by the nucleotide sequence shown in SEQ ID NO:2.

[0059] In this invention, the amino acid sequences of all heavy chain variable regions of the PD-L1 antibody are identical, as shown in SEQ ID NO:3 below, which is encoded by the nucleotide sequence shown in SEQ ID NO:4.

[0060] In this invention, the amino acid sequences of all light chain variable regions of the FasL antibody are identical, as shown in SEQ ID NO:5 below, which is encoded by the nucleotide sequence shown in SEQ ID NO:6.

[0061] In this invention, the amino acid sequences of all heavy chain variable regions of the FasL antibody are identical, as shown in SEQ ID NO:7 below, which is encoded by the nucleotide sequence shown in SEQ ID NO:8.

[0062] In this invention, the amino acid sequence of the Fas extracellular domain (Fas-ECD) is shown in SEQ ID NO:9, which is encoded by the nucleotide sequence of the amino acid shown in SEQ ID NO:10.

[0063] In this invention, the light chain constant regions of the PD-L1 antibody and the FasL antibody are identical and the amino acid sequence of the light chain constant region is shown in SEQ ID NO:11, which is encoded by the nucleotide sequence shown in SEQ ID NO:12.

[0064] Example 1: Cloning and Expression of Bispecific Antibody PD-L1 / FasL

[0065] Using anti-human PD-L1 antibody as the part of bispecific antibody that regulates the PD-1 / PD-L1 immune checkpoint, and using the extracellular domain of human tumor necrosis factor protein Fas or anti-human FasL antibody as the molecular part of bispecific antibody that regulates T cell apoptosis, a PD-L1 / FasL bispecific antibody is formed.

[0066] Plasmid construction: The plasmid is based on an intact antibody (PD-L1 antibody or FasL antibody). A variable region of the heavy chain of another target antibody of the bispecific antibody is attached to the N-terminus of the antibody heavy chain, with two domains linked by a (G4S)3 linker sequence. Similarly, a variable region of the light chain of another target antibody of the bispecific antibody is attached to the N-terminus of the antibody light chain, with two domains linked by a (G4S)3 linker sequence. One or two Fas extracellular domains (Fas-ECD) are tandemly connected to the C-terminus of the antibody heavy chain, with the Fc domain linked to the Fas extracellular domain by a (G4S)3 linker sequence. The two Fas extracellular domains are linked by a (G4S)3 linker sequence.

[0067] The amino acid and nucleotide sequences of the light and heavy chains of the PD-L1 or FasL antibody are shown in SEQ ID NO:1 to SEQ ID NO:16 in the sequence listing. The nucleotide sequence of the adapter sequence (G4S)3 is shown in SEQ ID NO:17, and the amino acid sequence is shown in SEQ ID NO:18. The nucleotide sequence of the signal peptide sequence is shown in SEQ ID NO:19, and the amino acid sequence is shown in SEQ ID NO:20. The signal peptide incorporates the Kozak sequence (GCCACC). All sequences were codon-optimized (for expression in mammalian cells) by Nanjing Genscript Biotech Co., Ltd., and then the whole genome was synthesized. The target fragment was digested with XbaI and EcoRV and ligated into the pCDNA3.4 expression vector. Sequencing confirmed the correctness of the sequence.

[0068] SEQ ID NO:17:

[0069] GGTGGTGGTGGTTCTGGTGGTGGTGGTTCTGGCGGCGGCGGCTCC

[0070] SEQ ID NO:18: GGGGSGGGGSGGGGS

[0071] SEQ ID NO:19:

[0072] ATGGGCTGGAGCTGCATCATCCTGTTCCTGGTGGCCACCGCCACAGGCGTGCACTCC

[0073] SEQ ID NO:20:MGWSCIILFLVATATGVHS

[0074] In this application, the correspondence between plasmid numbers and the protein portions they express is shown in Table 1 below.

[0075] Table 1. Plasmid number and the protein it expresses

[0076]

[0077]

[0078] The plasmid numbers and the corresponding amino acid and nucleotide sequences of the proteins they express are as follows:

[0079] PL101: PD-L1 light chain amino acid sequence (SEQ ID NO:21).

[0080] PL101: PD-L1 light chain nucleotide sequence (SEQ ID NO:22).

[0081] PL102: PD-L1 heavy chain amino acid sequence (SEQ ID NO:23).

[0082] PL102: PD-L1 heavy chain nucleotide sequence (SEQ ID NO:24).

[0083] PL141: FasL light chain amino acid sequence (SEQ ID NO:25).

[0084] PL141: FasL light chain nucleotide sequence (SEQ ID NO:26).

[0085] PL142: FasL heavy chain amino acid sequence (SEQ ID NO:27).

[0086] PL142: FasL heavy chain nucleotide sequence (SEQ ID NO:28).

[0087] PL060: FasL light chain amino acid sequence (SEQ ID NO:29).

[0088] PL060: FasL light chain nucleotide sequence (SEQ ID NO:30).

[0089] PL061: FasL heavy chain amino acid sequence (SEQ ID NO:31).

[0090] PL061: FasL heavy chain nucleotide sequence (SEQ ID NO:32).

[0091] PL162: PD-L1-FasL-LC amino acid sequence (SEQ ID NO:33).

[0092] PL162: PD-L1-FasL-LC nucleotide sequence (SEQ ID NO:34).

[0093] PL159: PD-L1-FasL-HC amino acid sequence (SEQ ID NO:35).

[0094] PL159: PD-L1-FasL-HC nucleotide sequence (SEQ ID NO:36).

[0095] PL165: PD-L1-FasL-HC-Fas ECD amino acid sequence (SEQ ID NO:37).

[0096] PL165: PD-L1-FasL-HC-Fas ECD nucleotide sequence (SEQ ID NO:38).

[0097] PL163: PD-L1-FasL-HC-2Fas ECD amino acid sequence (SEQ ID NO:39).

[0098] PL163: PD-L1-FasL-HC-2Fas ECD nucleotide sequence (SEQ ID NO:40).

[0099] PL167: PD-L1-HC-Fas ECD amino acid sequence (SEQ ID NO:41).

[0100] PL167: PD-L1-HC-Fas ECD nucleotide sequence (SEQ ID NO:42).

[0101] PL168: PD-L1-HC-2Fas ECD amino acid sequence (SEQ ID NO:43).

[0102] PL168: PD-L1-HC-2Fas ECD nucleotide sequence (SEQ ID NO:44).

[0103] PL161: FasL-PD-L1-LC amino acid sequence (SEQ ID NO:45).

[0104] PL161: FasL-PD-L1-LC nucleotide sequence (SEQ ID NO:46).

[0105] PL157: FasL-PD-L1-HC amino acid sequence (SEQ ID NO:47).

[0106] PL157: FasL-PD-L1-HC nucleotide sequence (SEQ ID NO:48).

[0107] PL166: FasL-PD-L1-HC-Fas ECD amino acid sequence (SEQ ID NO:49).

[0108] PL166: FasL-PD-L1-HC-Fas ECD nucleotide sequence (SEQ ID NO:50).

[0109] PL164: FasL-PD-L1-HC-2Fas ECD amino acid sequence (SEQ ID NO:51).

[0110] PL164: FasL-PD-L1-HC-2Fas ECD nucleotide sequence (SEQ ID NO:52).

[0111] Plasmid transformation was performed, and high-purity plasmids were extracted using an endotoxin-free large-scale extraction kit (purchased from Tiangen Biotech, catalog number DP117). Sequencing was performed to ensure the accuracy of the plasmid sequence. Cell transfection reagents were all purchased from Thermo Fisher Scientific, USA. For cell transfection (using 25 mL of culture medium as an example), the reagents were prepared one day prior to transfection at a concentration of 3 × 10⁻⁶. 6 ExpiCHO-S cells were seeded at a density of 6 × 10⁶ cells / mL until transfection. 6 Cells / mL, cell viability >95%. Before transfection, prepare the transfection complex by diluting 20 μg DNA in 1 mL of serum-free medium and mixing thoroughly to create a DNA dilution buffer; then prepare 80 μL of ExpiFectamine... TM CHO Reagent was diluted with 920 μL of serum-free medium and thoroughly mixed to prepare the transfection reagent dilution. Both dilutions were allowed to stand at room temperature for no more than 5 minutes. Then, both dilutions were thoroughly mixed and allowed to stand at room temperature for 5 minutes. Finally, the transfection complex was added to a 125 mL cell culture shake flask containing 25 mL of cells and serum-free complete medium, and gently mixed thoroughly. Cells were cultured for 12 days in a shaker at 95 rpm, 37°C, 90% humidity, and 8% CO2. 18-22 hours after transfection, 150 μL of LexpiFectamine was added. TM CHO Enhancer and 6mL ExpiCHO TM Feed.

[0112] Example 2: Purification and stability testing of PD-L1 / FasL bispecific antibody

[0113] Protein A agarose purification resin is an affinity resin (BBI Life Sciences, catalog number C600953) used for the purification and separation of IgG. Protein A is a cell wall protein isolated from Staphylococcus aureus that primarily binds to mammalian IgG via its Fc fragment.

[0114] Protein purification was performed in a cryogenic chromatography cabinet (purchased from Boyikang Company). Cells were collected and purified on day 12 after transfection. First, the cells were centrifuged at 1000 rpm for 3 min to remove cells, and then centrifuged at 8500 rpm for 30 min to remove impurities. Filtration was performed using a 0.22 μm membrane. Before loading the protein, the salt concentration was adjusted to 20 mM. When equilibrating the Protein A column, it was first rinsed with 10 column volumes of deionized water, and then rinsed with 10 column volumes of Binding Buffer (BBI Life Sciences, catalog number C600482). Then, the protein was loaded. After loading, it was eluted with 10 column volumes of Binding Buffer, and then eluted with Elution Buffer (BBI Life Sciences, catalog number C600481). 3 mL of eluent was collected per tube, and 12 tubes were connected (the pH of the protein eluent was pre-adjusted to approximately 7 using Neutralization Buffer (BBI Life Sciences, catalog number B548142)). After collecting the samples, the purification column was rinsed with 10 column volumes of Binding Buffer and deionized water to equilibrate it, and finally sealed with 20% ethanol at 4°C.

[0115] Protein concentration was performed using an ultrafiltration tube of appropriate size at 3800 rpm and 4°C. The protein buffer was replaced with PBS. After detecting the protein concentration using NanoDrop (purchased from Thermo Fisher Scientific, USA), the protein was filtered through a 0.22 μm filter membrane to obtain the PD-L1 / FasL bispecific antibody, which was stored at 4°C.

[0116] Protein expression was detected by SDS-PAGE and SEC-HPLC, and the results are as follows: Figure 7 and Figure 8 As shown, most proteins are stable, while proteins with Fas-ECD in their conformation are slightly less stable, with some aggregation and degradation.

[0117] In addition, to test the stability of the protein, the purified protein was tested using SYPRO. TMThe Tm value was determined using the Orange Protein GelStain kit (purchased from Sigma). A 10 μM protein sample was added to staining solution to prepare a 25 μL reaction system. The mixture was centrifuged to ensure homogeneity, kept in the dark, and then run on a qPCR instrument (purchased from Hongshi Technology). The temperature variation was set to 0.01℃ / s, with a range of 25-95℃. The analytical results are shown in Table 2. The Tm value of the naked antibody was relatively high. Some proteins in the purified bispecific antibody showed high Tm values, while the Tm values ​​of other proteins were significantly lower.

[0118] Table 2. Tm values ​​of PD-L1 / FasL protein detected by qPCR

[0119]

[0120] Note: In the table, the meanings of numbers such as 101 / 102, 60 / 61, and 141 / 142 are as follows: Taking 101 / 102 as an example, the number before the slash indicates the number of the protein (light chain) expressed by plasmid PL101, and the number after the slash indicates the number of the protein (heavy chain) expressed by plasmid PL102. The 101 / 102 number represents the protein expressed and purified after co-transfecting cells with plasmids PL101 and PL102.

[0121] As can be seen from the results in Table 2, the Tm values ​​of PD-L1 / FasL protein detected by qPCR show that the bispecific antibody has relatively high stability.

[0122] Example 3

[0123] PD-L1 / FasL bispecific antibodies block PD-1 / PD-L1 interaction at the molecular level.

[0124] The molecular-level blocking effect of PD-L1 / FasL bispecific antibodies was detected using the Cisbio Human PD1 / PD-L1 biochemical binding assay (purchased from PerkinElmer, catalog number 64ICP01PEH). The assay was performed using 384-well microplates (purchased from Corning). Each well contained 20 μL of the assay. First, 4 μL of Tag1-PD-L1 protein solution (working concentration 5 nM) was added. Next, 2 μL of bispecific antibodies at different dilutions were added, with each protein sample diluted 9 times at a ratio of 1:5. Then, 4 μL of Tag2-PD-1 protein solution (working concentration 50 nM) was added. After incubating at room temperature for 15 min, 10 μL of a mixture of anti-Tag1-Eu3+ and anti-Tag2-XL665 solution (first diluted to a 100× stock solution) was added. The sample was centrifuged at low speed for 2 min to ensure thorough mixing, and then incubated at room temperature for 2 hours. The fluorescence values ​​at 620 nm and 665 nm were detected using a multi-plate reader (purchased from BioTek). In this experiment, the PD-L1 antibody atezolizumab was used as a control. The analytical results are as follows: Figure 20 As shown, both PD-L1 and FasL bispecific antibodies can effectively block PD-1 / PD-L1 interaction. The IC50 concentrations calculated from the inhibition curves are shown in Table 3.

[0125] Table 3. IC50 concentrations of PD-L1 / FasL bispecific antibodies blocking PD-1 / PD-L1 interaction at the molecular level

[0126]

[0127] As shown in Table 3, the PD-L1 terminus of some bispecific antibodies of this invention functions at the level of PD-L1 monoclonal antibodies, effectively blocking PD-1 / PD-L1 interaction. Furthermore, this invention aims to employ a dual-target mechanism of PD-L1 and FasL, enabling the bispecific antibody to block PD-1 / PD-L1 interaction while effectively preventing Fas / FasL-mediated T cell apoptosis. By blocking the synergistic effect of these two signaling pathways, the overall anti-tumor effect of the body is enhanced. The FasL terminus function of the bispecific antibody is shown in the following examples. Additionally, the data in Table 3 also indicate that the more structures or sequences linked to the C-terminus of the bispecific antibody, the greater the steric hindrance of the protein, thus resulting in a slightly weaker inhibitory effect.

[0128] Example 4

[0129] PD-L1 / FasL bispecific antibody binding activity at the cellular level

[0130] The CHO-K1-PD-L1 stable cell line with high PD-L1 expression and the CHO-K1-FasL stable cell line with high FasL expression were cultured in DMEM / F12 medium (purchased from Thermo Fisher Scientific, USA) containing 10% FBS (purchased from Thermo Fisher Scientific, USA) and double antibiotics (purchased from Beyotime Biotechnology, Shanghai), and passaged every 2–3 days.

