CN111944052B - anti-TNF-alpha/PD-1 bispecific antibody and application thereof - Google Patents

Abstract

The invention discloses an anti-TNF-alpha/PD-1 bispecific antibody and application thereof. The invention uses anti-human PD-1 antibody and anti-human TNF-alpha antibody to construct a bispecific antibody, the bispecific antibody can neutralize TNF-alpha promoting tumor cell proliferation, and simultaneously is combined with PD-1 to block PD-1/PD-L1 signals between tumor cells and T cells, thereby effectively killing tumor cells and having good biological activity and application prospect.

Description

anti-TNF-alpha/PD-1 bispecific antibody and application thereof

Technical Field

The invention belongs to the field of biomedicine, and particularly relates to an anti-TNF-alpha/PD-1 bispecific antibody and application thereof.

Background

Bispecific antibodies (BsAbs) can simultaneously recognize two different epitopes on the same or different antigens, overcome the disadvantages of conventional mabs and improve their therapeutic efficacy, and can be used in a number of therapeutic areas, such as: cancer, chronic inflammatory diseases, autoimmune diseases, and the like. BsAbs have become hot spots in the field of antibody engineering at present, and have wide application prospects in immunotherapy of tumors. BsAbs have the following advantages: (1) BsAbs has two antigen binding arms, one of which is combined with a target antigen, and the other is combined with a labeled antigen on effector cells, and the labeled antigen can activate the effector cells to kill the tumor cells in a targeted manner; (2) double-target signal blocking, unique or overlapping functions are exerted, and drug resistance is effectively prevented; (3) has stronger specificity and targeting property and reduces off-target toxicity; (4) effectively reducing the cost of treatment, compared to combination therapy, BsAbs costs far less than two single agent combination therapies.

BsAbs can be divided into two major classes according to structure: BsAbs containing Fc region (IgG-like) and BsAbs without Fc region (non-IgG-like). BsAbs Prov for IgG-likeThe corresponding functions of the constant region are retained, and in addition, the molecular weight of the constant region is relatively large, so that the constant region has higher stability and longer half-life period, the purification of an antibody product is more facilitated, and the tissue permeability is relatively low. The major technical difficulty in producing BsAs is to obtain correctly paired BsAbs, and about 23 platform technologies for BsAs production have been developed, including DuoBody, DVD-Ig, DAF, KiH (Knob-into-hole) and CrossMab. The KiH technology is a breakthrough for solving the HC/HC matching problem, and KiH introduces mutation into the CH3 region of one heavy chain to form a convex structure similar to a pestle, and introduces mutation into the CH3 region of the other heavy chain to form a concave structure similar to a hole, and the pestle structure is designed to be beneficial to the correct matching of two heterologous antibody heavy chains. The CrossMab technology was developed by Roche and involves the exchange of the domains of one of the HC (heavy chain) and LC (light chain) pairs of the Fab region to solve the LC/HC mismatch problem, this exchange technology retaining the original antigen affinity, including CrossMab^CH1-CL、CrossMab^VH-VL、CrossMab^FabThree ways. Both the LC/HC and HC/HC mismatch problems can be overcome simultaneously using both the CrossMab and KiH techniques.

Tumor necrosis factor (TNF- α) belongs to the type II membrane protein, is secreted mainly by activated mononuclear macrophages, and acts as a trimer. TNF-alpha has double-edged sword function on tumor cells, low-concentration TNF-alpha has tumor promotion effect, and Egberts and the like use the TNF-alpha to treat a mouse pancreatic cancer model to cause the obvious growth and metastasis of tumors. A plurality of researches find that the trace TNF-alpha in the tumor microenvironment can also promote the growth and the diffusion of tumors in the thoracic cavity, the skin and the intestinal tract of animals. Rao et al found that mice receiving anti-TNF- α inhibitors improved intestinal lesions and that the mouse had significantly reduced intestinal epithelial hyperplasia and tumor infiltration compared to mice not treated with the antibody. The TNF-alpha inhibitor infliximab greatly reduces invasion and metastasis of metastatic breast cancer cell strain MDA-MB-231 cells and bone metastasis of a mouse breast cancer model. Clinical trials of advanced solid tumors have shown that plasma CCL2, IL-6, and serum CRP are reduced and approximately 20% of patients with advanced cancer have stable disease after infliximab treatment. In the secondary clinical study of the TNF-alpha inhibitor etanercept, 6 of 30 patients with advanced ovarian cancer showed long-term stable disease, and clinical trials of other TNF-alpha inhibitors also demonstrated their safety and effectiveness.

