Bispecific antibodies targeting pd-l1 and 4-1bb

Abstract

The application discloses a bispecific antibody targeting PD-L1 and 4-1BB. The bispecific antibody contains a PD-L1 antigen binding domain (HCDR1, HCDR2 and HCDR3 are sequentially located at positions 26-32, 52-56 and 98-107 of SEQ ID No. 1, and LCDR1, LCDR2 and LCDR3 are sequentially located at positions 24-36, 52-58 and 93-100 of SEQ ID No. 2) and a 4-1BB antigen binding domain (HCDR1, HCDR2 and HCDR3 are sequentially located at positions 31-35, 50-65 and 98-106 of SEQ ID No. 3, and LCDR1, LCDR2 and LCDR3 are sequentially located at positions 24-34, 50-56 and 89-97 of SEQ ID No. 4). The bispecific antibody has good stability and high safety, can block the combination of PD-1 and PD-L1, activate the human 4-1BB signal path, stimulate T cell activation, and can be applied to an immune enhancer or an immune regulator of T cell-mediated autoimmune diseases.

Description

Technical Field

[0001] This invention relates to the fields of tumor therapy and immunology, and more specifically to a bispecific antibody targeting PD-L1 and 4-1BB. Background Technology

[0002] Bispecific antibodies (BsAbs), also known as bifunctional antibodies, are antibodies that can simultaneously recognize and bind to two different targets or two different epitopes of the same target. They can perform specific biological functions, sometimes even superior to the synergistic effects of combining two monoclonal antibodies. For example, they can directly target effector cells to tumor cells, enhancing cytotoxicity, improving antibody selectivity and functionality, co-stimulating or inhibiting receptors, and preventing immune escape mechanisms, thereby improving treatment efficacy. Compared to the combined use of two monoclonal antibody drugs, BsAbs have stronger specificity, targeting ability, and reduced off-target toxicity. BsAbs also reduce the costs of drug development and clinical trials.

[0003] Bispecific antibodies do not exist in nature. Their technological platforms can be structurally classified into three categories: non-IgG-like, symmetric IgG-like, and asymmetric IgG-like. Bispecific antibodies are currently a hot topic in the biopharmaceutical industry for new drug development and an important branch of antibody drug development. Approximately 20% of antibody drugs currently in the preclinical stage globally are bispecific antibodies. In China, bispecific antibody projects are mainly concentrated in the pre-Phase II clinical trial stage, and no bispecific antibody drugs have yet been approved. In terms of disease distribution, Chinese bispecific antibody projects are mainly focused on oncology, with breast cancer and gastric cancer being the most common.

[0004] In recent years, new targets and pathways for immune activation in immunotherapy have been continuously discovered, leading to significant progress in tumor immunotherapy. Programmed cell death protein 1 (PD-1) is a member of the CD28 superfamily. As a T-cell inhibitory receptor, it can restrict the function of T-cell effectors in tumor cells and plays a crucial role in tumor immune escape. Blocking the interaction between PD-1 and PD-L1 can effectively restore the tumor-killing function of T cells; it can promote the proliferation of tumor antigen-specific T cells, enabling them to kill tumor cells and thus inhibit local tumor growth; PD-L1 monoclonal antibodies can upregulate infiltrating CD8+. +The secretion of IFN-γ by T cells indicates that blocking the PD-1 / PD-L1 signaling pathway plays a role in tumor immune responses aimed at inducing immune responses. Furthermore, PD-L1 can bind to B7-1 in vivo. Previous studies have shown that the PD-L1 / B7-1 complex is also a negative signal for T cell activation; their binding can lead to a decrease in the expression of T cell surface activation markers and inhibit T cell proliferation. Currently, it is widely believed that antibodies targeting the PD-L1 pathway will bring breakthrough progress in the treatment of various tumors, including non-small cell lung cancer, renal cell carcinoma, ovarian cancer, and melanoma. PD-1 / PD-L1 immunotherapy has become a new class of anti-cancer immunotherapies that has attracted worldwide attention and brought new hope to patients. However, PD-1 / PD-L1-targeted therapies have certain limitations: some patients experience rapid and durable tumor regression, but most patients experience little or no significant effect. To increase the response rate of immunotherapy to patients, researchers are attempting to develop new immunomodulatory targets and therapeutic strategies. One promising strategy is to stimulate the activation of immune cells by targeting immune stimulatory receptors. This “co-stimulation” strategy provides a mechanistic basis for a variety of agents in clinical development, including antibodies targeting OX40, CD27, CD40, GITR, and 4-1BB.

[0005] 4-1BB (CD137 / TNFRSF9) is a co-stimulatory molecule, a type I membrane glycoprotein, and a member of the tumor necrosis factor receptor (TNFRSF9) superfamily. It is primarily expressed on activated T cells and, after binding to its ligand 4-1BBL (CD137L), transmits signals via a trimer. The 4-1BB signaling pathway can enhance tumor-specific CD8+. + T cell function achieves anti-tumor effects, and can also enhance NK cells, DCs, and CD4. + T-cell immune function enhances CD8 +T-cell-mediated anti-tumor immune responses hold unique potential as therapeutic targets. In preclinical trials, the anti-tumor activity of anti-4-1BB monoclonal antibodies was validated in multiple mouse models of colon cancer (MC38, CT26), lung cancer (M109), breast cancer (EMT6), and B-cell lymphoma (A20). Furthermore, synergistic effects were observed with PD1 / PDL1, CTLA4 antibodies, chemotherapy, and other targeted therapies. The first anti-4-1BB treatment to enter clinical trials, Urelumab (BMS-663513), is a fully human IgG4 monoclonal antibody. While it has shown encouraging clinical efficacy, Phase I and Phase II data suggest that hepatotoxicity appears to be target- and dose-dependent, limiting its clinical development. The second drug to enter clinical trials, Utomilumab (PF-05082566), is a humanized IgG2 monoclonal antibody that activates 4-1BB while blocking binding to endogenous 4-1BBL. This antibody has better safety than Urelumab, but weaker agonistic activity. Therefore, developing a highly effective and low-toxicity anti-tumor drug targeting the 4-1BB site has become a focus for drug developers.

[0006] Currently, there are no effective PD-L1 / 4-1BB bispecific antibody drugs on the market. Summary of the Invention

[0007] The purpose of this invention is to provide a bispecific antibody targeting PD-L1 and 4-1BB. The development of this bispecific antibody is expected to fill the gaps in the current market for PD-1 / PD-L1 or 4-1BB tumor targeting and expand new indications. It can also serve as a next-generation PD-L1 immunotherapy product, not only for treating patients who have developed immune tolerance after existing PD-1 / PD-L1 treatments, or those with low response rates, but also for cancers with low PD-L1 expression that currently lack effective treatment.

[0008] In a first aspect, the present invention claims protection for a bispecific antibody targeting PD-L1 and 4-1BB, containing a PD-L1 antigen-binding domain and a 4-1BB antigen-binding domain.

[0009] The PD-L1 antigen-binding domain comprises a heavy chain variable region and a light chain variable region; the amino acid sequences of HCDR1, HCDR2, and HCDR3 in the heavy chain variable region are as shown at positions 26-32, 52-56, and 98-107 of SEQ ID No. 1, respectively; the amino acid sequences of LCDR1, LCDR2, and LCDR3 in the light chain variable region are as shown at positions 24-36, 52-58, and 93-100 of SEQ ID No. 2, respectively. Alternatively, the amino acid sequence of HCDR3 in the heavy chain variable region of the PD-L1 antigen-binding domain can also be as shown at positions 98-107 of SEQ ID No. 10 (by mutating DRPDGAATNL to DRPEGAATNL at positions 98-107 of SEQ ID No. 1).

[0010] The 4-1BB antigen-binding domain comprises a heavy chain variable region and a light chain variable region; the amino acid sequences of HCDR1, HCDR2, and HCDR3 in the heavy chain variable region are shown as positions 31-35, 50-65, and 98-106 of SEQ ID No. 3, respectively; the amino acid sequences of LCDR1, LCDR2, and LCDR3 in the light chain variable region are shown as positions 24-34, 50-56, and 89-97 of SEQ ID No. 4, respectively.

[0011] Further, in the PD-L1 antigen-binding domain, the amino acid sequence of the heavy chain variable region is positions 627-744 of SEQ ID No. 1 or SEQ ID No. 5 or positions 1-118 of SEQ ID No. 10, or has a 99% or higher, 95% or higher, 90% or higher, 85% or higher, 80% or higher, or 75% or higher similarity to positions 627-744 of SEQ ID No. 1 or SEQ ID No. 5 or positions 1-118 of SEQ ID No. 10 (the inconsistency is preferably in the frame region (FR)); the amino acid sequence of the light chain variable region is positions 497-606 of SEQ ID No. 2 or SEQ ID No. 5, or has a 99% or higher, 95% or higher, 90% or higher, 85% or higher, 80% or higher, or 75% or higher similarity to positions 497-606 of SEQ ID No. 2 or SEQ ID No. 5 (the inconsistency is preferably in the frame region (FR)).

[0012] Further, in the 4-1BB antigen-binding domain, the amino acid sequence of the heavy chain variable region is positions 128-244 of SEQ ID No. 3 or SEQ ID No. 5, or has a 99% or higher, 95% or higher, 90% or higher, 85% or higher, 80% or higher, or 75% or higher similarity to positions 128-244 of SEQ ID No. 3 or SEQ ID No. 5 (the inconsistency is preferably in the frame region (FR)); the amino acid sequence of the light chain variable region is positions 1-107 of SEQ ID No. 4 or SEQ ID No. 5, or has a 99% or higher, 95% or higher, 90% or higher, 85% or higher, 80% or higher, or 75% or higher similarity to positions 1-107 of SEQ ID No. 4 or SEQ ID No. 5 (the inconsistency is preferably in the frame region (FR)).

[0013] Furthermore, the structure of the bispecific antibody from the N-terminus to the C-terminus can be any of the following:

[0014] Structure A: 1 st scFv-L1-Fc-L2-2 nd scFv;

[0015] Structure B: 1 st scFv-L1-Fc-L2-2 nd Fab;

[0016] Structure D: 1 st Fab-Fc-L1-2 nd scFv;

[0017] Where, - represents a peptide bond; L1 and L2 represent linker peptides or independent peptide bonds; L1 and L2 may be different or the same; Fc represents the Fc segment of the antibody; 1 st scFv represents the scFv domain that can specifically bind to the first antigen; 1 st Fab represents the Fab domain that can specifically bind to the first antigen; 2 nd scFv represents the scFv domain that can specifically bind to a second antigen; 2 nd Fab represents the Fab domain that can specifically bind to the second antigen; one of the first antigen and the second antigen is PD-L1, and the other is 4-1BB.

[0018] The scFv structural domain can have a heavy chain variable region at the N end and a light chain variable region at the C end; or it can have a light chain variable region at the N end and a heavy chain variable region at the C end.

[0019] The linker can be selected from the following: A(EAAAK)4ALE, KVDKKVEPKSCDKTHT, G4S, (G4S)n. Wherein, n is a positive integer (e.g., 1, 2, 3, 4, 5 or 6), preferably, n = 4.

[0020] The bispecific antibody contains an Fc segment.

[0021] The Fc segment may or may not contain mutation sites.

[0022] The Fc segment is of type IgG1, IgG2, IgG3 or IgG4, preferably type IgG4.

[0023] Preferably, in the bispecific antibody of structure A in the embodiment, 1 st scFv recognizes 4-1BB antigen, 2 nd scFv recognizes the PD-L1 antigen.

[0024] Preferably, the bispecific antibody 1 with structure B in the embodiment st scFv recognizes 4-1BB antigen, 2 nd Fab recognizes the PD-L1 antigen.

[0025] Preferably, the bispecific antibody (named D1) with structure D in the embodiment is described. st Fab recognizes the PD-L1 antigen, 2 nd The scFv recognizes the 4-1BB antigen, L1 is the amino acid sequence “A(EAAAK)4ALE”, and Fc is preferably IgG4. In the examples, the bispecific antibody is mutated from D1 to D3 by mutating amino acids 61 and 101 from the N-terminus of the heavy chain SEQ ID No. 7, which can be mutated individually or simultaneously to glutamic acid “E”, or amino acids 62 and 102 from the N-terminus of the heavy chain SEQ ID No. 7 can be mutated individually or simultaneously to glycine “G” or alanine “A”.

[0026] Preferably, in another embodiment, the bispecific antibody (named D2) with structure D, 1 st Fab recognizes the PD-L1 antigen, 2 ndscFv recognizes the 4-1BB antigen, L1 is the "(G4S)3" amino acid sequence, and Fc is preferably IgG4. In the embodiment, the bispecific antibody is mutated from D2 to D6 by mutating amino acids 61 and 101 from the N-terminus of the heavy chain SEQ ID No. 8, which can be mutated individually or simultaneously to glutamic acid "E", or by mutating amino acids 62 and 102 from the N-terminus of the heavy chain SEQ ID No. 8, which can be mutated individually or simultaneously to glycine "G" or alanine "A".

[0027] In a specific embodiment of the present invention, the bispecific antibody is any one of the following:

[0028] (A) consists of two identical peptide chains, each with an amino acid sequence as shown in SEQ ID No. 5 (corresponding to structure A);

[0029] (B) consists of two heavy chains and two light chains; the amino acid sequences of the heavy chains are all as shown in SEQ ID No. 6, and the amino acid sequences of the light chains are all as shown in SEQ ID No. 11 (corresponding to structure B);

[0030] (C) consists of two heavy chains and two light chains; the amino acid sequences of the heavy chains are all as shown in SEQ ID No. 7, and the amino acid sequences of the light chains are all as shown in SEQ ID No. 11 (corresponding to structure D1);

[0031] (D) consists of two heavy chains and two light chains; the amino acid sequences of the heavy chains are all as shown in SEQ ID No. 9, and the amino acid sequences of the light chains are all as shown in SEQ ID No. 11 (corresponding to structure D3);

[0032] (E) consists of two heavy chains and two light chains; the amino acid sequences of the heavy chains are all as shown in SEQ ID No. 8, and the amino acid sequences of the light chains are all as shown in SEQ ID No. 11 (corresponding to structure D2);

[0033] (F) consists of two heavy chains and two light chains; the amino acid sequences of the heavy chains are all as shown in SEQ ID No. 10, and the amino acid sequences of the light chains are all as shown in SEQ ID No. 11 (corresponding to structure D6).

[0034] Secondly, the present invention claims protection for nucleic acid molecules encoding the bispecific antibodies described in the first aspect above.

[0035] In the nucleic acid molecule, the nucleotide sequences encoding HCDR1, HCDR2, and HCDR3 in the heavy chain variable region of the PD-L1 antigen-binding domain are shown in SEQ ID No. 27, positions 76-96, 154-168, and 292-321 from the 5' end, respectively; wherein, the nucleotide sequence encoding HCDR3 in the heavy chain variable region of the PD-L1 antigen-binding domain can also be shown in SEQ ID No. 16, positions 292-321. The nucleotide sequences encoding LCDR1, LCDR2, and LCDR3 in the light chain variable region of the PD-L1 antigen-binding domain are shown in SEQ ID No. 28, positions 70-108, 154-174, and 271-300 from the 5' end, respectively.

[0036] In the nucleic acid molecule, the nucleotide sequences encoding HCDR1, HCDR2, and HCDR3 in the heavy chain variable region of the 4-1BB antigen-binding domain are shown in SEQ ID No. 29, positions 91-105, 148-195, and 292-318 from the 5' end, respectively. In the nucleic acid molecule, the nucleotide sequences encoding LCDR1, LCDR2, and LCDR3 in the light chain variable region of the 4-1BB antigen-binding domain are shown in SEQ ID No. 30, positions 70-102, 148-168, and 265-291 from the 5' end, respectively.

[0037] Further, in the nucleic acid molecule, the nucleotide sequence encoding the heavy chain variable region in the PD-L1 antigen-binding domain is position 1879-2232 of SEQ ID No. 27 or SEQ ID No. 12 or position 1-354 of SEQ ID No. 16, or has a similarity of 99% or more, 95% or more, 90% or more, 85% or more, 80% or more, or 75% or more to position 1879-2232 of SEQ ID No. 12 or position 1-354 of SEQ ID No. 16 (the inconsistency is preferably in the frame region (FR)). In the nucleic acid molecule, the nucleotide sequence encoding the light chain variable region in the PD-L1 antigen-binding domain is position 1489-1818 of SEQ ID No. 28 or SEQ ID No. 12, or has a 99% or higher, 95% or higher, 90% or higher, 85% or higher, 80% or higher, or 75% or higher identity with position 1489-1818 of SEQ ID No. 28 or SEQ ID No. 12.