[0131] Stable cell lines in the logarithmic growth phase were harvested, and adherent cells were digested with trypsin and resuspended in complete culture medium. After cell counting, cell clumps were washed with 1×PBS buffer and centrifuged at 250×g for 5 minutes. Cells were resuspended in 1×PBS buffer and seeded in 96-well V-bottom plates (NEST Biotech) at a density of 5×10⁶ cells per well. 5 Cells / wells. Add 50 μL of serially diluted bispecific antibody to each well to achieve final protein concentrations of 100, 20, 4, 0.8, 0.16, 0.032, 0.0064, 0.00128, 0.000256, 0.0000512, and 0.00001024 nM. Wells without antibody served as negative controls. Incubate at 4°C for 1 hour. Wash cells twice with PBS, centrifuging at 250×g for 5 minutes each time. Add 100 μL of 1:200 diluted secondary antibody (Goat anti-Human IgG Fc Secondary Antibody PE, Thermo Fisher Scientific, catalog number 12-4998-82) to each well and incubate at 4°C for 30 minutes. Cells were washed twice with PBS, centrifuged at 250×g for 5 minutes each time, and resuspended in 120μL PBS. The binding activity of the bispecific antibody at the cellular level was measured using a CytoFLEX flow cytometer (purchased from Beckman Coulter).

[0132] The binding activity curves of PD-L1 / FasL bispecific antibodies at different concentrations with the target sites of CHO-K1-PD-L1 or CHO-K1-FasL cell lines are shown in the figure below. Figure 21 The EC50 concentrations calculated from the binding curves are shown in Table 4.

[0133] Table 4. EC50 concentrations of PD-L1 / FasL bispecific antibodies binding to the target at the cellular level.

[0134]

[0135]

[0136] Example 5

[0137] PD-L1 / FasL bispecific antibody inhibits free FasL-induced apoptosis in Jurkat cells

[0138] Jurkat cells (human peripheral blood leukemia T cells) were cultured in RPMI-1640 medium (purchased from Thermo Fisher Scientific, USA) containing 10% FBS (purchased from Thermo Fisher Scientific, USA) and penicillin antibiotics (purchased from Beyotime Biotechnology, Shanghai), and passaged every 2-3 days. Jurkat cells in the logarithmic growth phase were harvested, counted, and then adjusted to a cell concentration of 1.11 × 10⁻⁶ cells using complete medium. 5 Cells / mL, seeded in black 96-well plates (purchased from Corning Incorporated, USA), 90 μL per well (2 × 10⁻⁶ cells / mL). 4 Cells / well). Add 5 μL of serially diluted bispecific antibody to the cells and pre-incubate at 37°C for 30 minutes. Then add 5 μL of recombinant human FasL (CST Biotech, catalog number 5452) to bring the final FasL concentration to 80 ng / mL. The final concentrations of the bispecific antibody to be tested are 500, 100, 20, 4, 0.8, 0.16, 0.032, 0.0064, and 0.00128 nM. Wells without antibody serve as controls. Incubate the culture plate at 37°C for 24 hours, then allow it to equilibrate at room temperature for 10 minutes. Add 50 μL of CellTiter-Lumi to each well of the 96-well plate. TM Chemiluminescence assay reagent. Shake at room temperature for 2 minutes to promote cell lysis. Incubate at room temperature for 10 minutes to stabilize the luminescent signal. Perform chemiluminescence detection using a multi-functional microplate reader (BioTek).

[0139] The results are as follows Figure 22 As shown, both FasL / PD-L1 bispecific antibodies inhibit Fas / FasL-mediated Jurkat cell apoptosis by binding to free FasL, thereby increasing cell viability. The IC50 concentrations calculated from the inhibition curves are shown in Table 5.

[0140] Table 5. IC50 concentrations of PD-L1 / FasL bispecific antibodies in inhibiting free FasL-induced apoptosis in Jurkat cells.

[0141]

[0142] Example 6

[0143] Annexin V-FITC assay showed that FasL / PD-L1 bispecific antibody inhibited Jurkat cell apoptosis induced by CHO-K1-FasL cell line.

[0144] CHO-K1-FasL stable cell lines in logarithmic growth phase were collected, and adherent cells were digested with trypsin and resuspended in complete culture medium. After cell counting, the cells were centrifuged and the cell concentration was adjusted to 1.2 × 10⁻⁶ cells / mL using RPMI-1640 medium containing 4% FBS. 6 / mL, seeded into 50μL per well of a V-bottom 96-well cell culture plate (purchased from Corning Incorporated, USA). Add 50μL of serially diluted test antibody to each well and pre-incubate at 37°C for 2 hours. Follow the manufacturer's instructions using CellTracker. TM Jurkat cells were labeled with the Violet BMQC probe (purchased from Thermo Fisher Scientific, catalog number C10094), then resuspended in RPMI-1640 medium containing 4% FBS, and 50 μL (2 × 10⁶ cells / well) was added to each well of a 96-well cell culture plate. 4 Cells / well). In this co-culture system, the ratio of effector cells (CHO-K1-FasL cell line) to target cells (Jurkat cells) was 3:1, and the final concentrations of the antibody to be tested were 500, 100, 20, 4, and 0.8 nM. Wells without antibody and wells containing only one type of cell served as controls. The culture plates were incubated at 37°C for 20 hours.

[0145] The Annexin V-FITC apoptosis detection kit was purchased from Shanghai Beyotime Biotechnology Co., Ltd. (catalog number C1062L). After co-culture, cells were centrifuged at 250×g for 5 minutes and washed once with PBS. Cells were then centrifuged again at 250×g for 5 minutes and resuspended in 195 μL of binding buffer. 5 μL of Annexin V-FITC was added and gently mixed. 10 μL of propidium iodide (PI) staining solution was added and gently mixed. Cells were incubated at room temperature in the dark for 10-20 minutes, then placed on ice. The proportion of viable and apoptotic cells in Jurkat cells was detected using a CytoFLEX flow cytometer (Beckman Coulter). Jurkat cell populations were determined using purple fluorescence; Annexin V-FITC showed green fluorescence, and propidium iodide (PI) showed red fluorescence. Cells negative for both Annexin-V and PI were considered viable cells.

[0146] The results are as follows Figure 23As shown, the PD-L1 / FasL bispecific antibodies constructed in this invention can all block Jurkat cell apoptosis induced by the CHO-K1-FasL cell line. With increasing antibody concentration, the proportion of viable Jurkat target cells in the co-culture system gradually increases. The experimental results show that, compared with bispecific antibodies of the PD-L1 / FasL structure or the PD-L1 / Fas-ECD structure, the FasL / PD-L1 bispecific antibody exhibits better inhibitory activity. This is mainly because the PD-L1 / FasL structure is based on the FasL antibody, with the N-terminus of the FasL antibody connected to the variable region of the PD-L1 antibody; the PD-L1 / FasL structure is based on the PD-L1 antibody, with the N-terminus of the PD-L1 antibody connected to the variable region of the FasL antibody, and their positions can be interchanged. Generally, the antibody function at the outermost (N-terminus of the whole protein) is less affected because there is no steric hindrance, while the function of the main antibody may be partially lost due to steric hindrance.

[0147] Example 7

[0148] Lactate dehydrogenase (LDH) release assay to determine the effect of PD-L1 / FasL bispecific antibody on the inhibition of Jurkat cell apoptosis induced by CHO-K1-FasL cell line.

[0149] CHO-K1-FasL stable cell lines in logarithmic growth phase were collected, and adherent cells were digested with trypsin and resuspended in complete culture medium. After cell counting, the cells were centrifuged and the cell concentration was adjusted to 1.2 × 10⁻⁶ cells / mL using RPMI-1640 medium containing 4% FBS. 6 / mL, seeded into 50μL per well of a V-bottom 96-well cell culture plate (purchased from Corning Incorporated, USA). Add 50μL of serially diluted test antibody to each well and pre-incubate at 37°C for 2 hours. Take Jurkat cells in the logarithmic growth phase, centrifuge at 250×g for 5 minutes, resuspend in RPMI-1640 medium containing 4% FBS, and add 50μL (2×10⁶) to each well of a 96-well cell culture plate. 4 Cells / well). In this co-culture system, the ratio of effector cells (CHO-K1-FasL cell line) to target cells (Jurkat cells) was 3:1. The final concentrations of the bispecific antibodies to be tested were 500, 100, 20, 4, 0.8, 0.16, 0.032, 0.0064, and 0.00128 nM. Wells without antibodies and wells containing only one type of cell served as controls. Each treatment condition had 3 replicates.

[0150] The lactate dehydrogenase (LDH) release assay was used to determine CHO-K1-FasL-induced apoptosis in Jurkat cells. The LDH quantitative assay kit was purchased from Prometheus (catalog number G1780). The culture plate was removed 45 minutes before the end of the incubation period, and 15 μL of lysis buffer (10×) was added to the largest viable control well containing only Jurkat target cells. The plate was then incubated at 37°C for 45 minutes. After co-culture, the cells were centrifuged at 250×g for 5 minutes, and 50 μL of the supernatant was transferred to a 96-well microplate. 50 μL of substrate prepared with assay buffer was added to each well, and the plates were incubated at room temperature in the dark for 30 minutes. Then, 50 μL of stop solution was added to each well, and the absorbance was recorded at 490 nM using a multi-mode microplate reader.

[0151] Experimental results are as follows Figure 24 As shown, the PD-L1 / FasL bispecific antibodies constructed in this invention can block Jurkat cell apoptosis induced by the CHO-K1-FasL cell line, and the results are consistent with those obtained by the Annexin-V-FITC method. With increasing antibody concentration, the amount of lactate dehydrogenase released by Jurkat target cells in the co-culture system gradually decreased, indicating that the proportion of target cell apoptosis gradually decreased, and this trend showed a dose-dependent trend with the added antibody concentration. The IC50 concentrations calculated from the inhibition curves of the bispecific antibodies measured in this example are shown in Table 6.

[0152] Table 6. IC50 concentrations of FasL / PD-L1 bispecific antibody blocking CHO-K1-FasL-induced Jurkat cell apoptosis

[0153]

[0154] As can be seen from the experimental results in Table 6, the bispecific antibody with the FasL / PD-L1 structure exhibits better inhibitory activity compared with the bispecific antibody with the PD-L1 / FasL structure or the bispecific antibody with the PD-L1 / Fas-ECD structure.

[0155] Example 8

[0156] Neutralizing effect of PD-L1 / FasL bispecific antibody on free FasL expression in CHO-K1-FasL cell line

[0157] The cell co-culture system was established using the same method as in Example 7. The final concentrations of the bispecific antibodies to be tested were 500, 100, 20, 4, 0.8, 0.16, 0.032, and 0.0064 nM. Wells without antibodies and wells containing only one type of cell were used as controls. The culture plates were incubated at 37°C for 20 hours. After co-culture, the cells were centrifuged at 250×g for 5 minutes, and the cell supernatant was collected. The concentration of free FasL was determined using a human FasL ELISA kit (purchased from Boster Biological Engineering Co., Ltd., catalog number EK0337).

[0158] This experiment reflects the binding ability of the bispecific antibody to FasL by measuring the level of unbound free FasL in the system. The results are as follows: Figure 25 As shown, in the co-culture system, the PD-L1 / FasL bispecific antibody constructed in this invention can bind to free FasL expressed in the cell supernatant of CHO-K1-FasL-stabilized cell lines, thereby inhibiting CHO-K1-FasL-induced Jurkat cell apoptosis. With increasing antibody concentration, the concentration of free FasL in the cell co-culture system gradually decreases in a dose-dependent manner. The EC50 concentration calculated from the FasL neutralization curves of the antibodies measured in this example is shown in Table 7.

[0159] Table 7. EC50 concentration of free FasL expressed in CHO-K1-FasL cell lines bound to PD-L1 / FasL bispecific antibody

[0160]

[0161] Example 9

[0162] PD-L1 / FasL bispecific antibody inhibits CD8+ T cell apoptosis induced by CHO-K1-FasL cell line.

[0163] Human primary CD8-positive T cells (CD8+ T cells) were purchased from Guangzhou Redberry Biotechnology Co., Ltd. CD8 T cells were resuscitated and cultured in ImmunoCulturase containing 30 UI / mL human recombinant IL-2 (purchased from Beijing Sinocare). TM -XF medium (purchased from Stemcell, USA, catalog number 10981), with ImmunoCult added. TM Human CD3 / CD28 T cell activator (25 μL / mL) (purchased from Stemcell, Inc., USA, catalog number 10971), replace with fresh culture medium containing IL-2 every 2-3 days.

[0164] Log-phase CHO-K1-FasL-PD-L1 stable cell lines (simultaneously expressing FasL and PD-L1) were collected. Adherent cells were digested with trypsin, counted, and centrifuged. ImmunoCult was used for cell culture. TM Adjust the cell concentration with XF medium and seed 50 μL (1 × 10⁻⁶) into 96-well V-bottom cell culture plates. 4 (Cells / well). Add 50 μL of serially diluted PD-L1 / FasL bispecific antibody to be tested and incubate at 37°C for 2 hours.

[0165] Collect CD8+ T cells in the logarithmic growth phase, count the cells, centrifuge at 300×g for 10 minutes, and discard the supernatant. Wash once with PBS and use CellTrace. TM Cells were labeled using the Violet Cell Expansion Kit (Thermo Fisher Scientific, catalog number C34557). After labeling, cells were resuspended in ImmunoCult... TM XF medium was added to each well of a 96-well cell culture plate, 50 μL (3 × 10⁻⁶) 4 Cells / well). In this co-culture system, the ratio of CHO-K1-FasL-PD-L1 cell line to CD8+ T cells was 1:3. The final concentrations of the antibodies to be tested were 500, 100, 20, 4, and 0.8 nM, respectively. Wells without antibodies and wells containing only one type of cell were used as controls. The culture plates were incubated at 37°C for 72 hours.

[0166] After co-culture, cells were centrifuged at 300×g for 10 minutes, and the cell supernatant was collected. The concentration of IFN-γ in the culture system was determined using a human IFN-γ ELISA kit (BioLegend, Inc., Catalog No. 430104). The remaining cells were washed twice with pre-chilled PBS, centrifuged at 300×g for 10 minutes, the supernatant was discarded, and the cells were resuspended in 100 μL of binding buffer. 2.5 μL of Annexin-V-APC and 5 μL of PI (kit purchased from Shanghai Yisheng Biotechnology Co., Ltd., Catalog No. 40304) were added to each well. After incubation at room temperature for 15 minutes, 100 μL of PBS was added to each well, and CD8+ T cell viability was determined using a CytoFLEX flow cytometer (Beckman Coulter).

[0167] like Figure 26 As shown, in the co-culture system, the PD-L1 / FasL bispecific antibody constructed in this invention significantly inhibited apoptosis of activated CD8+ T cells induced by the CHO-K1-FasL-PD-L1 stable cell line. With increasing antibody concentration, the proportion of CD8+ T cells in the co-culture system (…) Figure 26 A) and cell viability ( Figure 26B) gradually increases, IFN-γ expression level ( Figure 26 C) The effect increased, and showed a dose-dependent trend with antibody concentration. Among them, compared with PD-L1 / FasL structure bispecific antibodies, FasL / PD-L1 structure bispecific antibodies showed better protective activity against CD8+ T cells.