Programmed cell death receptor 1 (PD-1) is one of CD28 superfamily members, mainly expresses in lymphocyte, also has been shown to express positively in some tumor tissues such as melanoma, non-small cell lung cancer, etc. Programmed cell death 1ligand 1 (PD-L1) on the surface of tumor cells interacts with PD-1 to inhibit the immune response of an organism, so that the blockage of a PD-1/PD-L1 related signal channel has important significance for relieving the immune inhibition of the organism and inhibiting the immune escape of the tumor. FDA approved nivolumab (trade name optivo) and palboclizumab (trade name keytroda) for the treatment of advanced melanoma, non-small cell lung cancer and renal cell carcinoma, their efficacy in various solid tumors and hematologic malignancies has been demonstrated in several studies, and tumor immunotherapy has now become an effective means against tumors.

Symptoms of nausea, vomiting, diarrhea, rash, itching, etc. occurring in some patients after receiving immunotherapy are called Immune-related adverse effects (irAEs) due to autoimmune diseases or inflammation caused by a highly active Immune response. It was found that ipilimumab (trade name Yervoy) in combination with nivolumab resulted in increased TNF-alpha concentration in intestinal tissue in a mouse model of colon cancer, and that colitis symptoms worsened and, when TNF-alpha was neutralized by TNF-alpha inhibitor, colitis symptoms alleviated and tumor-infiltrated CD8⁺T cell (CD 8)⁺TILs) and thereby improving the anti-tumor effect. Furthermore, administration of PD-1 inhibitors in a melanoma mouse model resulted in increased TNF-. alpha.concentrations, promoting CD8⁺T cell death induced by cell activation, increases PD-L1 expression, inhibits anti-tumor reaction, and inhibits CD8 if PD-1 and TNF-alpha are blocked simultaneously⁺T cell death and improved antitumor effect.

In conclusion, bispecific antibodies co-targeting PD-1 and TNF- α can be obtained by neutralizingTNF-alpha and blocking the PD-1/PD-L1 pathway effect antitumor effect. On the other hand, because the PD-1 antibody treatment can cause the concentration of TNF-alpha to rise, through neutralizing the TNF-alpha, irAEs can be reduced, and CD8 can be increased⁺T cell activity, improving the effect of the PD-1 antibody and realizing the synergistic anti-tumor effect of the combination of the TNF-alpha and the PD-1 antibodies. Therefore, the development of the bispecific antibody co-targeting PD-1 and TNF-alpha has important clinical significance and application value.

Disclosure of Invention

The purpose of the invention is as follows: the invention aims to provide an Anti-TNF-alpha/PD-1 bispecific antibody (Anti-TNF-alpha/PD-1 CrossMab) which co-targets PD-1 and TNF-alpha. The invention also provides the application of the anti-TNF-alpha/PD-1 bispecific antibody.

The technical scheme is as follows: the bispecific antibody comprises two heavy chains and two light chains, and the construction method is shown in figure 16, wherein an Fc section of the anti-human PD-1 heavy chain and an Fc section of the anti-human TNF-alpha heavy chain are exchanged with a CH1 domain of the anti-human PD-1 heavy chain and a CL domain of the anti-human PD-1 light chain respectively through site-directed mutagenesis (realization of 'Knob entrance hole', which is beneficial to correct matching of heterologous antibody heavy chains), so that correct assembly of the antibody light chain and the heavy chain is realized.

As an embodiment of the present invention, the heavy chain variable region of the anti-human PD-1 antibody contains an amino acid sequence shown in SEQ ID NO. 1, or the heavy chain variable region of the anti-human PD-1 antibody contains an amino acid sequence encoded by a nucleotide sequence shown in SEQ ID NO. 5, or the heavy chain variable region of the anti-human PD-1 antibody contains an amino acid sequence encoded by a nucleotide sequence similar to SEQ ID NO. 5 deduced according to the degeneracy principle of the amino acid codes.

As an embodiment of the present invention, the light chain variable region of the anti-human PD-1 antibody contains an amino acid sequence shown in SEQ ID NO. 2, or the light chain variable region of the anti-human PD-1 antibody contains an amino acid sequence encoded by a nucleotide sequence shown in SEQ ID NO. 6, or the light chain variable region of the anti-human PD-1 antibody contains an amino acid sequence encoded by a nucleotide sequence similar to SEQ ID NO. 6 deduced according to the degeneracy principle of amino acid encoding.

As an embodiment of the present invention, the heavy chain variable region of the anti-human TNF-alpha antibody comprises the amino acid sequence shown in SEQ ID NO. 3, or the heavy chain variable region of the anti-human TNF-alpha antibody comprises the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO. 7, or the heavy chain variable region of the anti-human TNF-alpha antibody comprises the amino acid sequence encoded by the nucleotide sequence similar to SEQ ID NO. 7 deduced according to the degeneracy principle of the amino acid codes.