[0038] Further, in the nucleic acid molecule, the nucleotide sequence encoding the heavy chain variable region of the 4-1BB antigen-binding domain is positions 382-732 of SEQ ID No. 29 or SEQ ID No. 12, or has a 99% or higher, 95% or higher, 90% or higher, 85% or higher, 80% or higher, or 75% or higher similarity to positions 382-732 of SEQ ID No. 29 or SEQ ID No. 12 (the inconsistency is preferably in the frame region (FR)). In the nucleic acid molecule, the nucleotide sequence encoding the light chain variable region of the 4-1BB antigen-binding domain is positions 1-321 of SEQ ID No. 30 or SEQ ID No. 12, or has a 99% or higher, 95% or higher, 90% or higher, 85% or higher, 80% or higher, or 75% or higher similarity to positions 1-321 of SEQ ID No. 30 or SEQ ID No. 12 (the inconsistency is preferably in the frame region (FR)).

[0039] Furthermore, the nucleic acid molecule can be any of the following:

[0040] (a) A nucleic acid molecule a encoding the peptide chain described in (A) above; the nucleotide sequence of said nucleic acid molecule a is SEQ ID No. 12 or has 99% or more, 95% or more, 90% or more, 85% or more, 80% or more or 75% or more similarity to SEQ ID No. 12 (the inconsistency is preferably in the frame region (FR)).

[0041] (b) Composed of nucleic acid molecule b1 encoding the heavy chain described in (B) above and nucleic acid molecule b2 encoding the light chain described in (B) above; the nucleotide sequence of nucleic acid molecule b1 is SEQ ID No. 13 or has 99% or more, 95% or more, 90% or more, 85% or more, 80% or more or 75% or more identity with SEQ ID No. 13 (the inconsistency is preferably in the frame region (FR)); the nucleotide sequence of nucleic acid molecule b2 is SEQ ID No. 18 or has 99% or more, 95% or more, 90% or more, 85% or more, 80% or more or 75% or more identity with SEQ ID No. 18 (the inconsistency is preferably in the frame region (FR)).

[0042] (c) Composed of a nucleic acid molecule c1 encoding the heavy chain described in (C) above and a nucleic acid molecule c2 encoding the light chain described in (C) above; the nucleotide sequence of the nucleic acid molecule c1 is SEQ ID No. 14 or has 99% or more, 95% or more, 90% or more, 85% or more, 80% or more or 75% or more identity with SEQ ID No. 14 (the inconsistency is preferably in the frame region (FR)); the nucleotide sequence of the nucleic acid molecule c2 is SEQ ID No. 18 or has 99% or more, 95% or more, 90% or more, 85% or more, 80% or more or 75% or more identity with SEQ ID No. 18 (the inconsistency is preferably in the frame region (FR)).

[0043] (d) Composed of a nucleic acid molecule d1 encoding the heavy chain described in (D) above and a nucleic acid molecule d2 encoding the light chain described in (D) above; the nucleotide sequence of the nucleic acid molecule d1 is SEQ ID No. 16 or has 99% or more, 95% or more, 90% or more, 85% or more, 80% or more or 75% or more identity with SEQ ID No. 16 (the inconsistency is preferably in the frame region (FR)); the nucleotide sequence of the nucleic acid molecule d2 is SEQ ID No. 18 or has 99% or more, 95% or more, 90% or more, 85% or more, 80% or more or 75% or more identity with SEQ ID No. 18 (the inconsistency is preferably in the frame region (FR)).

[0044] (e) Composed of nucleic acid molecule e1 encoding the heavy chain described in (E) above and nucleic acid molecule e2 encoding the light chain described in (E) above; the nucleotide sequence of nucleic acid molecule e1 is SEQ ID No. 15 or has 99% or more, 95% or more, 90% or more, 85% or more, 80% or more or 75% or more identity with SEQ ID No. 15 (the inconsistency is preferably in the frame region (FR)); the nucleotide sequence of nucleic acid molecule e2 is SEQ ID No. 18 or has 99% or more, 95% or more, 90% or more, 85% or more, 80% or more or 75% or more identity with SEQ ID No. 18 (the inconsistency is preferably in the frame region (FR)).

[0045] (f) consists of a nucleic acid molecule f1 encoding the heavy chain described in (F) above and a nucleic acid molecule f2 encoding the light chain described in (F) above; the nucleotide sequence of the nucleic acid molecule f1 is SEQ ID No. 17 or has 99% or more, 95% or more, 90% or more, 85% or more, 80% or more or 75% or more identity with SEQ ID No. 17 (the inconsistency is preferably in the frame region (FR)); the nucleotide sequence of the nucleic acid molecule f2 is SEQ ID No. 18 or has 99% or more, 95% or more, 90% or more, 85% or more, 80% or more or 75% or more identity with SEQ ID No. 18 (the inconsistency is preferably in the frame region (FR)).

[0046] Thirdly, the present invention claims protection for expression cassettes, recombinant vectors, recombinant bacteria or transgenic cell lines containing the nucleic acid molecules described above.

[0047] Fourthly, the present invention claims protection for a pharmaceutical composition.

[0048] The pharmaceutical composition claimed in this invention may comprise: (a1) the bispecific antibody described above; and (a2) a pharmaceutically acceptable excipient, diluent, or carrier.

[0049] Fifthly, the present invention claims protection for a method for preparing the bispecific antibody described in the first aspect above.

[0050] The method for preparing the bispecific antibody described in the first aspect above, as claimed in this invention, may include the following steps:

[0051] (1) The recombinant plasmid obtained by cloning the nucleic acid molecule described in the second aspect above into the pcDNA3.4 vector;

[0052] (2) Transfect the recombinant plasmid obtained in step (1) into the recipient cell to obtain recombinant cells, culture the recombinant cells, and obtain the bispecific antibody.

[0053] When the nucleic acid molecule involves both a heavy chain and a light chain, it is cloned into the pcDNA3.4 vector to obtain two recombinant plasmids, which are then co-transfected into recipient cells to obtain recombinant cells. The recombinant cells are then cultured to obtain the bispecific antibody.

[0054] The KD value of the binding affinity of the bispecific antibody to human PD-L1 is less than 10E-08M.

[0055] The KD value of the binding affinity of the bispecific antibody to human 4-1BB is less than 10E-08M.

[0056] The bispecific antibody can bind to both PD-L1 and 4-1BB simultaneously.

[0057] The bispecific antibody can block the binding of PD-1 to PD-L1.

[0058] The KD value of the binding affinity of the bispecific antibody to monkey PD-L1 is less than 10E-08M.

[0059] The KD value of the binding affinity of the bispecific antibody to monkey 4-1BB is less than 10E-08M.

[0060] The bispecific antibody can activate T cells and enhance the secretion levels of IL-2 and IFN-γ in MLR in vitro experiments.

[0061] The bispecific antibody's T-cell activation effect depends on PD-L1, has no activity in the absence of PD-L1, and has an effective dose similar to EC50 for different PD-L1 expression levels.

[0062] The bispecific antibody can activate the 4-1BB signaling pathway, and this activation depends on crosslinking.

[0063] The bispecific antibody has anti-tumor efficacy, and its efficacy is superior to that of the combination of PD-L1 and 4-1BB monoclonal antibodies.

[0064] The bispecific antibody exhibits good stability.

[0065] The bispecific antibody has a high safety profile, and no obvious abnormalities were observed after administration to rhesus monkeys.

[0066] The bispecific antibody described in this invention exhibits good stability and high safety. It can both block the binding of PD-1 and PD-L1 and activate the human 4-1BB signaling pathway, stimulating T cell activation and significantly increasing the expression levels of IL-2 and IFN-γ. This indicates that the antibody can regulate the immune system by modulating immune cell activity, making it suitable for use as an immune enhancer in anti-tumor or antiviral immune responses, or as an immunomodulator for T cell-mediated autoimmune diseases. It can also be used to prepare drugs for treating tumors. Therefore, the bispecific antibody provided in this invention has significant importance and application potential for the preparation of antibody-targeted drugs.

[0067] Terms and Definitions

[0068] BsAb: Bispecific antibody, also known as bispecific antibody.

[0069] ScFv: Single-chain variable region antibody fragment, also known as single-chain variable fragment.

[0070] FACS: Fluorescence-activated cell sorting, also known as flow cytometry.

[0071] In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory procedures used herein are all standard procedures widely used in their respective fields. To better understand this invention, definitions and explanations of relevant terms are provided below.

[0072] As used herein, references to the amino acid sequence of CD137 protein or 4-1BB protein (UniProt Q07011) include the full-length 4-1BB protein or the extracellular fragment 4-1BB-ECD of 4-1BB; and also to fusion proteins of 4-1BB-ECD, such as fragments fused with a fragment of the Fc protein (mFc or hFc) of mouse or human IgG. References to the amino acid sequence of PD-L1 protein (Uniprot#Q9NZQ7) include the full-length PD-L1 protein or the extracellular fragment PD-L1-ECD of PD-L1; and also to fusion proteins of PD-L1-ECD, such as fragments fused with a fragment of the Fc protein (mFc or hFc) of mouse or human IgG. However, those skilled in the art will understand that mutations or variations (including but not limited to substitutions, deletions, and / or additions) can be naturally generated or artificially introduced into the amino acid sequence of 4-1BB protein or PD-L1 without affecting its biological function. Therefore, in this invention, the terms "4-1BB protein" or "PD-L1 protein" should include all such sequences, including their natural or artificial variants. Furthermore, when describing sequence fragments of 4-1BB protein or PD-L1 protein, it also includes corresponding sequence fragments from their natural or artificial variants.

[0073] As used in this article, the term EC50 refers to the concentration for 50% of maximal effect, which is the concentration that produces 50% of the maximum effect.

[0074] As used in this article, the term R 2 In statistics, the correlation coefficient (R0) refers to the degree of agreement between experimental data and a fitted function. 2 The closer the value is to 1, the higher the degree of match; the closer it is to 0, the lower the degree of match.

[0075] As used in this article, the term MLR refers to Mixed Lymphocyte Reaction, which involves detecting the stimulatory effect of antibodies or other drugs on lymphocytes when two unrelated individuals with normal function are cultured together in vitro.

[0076] As used in this article, the term Linker refers to a protein linker or linker element, which is a single peptide chain that connects different target genes through a suitable nucleotide sequence, enabling them to be expressed as a single peptide chain in a suitable organism.

[0077] As used herein, the term "antibody" refers to an immunoglobulin molecule typically composed of two pairs of polypeptide chains (each pair consisting of one "light" (L) chain and one "heavy" (H) chain). Generally, the heavy chain can be understood as the larger polypeptide chain in an antibody, and the light chain as the smaller polypeptide chain. Light chains can be classified as κ and λ light chains. Heavy chains are typically classified as μ, δ, γ, α, or ε, and antibody isotypes are defined as IgM, IgD, etc., respectively. Antibodies contain IgG, IgA, and IgE. Within the light and heavy chains, variable and constant regions are linked by "J" regions of approximately 12 or more amino acids, and the heavy chain also contains "D" regions of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region consists of one domain, CL. Antibodies... Constant regions mediate the binding of immunoglobulins to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. VH and VL regions can be further subdivided into highly degenerated regions (called complementarity-determining regions (CDRs)) interspersed with more conserved regions called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4, from the amino terminus to the carboxyl terminus. Variable regions (VH and VL) of each heavy / light chain pair form antibody-binding sites. The term "antibody" is not limited to any particular method of antibody production. For example, it includes, in particular, recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. Antibodies can be different isotypes of antibodies, such as IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

[0078] As used in this article, the terms "bispecific antibody heavy chain" and "bispecific antibody light chain" refer to the fact that when there are two chains in the structure of a bispecific antibody, the chain with the larger molecular weight is the bispecific antibody heavy chain, and the chain with the smaller molecular weight is the bispecific antibody light chain.

[0079] As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which polynucleotides can be inserted. When a vector enables the expression of a protein encoded by the inserted polynucleotide, it is called an expression vector. Vectors can be introduced into host cells through transformation, transduction, or transfection, allowing the genetic material elements they carry to be expressed in the host cells. Vectors are well-known to those skilled in the art and include, but are not limited to: plasmids; phage particles; Cos plasmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, a vector may contain a replication initiation site.

[0080] As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells, or human cells.

[0081] As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its targeted antigen. In some embodiments of the invention, the term "targeting" refers to specific binding.

[0082] As used herein, the terms "monoclonal antibody" and "monoclonal antibody" have the same meaning and are used interchangeably; the terms "peptide" and "protein" have the same meaning and are used interchangeably. Furthermore, in this invention, amino acids are generally represented by single-letter abbreviations known in the art. For example, alanine can be represented by A. Attached Figure Description

[0083] Figure 1 This is a schematic diagram of the dual-characteristic antibody structure of the present invention.

[0084] Figure 2 The molecular weight and purity of the bispecific antibody of the present invention were detected under SDS-PAGE reduction and non-reduction conditions (R indicates reduction, NR indicates non-reduction).

[0085] Figure 3 The present invention relates to a bispecific antibody that binds to human PD-L1.

[0086] Figure 4 The bispecific antibody mutant candidate molecule of this invention binds to human PD-L1.

[0087] Figure 5 The present invention is a bispecific antibody that binds to human 4-1BB.

[0088] Figure 6 The bispecific antibody mutant candidate molecule of this invention binds to human 4-1BB.

[0089] Figure 7 The present invention provides a bispecific antibody that simultaneously binds to human PD-L1 and human 4-1BB.

[0090] Figure 8 The binding activity of the bispecific antibody of this invention to human PD-L1 was detected by FACS.

[0091] Figure 9 The binding activity of the bispecific antibody of this invention to human 4-1BB was detected by FACS.

[0092] Figure 10 The binding activity of the bispecific antibody of this invention to monkey PD-L1 was detected by ELISA.

[0093] Figure 11 The binding activity of the bispecific antibody of this invention to monkey 4-1BB was detected by ELISA.

[0094] Figure 12 MLR was used to detect the activation of T cells by the bispecific antibody of this invention. A shows the detection of IL-2 secretion level in cell supernatant; B shows the detection of IFN-γ secretion level in cell supernatant. In the figure, the concentrations of each drug administration group from left to right in the bar chart are 10 μg / ml, 2 μg / ml, 0.4 μg / ml, 0.08 μg / ml, and 0.016 μg / ml; the concentrations of IgG from left to right are 10 μg / ml, 0.4 μg / ml, and 0.016 μg / ml.

[0095] Figure 13 CD8 that HuPD-L1 depends on + T-cell activation activity assay.

[0096] Figure 14The activity of the bispecific antibody of this invention in activating 4-1BB / NFκB was detected by reporter gene assay.

[0097] Figure 15 The activity of the bispecific antibody blocking PD-1 / PD-L1 in this invention was detected by reporter gene assay.

[0098] Figure 16 This invention relates to the antitumor efficacy of bispecific antibodies. Detailed Implementation

[0099] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0100] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0101] In the following examples, unless otherwise specified, the first position of each nucleotide sequence in the sequence listing is the 5′ terminal nucleotide of the corresponding DNA, and the last position is the 3′ terminal nucleotide of the corresponding DNA.

[0102] The cell culture, molecular genetics, nucleic acid chemistry, and immunology laboratory procedures used in the following examples are all routine procedures widely used in their respective fields.

[0103] The following examples do not include a detailed description of conventional methods, such as those used for gene amplification, recombinant plasmid construction, and introduction of plasmids into host cells.

[0104] pcDNA3.4 vector: (Invitrogen, Cat: A14697)

[0105] Expi293F cells: ATCC cell bank.

[0106] CHO-K1 cells: ATCC cell bank.

[0107] MC38 cells: Nanjing Kebai Biotechnology Co., Ltd.

[0108] Jurkat cells: Nanjing Kebai Biotechnology Co., Ltd.

[0109] The standard equipment and reagents are as follows:

[0110] 1. 96-well microplate (Nunc);

[0111] 2. Plating buffer: 0.05M sodium bicarbonate aqueous solution;

[0112] 3. Washing solution: Phosphate buffer with a pH of 7.0 and a volume percentage of 0.05% Tween 20;

[0113] 4. Sealing solution: Washing solution containing only 10g / L BSA.

[0114] 5. Horseradish peroxidase-labeled avidin;

[0115] 6. Chromogenic substrate: Tetramethylbenzidine;

[0116] 7. Termination solution: 1M sulfuric acid.

[0117] The solvent of the phosphate buffer solution with pH 7.0 is water, and the solutes are sodium chloride, potassium chloride, potassium dihydrogen phosphate, and disodium hydrogen phosphate. The concentration of sodium chloride in the phosphate buffer solution with pH 7.0 is 135 mM, the concentration of potassium chloride in the phosphate buffer solution with pH 7.0 is 2.7 mM, the concentration of potassium dihydrogen phosphate in the phosphate buffer solution with pH 7.0 is 1.5 mM, and the concentration of disodium hydrogen phosphate in the phosphate buffer solution with pH 7.0 is 8 mM (i.e., the concentration of disodium hydrogen phosphate is 8 mM).