[0168] Example 10

[0169] Mixed lymphocyte reaction

[0170] Experimental methods: Primary mononuclear cells (CD14 positive) isolated from human peripheral blood were purchased from Guangzhou Redbell Biotechnology Co., Ltd., and dendritic cell (DC) induction kits (ImmunoCult) were used. TM The ACF Dendritic Cell Differentiation Kit was purchased from Stemcell, Inc., USA, catalog number 10985. After resuscitating CD14+ monocytes, the cell concentration was adjusted to 1×10⁻⁶ cells using DC cell culture medium containing 1% DC cell differentiation components. 6 / mL, and seeded into 6-well cell culture plates. On day 3 of culture, replace with fresh medium containing differentiation components. On day 6 of culture, immature DC cells (imDC) are obtained. Without changing the medium, directly add the maturation-promoting component (1:100 dilution). On day 8 of culture, mature DC cells (mDC) are obtained.

[0171] One day before the experiment, primary human CD8-positive T cells (CD8+ T cells) (purchased from Guangzhou Redbel Biotechnology Co., Ltd.) were revived. ImmunoCult was then added. TM -XF medium, adjust cell concentration to 1×10⁻⁶ 6 / mL, and seeded into 6-well cell culture plates, and incubated overnight at 37°C.

[0172] On the day of the experiment, DC cells were counted, centrifuged at 300×g for 10 minutes, and the cell concentration was adjusted using T cell expansion medium. Cells were then seeded into 96-well V-bottom cell culture plates at 50 μL (1×10⁻⁶) per well. 4 Cells / well). Add 50 μL of serially diluted test antibody to each well and pre-incubate at 37°C for 2 hours. Take CD8+ T cells that have been resuscitated the day before the experiment, count the cells, centrifuge at 300×g for 10 minutes, and discard the supernatant. Resuspend the cells in T cell expansion medium and add 50 μL (3×10⁶ cells / well) to each 96-well cell culture plate. 4Cells / well). After mixing, the culture plate was incubated at 37°C for 5 days. The ratio of DC cells to T cells in the system was 1:3. The final concentrations of the antibodies to be tested were 1000, 100, and 10 nM. Wells without antibodies, wells containing Human IgG1 (purchased from BioLegend, USA), and wells containing only one type of cell were used as negative controls. Wells containing CD3 / CD28 magnetic beads (purchased from Thermo Fisher Scientific, catalog number 11161D) were used as positive controls.

[0173] After 5 days of culture, the cells were centrifuged at 300×g for 10 minutes. The cell culture supernatant was collected, and the expression levels of IFN-γ and IL-2 in the culture system were measured using a human IFN-γ ELISA kit and a human IL-2 ELISA kit (both purchased from BioLegend, USA).

[0174] The mixed lymphocyte assay can be used to evaluate the PD-L1 end activity of PD-L1 / FasL bispecific antibodies, with atezolizumab serving as an activity control. Results are as follows: Figure 27 As shown in Figure A, after the addition of the PD-L1 / FasL bispecific antibody, the level of IFN-γ expression in CD8+ T cells was higher than that of atezolizumab, and the PD-L1 end activity of the PD-L1 / FasL protein was significantly higher than that of the FasL / PD-L1 protein. Figure 27 B shows the level of IL-2 in the co-culture system.