[0118] Example 1: Construction of a recombinant vector for anti-human PD-L1 / 4-1BB bispecific antibody

[0119] The sequence of the anti-human PD-L1 monoclonal antibody is derived from Chinese patent application CN 201910174374.8 of Anhui Anke Biotechnology Engineering (Group) Co., Ltd. (hereinafter referred to as Anke Biotechnology). The sequence of the anti-human 4-1BB monoclonal antibody is derived from Chinese patent application CN 201911105611.1 of Hefei Hanke Mabbio Biotechnology Co., Ltd., and international patent application PCT / CN2020 / 127993. A schematic diagram of the structure of the anti-human PD-L1 / 4-1BB bispecific antibody of this invention is shown below. Figure 1 As shown, all of them exhibit antibody activity.

[0120] Bispecific antibodies from N-terminus to C-terminus, such as Figure 1 Structure A is shown: 1 st scFv-L1-Fc-L2-2 nd scFv;

[0121] Bispecific antibodies from N-terminus to C-terminus, such as Figure 1 Structure B is shown: 1 st scFv-L1-Fc-L2-2 nd Fab;

[0122] Bispecific antibodies from N-terminus to C-terminus, such as Figure 1 Structure D is shown: 1 st Fab-Fc-L1-2 nd scFv;

[0123] Where "-" represents a peptide bond; L1 and L2 are independent peptide bonds or linker elements; Fc is the Fc segment of the antibody; 1 st It is the N-terminal first antigen scFv domain or Fab domain, 2 nd The first antigen scFv domain or Fab domain is the C-terminal second antigen scFv domain or Fab domain. The first antigen scFv domain or Fab domain and the second antigen scFv domain or Fab domain each have binding specificity for different antigens (e.g., PD-L1 or 4-1BB). The scFv structure can be heavy chain first (VH-VL) or light chain first (VL-VH). The linker sequence can be A(EAAAK)4ALE, KVDKKVEPKSCDKTHT, etc., with a preferred sequence being (G4S)n, where n is a positive integer (e.g., 1, 2, 3, 4, 5, or 6), preferably n = 4. The Fc segment can contain mutated or non-mutated sites, and the Fc segment can be of IgG1, IgG2, IgG3, or IgG4 type, with IgG4 type Fc being preferred.

[0124] Alternatively, in structure A, Fc is the human IgG4 subtype. The N-terminus of Fc is linked to a single-chain antibody (VH-(G4S)4-VL) against 4-1BB antibody via Linker ((G4S)2), and the C-terminus of Fc is linked to a single-chain antibody (VH-(G4S)4-VL) against PD-L1 antibody via Linker ((G4S)3). The amino acid sequence of structure A is shown in SEQ ID No. 5, and the corresponding nucleotide sequence is shown in SEQ ID No. 12.

[0125] Alternatively, in structure B, Fc is the human IgG4 subtype. One chain (bispecific antibody heavy chain) is the single-chain antibody (VH-(G4S)4-VL) against 4-1BB antibody linked to the N-terminus of Fc via Linker ((G4S)2), and the heavy chain against PD-L1 antibody linked to the C-terminus of Fc via Linker ((G4S)3). The amino acid sequence is shown in SEQ ID No. 6 (the corresponding nucleotide sequence is shown in SEQ ID No. 13). The other chain (bispecific antibody light chain) is the light chain against PD-L1 antibody. The amino acid sequence is shown in SEQ ID No. 11 (the corresponding nucleotide sequence is shown in SEQ ID No. 18).

[0126] Alternatively, in structure D, Fc represents the human IgG4 subtype, and a single-chain antibody (VH-(G4S)4-VL) against human 4-1BB is linked to the C-terminus of each of the two heavy chains of the anti-PD-L1 antibody via a linker (A(EAAAK)4ALE, KVDKKVEPKSCDKTHT, or (G4S)n). The other chain is the light chain of the anti-PD-L1 antibody.

[0127] D1: L1 is the amino acid sequence “A(EAAAK)4ALE”. The heavy chain amino acid sequence is shown in SEQ ID No. 7 (the corresponding nucleotide sequence is shown in SEQ ID No. 14), and the light chain amino acid sequence is shown in SEQ ID No. 11 (the corresponding nucleotide sequence is shown in SEQ ID No. 18).

[0128] D3: D3 is a mutation based on D1. The 61st and 101st amino acids of the heavy chain SEQ ID No. 7, starting from the N-terminus, are mutated to glutamic acid "E" (either individually or simultaneously), or the 62nd and 102nd amino acids of the heavy chain SEQ ID No. 7, starting from the N-terminus, are mutated to glycine "G" (either individually or simultaneously) or alanine "A". The heavy chain amino acid sequence is shown in SEQ ID No. 9 (the corresponding nucleotide sequence is shown in SEQ ID No. 16), and the light chain amino acid sequence is shown in SEQ ID No. 11 (the corresponding nucleotide sequence is shown in SEQ ID No. 18).

[0129] D2: L1 is the "(G4S)3" amino acid sequence. The heavy chain amino acid sequence is shown in SEQ ID No. 8 (the corresponding nucleotide sequence is shown in SEQ ID No. 15), and the light chain amino acid sequence is shown in SEQ ID No. 11 (the corresponding nucleotide sequence is shown in SEQ ID No. 18).

[0130] D6: D6 is a mutation based on D2. Mutations are made at amino acids 61 and 101 from the N-terminus of the heavy chain (SEQ ID No. 8), either individually or simultaneously, to glutamic acid ("E"), or at amino acids 62 and 102 from the N-terminus of the heavy chain ("G"), either individually or simultaneously, to glycine ("G") or alanine ("A"). The heavy chain amino acid sequence is shown in SEQ ID No. 10 (the corresponding nucleotide sequence is shown in SEQ ID No. 17), and the light chain amino acid sequence is shown in SEQ ID No. 11 (the corresponding nucleotide sequence is shown in SEQ ID No. 18).

[0131] The following is a detailed description of the construction process of the bispecific antibody recombinant expression vector.

[0132] The heavy chain nucleotide sequences of bispecific antibodies in structures A, B, and D (D1, D3, D2, or D6) were directly synthesized. During synthesis, an XbaI restriction site (TCTAGA), a Kozak co-recognition sequence (5'-GCCACC-3'), and a signal peptide sequence (5'-ATGGAGTTCGGCCTGTCCTGGCTGTTTCTGGTGGCCATCCTGAAGGGCGTGCAGTGC-3') were introduced at the N-terminus, and a stop codon and a HindIII restriction site (AAGCTT) were introduced at the C-terminus. The synthesized sequences were then double-digested with XbaI and HindIII and inserted into a similarly digested pcDNA3.4 vector to obtain the recombinant vector of the target gene of the bispecific antibody. The recombinant plasmids, after being verified by sequencing, were named pcDNA3.4-A, pcDNA3.4-BH, pcDNA3.4-D1-H, pcDNA3.4-D3-H, pcDNA3.4-D2-H, and pcDNA3.4-D6-H according to their different inserted sequences.

[0133] Simultaneously, a light chain gene for the bispecific antibody was artificially synthesized. During synthesis, an XbaI restriction site (TCTAGA), a Kozak co-recognition sequence (5'-GCCACC-3'), and a signal peptide sequence (5'-ATGGAGACAGACACACTCCTGCTATGGGTACTGCTGCTCTGGGTTCCAGGATCCACTGGT-3') were introduced at the N-terminus, and a stop codon and a HindIII restriction site (AAGCTT) were introduced at the C-terminus. The synthesized sequence was double-digested with XbaI and HindIII and cloned into a pcDNA3.4 vector that had also been double-digested, thus obtaining the recombinant vector of the light chain gene for the bispecific antibody. The recombinant plasmids, after being verified by sequencing, were named pcDNA3.4-BL, pcDNA3.4-D1-L, pcDNA3.4-D3-L, pcDNA3.4-D2-L, and pcDNA3.4-D6-L according to the different inserted sequences.

[0134] Example 2: Expression and purification of bispecific antibodies

[0135] The recombinant vectors corresponding to the structures described in Example 1 were used with the Expi293 expression system (Thermo Fisher) according to the product instructions. Expi Fectamine transfection reagent and DNA plasmid were added to OptiMEM to obtain solutions A and B, respectively. Solutions A and B were then mixed to obtain solution C. Solution C was added to Expi293 cells (Thermo Fisher) and cultured for 5 days. For structure A, only one recombinant plasmid (pcDNA3.4-A) was transfected; for structures B and D, two recombinant plasmids corresponding to the heavy and light chains were co-transfected. The cells were centrifuged (10000 rpm for 10 minutes), and the supernatant was collected. The supernatant was purified using a Protein A affinity chromatography column, followed by further purification using a Superdex 200 pg gel filtration system.

[0136] The specific procedure is as follows: First, equilibrate the Protein A column (GE) with PBS, then pass the culture supernatant through the column, equilibrate again, and then elute for 5 column volumes with affinity elution buffer (formulation: solvent is water, solute and concentration is: 50mM sodium acetate, pH 3.5), and collect the elution peak; equilibrate Superdex 200pg (GE) with PBS, then pass the affinity elution collection solution through the column at a ratio of 4%, collect the monomer peak, and then concentrate it using a 30KD centrifuge tube to obtain the target molecule.

[0137] The purified antibody was analyzed by SDS-PAGE under both reducing and non-reducing conditions to determine its molecular weight and purity. Results are as follows: Figure 2 As shown, under non-reducing conditions, structures A, B, and D appear as essentially single bands. Under reducing conditions, structure A is a single band with an analytical weight of approximately 80-100 kDa; structures B and D, under reducing conditions, appear as two bands with molecular weights of 80-100 kDa and 25-30 kDa, respectively, consistent with theoretical molecular weights.

[0138] The bispecific antibody of structure A prepared as described above consists of two identical peptide chains, the amino acid sequence of which is shown in SEQ ID No. 5 (the corresponding nucleotide sequence is shown in SEQ ID No. 12).

[0139] The B-structure bispecific antibody prepared as described above consists of two heavy chains and two light chains; the amino acid sequences of the heavy chains are all as shown in SEQ ID No. 6 (the corresponding nucleotide sequences are shown in SEQ ID No. 13), and the amino acid sequences of the light chains are all as shown in SEQ ID No. 11 (the corresponding nucleotide sequences are shown in SEQ ID No. 18).

[0140] The D1 structure bispecific antibody prepared as described above consists of two heavy chains and two light chains; the amino acid sequence of the heavy chains is shown in SEQ ID No. 7 (the corresponding nucleotide sequence is shown in SEQ ID No. 14), and the amino acid sequence of the light chains is shown in SEQ ID No. 11 (the corresponding nucleotide sequence is shown in SEQ ID No. 18).

[0141] The D3 structure bispecific antibody prepared as described above consists of two heavy chains and two light chains; the amino acid sequence of the heavy chains is shown in SEQ ID No. 9 (the corresponding nucleotide sequence is shown in SEQ ID No. 16), and the amino acid sequence of the light chains is shown in SEQ ID No. 11 (the corresponding nucleotide sequence is shown in SEQ ID No. 18).

[0142] The D2 structure bispecific antibody prepared as described above consists of two heavy chains and two light chains; the amino acid sequence of the heavy chains is shown in SEQ ID No. 8 (the corresponding nucleotide sequence is shown in SEQ ID No. 15), and the amino acid sequence of the light chains is shown in SEQ ID No. 11 (the corresponding nucleotide sequence is shown in SEQ ID No. 18).

[0143] The D6-structured bispecific antibody prepared as described above consists of two heavy chains and two light chains; the amino acid sequences of the heavy chains are all as shown in SEQ ID No. 10 (the corresponding nucleotide sequences are shown in SEQ ID No. 17), and the amino acid sequences of the light chains are all as shown in SEQ ID No. 11 (the corresponding nucleotide sequences are shown in SEQ ID No. 18).

[0144] Example 3: ELISA method for determining the binding activity of bispecific antibody to human PD-L1 antigen

[0145] The affinity of the bispecific antibody for human PD-L1 was assessed by ELISA. The full-length amino acid sequence of human PD-L1 is shown in SEQ ID No. 22 (Uniprot#Q9NZQ7). Similar to the antibody expression preparation described above, the extracellular segment (serial positions 19-238 from the N-terminus of SEQ ID No. 22) was used as the target sequence and inserted between the XbaⅠ and HindⅢ sites of the pCDNA3.4 vector. After confirmation by sequencing, the target plasmid was obtained and expressed by transient transfection using HEK293. Human PD-L1 was coated in 96-well plates (96-well microplate, Nunc) at a concentration of 350 ng / ml, 100 μl / well, and incubated overnight at 4°C. After washing three times, 300 μl of blocking buffer (formula above) was added to each well, and the plates were blocked at 37°C for 1 hour. The plate was washed three times with washing buffer (formula described above). The bispecific antibody structures A, B, D3, and D6 obtained in Example 2 of this invention were diluted to 5 nM with sample diluent (PBS-T with 1% bovine serum albumin), and then serially diluted 4-fold in 7 centrifuge tubes to different concentrations. Each concentration was used in duplicate, 100 μl / well, and incubated for 1 hour. The plate was washed three times with washing buffer, and 100 μl of horseradish peroxidase-labeled goat anti-human (goat anti-human-HRP, Thermo Fisher Scientific) diluted 8000 times was added to each well, and the plate was shaken for 0.5 hours. The plate was washed three times, and 100 μl of tetramethylbenzidine (TMB, Thermo Fisher Scientific) was added to each well. The plate was developed in the dark, and the reaction was terminated by adding 1 M sulfuric acid. The OD value was measured at 450 nm using a Versamax microplate reader (Molecular). The antibody-antigen reaction curve was plotted using a 4-parameter logistic regression method. The specific dilution concentrations and detection results are shown in Table 1.

[0146] The result curve is as follows: Figure 3 As shown, bispecific antibodies bind well to human PD-L1 antigen in a dose-dependent manner, and bispecific antibodies with different structures exhibit different binding activities to human PD-L1.

[0147] Table 1. ELISA detection of the binding activity of bispecific antibodies to human PD-L1

[0148]

[0149] Using the same method, the binding activity of different mutant molecules corresponding to the D structure with human PD-L1 was detected. Specific results are shown in Table 2, and the curve fitting results are as follows: Figure 4 As shown, bispecific antibody candidate molecules with the same structure do not show significant differences in binding activity with human PD-L1.

[0150] Table 2. ELISA detection of the binding activity of bispecific antibody mutant molecules to human PD-L1

[0151]

[0152]

[0153] Example 4: ELISA method for determining the binding activity of bispecific antibodies to human 4-1BB antigen.

[0154] The affinity of the bispecific antibody for human 4-1BB was assessed by ELISA. The full-length amino acid sequence of human 4-1BB is shown in SEQ ID No. 19 (Uniprot#Q07011). The preparation method was similar to that of the antibody protein and human PD-L1 protein. The extracellular segment (amino acid sequence shown in SEQ ID No. 19, positions 24-186 from the N-terminus) was used as the target sequence and inserted between the XbaⅠ and HindⅢ sites of the pCDNA3.4 vector. After confirmation by sequencing, the target plasmid was obtained and expressed via transient transfection using HEK293. The human 4-1BB antigen was diluted to 350 ng / ml with plating buffer, and 100 μl / well was added to an ELISA plate (Nunc) and incubated overnight at 4°C. The plate was washed three times, and 300 μl of blocking buffer (formulation described above) was added to each well, and the plate was blocked at 37°C for 1 hour. Bispecific antibodies A, B, D3, and D6 obtained in Example 2 of this invention were diluted to 10 nM with sample dilution buffer (PBS-T with 1% bovine serum albumin), and then serially diluted 4-fold in 7 centrifuge tubes to different concentrations, with two replicates for each concentration, 100 μl / well, and incubated for 1 hour. Horseradish peroxidase-labeled goat anti-human IgG (goat anti-human HRP, Thermo Fisher Scientific) diluted 1:8000 (v / v) was added to each well, 100 μl, and incubated at room temperature with shaking for 1 hour. The plate was washed three times, and 100 μl of tetramethylbenzidine (TMB, Thermo Fisher Scientific) was added to each well. The plate was developed in the dark, and the reaction was terminated by adding 1 M sulfuric acid. The OD value was measured at 450 nm using a Versamax molecular microplate reader. The antibody-antigen reaction curve was plotted using a 4-parameter logistic regression method. Specific dilution concentrations and detection results are shown in Table 3.