[0175] In summary, the PD-L1 and FasL bispecific antibody provided by this invention can simultaneously target PD-L1 and FasL, significantly improve the function of tumor-infiltrating T cells, and has a highly effective anti-tumor effect, providing a more effective treatment option for tumors or cancer. sequence list <110> Beijing Kangchen Pharmaceutical Co., Ltd. <120> Bispecific antibodies, pharmaceutical compositions, and their applications that simultaneously target PD-L1 and FasL <150> 202111165537X <151> 2021-09-30 <160> 52 <170> SIPOSequenceListing 1.0 <210> 1 <211> 106 <212> PRT <213> Artificial sequences <400> 1 Asp Ile Gln Ile Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Lys Met 20 25 30 Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Trp Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp 65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 2 <211> 318 <212> DNA <213> Artificial sequences <400> 2 gacatccaga tcacccagtc tcctagcaca ctgtccgcct ctgttggaga tagagtgaca 60 atcacctgta gcgccagcag ctccgtgtcc aaaatgaact ggtaccagca aaagcccggc 120 aaggccccta agagatggat ctacgacacc agcaaactgg ccagcggcgt gcccagtaga 180 ttcagcggca gcggatctgg cacagagtac accctgacca tcagcagcct gcaacctgat 240 gacttcgcca catactactg ccagcagtgg tcctctaatc ctctgacctt cggccagggc 300 accaagctgg aaatcaag 318 <210> 3 <211> 118 <212> PRT <213> Artificial sequences <400> 3 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 Ile Phe Thr Glu Tyr 20 25 30 Ile Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Trp Phe Tyr Pro Gly Ser Asp Asn Ile Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Asp Arg Ala Thr Leu 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 His Glu Thr Gly Tyr Phe Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 4 <211> 354 <212> DNA <213> Artificial sequences <400> 4[[ID=~17]] caggtgcagc tggttcagag cggagctgaa gtgaagaagc ccggcgccag cgtgaaggtg 60 tcctgcaagg cctctggata catcttcacc gagtacatca tccactgggt gcggcaggcc 120 cctggacaag gcctggaatg gatcggctgg ttctaccctg gcagcgacaa catcaagtac 180 aacgagaagt tcaaggatag agccaccctg accgcagata agtccaccag caccgtctac 240 atggaactga gcagcctgag aagcgaggat acagccgtgt actactgcgc cagacacgaa 300 accggctact tcttcgacta ctggggccag ggtacactgg tgaccgtgtc ctcc 354 <210> 5 <211> 112 <212> PRT <213> Artificial sequences <400> 5 Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Thr Lys Ser Leu Leu Asn Ser 20 25 30 Asp Gly Phe Thr Tyr Leu Gly Trp Cys Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Leu Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Ser 85 9� 95 Asn Tyr Leu Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 6 <211> 336 <212> DNA <213> Artificial sequences <400> 6 gacgtggtga tgacccagac ccctctgtct ctgcctgtga ccctcggcca gcctgcctcc 60 atctcctgca agtccaccaa aagcctgctg aattccgacg gctttaccta tctgggctgg 120 tgcctgcaga agcctggcca gagccctcag ctgctgatct acctggtgtc caaccggttt 180 tctggcgtgc ccgacagatt ctccggctcc ggatctggaa ccgatttcac cctcaagatc 240 tccagagtgg aagccgagga tgtgggcgtg tactactgct tccagtccaa ctacctgcct 300 ctgaccttcg gccaaggcac caagctggaa atcaag 336 <210> 7 <211> 119 <212> PRT <213> Artificial sequences <400> 7 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 Asn Tyr 20 25 30 Trp Ile Gly Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Leu Tyr Pro Gly Gly Leu Tyr Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Met Thr Ala Asp Thr Ser Thr Asn 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 Tyr Arg Asp Tyr Asp Tyr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser 115 <210> 8 <211> 357 <212> DNA <213> Artificial sequences <400> 8 caggttcagc tggtgcagtc cggagccgag gtgaagaagc ccggcgcttc tgtgaaggtg 60 tcttgtaagg cctctggcta caccttcacc aactactgga tcggctgggt gaagcaggcc 120 cctggccagg gcctggaatg gatcggctat ctgtaccctg gaggcctgta taccaattac 180 aacgagaagt tcaagggcaa ggccaccatg accgctgaca cctctaccaa cactgcctac 240 atggaactgt ccagcttgcg gtccgaggat accgctgtgt actactgcgc cagataccgg 300 gactatgact acgccatgga ctattgggga caaggcaccc tggtgacagt atcctcc 357 <210> 9 <211> 148 <212> PRT <213> Artificial sequences <400> 9 Gln Val Thr Asp Ile Asn Ser Lys Gly Leu Glu Leu Arg Lys Thr Val 1 5 10 15 Thr Thr Val Glu Thr Gln Asn Leu Glu Gly Leu His His Asp Gly Gln 20 25 30 Phe Cys His Lys Pro Cys Pro Pro Gly Glu Arg Lys Ala Arg Asp Cys 35 40 45 Thr Val Asn Gly Asp Glu Pro Asp Cys Val Pro Cys Gln Glu Gly Lys 50 55 60 Glu Tyr Thr Asp Lys Ala His Phe Ser Ser Lys Cys Arg Arg Cys Arg 65 70 75 80 [[ID=I5]]Leu Cys Asp Glu Gly His Gly Leu Glu Val Glu Ile Asn Cys Thr Arg 85 90 95 Thr Gln Asn Thr Lys Cys Arg Cys Lys Pro Asn Phe Phe Cys Asn Ser 100 105 110 Thr Val Cys Glu His Cys Asp Pro Cys Thr Lys Cys Glu His Gly Ile 115 120 125 Ile Lys Glu Cys Thr Leu Thr Ser Asn Thr Lys Cys Lys Glu Glu Gly 130 135 140 Ser Arg Ser Asn 145 <210> 10 <211> 444 <212> DNA <213> Artificial sequences <400> 10 caagtgactg acatcaactc caagggattg gaattgagga agactgttac tacagttgag 60 actcagaact tggaaggcct gcatcatgat ggccaattct gccataagcc ctgtcctcca 120 ggtgaaagga aagctaggga ctgcacagtc aatggggatg aaccagactg cgtgccctgc 180 caagaaggga aggagtacac agacaaagcc catttttctt ccaaatgcag aagatgtaga 240 ttgtgtgatg aaggacatgg cttagaagtg gaaataaact gcacccggac ccagaatacc 300 aagtgcagat gtaaaccaaa ctttttttgt aactctactg tatgtgaaca ctgtgaccct 360 tgcaccaaat gtgaacatgg aatcatcaag gaatgcacac tcaccagcaa caccaagtgc 420 aaagaggaag gatccagatc taac 444 <210> 11 <211> 107 <212> PRT <213> Artificial sequences <400> 11 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> 12 <211> 321 <212> DNA <213> Artificial sequences <400> 12 cggaccgtgg ccgccccttc tgtgttcatc ttccccccca gcgacgagca gctgaagagc 60 ggaaccgcca gcgtggtgtg cctgctcaac aacttctacc cgcgggaagc caaggtgcag 120 tggaaggtgg acaacgccct gcagagcggc aacagccagg agagcgtgac cgagcaggac 180 agcaaggact ctacatacag cctgagcagc accctgacac tgtctaaagc cgactacgag 240 aagcacaagg tgtacgcctg tgaagtgaca caccagggcc tgagcagccc tgtgaccaag 300 tcttttaacc ggggcgagtg c 321 <210> 13 <211> 330 <212> PRT <213> Artificial sequences <400> 13 Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys 1 5 10 15 Ser Thr Ser Gly Gly 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 Gln Thr 65 70 75 80 Tyr Ile Cys Asn Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95 Lys Val Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys 100 105 110 Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro 115 120 125 Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys 130 135 140 Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp 145 150 155 160 Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu 165 170 175 Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu 180 185 190 His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn 195 200 205 Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly 210 215 220 Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu 225 230 235 240 Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr 245 250 255 Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn 260 265 270 Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe 275 280 285 Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn 290 295 300 Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr 305 310 315 320 Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 325 330 <210> 14 <211> 990 <212> DNA <213> Artificial sequences <400> 14 gccagcacca agggccctag cgtctttcca ctggcccctt cttctaagag cacaagcggc 60 ggaaccgccg ctctgggttg tctggtcaaa gattacttcc ccgaacctgt gaccgtgtcc 120 tggaacagcg gcgccctgac atctggcgtg cacacattcc cagccgtgtt gcagagcagc 180 ggcctgtact ctctgtctag cgtcgtcacc gtgcccagca gcagcctggg aacacagacc 240 tacatctgca acgtgaacca caagcctagc aacaccaaag tggataagaa agtggaaccc 300 aagagctgcg acaagaccca cacctgtcct ccgtgccctg ctcctgagct gctgggcggc 360 cccagcgtgt tcctgttccc ccccaagcct aaggacaccc tgatgatcag ccgcacccct 420 gaggtgacat gcgtggtcgt cgacgtgtcc cacgaggacc ccgaggtgaa attcaactgg 480 tacgtggacg gcgtggaagt gcacaacgcc aagaccaagc caagagaaga gcagtacgcc 540 tctacataca gagtggtgtc cgtgctgacc gtgctgcacc aggactggct gaacggcaag 600 gaatacaagt gcaaggtgtc caacaaggcc ctgcccgctc ctatcgagaa gacaatctct 660 aaggctaaag gccagcctag agaacctcag gtttatacac tgcctcctag cagagaggaa 720 atgaccaaga accaggtgtc tctgacctgt ctggtgaagg gcttctatcc ttctgacatc 780 gccgtggaat gggagagcaa tggccaacct gagaacaact acaagacgac ccctccagtg 840 ctggacagcg acggcagttt tttcctgtac agcaagctga cagtcgacaa aagccggtgg 900 cagcagggca atgtgttcag ctgcagcgtg atgcacgagg ccctccataa tcactacacc 960 cagaagtccc tgagcctgag tcctggcaag 990 <210> 15 <211> 327 <212> PRT <213> Artificial sequences <400> 15 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 Leu 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 Ser Gln Glu Glu Met Thr Lys 225 230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp 245​​​​​​​​​​​​​​​​​​​​​​​​​​​​​<400> 16 gccagcacca agggccccag cgtgttccct ctggcccctt gtagccggag cacatcagag 60 agcaccgccg ctctgggttg tctggtgaaa gactacttcc cagaacccgt gaccgtgagc 120 tggaacagcg gcgccttaac aagcggcgtg cacacctttc cagccgtgct ccagagctct 180 ggcctgtata gcctctcctc tgtggtgacc gtgccctcta gcagcctggg gaccaagacc 240 tacacatgca acgtggacca caagcctagc aacactaagg tcgacaagag agtggaatct 300 aagtacggcc ctccatgccc cccctgtcct gcccctgagt tcctgggagg cccttccgtc 360 tttctgttcc cccccaagcc taggcacc ctgatgatca gccgcacccc tgaggtcaca 420 tgcgtggtgg tggatgtgag tcaggaggat cctgaggtgc agtttaactg gtacgtggac 480 ggcgtggaag tgcacaacgc caagacaaaa cctcgggaag agcagttcaa ttctacctac 540 cgggtggtca gcgtgctgac agtgctgcac caggactggc tgaacggaa ggaataacag 600 tgcaaggtgt ccaacaaggg cctgcccagc agcatcgaga aaaccatcag taaagccaag 660 ggccagccta gagagcctca ggtgtatacc ctgcctccta gccaggagga aatgaccaag 720 aaccaggtgt ccctgacctg cctggtgaag ggcttctacc ccagcgacat cgccgtggaa 780 cccctccagt gcttgattct 840. cccctccagt gcttgattct gatggcagct tcttcctgta ctctaggctg acagtggaca agagcagatg gcaggagggc 900 aacgttttca gctgcagcgt catgcacgag gccctgcata atcactacac acagaaaagc ctgtctctgt ctctgggcaa g <210> 17 <211> 45 <212> DNA <213> Artificial sequences (Artificial sequences) <400> 17 ggtggtggtg gttctggtgg tggtggttct ggcggcggcg gctcc <210> 18 <211> 15 <212> PRT <213> Artificial sequences (Artificial sequences) <400> 18 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 19 <211> 57 <212> DNA <213> Artificial sequences (Artificial sequences) <400> 19 atgggctgga gctgcatcat cctgttcctg gtggccaccg ccacaggcgt gcactcc 57 <210> 20 <211> 19 <212> PRT <213> Artificial sequences <400> 20 Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr Gly 1 5 10 15 Val His Ser <210> 21 <211> 213 <212> PRT <213> Artificial sequences <400> 21 Asp Ile Gln Ile Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Lys Met 20 25 30 Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Trp Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp 65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro 100 105 110 Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr 115 120 125 Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys 130 135 140 Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu 145 150 155 160 Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165 170 175 Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala 180 185 190 Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195 200 205 Asn Arg Gly Glu Cys 210 <210> 22 <211> 639 <212> DNA <213> Artificial sequences <400> 22 gacatccaga tcacccagtc tcctagcaca ctgtccgcct ctgttggaga tagagtgaca 60 atcacctgta gcgccagcag ctccgtgtcc aaaatgaact ggtaccagca aaagcccggc 120 atcacctgta gcgccagcag ctccgtgtcc aaaatgaact ggtaccagca aaagcccggc 120 aaggccccta agagatggat ctacgacacc agcaaactgg ccagcggcgt gcccagtaga 180 aaggccccta agagatggat ctacgacacc agcaaactgg ccagcggcgt gcccagtaga 180 ttcagcggca gcggatctgg cacagagtac accctgacca tcagcagcct gcaacctgat 240 ttcagcggca gcggatctgg cacagagtac accctgacca tcagcagcct gcaacctgat 240 gacttcgcca catactactg ccagcagtgg tcctctaatc ctctgacctt cggccagggc 300 gacttcgcca catactactg ccagcagtgg tcctctaatc ctctgacctt cggccagggc 300 accaagctgg aaatcaagcg gaccgtggcc gccccttctg tgttcatctt cccccccagc 360 accaagctgg aaatcaagcg gaccgtggcc gccccttctg tgttcatctt cccccccagc 360 gacgagcagc tgaagagcgg aaccgccagc gtggtgtgcc tgctcaacaa cttctacccg 420 gacgagcagc tgaagagcgg aaccgccagc gtggtgtgcc tgctcaacaa cttctacccg 420 cgggaagcca aggtgcagtg gaaggtggac aacgccctgc agagcggcaa cagccaggag 480 cgggaagcca aggtgcagtg gaaggtggac aacgccctgc agagcggcaa cagccaggag 480 agcgtgaccg agcaggacag caaggactct acatacagcc tgagcagcac cctgacactg 540 agcgtgaccg agcaggacag caaggactct acatacagcc tgagcagcac cctgacactg 540 tctaaagccg actacgagaa gcacaaggtg tacgcctgtg aagtgacaca ccagggcctg 600 tctaaagccg actacgagaa gcacaaggtg tacgcctgtg aagtgacaca ccagggcctg 600 agcagccctg tgaccaagtc ttttaaccgg ggcgagtgc 639 agcagccctg tgaccaagtc ttttaaccgg ggcgagtgc 639 <210> 23 <210> 23 <211> 448 <211> 448 <212> PRT <212> PRT <213> 人工序列(Artificial sequences) <213> Artificial sequences <400> 23 <400> 23 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ala 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 Ile Phe Thr Glu Tyr 20 25 30 Ile Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Trp Phe Tyr Pro Gly Ser Asp Asn Ile Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Asp Arg Ala Thr Leu 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 His Glu Thr Gly Tyr Phe Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 <210> 24 <211> 1344 <212> DNA <213> Artificial sequences <400> 24 caggtgcagc tggttcagag cggagctgaa gtgaagaagc ccggcgccag cgtgaaggtg 60 tcctgcaagg cctctggata catcttcacc gagtacatca tccactgggt gcggcaggcc 120 cctggacaag gcctggaatg gatcggctgg ttctaccctg gcagcgacaa catcaagtac 180 aacgagaagt tcaaggatag agccaccctg accgcagata agtccaccag caccgtctac 240 atggaactga gcagcctgag aagcgaggat acagccgtgt actactgcgc cagacacgaa 300 accggctact tcttcgacta ctggggccag ggtacactgg tgaccgtgtc ctccgccagc 360 accaagggcc ctagcgtctt tccactggcc ccttcttcta agagcacaag cggcggaacc 420 gccgctctgg gttgtctggt caaagattac ttccccgaac ctgtgaccgt gtcctggaac 480 agcggcgccc tgacatctgg cgtgcacaca ttcccagccg tgttgcagag cagcggcctg 540 tactctctgt ctagcgtcgt caccgtgccc agcagcagcc tgggaacaca gacctacatc 600 tgcaacgtga accacaagcc tagcaacacc aaagtggata agaaagtgga acccaagagc 660 tgcgacaaga cccacacctg tcctccgtgc cctgctcctg agctgctggg cggccccagc 720 gtgttcctgt tcccccccaa gcctaaggac accctgatga tcagccgcac ccctgaggtg 780 acatgcgtgg tcgtcgacgt gtcccacgag gaccccgagg tgaaattcaa ctggtacgtg 840 gacggcgtgg aagtgcacaa cgccaagacc aagccaagag aagagcagta cgcctctaca 900 tacagagtgg tgtccgtgct gaccgtgctg caccaggact ggctgaacgg caaggaatac 960 aagtgcaagg tgtccaacaa ggccctgccc gctcctatcg agaagacaat ctctaaggct 1020 aaaggccagc ctagagaacc tcaggtttat acactgcctc ctagcagaga ggaaatgacc 1080 aagaaccagg tgtctctgac ctgtctggtg aagggcttct atccttctga catcgccgtg 1140 gaatgggaga gcaatggcca acctgagaac aactacaaga cgacccctcc agtgctggac 1200 agcgacggca gttttttcct gtacagcaag ctgacagtcg acaaaagccg gtggcagcag 1260 ggcaatgtgt tcagctgcag cgtgatgcac gaggccctcc ataatcacta cacccagaag 1320 tccctgagcc tgagtcctgg caag 1344 <210> 25 <211> 219 <212> PRT <213> Artificial sequences <400> 25 Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Thr Lys Ser Leu Leu Asn Ser 20 25 30 Asp Gly Phe Thr Tyr Leu Gly Trp Cys Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Leu Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Ser 85 90 95 Asn Tyr Leu Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 26 <211> 657 <212> DNA <213> Artificial sequences <400> 26 gacgtggtga tgacccagac ccctctgtct ctgcctgtga ccctcggcca gcctgcctcc 60 atctcctgca agtccaccaa aagcctgctg aattccgacg gctttaccta tctgggctgg 120 tgcctgcaga agcctggcca gagccctcag ctgctgatct acctggtgtc caaccggttt 180 tctggcgtgc ccgacagatt ctccggctcc ggatctggaa ccgatttcac cctcaagatc 240 tccagagtgg aagccgagga tgtgggcgtg tactactgct tccagtccaa ctacctgcct 300 ctgaccttcg gccaaggcac caagctggaa atcaagcgga ccgtggctgc tccctctgtg 360 ttcatcttcc ctccttccga tgaacagctg aaatctggta ccgcttctgt cgtgtgtctg 420 ctgaacaact tctaccccag agaggccaag gtgcagtgga aggtggacaa cgccctgcag 480 tctggcaact cccaagagtc cgtgaccgag caggactcta aggactccac atactccctg 540 agctctaccc tgacactgtc caaggccgac tacgagaagc acaaggtcta cgcctgcgag 600 gtgacccacc agggcctgtc ctctccagtt acaaagtcct tcaacagagg cgagtgt 657 <210> 27 <211> 449 <212> PRT <213> Artificial sequences <400> 27 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 Asn Tyr 20 25 30 Trp Ile Gly Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Leu Tyr Pro Gly Gly Leu Tyr Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Met Thr Ala Asp Thr Ser Thr Asn 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 Tyr Arg Asp Tyr Asp Tyr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys 210 215 220 Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro 225 230 235 240 Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser 245 250 255 Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp 260 265 270 Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn 275 280 285 Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val 290 295 300 Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu 305 310 315 320 Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys 325 330 335 Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr 340 345 350 Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr 355 360 365 Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu 370 375 380 Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu 385 390 395 400 Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys 405 410 415 Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu 420 425 430 Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly 435 440 445 Lys <210> 28 <211> 1347 <212> DNA <213> Artificial sequences <400> 28 caggttcagc tggtgcagtc cggagccgag gtgaagaagc ccggcgcttc tgtgaaggtg 60 tcttgtaagg cctctggcta caccttcacc aactactgga tcggctgggt gaagcaggcc 120 cctggccagg gcctggaatg gatcggctat ctgtaccctg gaggcctgta taccaattac 180 aacgagaagt tcaagggcaa ggccaccatg accgctgaca cctctaccaa cactgcctac 240 atggaactgt ccagcttgcg gtccgaggat accgctgtgt actactgcgc cagataccgg 300 gactatgact acgccatgga ctattgggga caaggcaccc tggtgacagt atcctccgcc 360 It should be noted that in the above translation, there are some possible inaccuracies in the original text. For example, in the sequence "aacgagaagt tcaagggcaa ggccaccatg accgctgaca cctctaccaa cactgcctac 240", the "240" should probably be "240" for better clarity. And in "atggaactgt ccagcttgcg gtccgaggat accgctgtgt actactgcgc cagataccgg 300", the "300" should be "300". These are likely just input errors in the original text.tctaccaagg gcccatctgt gttccctctg gccccctcct ccaagtccac atccggcggc 420 acagctgctc tgggctgcct ggtcaaagat tacttcccag aacctgtgac cgtctcctgg 480 aactccggcg ccctgacctc cggtgtccat acctttcctg ccgtgctgca gtttctggc 540 ctgtacagcc tgagctctgt ggtgaccgtg cctagcagct ctctcggcac ccagacctac 600 atctgcaacg tgaaccacaa gccttctaac accaaagtgg ataagaaagt ggaacctaag 660 tcctgcgaca agacccatac ctgtcctcct tgtccagctc cagagctgct gggcggcccc 720 tccgtgtttc tgttcccccc taagcccaag gatacactga tgatctccccg gacccctgaa 780 gtgacctgcg tggtggttga cgtgtctcac gaggaccccg aggtgaagtt caactggtac 840 gtggatggag tggaagtgca caacgccaag accaagccta gagaggaaca gtacgcttct 900 acatacagag tggtctctgt gctgaccgtg ctgcaccagg actggctgaa cggaaaagag 960 tacaagtgca aggtctccaa caaggctctg cctgctccta tcgagaaaac catctctaag 1020 gccaagggcc agcctcggga gcctcaagtg tacaccctgc ctccttcccg cgaagagatg 1080 accaagaacc aggtgagcct cacatgtctg gtgaaaggct tctacccctc agacatcgcc 1140 gtcgagtggg agtcgaatgg ccagcctgag aacaactaca agaccacccc tcccgtgctg 1200 gactccgacg gctccttctt cctgtactct aagctgacag tggacaagtc cagatggcag 1260 cagggcaacg tgttctcctg ctccgtgatg cacgaggccc tgcacaatca ctacacccag 1320 aagtccctgt ccctgtctcc tggcaag 1347 <210> 29 <211> 219 <212> PRT <213> Artificial sequences <400> 29 Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Thr Lys Ser Leu Leu Asn Ser 20 25 30​​​​​​​​​​​​​Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Ser 85 90 95 Asn Tyr Leu Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 115 120 125 Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 130 135 140 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln 145 150 155 160 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser 165 170 175 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu 180 185 190 Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser 195 200 205 Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 30 <211> 657 <212> DNA <213> Artificial sequences <400> 30 60. gacgtggtga tgacccagac ccctctgagc ctgccagtga ccctgggaca gcctgcttct atcagctgta aaagcacaaa atctctgctg aacagcgatg gcttcaccta cctgggctgg 180. tgtctgcaga aacctggaca gagccctcag ctgctgatct acctggtgtc father tctggcgtcc ccgatagtt tagcggcagc ggctctggca ccgacttcac cctcaagatc agcagagtgg aagccgagga cgtggggcgtg tactactgct tccagagcaa ctacctgcca 360. ctgacattcg gacaaggcac aaagctgga atcaagcgca ccgtggccgc ccctagcgtg ttcatcttcc cccccagcga cgagcagctg aagtccggca ccgccagcgt cgtgtgcctg 420 ctgaacaact tctaccctcg ggaagccaag gtgcagtgga aggtggacaa cgccctgcaa tctggcaata gccaggagag cgttacagaa caggacagca aggacagcac ctattctctg agctccaccc tgaccctgtc caaggccgat tacgagaagc acaaggtgta cgcctgcgag 600. gtgacacacc agggcctgag ctcccctgtg acaaagagct tcaaccgggg cgagtgc 657 <210> 31 <211> 446 <212> PRT <213> Artificial sequences <400> 31 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 Asn Tyr 20 25 30 Trp Ile Gly Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Leu Tyr Pro Gly Gly Leu Tyr Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Met Thr Ala Asp Thr Ser Thr Asn 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 Tyr Arg Asp Tyr Asp Tyr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe 115 120 125 Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu<{ 130 135 140 Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp 145 150 155 160 Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu 165 170 175 Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser 180 185 190 Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro 195 200 205 Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro 210 215 220 Cys Pro Pro Cys Pro Ala Pro Glu Phe Leu Gly Gly Pro Ser Val Phe 225 230 235 240 Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro 245 250 255 Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val 260 265 270 Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr 275 280 285 Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val 290 295 300 Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys 305 310 315 320 Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser 325 330 335 Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro 340 345 350 Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val 355 360 365 Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly 370 375 380 Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp 385 390 395 400 Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp 405 410 415 Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His 420 425 430 Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Lys 435 440 445 <210> 32 <211> 1338 <212> DNA <213> Artificial sequences <400> 32 caggtgcagc tggtccagag cggcgccgag gtgaaaaaac ccggcgctag cgtgaaggtt 60 tcctgtaaag ccagcggata caccttcacc aattactgga tcggctgggt gaagcaagcc 120 cctggacagg gcctggaatg gattggctac ctgtaccctg gcggcctgta caccaactac 180 aacgagaagt tcaagggcaa ggctacaatg acagccgata ccagcacaaa taccgcctac 240 atggaactgt cgagcctgag atctgaggac acggccgtgt actactgcgc cagatacaga 300 gattacgact acgccatgga ctattggggc caaggcaccc tggtgacagt gagcagcgcc 360 agcaccaagg gccccagcgt gttccctctg gccccttgta gccggagcac atcagagagc 420 accgccgctc tgggttgtct ggtgaaagac tacttcccag aacccgtgac cgtgagctgg 480 aacagcggcg ccttaacaag cggcgtgcac acctttccag ccgtgctcca gagctctggc 540 ctgtatagcc tctcctctgt ggtgaccgtg ccctctagca gcctggggac caagacctac 600 acatgcaacg tggaccacaa gcctagcaac actaaggtcg acaagagagt ggaatctaag 660 tacggccctc catgcccccc ctgtcctgcc cctgagttcc tgggaggccc ttccgtcttt 720 ctgttccccc ccaagcctaa ggacaccctg atgatcagcc gcaccctga ggtcacatgc 780 gtggtggtgg atgtgagtca ggaggatcct gaggtgcagt ttactgta cgtggacggc 840 gtggaagtgc acaacgccaa gataaacct cgggaagagc agttcaatc tacctaccgg 900 gtggtcagcg tgctgacagt gctgcaccag gactggctga acggaaagga atacaagtgc 960 aaggtgtcca acaagggcct gcccagcagc atcgagaaaa ccatcagtaa agccaagggc 1020 cagcctagag agcctcaggt gtataccctg cctcctagcc aggaggaat gaccagaac 1080 caggtgtccc tgacctgcct ggtgaagggc ttctacccca gcgacatcgc cgtggaatgg 1140 gagagcaacg vakaacctga gacaacac aagaccaccc ctccagtgct tgattctgat 1200 ggcagcttct tcctgtactc taggctgaca gtggacaaga gcagatggca ggaggggcac 1260 gtttcagct gcagcgtcat gcacgaggcc ctgcataatc actacacaca gaaaagcctg 1320 tctctgtctc tggcaag 1338 <210> 33 <211> 340 <212> PRT <213> Artificial sequences <400> 33 Asp Ile Gln Ile Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Lys Met 20 25 30 Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Trp Ile Tyr 35 40 45 Asp Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp 65 70 75 80 Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr 85 90 95 Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly 100 105 110 Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Val Val Met Thr Gln Thr ​​​​​​Lys Ser Thr Lys Ser Leu Leu Asn Ser Asp Gly Phe Thr Tyr Leu Gly 145 150 155 160 Trp Cys Leu Gln Lys Pro Gly Gln Ser Pro Gln Leu Leu Ile Tyr Leu 165 170 175 Val Ser Asn Arg Phe Ser Gly Val Pro Asp Arg Phe Ser Gly Ser Gly 180 185 190 Ser Gly Thr Asp Phe Thr Leu Lys Ile Ser Arg Val Glu Ala Glu Asp 195 200 205 Val Gly Val Tyr Tyr Cys Phe Gln Ser Asn Tyr Leu Pro Leu Thr Phe 210 215 220 Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser 225 230 235 240 Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala 245 250 255 Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val 260 265 270 Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser 275 280 285 Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr 290 295 300 Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys 305 310 315 320 Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn 325 330 335 Arg Gly Glu Cys 340 <210> 34 <211> 1020 <212> DNA <213> Artificial sequences <400> 34 gacatccaga tcacccagtc tcctagcaca ctgtccgcct ctgttggaga tagagtgaca 60 atcacctgta gcgccagcag ctccgtgtcc aaaatgaact ggtaccagca aaagcccggc 120 aaggccccta agagatggat ctacgacacc agcaaactgg ccagcggcgt gcccagtaga 180 ttcagcggca gcggatctgg cacagagtac accctgacca tcagcagcct gcaacctgat 240 gacttcgcca catactactg ccagcagtgg tcctctaatc ctctgacctt cggccagggc 300 accaagctgg aaatcaaggg tggtggtggt tctggtggtg gtggttctgg cggcggcggc 360 tccgacgtgg tgatgaccca gacccctctg tctctgcctg tgaccctcgg ccagcctgcc 420 tccatctcct gcaagtccac caaaagcctg ctgaattccg acggctttac ctatctgggc 480 tggtgcctgc agaagcctgg ccagagccct cagctgctga tctacctggt gtccaaccgg 540 ttttctggcg tgcccgacag attctccggc tccggatctg gaaccgattt caccctcaag atctccagag tggaagccga ggatgtgggc gtgtactact gcttccagtc caactacctg 660 cctctgacct tcggccaagg caccaagctg gaatcaagc ggaccgtggc tgctccctct gtgttcatct tccctccttc cgatgaacag ctgaaatctg gtaccgcttc tgtcgtgtgt 780 ctgctgaaca acttctaccc cagagaggcc aaggtgcagt ggaaggtgga caacgccctg cagtctggca actcccaaga gtccgtgacc cagcaggact ctaaggactc cacatactcc ctgagctcta ccctgacact gtccaaggcc gactacgaga agcacaaggt ctacgcctgc 960 gaggtgaccc accagggcct gtcctctcca gttacaaagt ccttcaacag aggcgagtgt 1020 <210> 35 <211> 582 <212> PRT <213> Artificial sequences (Artificial sequences) <400> 35 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 Ile Phe Thr Glu Tyr 20 25 30 Ile Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Trp Phe Tyr Pro Gly Ser Asp Asn Ile Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Asp Arg Ala Thr Leu 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 His Glu Thr Gly Tyr Phe Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val 130 135 140 Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr 145 150 155 160 Thr Phe Thr Asn Tyr Trp Ile Gly Trp Val Lys Gln Ala Pro Gly Gln 165 170 175 Gly Leu Glu Trp Ile Gly Tyr Leu Tyr Pro Gly Gly Leu Tyr Thr Asn 180 185 190 Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Met Thr Ala Asp Thr Ser 195 200 205 Thr Asn Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr 210 215 220 Ala Val Tyr Tyr Cys Ala Arg Tyr Arg Asp Tyr Asp Tyr Ala Met Asp 225 230 235 240 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys 245 250 255 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 260 265 270 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 275 280 285 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 290 295 300 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 305 310 315 320 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 325 330 335 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro 340 345 350 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 355 360 365 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 370 375 380 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 385 390 395 400 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 405 410 415 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala 420 425 430 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 435 440 445 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 450 455 460 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 465 470 475 480 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 485 490 495 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 500 505 510 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 515 520 525<​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​cctggacaag gcctggaatg gatcggctgg ttctaccctg gcagcgacaa catcaagtac 180 aacgagaagt tcaaggatag agccaccctg accgcagata agtccaccag caccgtctac 240 atggaactga gcagcctgag aagcgaggat acagccgtgt actactgcgc cagacacgaa 300 accggctact tcttcgacta ctggggccag ggtacactgg tgaccgtgtc ctccggtggt 360 ggtggttctg gtggtggtgg ttctggcggc ggcggctccc aggttcagct ggtgcagtcc 420 ggagccgagg tgaagaagcc cggcgcttct gtgaaggtgt cttgtaaggc ctctggctac 480 accttcacca actactggat cggctgggtg aagcaggccc ctggccaggg cctggaatgg 540 atcggctatc tgtaccctgg aggcctgtat accaattaca acgagaagtt caagggcaag 600 gccaccatga ccgctgacac ctctaccaac actgcctaca tggaactgtc cagcttgcgg 660 tccgaggata ccgctgtgta ctactgcgcc agataccggg actatgacta cgccatggac 720 tattggggac aaggcaccct ggtgacagta tcctccgcct ctaccaaggg cccatctgtg 780 ttccctctgg ccccctcctc caagtccaca tccggcggca cagctgctct gggctgcctg 840 gtcaaagatt acttcccaga acctgtgacc gtctcctgga actccggcgc cctgacctcc 900 ggtgtccata cctttcctgc cgtgctgcag tcttctggcc tgtacagcct gagctctgtg 960 gtgaccgtgc ctagcagctc tctcggcacc cagacctaca tctgcaacgt gaaccacaag 1020 ccttctaaca ccaaagtgga taagaaagtg gaacctaagt cctgcgacaa gacccatacc 1080 tgtcctcctt gtccagctcc agagctgctg ggcggcccct ccgtgtttct gttcccccct 1140 aagcccaagg atacactgat gatctcccgg acccctgaag tgacctgcgt ggtggttgac 1200 gtgtctcacg aggaccccga ggtgaagttc aactggtacg tggatggagt ggaagtgcac 1260 aacgccaaga ccaagcctag agaggaacag tacgcttcta catacagagt ggtctctgtg 1320 ctgaccgtgc tgcaccagga ctggctgaac ggaaaagagt acaagtgcaa ggtctccaac 1380 aaggctctgc ctgctcctat cgagaaaacc atctctaagg ccaagggcca gcctcgggag 1440 cctcaagtgt acaccctgcc tccttcccgc gaagagatga ccaagaacca ggtgagcctc 1500 acatgtctgg tgaaaggctt ctacccctca gacatcgccg tcgagtggga gtcgaatggc 1560 cagcctgaga acaactacaa gaccacccct cccgtgctgg actccgacgg ctccttcttc 1620 ctgtactcta agctgacagt ggacaagtcc agatggcagc agggcaacgt gttctcctgc 1680 tccgtgatgc acgaggccct gcacaatcac tacacccaga agtccctgtc cctgtctcct 1740 ggcaag 1746 <210> 37 <211> 745 <212> PRT <213> Artificial sequences <400> 37 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 Ile Phe Thr Glu Tyr 20 25 30 Ile Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Trp Phe Tyr Pro Gly Ser Asp Asn Ile Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Asp Arg Ala Thr Leu 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 His Glu Thr Gly Tyr Phe Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val 130 135 140 Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr 145 150 155 160 Thr Phe Thr Asn Tyr Trp Ile Gly Trp Val Lys Gln Ala Pro Gly Gln 165 170 175 Gly Leu Glu Trp Ile Gly Tyr Leu Tyr Pro Gly Gly Leu Tyr Thr Asn 180 185 190 Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Met Thr Ala Asp Thr Ser 195 200 205 Thr Asn Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr 210 215 220 Ala Val Tyr Tyr Cys Ala Arg Tyr Arg Asp Tyr Asp Tyr Ala Met Asp 225 230 235 240 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys 245 250 255 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 260 265 270 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 275 280 285 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 290 295 300 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 305 310 315 320 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 325 330 335 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro 340 345 350 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 355 360 365 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 370 375 380 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 385 390 395 400 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 405 410 415 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala 420 425 430 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 435 440 445 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 450 455 460 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 465 470 475 480 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 485 490 495 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 500 505 510 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 515 520 525 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 530 535 540 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 545 550 555 560 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 565 570 575 Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 580 585 590 Gly Gly Gly Gly Ser Gln Val Thr Asp Ile Asn Ser Lys Gly Leu Glu 595 600 605 Leu Arg Lys Thr Val Thr Thr Val Glu Thr Gln Asn Leu Glu Gly Leu 610 615 620 His His Asp Gly Gln Phe Cys His Lys Pro Cys Pro Pro Gly Glu Arg 625 630 635 640 Lys Ala Arg Asp Cys Thr Val Asn Gly Asp Glu Pro Asp Cys Val Pro 645 650 655 Cys Gln Glu Gly Lys Glu Tyr Thr Asp Lys Ala His Phe Ser Ser Lys 660 665 670 Cys Arg Arg Cys Arg Leu Cys Asp Glu Gly His Gly Leu Glu Val Glu 675 680 685 Ile Asn Cys Thr Arg Thr Gln Asn Thr Lys Cys Arg Cys Lys Pro Asn 690 695 700 Phe Phe Cys Asn Ser Thr Val Cys Glu His Cys Asp Pro Cys Thr Lys 705 710 715 720 Cys Glu His Gly Ile Ile Lys Glu Cys Thr Leu Thr Ser Asn Thr Lys 725 730 735 Cys Lys Glu Glu Gly Ser Arg Ser Asn 740 745 <210> 38 <211> 2235 <212> DNA <213> Artificial sequences <400> 38 caggtgcagc tggttcagag cggagctgaa gtgaagaagc ccggcgccag cgtgaaggtg 60 tcctgcaagg cctctggata catcttcacc gagtacatca tccactgggt gcggcaggcc 120 cctggacaag gcctggaatg gatcggctgg ttctaccctg gcagcgacaa catcaagtac 180 aacgagaagt tcaaggatag agccaccctg accgcagata agtccaccag caccgtctac 240 atggaactga gcagcctgag aagcgaggat acagccgtgt actactgcgc cagacacgaa 300 accggctact tcttcgacta ctggggccag ggtacactgg tgaccgtgtc ctccggtggt 360 ggtggttctg gtggtggtgg ttctggcggc ggcggctccc aggttcagct ggtgcagtcc 420 ggagccgagg tgaagaagcc cggcgcttct gtgaaggtgt cttgtaaggc ctctggctac 480 accttcacca actactggat cggctgggtg aagcaggccc ctggccaggg cctggaatgg 540 atcggctatc tgtaccctgg aggcctgtat accaattaca acgagaagtt caggggcaag 600 gccaccatga ccgctgacac ctctaccaac actgcctaca tggaactgtc cagcttgcgg 660 tccgaggata ccgctgtgta ctactgcgcc agataccggg actatgacta cgccatggac 720 tattggggac aaggcaccct ggtgacagta tcctccgcct ctaccaaggg cccatctgtg 780 ttccctctgg ccccctctc