[0155] The result curve is as follows: Figure 5 As shown, bispecific antibodies bind well to human 4-1BB antigen in a dose-dependent manner, and bispecific antibodies with different structures exhibit different binding activities to human 4-1BB.

[0156] Table 3. ELISA detection of the binding activity of bispecific antibodies to human 4-1BB

[0157]

[0158]

[0159] Using the same method, the binding activity of different mutant molecules corresponding to the D structure with human 4-1BB was detected. Bispecific antibodies D1, D2, D3, and D6 were diluted to 5 nM with sample dilution buffer (PBS-T with 1% bovine serum albumin), and then serially diluted 4-fold in seven centrifuge tubes to different concentrations. Specific results are shown in Table 2, and curve fitting results are as follows: Figure 6 As shown, after mutation of bispecific antibodies with the same structure, the binding activities of candidate molecules D1 and D3, and D2 and D6 to human 4-1BB are not significantly different.

[0160] Table 4. ELISA detection of the binding activity of bispecific antibodies to human 4-1BB

[0161]

[0162] Example 5: ELISA method for detecting the simultaneous binding characteristics of bispecific antibodies to human PD-L1 and human 4-1BB antigens.

[0163] The binding activity of the bispecific antibodies to both human PD-L1 and human 4-1BB was assessed by ELISA. The extracellular fragment of human PD-L1 (amino acid sequence as shown in SEQ ID No. 22, positions 19-238 from the N-terminus, with a His tag; preparation method see Example 3) was diluted to 500 ng / mL with plating buffer, and 100 μl / well was added to an ELISA plate (Nunc), incubated overnight at 4°C. The plate was washed three times, and 300 μl of blocking buffer (formulation described above) was added to each well, and the plate was blocked at 37°C for 1 hour. The bispecific antibodies A, B, D3, and D6 obtained in Example 2 of this invention were diluted to 5 nM with sample diluent (PBS-T with 1% bovine serum albumin), and then serially diluted 5-fold in seven centrifuge tubes to different concentrations. Each concentration was used in duplicate, 100 μl / well, and incubated for 1 hour. Add 100 μl of human 4-1BB antigen (SEQ ID No. 19, positions 24-186 from the N-terminus, with a mouse Fc tag; preparation method see Example 4) at a detection concentration of 500 ng / ml to each well, and incubate at room temperature for 1 hour. Dilute goat anti-mouse-HRP 1:2000 with 1% BSA, add 100 μl of each well to an ELISA plate, and incubate with shaking at room temperature for 0.5 hours. Wash the plate three times, and add 100 μl of tetramethylbenzidine (TMB, Thermo Fisher Scientific) to each well. Develop the color in the dark, and stop the reaction with 1M sulfuric acid. Measure the OD value at 450 nm using a Versamax molecular microplate reader. Plot the antibody-antigen reaction curve using a 4-parameter logistic regression method. Specific dilution concentrations and detection results are shown in Table 5.

[0164] The result curve is as follows: Figure 7 As shown, bispecific antibodies can bind to both human PD-L1 and human 4-1BB simultaneously, and this binding is dose-dependent. Bispecific antibodies with different structures exhibit different binding activities to human 4-1BB.

[0165] Table 5. ELISA detection of the simultaneous binding activity of bispecific antibodies with human PD-L1 and human 4-1BB

[0166]

[0167] Example 6: Detection of the binding characteristics of bispecific antibodies to human PD-L1 antigen on cell surface using FACS method

[0168] Following conventional methods in the art, the full-length human PD-L1 sequence (SEQ ID No. 22) was inserted between the XbaⅠ and HindⅢ sites of the pCDNA3.4 vector. After sequencing verification, the resulting recombinant plasmid was introduced into wild-type CHO-K1 cells using Lipofectamine 3000 transfection reagent (Invitrogen) to obtain the CHO-K1 / hPD-L1 cell line, which highly expresses human PD-L1. CHO-K1 / hPD-L1 cells in the logarithmic growth phase were cultured and collected. After washing and resuspending in approximately 1 ml of buffer, the cells were centrifuged and aliquoted into centrifuge tubes at 2 × 10⁻⁶ cm⁻¹. 5 Cells per tube. The bispecific antibodies A, B, D3, and D6 obtained in Example 2 of this invention were diluted to 50 nM with PBS, and then serially diluted 3-fold to obtain six concentrations. 100 μl of each concentration sample was added sequentially to centrifuge tubes containing cells, with a blank control included. After incubation for 60 min, the cells were washed twice with 1 ml of washing buffer (PBS + 2% fetal bovine serum), then resuspended with goat anti-human FITC secondary antibody (Invitrogen, catalog number H10301), and incubated in the dark for 30 min. The cells were washed twice again with 1 ml of washing buffer, and then resuspended in 500 μl of PBS per tube, placed on ice in the dark, and analyzed. The antibody-antigen reaction curves were plotted using a 4-parameter logistic regression method. Specific dilution concentrations and detection results are shown in Table 6.

[0169] Table 6. FACS detection of the binding activity of bispecific antibody to human PD-L1

[0170] Concentration (nM) A B D3 D6 50 8731.7 8489.5 8872.2 8924.8 16.666 7997.6 8215.3 8711.1 8404.1 5.555 3337.1 4429.6 3816.1 2950.3 1.851 1197.8 1449.3 1394.8 1187.5 0.617 671.4 889.1 708 653.7 0.308 504.9 616.7 543.3 516 EC50(nM) 7.345 5.816 6.588 7.598 <![CDATA[R 2 ]]> 0.99 0.99 0.99 0.99

[0171] like Figure 8 As shown, bispecific antibodies bind well to human PD-L1 antigen in a dose-dependent manner, and bispecific antibodies with different structures exhibit different binding activities to human PD-L1.

[0172] Example 7: Detection of the binding characteristics of bispecific antibodies to human 4-1BB antigen on cell surface using FACS method

[0173] Following conventional methods in the art, the full-length human 4-1BB target sequence (SEQ ID No. 19) was inserted between the XbaⅠ and HindⅢ sites of the pCDNA3.4 vector. After sequencing verification, the resulting recombinant plasmid was introduced into wild-type CHO-K1 cells using Lipofectamine 3000 transfection reagent (Invitrogen) to obtain the CHO-K1 / h4-1BB cell line, which highly expresses human 4-1BB. CHO-K1 / h4-1BB cells in the logarithmic growth phase were cultured and collected. After washing and resuspending in approximately 1 ml of buffer, the cells were centrifuged and aliquoted into centrifuge tubes at 2 × 10⁻⁶ cm⁻¹. 5 Cells per tube. The bispecific antibodies A, B, D3, and D6 obtained in Example 2 of this invention were diluted to 50 nM with PBS, and then serially diluted 3-fold to obtain six concentrations. 100 μl of each concentration sample was added sequentially to centrifuge tubes containing cells, with a blank control included. After incubation for 60 min, the cells were washed twice with 1 ml of washing buffer (PBS + 2% fetal bovine serum), then resuspended with goat anti-human FITC secondary antibody (Invitrogen, catalog number H10301), and incubated in the dark for 30 min. The cells were washed twice again with 1 ml of washing buffer, and then resuspended in 500 μl of PBS per tube, placed on ice in the dark, and analyzed. The antibody-antigen reaction curves were plotted using a 4-parameter logistic regression method. Specific dilution concentrations and detection results are shown in Table 7.

[0174] Table 7. FACS detection of the binding activity of bispecific antibody to human 4-1BB

[0175]

[0176]

[0177] like Figure 9 As shown, bispecific antibodies bind well to human 4-1BB antigen in a dose-dependent manner, and bispecific antibodies with different structures exhibit different binding activities to human 4-1BB.

[0178] Example 8: Detection of the binding characteristics of bispecific antibodies to monkey PD-L1 antigen using ELISA method

[0179] The binding activity of the bispecific antibody to monkey PD-L1 was assessed by ELISA. The amino acid sequence of the extracellular segment of monkey PD-L1 is shown in SEQ ID No. 23 (Uniprot#G7PSE7). Similar to the antibody expression preparation described above, a plasmid was constructed using its extracellular segment as the target sequence and expressed via HEK293 transient transfection. A 96-well plate was coated with monkey PD-L1 at a concentration of 500 ng / ml, 100 μl / well, and incubated overnight at 4°C. The plate was washed three times, and 300 μl of blocking buffer (formulation described above) was added to each well, and the plate was blocked at 37°C for 1 hour. After washing the plate three times, the bispecific antibody D6 obtained in Example 2 of this invention was diluted to 1 μg / ml with sample dilution buffer (PBS-T with 1% bovine serum albumin), and then serially diluted 4-fold in 7 centrifuge tubes to different concentrations, with two replicates for each concentration, 100 μl / well, and incubated for 1 hour. Wash the plate three times with washing buffer. Add 100 μl of horseradish peroxidase-labeled goat anti-human (goat anti-human-HRP, Thermo Fisher Scientific) diluted 8000 times to each well and shake for 0.5 hours. Wash the plate three times again and add 100 μl of tetramethylbenzidine (TMB, Thermo Fisher Scientific) to each well. Develop the color in the dark and stop the reaction with 1M sulfuric acid. Measure the OD value at 450 nm using a Versamax molecular microplate reader. Plot the antibody-antigen reaction curve using a 4-parameter logistic regression method. Specific dilution concentrations and detection results are shown in Table 8. The experiment selected the parental antibody Anti-PD-L1 (an anti-human PD-L1 monoclonal antibody from Chinese patent application CN201910174374.8 of Anhui Anke Biotechnology (Group) Co., Ltd. (hereinafter referred to as Anke Biotechnology)) and Tercentriq (Roche) as control antibodies. Tercentriq was produced and expressed according to the accession number DB11595 in DrugBank (https: / / go.drugbank.com).

[0180] The result curve is as follows: Figure 10 As shown, the bispecific antibody binds well to the monkey PD-L1 antigen in a dose-dependent manner, and its binding activity is not significantly different from that of the parent antibody.

[0181] Table 8. ELISA detection of the binding activity of bispecific antibody to monkey PD-L1

[0182]

[0183]

[0184] Example 9: Detection of the binding characteristics of bispecific antibodies to monkey 4-1BB antigen using ELISA method

[0185] The binding activity of the bispecific antibody to monkey 4-1BB was assessed by ELISA. The amino acid sequence of the extracellular segment of monkey 4-1BB is shown in SEQ ID No. 20 (Uniprot#A9YYE7). Similar to the antibody expression preparation described above, a plasmid was constructed using its extracellular segment as the target sequence and expressed via HEK293 transient transfection. A 96-well plate was coated with monkey 4-1BB at a concentration of 500 ng / ml, 100 μl / well, and incubated overnight at 4°C. The plate was washed three times, and 300 μl of blocking buffer (formulation described above) was added to each well, and the plate was blocked at 37°C for 1 hour. After washing the plate three times, the bispecific antibody D6 obtained in Example 2 of this invention was diluted to 10 nM with sample dilution buffer (PBS-T with 1% bovine serum albumin), and then serially diluted 4-fold in 7 centrifuge tubes to different concentrations, with two replicates for each concentration, 100 μl / well, and incubated for 1 hour. Wash the plate three times with washing buffer, add 100 μl of horseradish peroxidase-labeled goat anti-human (goat anti-human-HRP, Thermo Fisher Scientific) diluted 8000 times to each well, and shake for 0.5 hours. Wash the plate three times, add 100 μl of tetramethylbenzidine (TMB, Thermo Fisher Scientific) to each well. Develop the color in the dark, and stop the reaction with 1M sulfuric acid. Measure the OD value at 450 nm using a Versamax microplate reader (Molecular). Plot the antibody-antigen reaction curve using a 4-parameter logistic regression method. Specific dilution concentrations and detection results are shown in Table 9. The parental antibody Anti-4-1BB (i.e., the anti-human 4-1BB monoclonal antibody Hanke10F4 from Chinese patent application CN 201911105611.1 of Hefei Hanke Maibo Biotechnology Co., Ltd.) was selected as the control antibody, and human IgG4 (Sino Biological, catalog number HG4K) was selected as the irrelevant control antibody.

[0186] The result curve is as follows: Figure 11 As shown, the bispecific antibody binds well to the monkey 4-1BB antigen in a dose-dependent manner, and its binding activity is not significantly different from that of the parent antibody.

[0187] Table 9. ELISA detection of the binding activity of bispecific antibody to monkey 4-1BB

[0188]

[0189]

[0190] Example 10: In vitro pharmacodynamic detection of the activation effect of bispecific antibodies on T lymphocytes

[0191] Whole blood was obtained from healthy person A, and PBMC cells were isolated using lymphocyte separation medium (Sigma) according to the manufacturer's instructions. Whole blood was obtained from healthy person B, and PBMC cells were isolated using lymphocyte separation medium, then processed using EasySep... TM DC cells were isolated using the Human CD14 Positive Selection Kit II (Stemcell) and then resuspended in a medium containing 10 ng / mL IL-6, 10 ng / mL IL-1β, 10 ng / mL TNF-α and 1 μg / mL PGE2 to induce maturation.

[0192] The bispecific antibody D6 prepared in Example 2 was diluted to 10 μg / ml with the parental antibodies Anti-4-1BB (i.e., the anti-human 4-1BB monoclonal antibody Hanke10F4 from Chinese patent application CN 201911105611.1 of Hefei Hanke Mabbio Biotechnology Co., Ltd.) and Anti-PD-L1 (the anti-human PD-L1 monoclonal antibody from Chinese patent application CN 201910174374.8 of Anhui Anke Biotechnology (Group) Co., Ltd. (hereinafter referred to as Anke Biotechnology) and five concentrations (10 μg / ml, 2 μg / ml, 0.4 μg / ml, 0.08 μg / ml, and 0.016 μg / ml) in a 5-fold serial dilution. The irrelevant antibody human IgG4 (Sino Biological, catalog number HG4K) was also included, with concentrations of 10 μg / ml, 0.4 μg / ml, and 0.016 μg / ml. The prepared PBMCs and DC cells were added to a 96-well plate at a ratio of 10:1, with 1 × 10⁶ PBMCs. 5 Each well contains 100 μl of diluted antibody. After incubation for 3 days, the secretion levels of IFN-γ and IL-2 in the supernatant are measured.

[0193] The results are as follows Figure 12 As shown, the bispecific antibody D6, compared to the two parental monoclonal antibodies and the negative unrelated control antibody, can activate T cells in the mixed lymphocyte reaction system and further increase the secretion levels of IFN-γ and IL-2 in the cell supernatant.

[0194] Example 11: In vitro pharmacodynamics detection: The activation effect of bispecific antibody on T cells depends on PD-L1.

[0195] As mentioned above, whole blood is obtained from healthy individuals, PBMCs are separated, and then processed using human CD8... + T-cell magnetic beads (BD Biosciences, catalog number 557766) were used to isolate and purify human CD8 cells according to the manufacturer's instructions. + T cells, ready for use.

[0196] Anti-CD3 antibody (Biolegend, catalog number 317325) was diluted to 0.5 μg / ml with PBS and added to 96-well Corning plates at 60 μl / well. The plates were incubated at 37°C for 1 hour. After washing with PBS, CHO-K1 / hPD-L1 (see Example 6) and CHO-K1 cells were added to 96-well plates at different ratios (0:4; 1:3; 2:2; 3:1; 4:0), at 100 μl / well, for a total of 5000 cells / well. The plates were then incubated in a cell culture incubator for 6 hours. The cell supernatant was aspirated, and 100 μl of human CD8+ was added to each well. + T cells, 2.5 × 10 4 100 μl / well. Dilute the antibody (D6) to be tested to 2 μg / ml with culture medium, and then perform 20-fold serial dilutions to obtain 5 concentrations. Add the diluted antibody to a 96-well plate at 100 μl / well, with 3 replicates for each concentration. Incubate the 96-well plate in a cell culture incubator for 3 days, and detect the secretion of cytokine IFN-γ in the cell culture supernatant using ELISA.

[0197] The results are as follows Figure 13 As shown, bispecific antibodies can activate CD8. + T cell activation depends on PD-L1. When PD-L1 is absent in the system, the dual antibody cannot activate CD8 cells. + T cells, with increased PD-L1 expression, double antibody activation of CD8 + The maximum capacity of T cells is increasing, but the half-effective concentration of antibodies is not changing much.