caagtccaca tccggcggca cagctgctct gggctgcctg 840 gtcaaagatt acttcccaga acctgtgacc gtctcctgga actccggcgc cctgacctcc 900 ggtgtccata cctttcctgc cgtgctgcag tcttctggcc tgtacagcct gagctctgtg 960 gtgaccgtgc ctagcagctc tctcggcacc cagacctaca tctgcaacgt gaaccaag 1020 ccttctaaca ccaaagtgga taagaaagtg gaacctaagt cctgcgacaa gacccatacc 1080 tgtcctcctt gtccagctcc agagctgctg ggcggcccct ccgtgtttct gttcccccct 1140 aagcccaagg atacactgat gatctccccgg acccctgaag tgacctgcgt ggtggttgac 1200 gtgtctcacg aggaccccga ggtgaagttc aactggtacg tggatggagt ggaagtgcac 1260 aacgccaaga ccaagcctag agaggaacag tacgcttcta catacagagt ggtctctgtg 1320 ctgaccgtgc tgcaccagga ctggctgaac ggaaaagagt acaagtgcaa ggtctccaac 1380 aaggctctgc ctgctcctat cgagaaaacc atctctaagg ccaagggcca gcctcgggag 1440 cctcaagtgt acaccctgcc tccttcccgc gaagagatga ccaagaacca ggtgagcctc 1500 acatgtctgg tgaaaggctt ctacccctca gacatcgccg tcgagtggga gtcgaatggc 1560 cagcctgaga acaactacaa gaccacccct cccgtgctgg actccgacgg ctccttcttc 1620 ctgtaccta agctgacagt ggacaagtcc agatggcagc agggcaacgt gttctcctgc 1680 tccgtgatgc acgaggccct gcacaatcac tacacccaga agtccctgtc cctgtctcct 1740 ggcaagggtg gtggtggttc tggtggtggt ggttctggcg gcggcggctc ccaagtgact 1800 gacatcaact ccaagggatt ggaattgagg aagactgtta ctacagttga gactcagaac 1860 ttggaaggcc tgcatcatga tggccaattc tgccataagc cctgtcctcc aggtgaaagg 1920 aaagctaggg actgcacagt caatggggat gaaccagact gcgtgccctg ccaagaaggg 1980 aaggagtaca cagacaaagc ccatttttct tccaaatgca gaagatgtag attgtgtgat 2040 gaaggacatg gcttagaagt ggaaataaac tgcacccgga cccagaatac caagtgcaga 2100 tgtaaaccaa actttttttg taactctact gtatgtgaac actgtgaccc ttgcaccaaa 2160 tgtgaacatg gaatcatcaa ggaatgcaca ctcaccagca acaccaagtg caaagaggaa 2220 ggatccagat ctaac 2235 <210> 39 <211> 908 <212> PRT <213> Artificial sequences <400> 39 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 Ile Phe Thr Glu Tyr 20 25 30 Ile Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Trp Phe Tyr Pro Gly Ser Asp Asn Ile Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Asp Arg Ala Thr Leu 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 His Glu Thr Gly Tyr Phe Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val 130 135 140 Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr 145 150 155 160 Thr Phe Thr Asn Tyr Trp Ile Gly Trp Val Lys Gln Ala Pro Gly Gln 165 170 175 Gly Leu Glu Trp Ile Gly Tyr Leu Tyr Pro Gly Gly Leu Tyr Thr Asn 180 185 190 Tyr Asn Glu Lys Phe Lys Gly Lys Ala Thr Met Thr Ala Asp Thr Ser 195 200 205 Thr Asn Thr Ala Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr 210 215 220 Ala Val Tyr Tyr Cys Ala Arg Tyr Arg Asp Tyr Asp Tyr Ala Met Asp 225 230 235 240 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys 245 250 255 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 260 265 270 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 275 280 285 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 290 295 300 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 305 310 315 320 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 325 330 335 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro 340 345 350 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 355 360 365 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 370 375 380 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 385 390 395 400 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 405 410 415 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala 420 425 430 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 435 440 445 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 450 455 460 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 465 470 475 480 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 485 490 495 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 500 505 510 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 515 520 525 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 530 535 540 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 545 550 555 560 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 565 570 575 Ser Leu Ser Pro Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 580 585 590 Gly Gly Gly Gly Ser Gln Val Thr Asp Ile Asn Ser Lys Gly Leu Glu 595 600 605 Leu Arg Lys Thr Val Thr Thr Val Glu Thr Gln Asn Leu Glu Gly Leu 610 615 620 His His Asp Gly Gln Phe Cys His Lys Pro Cys Pro Pro Gly Glu Arg 625 630 635 640 Lys Ala Arg Asp Cys Thr Val Asn Gly Asp Glu Pro Asp Cys Val Pro 645 650 655 Cys Gln Glu Gly Lys Glu Tyr Thr Asp Lys Ala His Phe Ser Ser Lys 660 665 670 Cys Arg Arg Cys Arg Leu Cys Asp Glu Gly His Gly Leu Glu Val Glu 675 680 685 Ile Asn Cys Thr Arg Thr Gln Asn Thr Lys Cys Arg Cys Lys Pro Asn 690 695 700 Phe Phe Cys Asn Ser Thr Val Cys Glu His Cys Asp Pro Cys Thr Lys 705 710 715 720 Cys Glu His Gly Ile Ile Lys Glu Cys Thr Leu Thr Ser Asn Thr Lys 725 730 735 Cys Lys Glu Glu Gly Ser Arg Ser Asn Gly Gly Gly Gly Ser Gly Gly 740 745 750 Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Thr Asp Ile Asn Ser Lys 755 760 765 Gly Leu Glu Leu Arg Lys Thr Val Thr Thr Val Glu Thr Gln Asn Leu 770 775 780 Glu Gly Leu His His Asp Gly Gln Phe Cys His Lys Pro Cys Pro Pro 785 790 795 800 Gly Glu Arg Lys Ala Arg Asp Cys Thr Val Asn Gly Asp Glu Pro Asp 805 810 815 Cys Val Pro Cys Gln Glu Gly Lys Glu Tyr Thr Asp Lys Ala His Phe 820 825 830 Ser Ser Lys Cys Arg Arg Cys Arg Leu Cys Asp Glu Gly His Gly Leu 835 840 845 Glu Val Glu Ile Asn Cys Thr Arg Thr Gln Asn Thr Lys Cys Arg Cys 850 855 860 Lys Pro Asn Phe Phe Cys Asn Ser Thr Val Cys Glu His Cys Asp Pro 865 870 875 880 Cys Thr Lys Cys Glu His Gly Ile Ile Lys Glu Cys Thr Leu Thr Ser 885 890 895 Asn Thr Lys Cys Lys Glu Glu Gly Ser Arg Ser Asn 900 905 <210> 40 <211> 2724 <212> DNA <213> Artificial sequences <400> 40 caggtgcagc tggttcagag cggagctgaa gtgaagaagc ccggcgccag cgtgaaggtg 60 tcctgcaagg cctctggata catcttcacc gagtacatca tccactgggt gcggcaggcc 120 cctggacaag gcctggaatg gatcggctgg ttctaccctg gcagcgacaa catcaagtac (应为“cctggacaag gcctggaatg gatcggctgg ttctaccctg gcagcgacaa catcaagtac”,原内容中“180”位置错误,推测是想表达这一行内容)180 aacgagaagt tcaaggatag agccaccctg accgcagata agtccaccag caccgtctac 240 atggaactga gcagcctgag aagcgaggat acagccgtgt actactgcgc cagacacgaa 300 accggctact tcttcgacta ctggggccag ggtacactgg tgaccgtgtc ctccggtggt 360 ggtggttctg gtggtggtgg ttctggcggc ggcggctccc aggttcagct ggtgcagtcc 420 ggagccgagg tgaagaagcc cggcgcttct gtgaaggtgt cttgtaaggc ctctggctac 480 accttcacca actactggat cggctgggtg aagcaggccc ctggccaggg cctggaatgg 540 atcggctatc tgtaccctgg aggcctgtat accaattaca acgagaagtt caggggcaag 600 gccaccatga ccgctgacac ctctaccaac actgcctaca tggaactgtc cagcttgcgg 660 tccgaggata ccgctgtgta ctactgcgcc agataccggg actatgacta cgccatggac 720 tattggggac aaggcaccct ggtgacagta tcctccgcct ctaccaaggg cccatctgtg 780 ttccctctgg ccccctctc caagtccaca tccggcggca cagctgctct gggctgcctg 840 gtcaaagatt acttcccaga acctgtgacc gtctcctgga actccggcgc cctgacctcc 900 ggtgtccata cctttcctgc cgtgctgcag tcttctggcc tgtacagcct gagctctgtg 960 gtgaccgtgc ctagcagctc tctcggcacc cagacctaca tctgcaacgt gaaccaag 1020 ccttctaaca ccaaagtgga taagaaagtg gaacctaagt cctgcgacaa gacccatacc 1080 tgtcctcctt gtccagctcc agagctgctg ggcggcccct ccgtgtttct gttcccccct 1140 aagcccaagg atacactgat gatctcccgg acccctgaag tgacctgcgt ggtggttgac 1200 gtgtctcacg aggaccccga ggtgaagttc aactggtacg tggatggagt ggaagtgcac 1260 aacgccaaga ccaagcctag agaggaacag tacgcttcta catacagagt ggtctctgtg 1320 ctgaccgtgc tgcaccagga ctggctgaac ggaaaagagt acaagtgcaa ggtctccaac 1380 aaggctctgc ctgctcctat cgagaaaacc atctctaagg ccaagggcca gcctcgggag 1440 cctcaagtgt acaccctgcc tccttcccgc gaagagatga ccaagaacca ggtgagcctc 1500 acatgtctgg tgaaaggctt ctacccctca gacatcgccg tcgagtggga gtcgaatggc 1560 cagcctgaga acaactacaa gaccacccct cccgtgctgg actccgacgg ctccttcttc 1620 ctgtaccta agctgacagt ggacaagtcc agatggcagc agggcaacgt gttctcctgc 1680 tccgtgatgc acgaggccct gcacaatcac tacacccaga agtccctgtc cctgtctcct 1740 ggcaagggtg gtggtggttc tggtggtggt ggttctggcg gcggcggctc ccaagtgact 1800 gacatcaact ccaagggatt ggaattgagg aagactgtta ctacagttga gactcagaac 1860 ttggaaggcc tgcatcatga tggccaattc tgccataagc cctgtcctcc aggtgaaagg 1920 aaagctaggg actgcacagt caatggggat gaaccagact gcgtgccctg ccaagaaggg 1980 aaggatca cagacaaagc ccatttttct tccaaatgca gaagatgtag attgtgtgat 2040 2100 tgtaaaccaa actttttttg taactctact gtatgtgaac actgtgaccc ttgcaccaaa 2160 2220 ggatccagat ctaacggtgg tggtggttct ggtggtggtg gttctggcgg cggcggctcc 2280 2340 actcagaact tggaaggcct gcatcatgat ggccaattct gccataagcc ctgtcctcca 2400 ggtgaaagga aagctaggga ctgcacagtc aatggggatg aaccagactg cgtgccctgc 2460 caagaaggga aggagtacaac agacaaagcc catttttctt ccaaatgcag aagatgtaga 2520 ttgtgtgatg aaggacatgg cttagaagtg gaaataaact gcacccggac ccagaatacc 2580 aagtgcagat gtaaaccaaa ctttttttgt aactctactg tatgtgaaca ctgtgaccct 2640 tgcaccaaat gtgaacatgg aatcatcaag gaatgcacac tcaccagcaa caccaagtgc 2700 aaagaggaag gatccagatc taac 2724 <210> 41 <211> 611 <212> PRT <213> Artificial sequences <400> 41 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 Ile Phe Thr Glu Tyr 20 25 30 Ile Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile [[ID=]28]35 40 45 Gly Trp Phe Tyr Pro Gly Ser Asp Asn Ile Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Asp Arg Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Val Tyr 65 70 及 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Note: There seems to be a formatting issue in the original text where "35 40 45" and "65 70 75 80" are not separated as expected. In the translation, I've tried to make sense of it as best as possible. Also, "及" in "65 70 及 75 80" is translated as "and" in the context to make the English more understandable. If this is not what you intended, please clarify. Ala Arg His Glu Thr Gly Tyr Phe Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln 450 455 460 Val Thr Asp Ile Asn Ser Lys Gly Leu Glu Leu Arg Lys Thr Val Thr 465 470 475 480 Thr Val Glu Thr Gln Asn Leu Glu Gly Leu His His Asp Gly Gln Phe 485 490 495 Cys His Lys Pro Cys Pro Pro Gly Glu Arg Lys Ala Arg Asp Cys Thr 500 505 510 Val Asn Gly Asp Glu Pro Asp Cys Val Pro Cys Gln Glu Gly Lys Glu 515 520 525 Tyr Thr Asp Lys Ala His Phe Ser Ser Lys Cys Arg Arg Cys Arg Leu 530 535 540 Cys Asp Glu Gly His Gly Leu Glu Val Glu Ile Asn Cys Thr Arg Thr 545 550 555 560 Gln Asn Thr Lys Cys Arg Cys Lys Pro Asn Phe Phe Cys Asn Ser Thr 565 570 575 Val Cys Glu His Cys Asp Pro Cys Thr Lys Cys Glu His Gly Ile Ile 580 585 590 Lys Glu Cys Thr Leu Thr Ser Asn Thr Lys Cys Lys Glu Glu Gly Ser 595 600 605 Arg Ser Asn 610 <210> 42 <211> 1833 <212> DNA <213> Artificial sequences <400> 42 caggtgcagc tggttcagag cggagctgaa gtgaagaagc ccggcgccag cgtgaaggtg 60 tcctgcaagg cctctggata catcttcacc gagtacatca tccactgggt gcggcaggcc 120 cctggacaag gcctggaatg gatcggctgg ttctaccctg gcagcgacaa catcaagtac 180 aacgagaagt tcaaggatag agccaccctg accgcagata agtccaccag caccgtctac 240 atggaactga gcagcctgag aagcgaggat acagccgtgt actactgcgc cagacacgaa 300 accggctact tcttcgacta ctggggccag ggtacactgg tgaccgtgtc ctccgccagc 360 accaagggcc ctagcgtctt tccactggcc ccttcttcta agagcacaag cggcggaacc 420 gccgctctgg gttgtctggt caaagattac ttccccgaac ctgtgaccgt gtcctggaac 480 agcggcgccc tgacatctgg cgtgcacaca ttcccagccg tgttgcagag cagcggcctg 540 tactctctgt ctagcgtcgt caccgtgccc agcagcagcc tgggaacaca gacctacatc 600 tgcaacgtga accacaagcc tagcaacacc aaagtggata agaaagtgga acccaagagc 660 tgcgacaaga cccacacctg tcctccgtgc cctgctcctg agctgctggg cggccccagc 720 gtgttcctgt tcccccccaa gcctaaggac accctgatga tcagccgcac ccctgaggtg 780 acatgcgtgg tcgtcgacgt gtcccacgag gaccccgagg tgaaattcaa ctggtacgtg 840 gacggcgtgg aagtgcacaa cgccaagacc aagccaagag aagagcagta cgcctctaca 900 tacagagtgg tgtccgtgct gaccgtgctg caccaggact ggctgaacgg caaggaatac 960 aagtgcaagg tgtccaacaa ggccctgccc gctcctatcg agaagacaat ctctaaggct 1020 aaaggccagc ctagagaacc tcaggtttat acactgcctc ctagcagaga ggaaatgacc 1080 aagaaccagg tgtctctgac ctgtctggtg aagggcttct atccttctga catcgccgtg 1140 gaatgggaga gcaatggcca acctgagaac aactacaaga cgacccctcc agtgctggac 1200 agcgacggca gttttttcct gtacagcaag ctgacagtcg acaaaagccg gtggcagcag 1260 ggcaatgtgt tcagctgcag cgtgatgcac gaggccctcc ataatcacta cacccagaag 1320 tccctgagcc tgagtcctgg caagggtggt ggtggttctg gtggtggtgg ttctggcggc 1380 ggcggctccc aagtgactga catcaactcc aagggattgg aattgaggaa gactgttact 1440 acagttgaga ctcagaactt ggaaggcctg catcatgatg gccaattctg ccataagccc 1500 tgtcctccag gtgaaaggaa agctagggac tgcacagtca atggggatga accagactgc 1560 gtgccctgcc aagaagggaa ggagtacaca gacaaagccc atttttcttc caaatgcaga 1620 agatgtagat tgtgtgatga aggacatggc ttagaagtgg aaataaactg cacccggacc 1680 cagaatacca agtgcagatg taaaccaaac tttttttgta actctactgt atgtgaacac 1740 tgtgaccctt gcaccaaatg tgaacatgga atcatcaagg aatgcacact caccagcaac 1800 accaagtgca aagaggaagg atccagatct aac 1833 <210> 43 <211> 774 <212> PRT <213> Artificial sequences <400> 43 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 Ile Phe Thr Glu Tyr 20 25 30 Ile Ile His Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Trp Phe Tyr Pro Gly Ser Asp Asn Ile Lys Tyr Asn Glu Lys Phe 50 55 60 Lys Asp Arg Ala Thr Leu 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 His Glu Thr Gly Tyr Phe Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 0]Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 115 120 125 Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly 130 135 140 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 145 150 155 160 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 165 170 175 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 180 185 190 Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr 210 215 220 His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser 225 230 235 240 Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg 245 250 255 Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro 260 265 270 Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 275 280 285 Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala Ser Thr Tyr Arg Val Val 290 295 300 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr 305 310 315 320 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr 325 330 335 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu 340 345 350 Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys 355 360 365 Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 370 375 380 Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 385 390 395 400 Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 405 410 415 Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 420 425 430 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 435 440 445 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln 450 455 460 Val Thr Asp Ile Asn Ser Lys Gly Leu Glu Leu Arg Lys Thr Val Thr 465 470 475 480 Thr Val Glu Thr Gln Asn Leu Glu Gly Leu His His Asp Gly Gln Phe 485 490 495 Cys His Lys Pro Cys Pro Pro Gly Glu Arg Lys Ala Arg Asp Cys Thr 500 505 510 Val Asn Gly Asp Glu Pro Asp Cys Val Pro Cys Gln Glu Gly Lys Glu 515 520 525 Tyr Thr Asp Lys Ala His Phe Ser Ser Lys Cys Arg Arg Cys Arg Leu 530 535 540 Cys Asp Glu Gly His Gly Leu Glu Val Glu Ile Asn Cys Thr Arg Thr 545 550 555 560 Gln Asn Thr Lys Cys Arg Cys Lys Pro Asn Phe Phe Cys Asn Ser Thr 565 570 575 Val Cys Glu His Cys Asp Pro Cys Thr Lys Cys Glu His Gly Ile Ile 580 585 590 Lys Glu Cys Thr Leu Thr Ser Asn Thr Lys Cys Lys Glu Glu Gly Ser 595 600 605 Arg Ser Asn Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 610 615 620 Gly Ser Gln Val Thr Asp Ile Asn Ser Lys Gly Leu Glu Leu Arg Lys 625 630 635 640 Thr Val Thr Thr Val Glu Thr Gln Asn Leu Glu Gly Leu His His Asp 645 650 655 Gly Gln Phe Cys His Lys Pro Cys Pro Pro Gly Glu Arg Lys Ala Arg 660 665 670 Asp Cys Thr Val Asn Gly Asp Glu Pro Asp Cys Val Pro Cys Gln Glu 675 680 685 Gly Lys Glu Tyr Thr Asp Lys Ala His Phe Ser Ser Lys Cys Arg Arg 690 695 700 Cys Arg Leu Cys Asp Glu Gly His Gly Leu Glu Val Glu Ile Asn Cys 705 710 715 720 Thr Arg Thr Gln Asn Thr Lys Cys Arg Cys Lys Pro Asn Phe Phe Cys 725 730 735 Asn Ser Thr Val Cys Glu His Cys Asp Pro Cys Thr Lys Cys Glu His 740 745 750 Gly Ile Ile Lys Glu Cys Thr Leu Thr Ser Asn Thr Lys Cys Lys Glu 755 760 765 Glu Gly Ser Arg Ser Asn 770 <210> 44 <211> 2322 <212> DNA <213> Artificial sequences <400> 44 caggtgcagc tggttcagag cggagctgaa gtgaagaagc ccggcgccag cgtgaaggtg 60 tcctgcaagg cctctggata catcttcacc gagtacatca tccactgggt gcggcaggcc 120[[ID=,21]] cctggacaag gcctggaatg gatcggctgg ttctaccctg gcagcgacaa catcaagtac 180 aacgagaagt tcaaggatag agccaccctg accgcagata agtccaccag caccgtctac 240 atggaactga gcagcctgag aagcgaggat acagccgtgt actactgcgc cagacacgaa 300 accggctact tcttcgacta ctggggccag ggtacactgg tgaccgtgtc ctccgccagc 360 accaagggcc ctagcgtctt tccactggcc ccttcttcta agagcacaag cggcggaacc 420 gccgctctgg gttgtctggt caaagattac ttccccgaac ctgtgaccgt gtcctggaac 480 agcggcgccc tgacatctgg cgtgcacaca ttcccagccg tgttgcagag cagcggcctg 540 tactctctgt ctagcgtcgt caccgtgccc agcagcagcc tgggaacaca gacctacatc 600 tgcaacgtga accacaagcc tagcaacacc aaagtggata agaaagtgga acccaagagc 660 tgcgacaaga cccacacctg tcctccgtgc cctgctcctg agctgctggg cggccccagc 720 gtgttcctgt tcccccccaa gcctaaggac accctgatga tcagccgcac ccctgaggtg 780 acatgcgtgg tcgtcgacgt gtcccacgag gaccccgagg tgaaattcaa ctggtacgtg 840 gacggcgtgg aagtgcacaa cgccaagacc aagccaagag aagagcagta cgcctctaca 900 tacagagtgg tgtccgtgct gaccgtgctg caccaggact ggctgaacgg caaggaatac 960 aagtgcaagg tgtccaacaa ggccctgccc gctcctatcg agaagacaat ctctaaggct 1020 aaaggccagc ctagagaacc tcaggtttat acactgcctc ctagcagaga ggaaatgacc 1080 aagaaccagg tgtctctgac ctgtctggtg aagggcttct atccttctga catcgccgtg 1140 gaatgggaga gcaatggcca acctgagaac aactacaaga cgacccctcc agtgctggac 1200 agcgacggca gtttttcct gtacagcaag ctgacagtcg acaaaagccg gtggcagcag 1260 ggcaatgtgt tcagctgcag cgtgatgcac gaggccctcc ataatcacta cacccagaag 1320 tccctgagcc tgagtcctgg caagggtggt ggtggttctg gtggtggtgg ttctggcggc 1380 ggcggctccc aagtgactga