[0198] Example 12: Detection of bispecific antibody activity using reporter gene assay

[0199] The full-length human 4-1BB sequence was inserted as the target gene between the XbaⅠ and HindⅢ sites of the pCDNA3.4 vector (see Example 7). After sequencing verification, plasmid A was obtained. Simultaneously, plasmid B (pNF-κB-Luc, UBO Biotechnology, product number VT1588), containing the NFκB element sequence (as shown in SEQ ID No. 25) and the luciferase gene (as shown in SEQ ID No. 26), was also obtained. Then, plasmids A and B were introduced together into HEK293 cells (Shanghai Cell Bank, Chinese Academy of Sciences) using Lipofectamine 3000 transfection reagent (Invitrogen). Through pressure selection, HEK-293 / NFκB-Luci / 4-1BB was obtained.

[0200] HEK-293 / NFκB-Luci / 4-1BB cells and CHO-K1 / hPD-L1 cells in logarithmic growth phase (see Example 6) were added to 50 μl of each type of cell in a 96-well plate (Corning, 3917), with 3 × 10⁻⁶ cells for each type. 4 Dilute the test antibody (D6) to 20 μg / ml (a control was set up using the parental antibodies Anti 4-1BB and Anti PD-L1, where Anti-4-1BB is the anti-human 4-1BB monoclonal antibody Hanke10F4 from Chinese patent application CN201911105611.1 of Hefei Hanke Mabpharm Biotechnology Co., Ltd.; Anti-PD-L1 is the anti-human PD-L1 monoclonal antibody from Chinese patent application CN 201910174374.8 of Anhui Anke Biotechnology (Group) Co., Ltd. (hereinafter referred to as Anke Biotechnology). After serially diluting 9 concentrations by 5-fold, add 100 μl / well to each well of a 96-well plate. After standing in an incubator for 18-24 h, add 100 μl of ONE-Glo Luciferase assay systytem reagent (Promega) and incubate at room temperature for 10 min, then detect the chemiluminescence value.

[0201] The results are as follows Figure 14 As shown, the bispecific antibody can activate the downstream NFκB signaling pathway of 4-1BB in PD-L1 dependence, thereby enhancing the detection signal value. However, when the two parental monoclonal antibodies are used in combination, the anti-4-1BB monoclonal antibody loses its crosslinking function and cannot activate the downstream NFκB signaling pathway. The PD-L1 monoclonal antibody can only bind to CHO-K1 / hPD-L1 and cannot act on this signaling pathway.

[0202] Using a similar method, Jurkat / NFAT-Luci / PD1 cells stably expressing human PD-1 and NFAT elements, and CHO-K1 / PDL1 / TCR cells expressing human PD-L1 and TCR activating protein CHO-K1, were constructed. The two cell lines were added to 96-well plates at a ratio of 50,000:25,000. The test antibody D6 was diluted to 12 μg / ml, and then serially diluted 3-fold to nine concentrations, with 100 μl added to each well of the 96-well plate. After incubation for 18–24 h, 100 μl of ONE-Glo Luciferase assay system reagent (Promega) was added, and the plates were incubated at room temperature for 10 min. Chemiluminescence values ​​were then measured.

[0203] The results are as follows Figure 15 As shown, bispecific antibodies can block the PD-1 / PD-L1 signaling pathway, indicating that bispecific antibodies can also exert the inhibitory function of PD-L1 antibodies.

[0204] Example 13: Inhibitory effect of bispecific antibodies on tumor growth

[0205] The experiment used PD-1 / 4-1BB double knock-in mice (B-hPD-1 / h4-1BB mice) to detect the bispecific in vivo antitumor efficacy. The B-hPD-1 / h4-1BB mouse model is a genetically engineered mouse model, in which the genome of a C57BL / 6 mouse with a genetic background is chimeric with the human h4-1BB gene and human PD-L1, derived from Biocytogen (catalog number 110004).

[0206] Mouse colon cancer MC38 cell line was subcutaneously injected into the back (shaved side) of the test mice (5 × 10⁶ cells per mouse). 5 Cells, 100 μl). When the average tumor volume of tumor-bearing mice reached 100 mm². 3 Mice were randomly assigned to three groups of five each, according to the experimental design. After tumor inoculation, the animals' survival and activity levels were checked twice weekly, including tumor growth, activity level, diet, weight, and any abnormal behaviors. The medication was administered twice weekly, and tumor volume was measured. The volume was calculated using the formula 1 / 2 × length × width × width (mm). 3 The day of administration for each group is defined as day 0. Grouping details and administration regimens are shown in Table 10.

[0207] Table 10. Grouping and Dosing Regimens

[0208]

[0209] Note: N is the number of animals in each group. Vehicle is the saline control group; Anti PD-L1+Anti 4-1BB is the control group using the parental antibody Anti 4-1BB and Anti PD-L1 in combination; D1 is the bispecific antibody D1 prepared in Example 2.

[0210] Experimental results are as follows Figure 16 As shown, compared with the control group Vehicle and the two monoclonal antibody combination group, the tumor growth of mice was effectively inhibited after administration of bispecific antibody D1.

[0211] Example 14: In vivo toxicological detection of bispecific antibodies in rhesus monkeys

[0212] Four rhesus monkeys were selected for the experiment to evaluate the toxic side effects of the bispecific antibody in monkeys. The monkeys were divided into high-dose and low-dose groups, with one male and one female in each group. The high-dose group received 50 mg / kg, and the low-dose group received 5 mg / kg. The specific administration sample was D6. Administered intravenously once a week for four weeks, for a total of four administrations. During the adaptation period, observations were conducted at least twice daily, once in the morning and once in the afternoon. On the day of administration, observations were conducted once before administration and once in the afternoon after administration. On non-administration days, observations were conducted at least twice daily, once in the morning and once in the afternoon. If animals exhibited obvious toxic symptoms, the observation frequency was increased, and the time was recorded. The results are shown in Table 11. After four weeks of repeated administration of both high and low doses, the ALT (alanine aminotransferase) and AST (aspartate aminotransferase) levels in the rhesus monkeys did not significantly increase compared to the adaptation period, indicating good tolerability. On the other hand, the number of white blood cells and the proportion of lymphocytes in the peripheral blood of rhesus monkeys increased slightly, but both were within the normal range. Throughout the experimental period, the monkeys' general condition, respiration, heart rate, hematology, liver function, and kidney function were all normal, and the animals tolerated the test well.

[0213] Table 11. Toxicological tests of repeated-dose administration in rhesus monkeys over 4 weeks

[0214]

[0215] Note: Day 1 of the Pretest Phase (P1) is defined as the first day of administration, and Day 1 of the Dosing Phase (D1) is defined as the first day of administration. WBC is the white blood cell count, and %LYMPH is the percentage of lymphocytes.

[0216] The present invention has been described in detail above. For those skilled in the art, the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. Although specific embodiments have been given, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein. Some of the essential features can be applied within the scope of the following appended claims. <110> Hefei Hanke Maibo Biotechnology Co., Ltd.; Anhui Anke Biotechnology Engineering (Group) Co., Ltd. <120> Bispecific antibodies targeting PD-L1 and 4-1BB <130> GNCLN211752 <160> 30 <170> PatentIn version 3.5 <210> 1 <211> 118 <212> PRT <213> Artificial sequence <400> 1 Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Val Ser Gly Ile Asp Leu Ser Ser Tyr 20 25 30 Asp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Ile 35 40 45 Gly Tyr Ile Ser Tyr Val Ser Arg Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Lys Asp Thr Ser Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp Arg Pro Asp Gly Ala Ala Thr Asn Leu Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 2 <211> 110 <212> PRT <213> Artificial sequence <400> 2 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ser Ser Gln Asn Val Tyr Ser Asn 20 25 30 Asn Arg Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Trp Thr Ser Phe Leu Ala Ser Gly Val Pro Ser Arg Phe 50 55 60 Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu 65 70 75 80 Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Ala Gly Gly Tyr Ser Gly 85 90 95 Asn Leu Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 110 <210> 3 <211> 117 <212> PRT <213> Artificial sequence <400> 3 Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu 1 5 10 15 Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Ser Ser Leu Thr Ser Tyr 20 25 30 Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Gly Leu Glu Gly Leu 35 40 45 Gly Val Ile Trp Pro Gly Gly Ser Thr Asn Tyr Asn Ser Ala Leu Met 50 55 60 Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Ser Leu 65 70 75 80 Lys Met Ser Ser Leu Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Val Thr Gly Thr Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Thr 100 105 110 Val Thr Val Ser Ser 115 <210> 4 <211> 107 <212> PRT <213> Artificial sequence <400> 4 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Ser Ala Ser Gln Gly Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Thr Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Lys Leu Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 5 <211> 744 <212> PRT <213> Artificial sequence <400> 5 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Ser Ala Ser Gln Gly Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Thr Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Lys Leu Pro Trp 85 90 95 Thr Phe Gly Cys Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln 115 120 125 Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr 130 135 140 Leu Ser Leu Thr Cys Thr Val Ser Gly Ser Ser Leu Thr Ser Tyr Gly 145 150 155 160 Val His Trp Val Arg Gln Pro Pro Gly Lys Cys Leu Glu Gly Leu Gly 165 170 175 Val Ile Trp Pro Gly Gly Ser Thr Asn Tyr Asn Ser Ala Leu Met Ser 180 185 190 Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Ser Leu Lys 195 200 205 Met Ser Ser Leu Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg 210 215 220 Val Thr Gly Thr Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Thr Val 225 230 235 240 Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ser 245 250 255 Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 260 265 270 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 275 280 285 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln 290 295 300 Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val 305 310 315 320 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr 325 330 335 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 340 345 350 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile 355 360 365 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 370 375 380 Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser 385 390 395 400 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 405 410 415 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 420 425 430 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val 435 440 445 Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met 450 455 460 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 465 470 475 480 Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 485 490 495 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 500 505 510 Asp Arg Val Thr Ile Thr Cys Gln Ser Ser Gln Asn Val Tyr Ser Asn 515 520 525 Asn Arg Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu 530 535 540 Leu Ile Tyr Trp Thr Ser Phe Leu Ala Ser Gly Val Pro Ser Arg Phe 545 550 555 560 Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu 565 570 575 Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Ala Gly Gly Tyr Ser Gly 580 585 590 Asn Leu Tyr Thr Phe Gly Cys Gly Thr Lys Leu Glu Ile Lys Gly Gly 595 600 605 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly 610 615 620 Gly Ser Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro 625 630 635 640 Gly Gly Ser Leu Arg Leu Ser Cys Thr Val Ser Gly Ile Asp Leu Ser 645 650 655 Ser Tyr Asp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Cys Leu Glu 660 665 670 Tyr Ile Gly Tyr Ile Ser Tyr Val Ser Arg Thr Tyr Tyr Ala Asp Ser 675 680 685 Val Lys Gly Arg Phe Thr Ile Ser Lys Asp Thr Ser Lys Asn Thr Val 690 695 700 Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr 705 710 715 720 Cys Ala Arg Asp Arg Pro Asp Gly Ala Ala Thr Asn Leu Trp Gly Gln 725 730 735 Gly Thr Leu Val Thr Val Ser Ser 740 <210> 6 <211> 712 <212> PRT <213> Artificial sequence <400> 6 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr Ile Ser Cys Ser Ala Ser Gln Gly Ile Ser Asn Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile 35 40 45 Tyr Tyr Thr Ser Thr Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Ile Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Lys Leu Pro Trp 85 90 95 Thr Phe Gly Cys Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser 100 105 110 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln 115 120 125 Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr 130 135 140 Leu Ser Leu Thr Cys Thr Val Ser Gly Ser Ser Leu Thr Ser Tyr Gly 145 150 155 160 Val His Trp Val Arg Gln Pro Pro Gly Lys Cys Leu Glu Gly Leu Gly 165 170 175 Val Ile Trp Pro Gly Gly Ser Thr Asn Tyr Asn Ser Ala Leu Met Ser 180 185 190 Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Ser Gln Val Ser Leu Lys 195 200 205 Met Ser Ser Leu Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg 210 215 220 Val Thr Gly Thr Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Thr Val 225 230 235 240 Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Glu Ser 245 250 255 Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly 260 265 270 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met 275 280 285 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln 290 295 300 Glu Asp Pro Glu Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val 305 310 315 320 His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr 325 330 335 Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 340 345 350 Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile 355 360 365 Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 370 375 380 Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser 385 390 395 400 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu 405 410 415 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro 420 425 430 Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val 435 440 445 Asp Lys Ser Arg Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met 450 455 460 His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 465 470 475 480 Leu Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 485 490 495 Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 500 505 510 Ser Leu Arg Leu Ser Cys Thr Val Ser Gly Ile Asp Leu Ser Ser Tyr 515 520 525 Asp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Ile 530 535 540 Gly Tyr Ile Ser Tyr Val Ser Arg Thr Tyr Tyr Ala Asp Ser Val Lys 545 550 555 560 Gly Arg Phe Thr Ile Ser Lys Asp Thr Ser Lys Asn Thr Val Tyr Leu 565 570 575 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 580 585 590 Arg Asp Arg Pro Asp Gly Ala Ala Thr Asn Leu Trp Gly Gln Gly Thr 595 600 605 Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe Pro 610 615 620 Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly 625 630 635 640 Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn 645 650 655 Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln 660 665 670 Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser 675 680 685 Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser 690 695 700 Asn Thr Lys Val Asp Lys Arg Val 705 710 <210> 7 <211> 707 <212> PRT <213> Artificial sequence <400> 7 Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Val Ser Gly Ile Asp Leu Ser Ser Tyr 20 25 30 Asp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Ile 35 40 45 Gly Tyr Ile Ser Tyr Val Ser Arg Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Lys Asp Thr Ser Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp Arg Pro Asp Gly Ala Ala Thr Asn Leu 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 Cys Ser Arg Ser Thr Ser Glu Ser 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 Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys 210 215 220 Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu 225 230 235 240 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255 Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln 260 265 270 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285 Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu 290 295 300 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305 310 315 320 Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys 325 330 335 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350 Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360 365 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375 380 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 385 390 395 400 Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415 Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Ala Glu Ala Ala 435 440 445 Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala 450 455 460 Lys Ala Leu Glu Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val 465 470 475 480 Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Ser Ser 485 490 495 Leu Thr Ser Tyr Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Cys 500 505 510 Leu Glu Gly Leu Gly Val Ile Trp Pro Gly Gly Ser Thr Asn Tyr Asn 515 520 525 Ser Ala Leu Met Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Ser 530 535 540 Gln Val Ser Leu Lys Met Ser Ser Leu Thr Ala Ala Asp Thr Ala Val 545 550 555 560 Tyr Tyr Cys Ala Arg Val Thr Gly Thr Trp Tyr Phe Asp Val Trp Gly 565 570 575 Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 580 585 590 Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro 595 600 605 Ser Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Ser 610 615 620 Ala Ser Gln Gly Ile Ser Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro 625 630 635 640 Asp Gly Thr Val Lys Leu Leu Ile Tyr Tyr Thr Ser Thr Leu His Ser 645 650 655 Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr 660 665 670 Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys 675 680 685 Gln Gln Tyr Ser Lys Leu Pro Trp Thr Phe Gly Cys Gly Thr Lys Leu 690 695 700 Glu Ile Lys 705 <210> 8 <211> 704 <212> PRT <213> Artificial sequence <400> 8 Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Val Ser Gly Ile Asp Leu Ser Ser Tyr 20 25 30 Asp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Ile 35 40 45 Gly Tyr Ile Ser Tyr Val Ser Arg Thr Tyr Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Lys Asp Thr Ser Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp Arg Pro Asp Gly Ala Ala Thr Asn Leu Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser Ala Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Cys Ser Arg Ser Thr Ser Glu Ser Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185 190 Ser Ser Ser Leu Gly Thr Lys Thr Tyr Thr Cys Asn Val Asp His Lys 195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro 210 215 220 Pro Cys Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val 225 230 235 240 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr 245 250 255 Pro Glu Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu 260 265 270 Val Gln Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 275 280 285 Thr Lys Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser 290 295 300 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 305 310 315 320 Cys Lys Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile 325 330 335 Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 340 345 350 Pro Ser Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu 355 360 365 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn 370 375 380 Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 385 390 395 400 Asp Gly Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg 405 410 415 Trp Gln Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 420 425 430 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly 435 440 445 Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met 450 455 460 Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr 465 470 475 480 Ile Ser Cys Ser Ala Ser Gln Gly Ile Ser Asn Tyr Leu Asn Trp Tyr 485 490 495 Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile Tyr Tyr Thr Ser 500 505 510 Thr Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly 515 520 525 Thr Asp Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala 530 535 540 Thr Tyr Tyr Cys Gln Gln Tyr Ser Lys Leu Pro Trp Thr Phe Gly Cys 545 550 555 560 Gly Thr Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly 565 570 575 Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln 580 585 590 Glu Ser Gly Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr 595 600 605 Cys Thr Val Ser Gly Ser Ser Leu Thr Ser Tyr Gly Val His Trp Val 610 615 620 Arg Gln Pro Pro Gly Lys Cys Leu Glu Gly Leu Gly Val Ile Trp Pro 625 630 635 640 Gly Gly Ser Thr Asn Tyr Asn Ser Ala Leu Met Ser Arg Val Thr Ile 645 650 655 Ser Lys Asp Asn Ser Lys Ser Gln Val Ser Leu Lys Met Ser Ser Leu 660 665 670 Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Val Thr Gly Thr 675 680 685 Trp Tyr Phe Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 690 695 700 <210> 9 <211> 707 <212> PRT <213> Artificial sequence <400> 9 Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Val Ser Gly Ile Asp Leu Ser Ser Tyr 20 25 30 Asp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Ile 35 40 45 Gly Tyr Ile Ser Tyr Val Ser Arg Thr Tyr Tyr Ala Glu Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Lys Asp Thr Ser Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp Arg Pro Glu Gly Ala Ala Thr Asn Leu 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 Cys Ser Arg Ser Thr Ser Glu Ser 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 Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys 210 215 220 Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu 225 230 235 240 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255 Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln 260 265 270 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285 Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu 290 295 300 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305 310 315 320 Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys 325 330 335 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350 Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360 365 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375 380 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 385 390 395 400 Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415 Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Ala Glu Ala Ala 435 440 445 Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala Lys Glu Ala Ala Ala 450 455 460 Lys Ala Leu Glu Gln Val Gln Leu Gln Glu Ser Gly Pro Gly Leu Val 465 470 475 480 Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr Val Ser Gly Ser Ser 485 490 495 Leu Thr Ser Tyr Gly Val His Trp Val Arg Gln Pro Pro Gly Lys Cys 500 505 510 Leu Glu Gly Leu Gly Val Ile Trp Pro Gly Gly Ser Thr Asn Tyr Asn 515 520 525 Ser Ala Leu Met Ser Arg Val Thr Ile Ser Lys Asp Asn Ser Lys Ser 530 535 540 Gln Val Ser Leu Lys Met Ser Ser Leu Thr Ala Ala Asp Thr Ala Val 545 550 555 560 Tyr Tyr Cys Ala Arg Val Thr Gly Thr Trp Tyr Phe Asp Val Trp Gly 565 570 575 Gln Gly Thr Thr Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 580 585 590 Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro 595 600 605 Ser Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Ser 610 615 620 Ala Ser Gln Gly Ile Ser Asn Tyr Leu Asn Trp Tyr Gln Gln Lys Pro 625 630 635 640 Asp Gly Thr Val Lys Leu Leu Ile Tyr Tyr Thr Ser Thr Leu His Ser 645 650 655 Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Thr 660 665 670 Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr Tyr Cys 675 680 685 Gln Gln Tyr Ser Lys Leu Pro Trp Thr Phe Gly Cys Gly Thr Lys Leu 690 695 700 Glu Ile Lys 705 <210> 10 <211> 702 <212> PRT <213> Artificial sequence <400> 10 Asp Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Thr Val Ser Gly Ile Asp Leu Ser Ser Tyr 20 25 30 Asp Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Tyr Ile 35 40 45 Gly Tyr Ile Ser Tyr Val Ser Arg Thr Tyr Tyr Ala Glu Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Lys Asp Thr Ser Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Asp Arg Pro Glu Gly Ala Ala Thr Asn Leu 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 Cys Ser Arg Ser Thr Ser Glu Ser 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 Lys Thr Tyr Thr Cys Asn Val Asp His Lys Pro Ser 195 200 205 Asn Thr Lys Val Asp Lys Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys 210 215 220 Pro Pro Cys Pro Ala Pro Glu Ala Ala Gly Gly Pro Ser Val Phe Leu 225 230 235 240 Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu 245 250 255 Val Thr Cys Val Val Val Asp Val Ser Gln Glu Asp Pro Glu Val Gln 260 265 270 Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys 275 280 285 Pro Arg Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu 290 295 300 Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys 305 310 315 320 Val Ser Asn Lys Gly Leu Pro Ser Ser Ile Glu Lys Thr Ile Ser Lys 325 330 335 Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser 340 345 350 Gln Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys 355 360 365 Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln 370 375 380 Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly 385 390 395 400 Ser Phe Phe Leu Tyr Ser Arg Leu Thr Val Asp Lys Ser Arg Trp Gln 405 410 415 Glu Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn 420 425 430 His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Leu Gly Gly Gly Gly Ser 435 440 445 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln 450 455 460 Ser Pro Ser Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser 465 470 475 480 Cys Ser Ala Ser Gln Gly Ile Ser Asn Tyr Leu Asn Trp Tyr Gln Gln 485 490 495 Lys Pro Asp Gly Thr Val Lys Leu Leu Ile Tyr Tyr Thr Ser Thr Leu 500 505 510 His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp 515 520 525 Tyr Thr Leu Thr Ile Ser Ser Leu Gln Pro Glu Asp Ile Ala Thr Tyr 530 535 540 Tyr Cys Gln Gln Tyr Ser Lys Leu Pro Trp Thr Phe Gly Cys Gly Thr 545 550 555 560 Lys Leu Glu Ile Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly 565 570 575 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gln Val Gln Leu Gln Glu Ser 580 585 590 Gly Pro Gly Leu Val Lys Pro Ser Glu Thr Leu Ser Leu Thr Cys Thr 595 600 605 Val Ser Gly Ser Ser Leu Thr Ser Tyr Gly Val His Trp Val Arg Gln 610 615 620 Pro Pro Gly Lys Cys Leu Glu Gly Leu Gly Val Ile Trp Pro Gly Gly 625 630 635 640 Ser Thr Asn Tyr Asn Ser Ala Leu Met Ser Arg Val Thr Ile Ser Lys 645 650 655 Asp Asn Ser Lys Ser Gln Val Ser Leu Lys Met Ser Ser Leu Thr Ala 660 665 670 Ala Asp Thr Ala Val Tyr Tyr Cys Ala Arg Val Thr Gly Thr Trp Tyr 675 680 685 Phe Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 690 695 700 <210> 11 <211> 217 <212> PRT <213> Artificial sequence <400> 11 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Gln Ser Ser Gln Asn Val Tyr Ser Asn 20 25 30 Asn Arg Leu Ser Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu 35 40 45 Leu Ile Tyr Trp Thr Ser Phe Leu Ala Ser Gly Val Pro Ser Arg Phe 50 55 60 Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu 65 70 75 80 Gln Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Ala Gly Gly Tyr Ser Gly 85 90 95 Asn Leu Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys Arg Thr 100 105 110 Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu 115 120 125 Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro 130 135 140 Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly 145 150 155 160 Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr 165 170 175 Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His 180 185 190 Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val 195 200 205 Thr Lys Ser Phe Asn Arg Gly Glu Cys 210 215 <210> 12 <211> 2232 <212> DNA <213> Artificial sequence <400> 12 gacattcaga tgactcagtc tccctcttcc ctgtctgcct ccctgggcga tagggtgaca 60 atcagctgtt ctgcttccca gggcatctcc aactacctga actggtacca gcagaagcca 120 gatggcaccg tgaagctgct gatctactat acctctacac tgcactctgg cgtgccaagc 180 cggtttagcg gatctggatc tggaaccgac tatactctga ccattagctc tctgcagccc 240 gaggatatcg ccacatacta ttgccagcag tatagcaagc tgccttggac cttcggctgt 300 ggcacaaagc tggagatcaa gggtggtggt ggttccggtg gtggtggttc cggtggcggc 360 ggctcaggcg gaggggaag ccaggtgcag ctgcaggaga gcggaccagg actggtgaag 420 ccaagcgaga ccctgtctct gacctgcaca gtgagcggct cttccctgac atcttacggc 480 gtgcattggg tgagacagcc acctggcaag tgtctggagg gactgggcgt gatctggcca 540 ggaggctcta ccaactataa ttccgctctg atgagccgcg tgacaatcag caaggacaac 600 tccaagagcc aggtgtctct gaagatgagc tctctgaccg ccgctgatac cgccgtgtac 660 tattgcgcta gggtgaccgg cacatggtac ttcgacgtgt ggggccaggg caccacagtg 720 accgtgtcct ctggcggagg aggatccgga ggaggtggat ctgagagcaa gtatgggccc 780 ccatgccctc catgtccagc tccagaggct gctggaggcc cttccgtgtt cctgtttccc 840 cctaagccaa aggacaccct gatgatcagc agaacccctg aggtgacatg cgtggtggtg gacgtgtctc aggaggaccc agaggtgcag tttaactggt acgtggatgg cgtggaggtg 960 cacaatgcta agaccaagcc cagggaggag cagttcaatt ctacctaccg ggtggtgtcc gtgctgacag tgctgcatca ggattggctg aacggcaagg agtataagtg caaggtgtcc aataagggcc tgccttccag catcgagaag accatcagca aggctaaggg acagcctaga gagccacagg tgtacacact gccaccctcc caggaga tgaccaagaa ccaggtgagc ctgacatgtc tggtgaaggg cttttatcca tctgacatcg ctgtggagtg ggagtccaat ggccagcccg agaacaatta caagaccaca cctccagtgc tggacagcga tggctctttc tttctgtatt ccaggctgac cgtggataag agccggtggc aggagggcaa cgtgttcagc tgctctgtga tgcacgaggc cctgcacaat cattacacac agaagtccct gagcctgtct ctgggcggcg gcggctctgg aggagga tccggtggag gtggatctga catccagatg acacagagcc cttccaccct gtccgccagc gtgggagaca gagtgaccat cacttgccag tccagccaga acgtgtactc taacaataga ctgtcatggt atcagcaga gccagggaag gctcctaagc tcctgatcta ttggaccagc ttcctcgcct ccggagtgcc atcaaggttc 1680. agcggcagtg gatctgggac agaatttact ctgaccatca gctccctgca gcccgacgat 1740. tttgccactt attactgcgc tggcggatac agcggcaacc tgtacacctt cggttgcggt 1800. accaagttgg aaattaaggg tggtggtggt tccggtggtg gtggatctgg cggaggagga 1860. agcggtggag gtggatctga cgtgcagctt gtggagagcg gaggaggcct ggtgcagcct ggaggctctc tgagactctc ctgtaccgtg tctggcatcg acctgagctc ctatgacatg acctgggtcc gccaggctcc agggaagtgc ctggagtaca tcggctacat tagctacgtg tctcggacat actatgccga cagcgtgaag ggcagattca ccatctccaa ggacacctcc aagaacacgg tgtatctgca gatgaacagc ctgagagccg aggacacggc cgtgtattac tgtgccagag acaggcccga cggcgctgcc accaacctgt ggggacaggg taccctggtg 2220 accgtgagct cc 2232 <210> 13 <211> 2136 <212> DNA <213> Artificial sequence <400> 13 gacattcaga tgactcagtc tccctctc ctgtctgcct