catcaactcc aagggattgg aattgaggaa gactgttact 1440 acagttgaga ctcagaactt ggaaggcctg catcatgatg gccaattctg ccataagccc 1500 tgtcctccag gtgaaaggaa agctagggac tgcacagtca atggggatga accagactgc 1560 1620 1680 cagaatacca agtgcagatg taaaccaaac tttttttgta actctactgt atgtgaacac 1740 tgtgaccctt gcaccaaatg tgacatgga atcatcaagg aatgcacact caccagcaac 1800 accaagtgca aaggaagg atccagatct aacggtggtg gtggttctgg tggtggtggt 1860 tctggcggcg gcggctccca agtgactgac atcaactcca agggattgga attgaggaag 1920 actgttacta cagttgagac tcagaacttg gaaggcctgc atcatgatgg ccaattctgc 1980 cataagccct gtcctccagg tgaaaggaaa gctagggact gcacagtcaa tggggatgaa 2040 ccagactgcg tgccctgcca agaagggaag gagtacacag acaaagccca tttttcttcc 2100 aaatgcagaa gatgtagatt gtgtgatgaa ggacatggct tagaagtgga aataaactgc 2160 acccggaccc agaataccaa gtgcagatgt aaaccaaact ttttttgtaa ctctactgta 2220 tgtgaacact gtgacccttg caccaaatgt gaacatggaa tcatcaagga atgcacactc 2280 accagcaaca ccaagtgcaa agaggaagga tccagatcta ac 2322 <210> 45 <211> 340 <212> PRT <213> Artificial sequences <400> 45 Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Thr Lys Ser Leu Leu Asn Ser 20 25 30 Asp Gly Phe Thr Tyr Leu Gly Trp Cys Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Leu Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Ser 85 90 95 Asn Tyr Leu Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp 115 120 125 Ile Gln Ile Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly Asp 130 135 140 Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Lys Met Asn 145 150 155 160 Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Trp Ile Tyr Asp 165 170 175 Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly 180 185 190 Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp Asp 195 200 205 Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe 210 215 220 Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser 225 230 235 240 Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala 245 250 255 Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val 260 265 270 Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser 275 280 285 Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr 290 295 300 Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys 305 310 315 320 Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn 325 330 335 Arg Gly Glu Cys 340 <210> 46 <211> 1020 <212> DNA <213> Artificial sequences <400> 46 gacgtggtga tgacccagac ccctctgtct ctgcctgtga ccctcggcca gcctgcctcc 60 atctcctgca agtccaccaa aagcctgctg aattccgacg gctttaccta tctgggctgg 120 tgcctgcaga agcctggcca gagccctcag ctgctgatct acctggtgtc caaccggttt 180 tctggcgtgc ccgacagatt ctccggctcc ggatctggaa ccgatttcac cctcaagatc 240 tccagagtgg aagccgagga tgtgggcgtg tactactgct tccagtccaa ctacctgcct 300 ctgaccttcg gccaaggcac caagctggaa atcaagggtg gtggtggttc tggtggtggt 360 ggttctggcg gcggcggctc cgacatccag atcacccagt ctcctagcac actgtccgcc 420 tctgttggag atagagtgac aatcacctgt agcgccagca gctccgtgtc caaaatgaac 480 tggtaccagc aaaagcccgg caaggcccct aagagatgga tctacgacac cagcaaactg 540 gccagcggcg tgcccagtag attcagcggc agcggatctg gcacagagta caccctgacc 600 atcagcagcc tgcaacctga tgacttcgcc acatactact gccagcagtg gtcctctaat 660 cctctgacct tcggccaggg caccaagctg gaaatcaagc ggaccgtggc cgccccttct 720 gtgttcatct tcccccccag cgacgagcag ctgaagagcg gaaccgccag cgtggtgtgc 780 ctgctcaaca acttctaccc gcgggaagcc aaggtgcagt ggaaggtgga caacgccctg 840 cagagcggca acagccagga gagcgtgacc gagcaggaca gcaaggactc tacatacagc 900 ctgagcagca ccctgacact gtctaaagcc gactacgaga agcacaaggt gtacgcctgt 960 gaagtgacac accagggcct gagcagccct gtgaccaagt cttttaaccg gggcgagtgc 1020 <210> 47 <211> 582 <212> PRT <213> Artificial sequences <400> 47 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 Asn Tyr 20 25 30 Trp Ile Gly Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile 35 40 45 Gly Tyr Leu Tyr Pro Gly Gly Leu Tyr Thr Asn Tyr Asn Glu Lys Phe 50 55 60 Lys Gly Lys Ala Thr Met Thr Ala Asp Thr Ser Thr Asn 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 Tyr Arg Asp Tyr Asp Tyr Ala Met Asp Tyr Trp Gly Gln Gly 100 105 110 Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly 115 120 125 Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Val Gln Ser Gly Ala Glu 130 135 140 Val Lys Lys Pro Gly Ala Ser Val Lys Val Ser Cys Lys Ala Ser Gly 145 150 155 160 Tyr Ile Phe Thr Glu Tyr Ile Ile His Trp Val Arg Gln Ala Pro Gly 165 170 175 Gln Gly Leu Glu Trp Ile Gly Trp Phe Tyr Pro Gly Ser Asp Asn Ile 180 185 190 Lys Tyr Asn Glu Lys Phe Lys Asp Arg Ala Thr Leu Thr Ala Asp Lys 195 200 205 Ser Thr Ser Thr Val Tyr Met Glu Leu Ser Ser Leu Arg Ser Glu Asp 210 215 220 Thr Ala Val Tyr Tyr Cys Ala Arg His Glu Thr Gly Tyr Phe Phe Asp 225 230 235 240 Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys 245 250 255 Gly Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly 260 265 270 Gly Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro 275 280 285 Val Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr 290 295 300 Phe Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val 305 310 315 320 Val Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn 325 330 335 Val Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro 340 345 350 Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu 355 360 365 Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp 370 375 380 Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp 385 390 395 400 Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly 405 410 415 Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Ala 420 425 430 Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp 435 440 445 Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro 450 455 460 Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu 465 470 475 480 Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn 485 490 495 Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile 500 505 510 Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr 515 520 525 Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys 530 535 540 Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys 545 550 555 560 Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu 565 570 575 Ser Leu Ser Pro Gly Lys 580 <210> 48 <211> 1746 [[ID=,17]]<212> DNA <213> Artificial sequences <400> 48 caggtccaac tggtgcaatc aggcgccgag gtcaagaagc cgggcgcttc cgttaaggtg 60 tcctgcaagg cctccggcta caccttcacc aactactgga tcggctgggt gaagcaggcc 120 cccggccagg gcctggaatg gatcggatac ctgtaccccg gcggcctgta caccaactac 180 aacgagaagt tcaagggcaa ggctaccatg acagccgata catctaccaa taccgcctac 240 atggaactga gctccctgag atccgaggac accgccgtgt actactgcgc tcggtaccgg 300 gattatgact acgccatgga ctattggggc cagggcaccc tcgttacagt ctcctctgga 360 ggcggcggct ctggtggcgg cggctccggc ggagggggct ctcaggtgca gctggtgcag 420 agtggcgctg aggtgaagaa gcctggcgcc tccgtgaaag tgtcttgcaa ggcctctggc 480 tacattttca ccgagtacat catccactgg gtgcggcagg ctcctggaca gggcctggag 540 tggatcggct ggttctaccc cggatctgac aacatcaagt aacagagaa atttaaggac 600 agagccaccc tgaccgccga caagtccacc tctaccgtgt acatggaact gtccagcctg 660 cggtctgagg acacagccgt gtactactgt gccagacacg agaccggcta tttcttgac 720 tactggggac aaggcacact ggtgaccgtg tccagcgcct ccaccaaggg cccatctgtg 780 tttcctctgg ccccatcttc caagagcacc agcggggggca ccgctgctct gggctgcctg 840 gtgaaggact acttccctga gcctgtgaca gtgtcctgga actccggcgc tctgacctct 900 ggcgtgcata cctttccagc tgtgctgcag tccagcggcc tgtactccct gagttccgtg 960 gtaaccgtgc cctcttcttc tctgggcacc cagacctaca tctgcaatgt gaaccaag 1020 ccttccaaca ccaaggtgga taagaaagtg gaacctaagt cctgcgacaa gacccacacc 1080 tgccctcctt gtcctgctcc tgagctgctt ggaggcccta gcgttttcct gttccctccc 1140 aagcccaagg ataccctgat gatctcccgg accccagaag tgacctgtgt ggtggtggac 1200 gtgtctcatg aggatcctga ggtgaagttc aactggtacg tggatggcgt ggaagtgcac 1260 aacgccaaga ccaaacctag agaggaacag tacgcctcta cctacagagt ggtctccgtg 1320 ctaacagtgc tgcaccagga ctggctgaac ggcaaagagt ataagtgcaa agtgtctaac 1380 aaggctctgc ctgctcctat cgagaaaacc atctccaagg ccaaggggca gcctcgcgaa 1440 cctcaggtgt acactctgcc tccttccaga gaggagatga ccaagaacca ggtttccctg 1500 acctgtctgg tgaagggctt ctatccttcc gacatcgcag tggaatggga gtccaatggc 1560 cagcctgaga acaactacaa gacaacgccc cccgtgctgg actccgacgg ctccttcttc 1620 ctgtacagca agctgacagt ggacaagagc cggtggcaac agggcaacgt gttctcctgc 1680 tccgtgatgc acgaggccct gcacaaccac tacacccaga aatctctctc cctgtcccct 1740 ggcaag 1746 <210> 49 <211> 503 <212> PRT <213> Artificial sequences <400> 49 Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Thr Lys Ser Leu Leu Asn Ser 20 25 30 Asp Gly Phe Thr Tyr Leu Gly Trp Cys Leu Gln Lys Pro Gly Gln Ser 35 40 45 Pro Gln Leu Leu Ile Tyr Leu Val Ser Asn Arg Phe Ser Gly Val Pro 50 55 60 Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Ser 85 90 95 Asn Tyr Leu Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp 115 120 125 Ile Gln Ile Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly Asp 130 135 140 Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Lys Met Asn 145 150 155 160 Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Trp Ile Tyr Asp 165 170 175 Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly 180 185 190 Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp Asp 195 200 205 Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe 210 215 220 Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser 225 230 235 240 Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala 245 250 255 Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val 260 265 270 Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser 275 280 285 Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr 290 295 300 Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys 305 310 315 320 Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn 325 330 335 Arg Gly Glu Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 340 345 350 Gly Gly Ser Gln Val Thr Asp Ile Asn Ser Lys Gly Leu Glu Leu Arg 355 360 365 Lys Thr Val Thr Thr Val Glu Thr Gln Asn Leu Glu Gly Leu His His 370 375 380 Asp Gly Gln Phe Cys His Lys Pro Cys Pro Pro Gly Glu Arg Lys Ala 385 390 395 400 Arg Asp Cys Thr Val Asn Gly Asp Glu Pro Asp Cys Val Pro Cys Gln 405 410 415 Glu Gly Lys Glu Tyr Thr Asp Lys Ala His Phe Ser Ser Lys Cys Arg 420 425 430 Arg Cys Arg Leu Cys Asp Glu Gly His Gly Leu Glu Val Glu Ile Asn 435 440 445 Cys Thr Arg Thr Gln Asn Thr Lys Cys Arg Cys Lys Pro Asn Phe Phe 450 455 460 Cys Asn Ser Thr Val Cys Glu His Cys Asp Pro Cys Thr Lys Cys Glu 465 470 475 480 His Gly Ile Ile Lys Glu Cys Thr Leu Thr Ser Asn Thr Lys Cys Lys 485 490 495 Glu Glu Gly Ser Arg Ser Asn 500 <210> 50 <211> 1509 <212> DNA <213> Artificial sequences <400> 50 gacgtggtga tgacccagac ccctctgtct ctgcctgtga ccctcggcca gcctgcctcc 60 atctcctgca agtccaccaa aagcctgctg aattccgacg gctttaccta tctgggctgg 120 tgcctgcaga agcctggcca gagccctcag ctgctgatct acctggtgtc caaccggttt 180 tctggcgtgc ccgacagatt ctccggctcc ggatctggaa ccgatttcac cctcaagatc 240 tccagagtgg aagccgagga tgtgggcgtg tactactgct tccagtccaa ctacctgcct 300 ctgaccttcg gccaaggcac caagctggaa atcaagggtg gtggtggttc tggtggtggt 360 ggttctggcg gcggcggctc cgacatccag atcacccagt ctcctagcac actgtccgcc 420 tctgttggag atagagtgac aatcacctgt agcgccagca gctccgtgtc caaaatgaac 480 tgtaccacc aaagcccgg caggcccct aagagatgga tcgacac cagcaactg 540 gccagcggcg tgcccagtag attcagcggc agcggatg gcacagagta caccctgacc 600 atcagcagcc tgcaacctga tgactcgcc acatactact gccagcagtg gtcctctaat 660 cctctgacct tcggccagggg caccaagctg gaatcaagc ggaccgtggc cgccccttct 720 gtgttcatct tccccccag cgacgagcag ctgaagagcg gaaccgccag cgtggtgtgc 780 ctgctcaaca acttctaccc gcgggaagcc aaggtgcagt gggaggtgga caacgccctg 840 cagagcggca acagccagga gagcgtgacc gagcaggaca ghaaggactc tacatacagc 900 ctgagcagca ccctgacact gtctaaagcc gactacgaga agcacaggt gtacgcctgt 960 gaagtgacac accaggcct gagcagccct gtgaccaagt ctttaaccg gggcgagtgc 1020 ggtggtgtg gttctgtgg tggtggttct ggcggcggcg gctcccaagt gactgacatc 1080 aactccaagg gattggaatt gaggagact gttactacag ttgagactca gaacttggaa 1140 ggcctgcatc atgatggcca attctgccat aagccctgtc ctccaggtga aaggaaagct 1200 agggactgca cagtcaatgg ggatgaacca gactgcgtgc cctgccaaga agggaaggag 1260 tacacagaca aagcccattt ttcttccaaa tgcagaagat gtagattgtg tgatgaagga 1320 catggcttag aagtggaaat aaactgcacc cggacccaga ataccaagtg cagatgtaaa 1380 ccaaactttt tttgtaactc tactgtatgt gaacactgtg acccttgcac caaatgtgaa 1440 catggaatca tcaaggaatg cacactcacc agcaacacca agtgcaaaga ggaaggatcc 1500 agatctaac 1509 <210> 51 <211> 666 <212> PRT <213> Artificial sequences <400> 51 Asp Val Val Met Thr Gln Thr Pro Leu Ser Leu Pro Val Thr Leu Gly 1 5 10 15 Gln Pro Ala Ser Ile Ser Cys Lys Ser Thr Lys Ser Leu Leu Asn Ser<​​​​​​​​​​Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys Ile 65 70 75 80 Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Phe Gln Ser 85 90 95 Asn Tyr Leu Pro Leu Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp 115 120 125 Ile Gln Ile Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly Asp 130 135 140 Arg Val Thr Ile Thr Cys Ser Ala Ser Ser Ser Val Ser Lys Met Asn 145 150 155 160 Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Arg Trp Ile Tyr Asp 165 170 175 Thr Ser Lys Leu Ala Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly 180 185 190 Ser Gly Thr Glu Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Asp Asp 195 200 205 Phe Ala Thr Tyr Tyr Cys Gln Gln Trp Ser Ser Asn Pro Leu Thr Phe 210 215 220 Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr Val Ala Ala Pro Ser 225 230 235 240 Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly Thr Ala 245 250 255 Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala Lys Val 260 265 270 Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln Glu Ser 275 280 285 Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser Thr 290 295 300 Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala Cys 305 310 315 320 Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe Asn 325 330 335 Arg Gly Glu Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly 340 345 350 Gly Gly Ser Gln Val Thr Asp Ile Asn Ser Lys Gly Leu Glu Leu Arg 355 360 365 Lys Thr Val Thr Thr Val Glu Thr Gln Asn Leu Glu Gly Leu His His 370 375 380 Asp Gly Gln Phe Cys His Lys Pro Cys Pro Pro Gly Glu Arg Lys Ala 385 390 395 400 Arg Asp Cys Thr Val Asn Gly Asp Glu Pro Asp Cys Val Pro Cys Gln 405 410 415 Glu Gly Lys Glu Tyr Thr Asp Lys Ala His Phe Ser Ser Lys Cys Arg 420 425 430 Arg Cys Arg Leu Cys Asp Glu Gly His Gly Leu Glu Val Glu Ile Asn 435 440 445 Cys Thr Arg Thr Gln Asn Thr Lys Cys Arg Cys Lys Pro Asn Phe Phe 450 455 460 Cys Asn Ser Thr Val Cys Glu His Cys Asp Pro Cys Thr Lys Cys Glu 465 470 475 480 His Gly Ile Ile Lys Glu Cys Thr Leu Thr Ser Asn Thr Lys Cys Lys 485 490 495 Glu Glu Gly Ser Arg Ser Asn Gly Gly Gly Gly Ser Gly Gly Gly Gly 500 505 510 Ser Gly Gly Gly Gly Ser Gln Val Thr Asp Ile Asn Ser Lys Gly Leu 515 520 525 Glu Leu Arg Lys Thr Val Thr Thr Val Glu Thr Gln Asn Leu Glu Gly 530 535 540 Leu His His Asp Gly Gln Phe Cys His Lys Pro Cys Pro Pro Gly Glu 545 550 555 560 Arg Lys Ala Arg Asp Cys Thr Val Asn Gly Asp Glu Pro Asp Cys Val 565 570 575 Pro Cys Gln Glu Gly Lys Glu Tyr Thr Asp Lys Ala His Phe Ser Ser 580 585 590 Lys Cys Arg Arg Cys Arg Leu Cys Asp Glu Gly His Gly Leu Glu Val 595 600 605 Glu Ile Asn Cys Thr Arg Thr Gln Asn Thr Lys Cys Arg Cys Lys Pro 610 615 620 Asn Phe Phe Cys Asn Ser Thr Val Cys Glu His Cys Asp Pro Cys Thr 625 630 635 640 Lys Cys Glu His Gly Ile Ile Lys Glu Cys Thr Leu Thr Ser Asn Thr 645 650 655 Lys Cys Lys Glu Glu Gly Ser Arg Ser Asn 660 665 <210> 52 <211> 1998 <212> DNA <213> Artificial sequences <400> 52<00gacgtggtga tgacccagac ccctctgtct ctgcctgtga ccctcggcca gcctgcctcc 60 atctcctgca agtccaccaa aagcctgctg aattccgacg gctttaccta tctgggctgg 120 tgcctgcaga agcctggcca gagccctcag ctgctgatct acctggtgtc caaccggttt 180 tctggcgtgc ccgacagatt ctccggctcc ggatctggaa ccgatttcac cctcaagatc 240 tccagagtgg aagccgagga tgtgggcgtg tactactgct tccagtccaa ctacctgcct 300 ctgaccttcg gccaaggcac caagctggaa atcaagggtg gtggtggttc tggtggtggt 360 ggttctggcg gcggcggctc cgacatccag atcacccagt ctcctagcac actgtccgcc 420 tctgttggag atagagtgac aatcacctgt agcgccagca gctccgtgtc caaaatgaac 480 tggtaccagc aaaagcccgg caaggcccct aagagatgga tctacgacac cagcaaactg 540 gccagcggcg tgcccagtag attcagcggc agcggatctg gcacagagta caccctgacc 600 atcagcagcc tgcaacctga tgacttcgcc acatactact gccagcagtg gtcctctaat 660 cctctgacct tcggccaggg caccaagctg gaaatcaagc ggaccgtggc cgccccttct 720 gtgttcatct tccccccag cgacgagcag ctgaagagcg gaaccgccag cgtggtgtgc 780 ctgctcaaca acttctaccc gcgggaagcc aaggtgcagt gggaggtgga caacgccctg 840 cagagcggca acagccagga gagcgtgacc gagcaggaca ghaaggactc tacatacagc 900 ctgagcagca ccctgacact gtctaaagcc gactacgaga agcacaggt gtacgcctgt 960 gaagtgacac accaggcct gagcagccct gtgaccaagt ctttaaccg gggcgagtgc 1020 ggtggtgtg gttctgtgg tggtggttct ggcggcggcg gctcccaagt gactgacatc 1080 aactccaagg gattggaatt gaggagact gttactacag ttgagactca gaacttggaa 1140 ggcctgcatc atgatggcca attctgccat aagccctgtc ctccaggtga aaggaaagct 1200 agggactgca cagtcaatgg ggatgaacca gactgcgtgc cctgccaaga agggaggg 1260 tacacagaca aagcccattt ttctccaaa tgcagaagat gtagattgtg tgatgaagga 1320 catggcttag aagtggaat aactgcacc cggacccaga ataccaagtg cagatgtaaa 1380 ccaaactttt ttgtaactc tactgtatgt gaacactgtg acccttgcac caatgtgaa 1440 spacecraft spacecraft spacecaccacc space space space space space space 1500 agatctaacg gtggtggtgg tctgtgt ggtggttctg gcggcggcgg ctcccaagtg 1560 actgacatca actccaaggg attggaattg aggagactg ttactacagt tgagactcag 1620 aacttggaag gcctgcatca tgatggccaa ttctgccata agccctgtcc tccaggtgaa 1680 aggaaagcta gggactgcac agtcaatggg gatgaccag actgcgtgcc ctgccaagaa 1740 gggaaggagt acacagacaa agcccatttt tcttccaat gcagaagatg tagattgtgt 1800 gatgaggac atggcttaga agtggaata aactgcaccc ggaccaggaa taccaagtgc 1860 agatgtaaac caacttttt ttgtaactct actgtatgtg aacactgtga cccttgcacc 1920 aaatgtgaac atggaatcat caggaatgc acactcacca gcacacca gtgcaaag 1980 gagggatcca gatctaac 1998