ccctgggcga tagggtgaca 60 atcagctgtt ctgcttccca gggcatctcc aactacctga actggtacca gcagaagcca 120 gatggcaccg tgaagctgct gatctactat acctctacac tgcactctgg cgtgccaagc 180 cggtttagcg gatctggatc tggaaccgac tatactctga ccattagctc tctgcagccc 240 gaggatatcg ccacatacta ttgccagcag tatagcaagc tgccttggac cttcggctgt 300 ggcacaaagc tggagatcaa gggtggtggt ggttccggtg gtggtggttc cggtggcggc 360 ggctcaggcg gaggaggaag ccaggtgcag ctgcaggaga gcggaccagg actggtgaag 420 ccaagcgaga ccctgtctct gacctgcaca gtgagcggct cttccctgac atcttacggc 480 gtgcattggg tgagacagcc acctggcaag tgtctggagg gactgggcgt gatctggcca 540 ggaggctcta ccaactataa ttccgctctg atgagccgcg tgacaatcag caaggacaac tccaagagcc aggtgtctct gaagatgagc tctctgaccg ccgctgatac cgccgtgtac tattgcgcta gggtgaccgg cacatggtac ttcgacgtgt ggggccaggg caccacagtg 720 accgtgtcct ctggcggagg aggatccgga ggaggtggat ctgagagcaa gtatgggccc 780 ccatgccctc catgtccagc tccagaggct gctggaggcc cttccgtgtt cctgtttccc 840 cctaagccaa aggacaccct gatgatcagc agaacccctg aggtgacatg cgtggtggtg gacgtgtctc aggaggaccc agaggtgcag tttaactggt acgtggatgg cgtggaggtg 960 cacaatgcta agaccaagcc cagggaggag cagttcaatt ctacctaccg ggtggtgtcc gtgctgacag tgctgcatca ggattggctg aacggcaagg agtataagtg caaggtgtcc aataagggcc tgccttccag catcgagaag accatcagca aggctaaggg acagcctaga gagccacagg tgtacacact gccaccctcc caggaga tgaccaagaa ccaggtgagc ctgacatgtc tggtgaaggg cttttatcca tctgacatcg ctgtggagtg ggagtccaat ggccagcccg agaacaatta caagaccaca cctccagtgc tggacagcga tggctctttc tttctgtatt ccaggctgac cgtggataag agccggtggc aggagggcaa cgtgttcagc tgctctgtga tgcacgaggc cctgcacaat cattacacac agaagtccct gagcctgtct ctgggcggcg gcggctctgg aggagga tccggtggag gtggatctga cgtgcagctt gtggagagcg gaggaggcct ggtgcagcct ggaggctctc tgagactctc ctgtaccgtg tctggcatcg acctgagctc ctatgacatg acctgggtcc gccaggctcc agggaagggg ctggagtaca tcggctacat tagctcgtg tctcggacat actatgccga cagcgtgaag ggcagattca ccatctccaa ggacacctcc aagaacacgg tgtatctgca gatgaacagc ctgagagccg aggacacggc cgtgtattac tgtgccagag acaggcccga cggcgctgcc accaacctgt ggggacaggg taccctggtg accgtgagct ccgctagcac aaagggacca tccgtgtttc ccctggctcc ttgcagccgc tctacatccg agagcaccgc cgctctggga tgtctggtga aggactactt ccccgagcct gtgaccgtgt cttggaactc cggcgccctg acaagcggag tgcacacctt tcccgctgtg ctgcagtctt ccggcctgta ctctctgagc 2040 tctgtggtga cagtgccttc cagctctctg ggcaccaaga catatacctg caacgtggac 2100 cataagccaa gcaataccaa ggtggataag agagtg 2136 <210> 14 <211> 2121 <212> DNA <213> Artificial sequence <400> 14 gacgtgcagc ttgtggagag cggaggaggc ctggtgcagc ctggaggctc tctgagactc 60 tcctgtaccg tgtctggcat cgacctgagc tcctatgaca tgacctgggt ccgccaggct 120 ccagggaagg ggctggagta catcggctac attagctacg tgtctcggac atactatgcc 180 gacagcgtga agggcagatt caccatctcc aaggacacct ccaagaacac ggtgtatctg 240 cagatgaaca gcctgagagc cgaggacacg gccgtgtatt actgtgccag agacaggccc 300 gatggcgctg ccaccaacct gtggggacag ggtaccctgg tgaccgtgag ctccgctagc 360 acaaagggac catccgtgtt tcccctggct ccttgcagcc gctctacatc cgagagcacc 420 gccgctctgg gatgtctggt gaaggactac ttccccgagc ctgtgaccgt gtcttggaac 480 tccggcgccc tgacaagcgg agtgcacacc tttcccgctg tgctgcagtc ttccggcctg 540 tactctctga gctctgtggt gacagtgcct tccagctctc tgggcaccaa gacatatacc tgcaacgtgg accataagcc aagcaatacc aaggtggata agagagtgga gagcaagtat gggcccccat gccctccatg tccagctcca gaggctgctg gaggcccttc cgtgttcctg 720 tttcccccta agccaaagga caccctgatg atcagcagaa cccctgaggt gacatgcgtg gtggtggacg tgtctcagga ggacccagag gtgcagttta actggtacgt ggatggcgtg 840 gaggtgcaca atgctaagac caagcccagg gaggagcagt tcaattctac ctaccgggtg gtgtccgtgc tgacagtgct gcatcaggat tggctgaacg gcaaggagta tagtgcaag gtgtccaata agggcctgcc ttccagcatc gagagacca tcagcaaggc taaggacag cctagagagc cacaggtgta cacactgcca ccctcccagg aggagatgac caagaaccag gtgagcctga catgtctggt gaagggcttt tatccatctg acatcgctgt ggagtggggag tccaatggcc agcccgagaa caattacaag accacacctc cagtgctgga cagcgatggc tctttctttc tgtattccag gctgaccgtg gataagagcc ggtggcagga gggcaacgtg 1260 ttcagctgct ctgtgatgca cgaggccctg cacaatcatt acacacagaa gtccctgagc 1320 ctgtctctgg gcgccgaggc tgctgctaag gaggccgctg ccaaggaggc tgccgctaag 1380 gaggctgctg ctaaggccct cgagcaggtg cagctgcagg agagcggacc aggactggtg 1440 aagccaagcg agaccctgtc tctgacctgc acagtgagcg gctcttccct gacatcttac 1500 ggcgtgcatt gggtgagaca gccacctggc aagtgtctgg agggactggg cgtgatctgg 1560 ccaggaggct ctaccaacta taattccgct ctgatgagcc gcgtgacaat cagcaaggac 1620 aactccaaga gccaggtgtc tctgaagatg agctctctga ccgccgctga taccgccgtg 1680 tactattgcg ctagggtgac cggcacatgg tacttcgacg tgtggggcca gggcaccaca 1740 gtgaccgtgt cctctggtgg tggtggttcc ggtggcggcg gctcaggcgg aggaggaagc 1800 gacattcaga tgactcagtc tccctcttcc ctgtctgcct ccctgggcga tagggtgaca 1860 atcagctgtt ctgcttccca gggcatctcc aactacctga actggtacca gcagaagcca 1920 gatggcaccg tgaagctgct gatctactat acctctacac tgcactccgg agtgccaagc 1980 cggtttagcg gatctggatc cggaaccgac tatactctga ccattagctc tctgcagccc 2040 gaggatatcg ccacatacta ttgccagcag tatagcaagc tgccttggac cttcggctgt 2100 ggcacaaagc tggagatcaa g 2121 <210> 15 <211> 2112 <212> DNA <213> Artificial sequence <400> 15 gacgtgcagc ttgtggagag cggaggaggc ctggtgcagc ctggaggctc tctgagactc 60 tcctgtaccg tgtctggcat cgacctgagc tcctatgaca tgacctgggt ccgccaggct 120 ccagggaagg ggctggagta catcggctac attagctacg tgtctcggac atactatgcc 180 gacagcgtga agggcagatt caccatctcc aaggacacct ccaagaacac ggtgtatctg 240 cagatgaaca gcctgagagc cgaggacacg gccgtgtatt actgtgccag agacaggccc 300 gatggcgctg ccaccaacct gtggggacag ggtaccctgg tgaccgtgag ctccgctagc 360 gcctacaa agggaccatc cgtgtttccc ctggctcctt gcagccgctc tacatccgag 420 agcaccgccg ctctgggatg tctggtgaag gactacttcc ccgagcctgt gaccgtgtct 480 tggaactccg gcgccctgac aagcggagtg cacacctttc ccgctgtgct gcagtcttcc 540 ggcctgtact ctctgagctc tgtggtgaca gtgccttcca gctctctggg caccaagaca 600 tatacctgca acgtggacca taagccaagc aataccaagg tggataagag agtggagagc 660 aagtatgggc ccccatgccc tccatgtcca gctccagagg ctgctgggagg cccttccgtg 720 ttcctgtttc cccctaagcc aaaggacacc ctgatgatca gcagaacccc tgaggtgaca 780 tgcgtggtgg tggacgtgtc tcaggaggac ccagaggtgc agtttaactg gtacgtggat 840 ggcgtggagg tgcacaatgc taagaccaag cccagggagg agcagttcaa ttctacctac 900 cgggtggtgt ccgtgctgac agtgctgcat caggattggc tgaacggcaa ggagtataag 960 tgcaaggtgt ccaataaggg cctgccttcc agcatcgaga agaccatcag caaggctaag 1020 ggacagccta gagagccaca ggtgtacaca ctgccaccct cccaggagga gatgaccaag 1080 aaccaggtga gcctgacatg tctggtgaag ggcttttatc catctgacat cgctgtggag 1140 tgggagtcca atggccagcc cgagaacaat tacaagacca cacctccagt gctggacagc 1200 gatggctctt tctttctgta ttccaggctg accgtggata agagccggtg gcaggagggc 1260 aacgtgttca gctgctctgt gatgcacgag gccctgcaca atcattacac acagaagtcc 1320 ctgagcctgt ctctgggcgg cggcggctct ggaggaggag gatccggtgg aggtggatct 1380 gacattcaga tgactcagtc tccctcttcc ctgtctgcct ccctgggcga tagggtgaca 1440 atcagctgtt ctgcttccca gggcatctcc aactacctga actggtacca gcagaagcca 1500 gatggcaccg tgaagctgct gatctactat acctctacac tgcactccgg agtgccaagc 1560 cggtttagcg gatctggatc tggaaccgac tatactctga ccattagctc tctgcagccc 1620 gaggatatcg ccacatacta ttgccagcag tatagcaagc tgccttggac cttcggctgt 1680 ggcacaaagc tggagatcaa gggtggtggt ggttccggtg gtggtggttc cggtggcggc 1740 ggctcaggcg gaggaggaag ccaggtgcag ctgcaggaga gcggaccagg actggtgaag 1800 ccaagcgaga ccctgtctct gacctgcaca gtgagcggct cttccctgac atcttacggc 1860 gtgcattggg tgagacagcc acctggcaag tgtctggagg gactgggcgt gatctggcca 1920 ggaggctcta ccaactataa ttccgctctg atgagccgcg tgacaatcag caaggacaac 1980 tccaagagcc aggtgtctct gaagatgagc tctctgaccg ccgctgatac cgccgtgtac 2040 tattgcgcta gggtgaccgg cacatggtac ttcgacgtgt ggggccaggg caccacagtg 2100 accgtgtcct ct 2112 <210> 16 <211> 2121 <212> DNA <213> Artificial sequence <400> 16 gacgtgcagc ttgtggagag cggaggaggc ctggtgcagc ctggaggctc tctgagactc 60 tcctgtaccg tgtctggcat cgacctgagc tcctatgaca tgacctgggt ccgccaggct 120 ccagggaagg ggctggagta catcggctac attagctacg tgtctcggac atactatgcc 180 gagagcgtga agggcagatt caccatctcc aaggacacct ccaagaacac ggtgtatctg 240 cagatgaaca gcctgagagc cgaggacacg gccgtgtatt actgtgccag agacaggccc 300 gagggcgctg ccaccaacct gtggggacag ggtaccctgg tgaccgtgag ctccgctagc 360 acaaagggac catccgtgtt tcccctggct ccttgcagcc gctctacatc cgagagcacc 420 gccgctctgg gatgtctggt gaaggactac ttccccgagc ctgtgaccgt gtcttggaac 480 tccggcgccc tgacaagcgg agtgcacacc tttcccgctg tgctgcagtc ttccggcctg 540 tactctctga gctctgtggt gacagtgcct tccagctctc tgggcaccaa gacatatacc 600 tgcaacgtgg accataagcc aagcaatacc aaggtggata agagagtgga gagcaagtat 660 gggcccccat gccctccatg tccagctcca gaggctgctg gaggcccttc cgtgttcctg 720 tttcccccta agccaaagga caccctgatg atcagcagaa cccctgaggt gacatgcgtg 780 gtggtggacg tgtctcagga ggacccagag gtgcagttta actggtacgt ggatggcgtg 840 gaggtgcaca atgctaagac caagcccagg gaggagcagt tcaattctac ctaccgggtg 900 gtgtccgtgc tgacagtgct gcatcaggat tggctgaacg gcaaggagta taagtgcaag 960 gtgtccaata agggcctgcc ttccagcatc gagaagacca tcagcaaggc taagggacag 1020 cctagagagc cacaggtgta cacactgcca ccctcccagg aggagatgac caagaaccag 1080 gtgagcctga catgtctggt gaagggcttt tatccatctg acatcgctgt ggagtgggag 1140 tccaatggcc agcccgagaa caattacaag accacacctc cagtgctgga cagcgatggc 1200 tctttctttc tgtattccag gctgaccgtg gataagagcc ggtggcagga gggcaacgtg 1260 ttcagctgct ctgtgatgca cgaggccctg cacaatcatt acacacagaa gtccctgagc 1320 ctgtctctgg gcgccgaggc tgctgctaag gaggccgctg ccaaggaggc tgccgctaag 1380 gaggctgctg ctaaggccct cgagcaggtg cagctgcagg agagcggacc aggactggtg 1440 aagccaagcg agaccctgtc tctgacctgc acagtgagcg gctcttccct gacatcttac 1500 ggcgtgcatt gggtgagaca gccacctggc aagtgtctgg agggactggg cgtgatctgg 1560 ccaggaggct ctaccaacta taattccgct ctgatgagcc gcgtgacaat cagcaaggac 1620 aactccaaga gccaggtgtc tctgaagatg agctctctga ccgccgctga taccgccgtg 1680 tactattgcg ctagggtgac cggcacatgg tacttcgacg tgtggggcca gggcaccaca 1740 gtgaccgtgt cctctggtgg tggtggttcc ggtggcggcg gctcaggcgg aggaggaagc 1800 gacattcaga tgactcagtc tccctcttcc ctgtctgcct ccctgggcga tagggtgaca 1860 atcagctgtt ctgcttccca gggcatctcc aactacctga actggtacca gcagaagcca 1920 gatggcaccg tgaagctgct gatctactat acctctacac tgcactccgg agtgccaagc 1980 cggtttagcg gatctggatc cggaaccgac tatactctga ccattagctc tctgcagccc 2040 gaggatatcg ccacatacta ttgccagcag tatagcaagc tgccttggac cttcggctgt 2100 ggcacaaagc tggagatcaa g 2121 <210> 17 <211> 2106 <212> DNA <213> Artificial sequence <400> 17 gacgtgcagc ttgtggagag cggaggaggc ctggtgcagc ctggaggctc tctgagactc 60 tcctgtaccg tgtctggcat cgacctgagc tcctatgaca tgacctgggt ccgccaggct 120 ccagggaagg ggctggagta catcggctac attagctacg tgtctcggac atactatgcc 180 gagagcgtga agggcagatt caccatctcc aaggacacct ccaagaacac ggtgtatctg 240 cagatgaaca gcctgagagc cgaggacacg gccgtgtatt actgtgccag agacaggccc 300 gagggcgctg ccaccaacct gtggggacag ggtaccctgg tgaccgtgag ctccgctagc 360 acaaagggac catccgtgtt tcccctggct ccttgcagcc gctctacatc cgagagcacc 420 gccgctctgg gatgtctggt gaaggactac ttccccgagc ctgtgaccgt gtcttggaac 480 tccggcgccc tgacaagcgg agtgcacacc tttcccgctg tgctgcagtc ttccggcctg 540 tactctctga gctctgtggt gacagtgcct tccagctctc tgggcaccaa gacatatacc 600 tgcaacgtgg accataagcc aagcaatacc aaggtggata agagagtgga gagcaagtat 660 gggcccccat gccctccatg tccagctcca gaggctgctg gaggcccttc cgtgttcctg 720 tttcccccta agccaaagga caccctgatg atcagcagaa cccctgaggt gacatgcgtg 780 gtggtggacg tgtctcagga ggacccagag gtgcagttta actggtacgt ggatggcgtg 840 gaggtgcaca atgctaagac caagcccagg gaggagcagt tcaattctac ctaccgggtg 900 gtgtccgtgc tgacagtgct gcatcaggat tggctgaacg gcaaggagta taagtgcaag 960 gtgtccaata agggcctgcc ttccagcatc gagaagacca tcagcaaggc taagggacag 1020 cctagagagc cacaggtgta cacactgcca ccctcccagg aggagatgac caagaaccag 1080 gtgagcctga catgtctggt gaagggcttt tatccatctg acatcgctgt ggagtgggag 1140 tccaatggcc agcccgagaa caattacaag accacacctc cagtgctgga cagcgatggc 1200 tctttctttc tgtattccag gctgaccgtg gataagagcc ggtggcagga gggcaacgtg 1260 ttcagctgct ctgtgatgca cgaggccctg cacaatcatt acacacagaa gtccctgagc 1320 ctgtctctgg gcggcggcgg ctctggagga ggaggatccg gtggaggtgg atctgacatt 1380 cagatgactc agtctccctc ttccctgtct gcctccctgg gcgatagggt gacaatcagc 1440 tgttctgctt cccagggcat ctccaactac ctgaactggt accagcagaa gccagatggc 1500 accgtgaagc tgctgatcta ctatacctct acactgcact ccggagtgcc aagccggttt 1560 agcggatctg gatctggaac cgactatact ctgaccatta gctctctgca gcccgaggat 1620 atcgccacat actattgcca gcagtatagc aagctgcctt ggaccttcgg ctgtggcaca 1680 aagctggaga tcaagggtgg tggtggttcc ggtggtggtg gttccggtgg cggcggctca 1740 ggcggaggag gaagccaggt gcagctgcag gagagcggac caggactggt gaagccaagc 1800 gagaccctgt ctctgacctg cacagtgagc ggctcttccc tgacatctta cggcgtgcat 1860 tgggtgagac agccacctgg caagtgtctg gagggactgg gcgtgatctg gccaggaggc 1920 tctaccaact ataattccgc tctgatgagc cgcgtgacaa tcagcaagga caactccaag 1980 agccaggtgt ctctgaagat gagctctctg accgccgctg ataccgccgt gtactattgc 2040 gctagggtga ccggcacatg gtacttcgac gtgtggggcc agggcaccac agtgaccgtg 2100 tcctct 2106 <210> 18 <211> 651 <212> DNA <213> Artificial sequence <400> 18 gacatccaga tgacacagag cccttccacc ctgtccgcca gcgtgggaga cagagtgacc 60 atcacttgcc agtccagcca gaacgtgtac tctaacaata gactgtcatg gtatcagcag 120 aagccaggga aggctcctaa gctcctgatc tattggacca gcttcctcgc ctccggagtg 180 ccatcaaggt tcagcggcag tggatctggg acagaattta ctctgaccat cagctccctg 240 cagcccgacg attttgccac ttattactgc gctggcggat acagcggcaa cctgtacacc 300 ttcggtcagg gtaccaagtt ggaaattaag cgtacggtgg ctgcaccatc tgtcttcatc 360 ttcccgccat ctgatgagca gttgaaatct ggaactgcct ctgttgtgtg cctgctgaat 420 aacttctatc ccagagaggc caaagtacag tggaaggtgg ataacgccct ccaatcgggt 480 aactcccagg agagtgtcac agagcaggac agcaaggaca gcacctacag cctcagcagc 540 accctgacgc tgagcaaagc agactacgag aaacacaaag tctacgcctg cgaagtcacc 600 catcaggggcc tgagctcgcc cgtcacaaag agcttcaaca ggggagagtg c 651 <210> 19 <211> 255 <212> PRT <213> Artificial sequence <400> 19 Met Gly Asn Ser Cys Tyr Asn Ile Val Ala Thr Leu Leu Leu Val Leu 1 5 10 15 Asn Phe Glu Arg Thr Arg Ser Leu Gln Asp Pro Cys Ser Asn Cys Pro 20 25 30 Ala Gly Thr Phe Cys Asp Asn Asn Arg Asn Gln Ile Cys Ser Pro Cys 35 40 45 Pro Pro Asn Ser Phe Ser Ser Ala Gly Gly Gln Arg Thr Cys Asp Ile 50 55 60 Cys Arg Gln Cys Lys Gly Val Phe Arg Thr Arg Lys Glu Cys Ser Ser 65 70 75 80 Thr Ser Asn Ala Glu Cys Asp Cys Thr Pro Gly Phe His Cys Leu Gly 85 90 95 Ala Gly Cys Ser Met Cys Glu Gln Asp Cys Lys Gln Gly Gln Glu Leu 100 105 110 Thr Lys Lys Gly Cys Lys Asp Cys Cys Phe Gly Thr Phe Asn Asp Gln 115 120 125 Lys Arg Gly Ile Cys Arg Pro Trp Thr Asn Cys Ser Leu Asp Gly Lys 130 135 140 Ser Val Leu Val Asn Gly Thr Lys Glu Arg Asp Val Val Cys Gly Pro 145 150 155 160 Ser Pro Ala Asp Leu Ser Pro Gly Ala Ser Ser Val Thr Pro Pro Ala 165 170 175 Pro Ala Arg Glu Pro Gly His Ser Pro Gln Ile Ile Ser Phe Phe Leu 180 185 190 Ala Leu Thr Ser Thr Ala Leu Leu Phe Leu Leu Phe Phe Leu Thr Leu 195 200 205 Arg Phe Ser Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe 210 215 220 Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly 225 230 235 240 Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu 245 250 255 <210> 20 <211> 163 <212> PRT <213> Artificial sequence <400> 20 Leu Gln Asp Leu Cys Ser Asn Cys Pro Ala Gly Thr Phe Cys Asp Asn 1 5 10 15 Asn Arg Ser Gln Ile Cys Ser Pro Cys Pro Pro Asn Ser Phe Ser Ser 20 25 30 Ala Gly Gly Gln Arg Thr Cys Asp Ile Cys Arg Gln Cys Lys Gly Val 35 40 45 Phe Lys Thr Arg Lys Glu Cys Ser Ser Thr Ser Asn Ala Glu Cys Asp 50 55 60 Cys Ile Ser Gly Tyr His Cys Leu Gly Ala Glu Cys Ser Met Cys Glu 65 70 75 80 Gln Asp Cys Lys Gln Gly Gln Glu Leu Thr Lys Lys Gly Cys Lys Asp 85 90 95 Cys Cys Phe Gly Thr Phe Asn Asp Gln Lys Arg Gly Ile Cys Arg Pro 100 105 110 Trp Thr Asn Cys Ser Leu Asp Gly Lys Ser Val Leu Val Asn Gly Thr 115 120 125 Lys Glu Arg Asp Val Val Cys Gly Pro Ser Pro Ala Asp Leu Ser Pro 130 135 140 Gly Ala Ser Ser Ala Thr Pro Pro Ala Pro Ala Arg Glu Pro Gly His 145 150 155 160 Ser Pro Gln <210> 21 <211> 164 <212> PRT <213> Artificial sequence <400> 21 Val Gln Asn Ser Cys Asp Asn Cys Gln Pro Gly Thr Phe Cys Arg Lys 1 5 10 15 Tyr Asn Pro Val Cys Lys Ser Cys Pro Pro Ser Thr Phe Ser Ser Ile 20 25 30 Gly Gly Gln Pro Asn Cys Asn Ile Cys Arg Val Cys Ala Gly Tyr Phe 35 40 45 Arg Phe Lys Lys Phe Cys Ser Ser Thr His Asn Ala Glu Cys Glu Cys 50 55 60 Ile Glu Gly Phe His Cys Leu Gly Pro Gln Cys Thr Arg Cys Glu Lys 65 70 75 80 Asp Cys Arg Pro Gly Gln Glu Leu Thr Lys Gln Gly Cys Lys Thr Cys 85 90 95 Ser Leu Gly Thr Phe Asn Asp Gln Asn Gly Thr Gly Val Cys Arg Pro 100 105 110 Trp Thr Asn Cys Ser Leu Asp Gly Arg Ser Val Leu Lys Thr Gly Thr 115 120 125 Thr Glu Lys Asp Val Val Cys Gly Pro Pro Val Val Ser Phe Ser Pro 130 135 140 Ser Thr Thr Ile Ser Val Thr Pro Glu Gly Gly Pro Gly Gly His Ser 145 150 155 160 Leu Gln Val Leu <210> 22 <211> 290 <212> PRT <213> Artificial sequence <400> 22 Met Arg Ile Phe Ala Val Phe Ile Phe Met Thr Tyr Trp His Leu Leu 1 5 10 15 Asn Ala Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr 20 25 30 Gly Ser Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu 35 40 45 Asp Leu Ala Ala Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile 50 55 60 Ile Gln Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser 65 70 75 80 Tyr Arg Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn 85 90 95 Ala Ala Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr 100 105 110 Arg Cys Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val 115 120 125 Lys Val Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val 130 135 140 Asp Pro Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr 145 150 155 160 Pro Lys Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser 165 170 175 Gly Lys Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn 180 185 190 Val Thr Ser Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr 195 200 205 Cys Thr Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu 210 215 220 Val Ile Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Thr His 225 230 235 240 Leu Val Ile Leu Gly Ala Ile Leu Leu Cys Leu Gly Val Ala Leu Thr 245 250 255 Phe Ile Phe Arg Leu Arg Lys Gly Arg Met Met Asp Val Lys Lys Cys 260 265 270 Gly Ile Gln Asp Thr Asn Ser Lys Lys Gln Ser Asp Thr His Leu Glu 275 280 285 Glu Thr 290 <210> 23 <211> 220 <212> PRT <213> Artificial sequence <400> 23 Phe Thr Val Thr Val Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser 1 5 10 15 Asn Met Thr Ile Glu Cys Lys Phe Pro Val Glu Lys Gln Leu Asp Leu 20 25 30 Thr Ser Leu Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile Ile Gln 35 40 45 Phe Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Asn Tyr Arg 50 55 60 Gln Arg Ala Gln Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala 65 70 75 80 Leu Arg Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Arg Cys 85 90 95 Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys Val 100 105 110 Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val Val Asp Pro 115 120 125 Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala Glu Gly Tyr Pro Lys 130 135 140 Ala Glu Val Ile Trp Thr Ser Ser Asp His Gln Val Leu Ser Gly Lys 145 150 155 160 Thr Thr Thr Thr Asn Ser Lys Arg Glu Glu Lys Leu Leu Asn Val Thr 165 170 175 Ser Thr Leu Arg Ile Asn Thr Thr Ala Asn Glu Ile Phe Tyr Cys Ile 180 185 190 Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile 195 200 205 Pro Glu Leu Pro Leu Ala Leu Pro Pro Asn Glu Arg 210 215 220 <210> 24 <211> 221 <212> PRT <213> Artificial sequence <400> 24 Phe Thr Ile Thr Ala Pro Lys Asp Leu Tyr Val Val Glu Tyr Gly Ser 1 5 10 15 Asn Val Thr Met Glu Cys Arg Phe Pro Val Glu Arg Glu Leu Asp Leu 20 25 30 Leu Ala Leu Val Val Tyr Trp Glu Lys Glu Asp Glu Gln Val Ile Gln 35 40 45 Phe Val Ala Gly Glu Glu Asp Leu Lys Pro Gln His Ser Asn Phe Arg 50 55 60 Gly Arg Ala Ser Leu Pro Lys Asp Gln Leu Leu Lys Gly Asn Ala Ala 65 70 75 80 Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Cys Cys 85 90 95 Ile Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Leu Lys Val 100 105 110 Asn Ala Pro Tyr Arg Lys Ile Asn Gln Arg Ile Ser Val Asp Pro Ala 115 120 125 Thr Ser Glu His Glu Leu Ile Cys Gln Ala Glu Gly Tyr Pro Glu Ala 130 135 140 Glu Val Ile Trp Thr Asn Ser Asp His Gln Pro Val Ser Gly Lys Arg 145 150 155 160 Ser Val Thr Thr Ser Arg Thr Glu Gly Met Leu Leu Asn Val Thr Ser 165 170 175 Ser Leu Arg Val Asn Ala Thr Ala Asn Asp Val Phe Tyr Cys Thr Phe 180 185 190 Trp Arg Ser Gln Pro Gly Gln Asn His Thr Ala Glu Leu Ile Ile Pro 195 200 205 Glu Leu Pro Ala Thr His Pro Pro Gln Asn Arg Thr His 210 215 220 <210> 25 <211> 70 <212> DNA <213> Artificial sequence <400> 25 tggggacttt ccgctgggga ctttccgctg gggactttcc gctggggact ttccgctggg 60 gactttccgc 70 <210> 26 <211> 1653 <212> DNA <213> Artificial sequence <400> 26 atggaagatg ccaaaacat taagaagggc ccagcgccat tctacccact cgaagacggg 60 accgccggcg agcagctgca caaagccatg aagcgctacg ccctggtgcc cggcaccatc 120 gcctttaccg acgcacatat cgaggtggac attacctacg ccgagtactt cgagatgagc 180 gttcggctgg cagaagctat gaagcgctat gggctgaata caaaccatcg gatcgtggtg 240 tgcagcgaga atagcttgca gttcttcatg cccgtgttgg gtgccctgtt catcggtgtg 300 gctgtggccc cagctaacga catctacaac gagcgcgagc tgctgaacag catgggcatc 360 agccagccca ccgtcgtatt cgtgagcaag aaagggctgc aaaagatcct caacgtgcaa 420 aagaagctac cgatcataca aaagatcatc atcatggata gcaagaccga ctaccagggc 480 ttccaaagca tgtacacctt cgtgacttcc catttgccac ccggcttcaa cgagtacgac 540 ttcgtgcccg agagcttcga ccgggacaaa accatcgccc tgatcatgaa cagtagtggc 600 agtaccggat tgcccaaggg cgtagcccta ccgcaccgca ccgcttgtgt ccgattcagt 660 catgcccgcg acccccatctt cggcaaccag atcatccccg acaccgctat cctcagcgtg 720 gtgccatttc accacggctt cggcatgttc accacgctgg gctacttgat ctgcggcttt 780 cgggtcgtgc tcatgtaccg cttcgaggag gagctattct tgcgcagctt gcaagactat 840 aagattcaat ctgccctgct ggtgcccaca ctatttagct tcttcgctaa gagcactctc 900 atcgacaagt acgacctaag caacttgcac gagatcgcca gcggcggggc gccgctcagc 960 aaagggtag gtgaggccgt ggccaaacgc ttccacctac caggcatccg ccagggctac 1020 ggcctgacag aaaaaccag cgccattctg atcaccccccg aaggggacga caagcctggc 1080 gcagtaggca aggtggtgcc cttcttcgag gctaaggtgg tggacttgga caccggtaag 1140 acactgggtg tgaaccagcg cggcgagctg tgcgtccgtg gccccatgat catgagcggc 1200 tacgttaaca accccgaggc tacaaacgct ctcatcgaca aggacggctg gctgcacagc 1260 ggcgacatcg cctactggga cgaggacgag cacttcttca tcgtggaccg gctgaagagc 1320 ctgatcaaat acaagggcta ccaggtagcc ccagccgaac tggagagcat cctgctgcaa 1380 caccccaaca tcttcgacgc cggggtcgcc ggcctgcccg acgacgatgc cggcgagctg 1440 cccgccgcag tcgtcgtgct ggaacacggt aaaaccatga ccgagaagga gatcgtggac 1500 tatgtggcca gccaggttac aaccgccaag aagctgcgcg gtggtgttgt gttcgtggac 1560 gaggtgccta aaggactgac cggcaagttg gacgcccgca agatccgcga gattctcatt 1620 aaggccaaga agggcggcaa gatcgccgtg taa 1653 <210> 27 <211> 354 <212> DNA <213> Artificial sequence <400> 27 gacgtgcagc ttgtggagag cggaggaggc ctggtgcagc ctggaggctc tctgagactc 60 tcctgtaccg tgtctggcat cgacctgagc tcctatgaca tgacctgggt ccgccaggct 120 ccagggaagg ggctggagta catcggctac attagctacg tgtctcggac atactatgcc 180 gacagcgtga agggcagatt caccatctcc aaggacacct ccaagaacac ggtgtatctg 240 cagatgaaca gcctgagagc cgaggacacg gccgtgtatt actgtgccag agacaggccc 300 gatggcgctg ccaccaacct gtggggacag ggtaccctgg tgaccgtgag ctcc 354 <210> 28 <211> 330 <212> DNA <213> Artificial sequence <400> 28 gacatccaga tgacacagag cccttccacc ctgtccgcca gcgtgggaga cagagtgacc 60 atcacttgcc agtccagcca gaacgtgtac tctaacaata gactgtcatg gtatcagcag 120 aagccaggga aggctcctaa gctcctgatc tattggacca gcttcctcgc ctctggcgtg 180 ccatcaaggt tcagcggcag tggatctggg acagaattta ctctgaccat cagctccctg 240 cagcccgacg attttgccac ttattactgc gctggcggat acagcggcaa cctgtacacc 300 ttcggtcagg gtaccaagtt ggaaattaag 330 <210> 29 <211> 351 <212> DNA <213> Artificial sequence <400> 29 caggtgcagc tgcaggagtc cggaccagga ctggtgaagc catccgagac actgagcctg 60 acctgtacag tgtccggatc cagcctgacc agctacggag tgcactgggt gaggcagcca 120 cctggcaagg gactggaggg cctgggcgtg atctggcctg gcggcagcac aaactataat 180 tctgctctga tgtcccgggt gaccatctct aaggacaact ccaagagcca ggtgtccctg 240 aagatgtctt ccctgacagc cgctgacacc gccgtgtact attgcgctag agtgaccggc 300 acatggtact tcgacgtgtg gggccagggc accacagtga cagtgagctc t 351 <210> 30 <211> 321 <212> DNA <213> Artificial sequence <400> 30 gacatccaga tgacacagtc cccatccagc ctgtctgcct ccctgggcga tagagtgacc 60 atcagctgct ctgcttccca gggcatctcc aactacctga attggtatca gcagaagccc 120 gatggcaccg tgaagctgct gatctactat accagcacac tgcactctgg agtgccttcc 180 cgcttcagcg gatctggatc cggaaccgac tacaccctga caatctcttc cctgcagcct 240 gaggacatcg ccacatacta ttgccagcag tattccaagc tgccatggac ctttggcggc 300 ggcacaaagc tggagatcaa g 321

Claims

1. A bispecific antibody targeting PD-L1 and 4-1BB, characterized in that: The bispecific antibody is any one of the following: (C) It consists of two heavy chains and two light chains; the amino acid sequences of the heavy chains are all as shown in SEQ ID No. 7, and the amino acid sequences of the light chains are all as shown in SEQ ID No. 11; (D) consists of two heavy chains and two light chains; the amino acid sequences of the heavy chains are all as shown in SEQ ID No. 9, and the amino acid sequences of the light chains are all as shown in SEQ ID No. 11; (E) consists of two heavy chains and two light chains; the amino acid sequences of the heavy chains are all as shown in SEQ ID No. 8, and the amino acid sequences of the light chains are all as shown in SEQ ID No. 11; (F) consists of two heavy chains and two light chains; the amino acid sequences of the heavy chains are all as shown in SEQ ID No. 10, and the amino acid sequences of the light chains are all as shown in SEQ ID No.

11.

2. A nucleic acid molecule encoding the bispecific antibody of claim 1.

3. The nucleic acid molecule according to claim 2, characterized in that: The nucleic acid molecule is any one of the following: (c) Composed of a nucleic acid molecule c1 encoding the heavy chain of claim 1 (C) and a nucleic acid molecule c2 encoding the light chain of claim 1 (C); wherein the nucleotide sequence of the nucleic acid molecule c1 is SEQ ID No. 14 or has a nucleotide sequence of 99% or more, 95% or more, 90% or more, 85% or more, 80% or more, or 75% or more identical to SEQ ID No. 14; and the nucleotide sequence of the nucleic acid molecule c2 is SEQ ID No. 18 or has a nucleotide sequence of 99% or more, 95% or more, 90% or more, 85% or more, 80% or more, or 75% or more identical to SEQ ID No.

18. (d) Composed of a nucleic acid molecule d1 encoding the heavy chain of claim 1 (D) and a nucleic acid molecule d2 encoding the light chain of claim 1 (D); the nucleotide sequence of the nucleic acid molecule d1 is SEQ ID No. 16 or has a nucleotide sequence of 99% or more, 95% or more, 90% or more, 85% or more, 80% or more, or 75% or more identical to SEQ ID No. 16; the nucleotide sequence of the nucleic acid molecule d2 is SEQ ID No. 18 or has a nucleotide sequence of 99% or more, 95% or more, 90% or more, 85% or more, 80% or more, or 75% or more identical to SEQ ID No. 18; (e) Composed of a nucleic acid molecule e1 encoding the heavy chain of claim 1 (E) and a nucleic acid molecule e2 encoding the light chain of claim 1 (E); the nucleotide sequence of the nucleic acid molecule e1 is SEQ ID No. 15 or has a nucleotide sequence identity of 99% or more, 95% or more, 90% or more, 85% or more, 80% or more, or 75% or more; the nucleotide sequence of the nucleic acid molecule e2 is SEQ ID No. 18 or has a nucleotide sequence identity of 99% or more, 95% or more, 90% or more, 85% or more, 80% or more, or 75% or more. (f) Composed of a nucleic acid molecule f1 encoding the heavy chain of claim 1 (F) and a nucleic acid molecule f2 encoding the light chain of claim 1 (F); the nucleotide sequence of the nucleic acid molecule f1 is SEQ ID No. 17 or has a nucleotide sequence of 99% or more, 95% or more, 90% or more, 85% or more, 80% or more, or 75% or more identical to SEQ ID No. 17; the nucleotide sequence of the nucleic acid molecule f2 is SEQ ID No. 18 or has a nucleotide sequence of 99% or more, 95% or more, 90% or more, 85% or more, 80% or more, or 75% or more identical to SEQ ID No.

18.

4. An expression cassette, recombinant vector, recombinant bacteria, or transgenic cell line containing the nucleic acid molecule described in claim 2 or 3.

5. A pharmaceutical composition comprising: (a1) the bispecific antibody of claim 1; and (a2) a pharmaceutically acceptable excipient, diluent, or carrier.

6. A method for preparing the bispecific antibody of claim 1, comprising the following steps: (1) The recombinant plasmid obtained by cloning the nucleic acid molecule described in claim 2 or 3 into the pcDNA3.4 vector; (2) Transfect the recipient cells with the recombinant plasmid obtained in step (1) to obtain recombinant cells, culture the recombinant cells, and obtain the bispecific antibody.

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