Claims

1. A bispecific antibody, characterized in that, The bispecific antibody includes: (a) PD-L1 antibodies that can specifically bind to PD-L1, and (b) FasL antibodies or Fas extracellular domains that can specifically bind to FasL; The heavy chain variable region and light chain variable region of the FasL antibody are respectively connected to the heavy chain variable region and light chain variable region of the PD-L1 antibody through a linker sequence, or the Fas extracellular domain is connected to the constant region of the PD-L1 antibody through a linker sequence. The amino acid sequence of the light chain variable region of the PD-L1 antibody is shown in SEQ ID NO: 1, which is encoded by the nucleotide sequence shown in SEQ ID NO: 2; the amino acid sequence of the heavy chain variable region of the PD-L1 antibody is shown in SEQ ID NO: 3, which is encoded by the nucleotide sequence shown in SEQ ID NO:

4. The amino acid sequence of the light chain variable region of the FasL antibody is shown in SEQ ID NO: 5, which is encoded by the nucleotide sequence shown in SEQ ID NO: 6; the amino acid sequence of the heavy chain variable region of the FasL antibody is shown in SEQ ID NO: 7, which is encoded by the nucleotide sequence shown in SEQ ID NO:

8. The amino acid sequence of the extracellular domain of Fas is shown in SEQ ID NO: 9, which is encoded by the nucleotide sequence shown in SEQ ID NO:

10.

2. The bispecific antibody according to claim 1, characterized in that, The bispecific antibody includes a PD-L1 antibody and one or two Fas extracellular domains connected by adapter sequences on both sides of the heavy chain constant region at the C-terminus of the PD-L1 antibody.

3. The bispecific antibody according to claim 1, characterized in that, The bispecific antibody includes a PD-L1 antibody and a FasL antibody, wherein the N-terminal heavy chain variable region and light chain variable region of the PD-L1 antibody are respectively connected to the N-terminal heavy chain variable region and light chain variable region of the FasL antibody through a linker sequence. Alternatively, the heavy chain variable region and light chain variable region at the N-terminus of the FasL antibody may be connected to the heavy chain variable region and light chain variable region at the N-terminus of the PD-L1 antibody via adapter sequences.

4. The bispecific antibody according to claim 1, characterized in that, The bispecific antibody includes a PD-L1 antibody, a FasL antibody, and a Fas extracellular domain. The N-terminal heavy chain variable region and light chain variable region of the PD-L1 antibody are respectively connected to the N-terminal heavy chain variable region and light chain variable region of the FasL antibody via adapter sequences. One or two Fas extracellular domains are respectively connected to both sides of the C-terminal heavy chain constant region of the bispecific antibody formed by the connection of the PD-L1 antibody and the FasL antibody via adapter sequences. Alternatively, the N-terminal heavy chain variable region and light chain variable region of the FasL antibody may be connected to the N-terminal heavy chain variable region and light chain variable region of the PD-L1 antibody via adapter sequences, and one or two Fas extracellular domains may be connected to both sides of the C-terminal heavy chain constant region of the bispecific antibody formed by the connection of the FasL antibody and the PD-L1 antibody via adapter sequences.

5. A pharmaceutical composition comprising the bispecific antibody of any one of claims 1 to 4 and optionally a pharmaceutically acceptable carrier or excipient.

6. The use of the bispecific antibody according to any one of claims 1 to 4 in the preparation of a medicament for treating cancer, characterized in that, The cancer is selected from one or more of the following: urothelial carcinoma, small cell lung cancer, liver cancer, melanoma, pancreatic cancer, gastric cancer, ovarian cancer, kidney cancer, colorectal cancer, breast cancer, and prostate cancer. The breast cancer includes triple-negative breast cancer, and the urothelial carcinoma includes bladder cancer.

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