As an embodiment of the present invention, the light chain variable region of the anti-human TNF-alpha antibody contains the amino acid sequence shown in SEQ ID NO. 4, or the light chain variable region of the anti-human TNF-alpha antibody contains the amino acid sequence encoded by the nucleotide sequence shown in SEQ ID NO. 8, or the light chain variable region of the anti-human TNF-alpha antibody contains the amino acid sequence encoded by the nucleotide sequence similar to SEQ ID NO. 8 deduced according to the degeneracy principle of the amino acid codes.

As one embodiment of the present invention, the light chain coding sequence of the anti-human PD-1 antibody contains the nucleotide sequence shown in SEQ ID NO. 10.

As an embodiment of the invention, the heavy chain coding sequence of the anti-human PD-1 antibody contains a nucleotide sequence shown as SEQ ID NO. 9.

As one embodiment of the present invention, the light chain coding sequence of the anti-human TNF- α antibody comprises the nucleotide sequence as set forth in SEQ ID NO. 12.

As one embodiment of the present invention, the heavy chain coding sequence of the anti-human TNF- α antibody contains the nucleotide sequence shown in SEQ ID NO. 11.

In a second aspect, the invention provides a polynucleotide encoding said bispecific antibody.

In the third aspect of the invention, the expression vector is used for expressing the bispecific antibody, wherein the heavy chain variable region of the anti-human PD-1 antibody comprises an amino acid sequence shown as SEQ ID NO. 1, the light chain variable region of the anti-human PD-1 antibody comprises an amino acid sequence shown as SEQ ID NO. 2, the heavy chain variable region of the anti-human TNF-alpha antibody comprises an amino acid sequence shown as SEQ ID NO. 3, and the light chain variable region of the anti-human TNF-alpha antibody comprises an amino acid sequence shown as SEQ ID NO. 4.

The expression vector for expressing the bispecific antibody comprises a nucleotide sequence for encoding the bispecific antibody, and is selected from the group consisting of: DNA, RNA, viral vectors, plasmids, transposons, other gene transfer systems, or combinations thereof, preferably mammalian cell expression vectors.

In a fourth aspect, the invention provides a host cell comprising said polynucleotide or said expression vector.

The invention also provides application of the bispecific antibody in preparing a medicament for treating tumor cells expressing PD-L1 and promoted to proliferate by TNF-alpha.

The fifth aspect of the invention provides the application of the bispecific antibody in the preparation of drugs for treating PD-L1 positive tumors or drugs for treating tumors which are promoted to proliferate by TNF-alpha.

The bispecific antibody of the invention is used for killing tumor cells expressing PD-L1, and the bispecific antibody of the invention can also be used for inhibiting tumor cells which are promoted to proliferate by TNF-alpha.

The sixth aspect of the invention provides the application of the bispecific antibody in the preparation of anti-cancer or cancer detection products.

In a seventh aspect, the invention provides a pharmaceutical composition comprising said bispecific antibody.

The bispecific antibody of the invention is prepared by the following method:

(1) culturing the host cell according to the fourth aspect of the invention under suitable conditions, thereby obtaining a culture comprising the bispecific antibody;

(2) purifying and/or isolating the culture obtained in step (1) to obtain the bispecific antibody.

In another preferred embodiment, the bispecific antibody can be purified by Protein A affinity chromatography to obtain the desired antibody.

Has the advantages that: (1) the bispecific antibody of the invention has good antigen binding activity with PD-1 and TNF-alpha. (2) The bispecific antibody of the invention can block the binding of PD-1/PD-L1 and activate the activity of T cells. (3) The bispecific antibody can neutralize TNF-alpha increased by immunotherapy by blocking the combination of PD-1/PD-L1, plays a role in synergy anti-tumor, and has the potential of being developed into anti-tumor drugs.

Drawings

FIG. 1 is an agarose gel electrophoresis of Anti-TNF- α plasmid construction for bispecific antibodies; in the figure, lanes 1-4 of panel A are PCR amplified Anti-TNF-alpha VL region genes, and lanes 5-8 are PCR amplified Kappa gene fragments; b, the graph is that overlap PCR is used for connecting an Anti-TNF-alpha VL region gene and a Kappa region gene; panel C shows the PCR amplification of pBudCE4.1 vector fragment; lanes 1-4 of the diagram are the lanes of bacteria liquid PCR identification of Anti-TNF-alpha light chain gene, and lanes 5-9 are the lanes of bacteria liquid PCR identification of Anti-TNF-alpha heavy chain gene; lanes 1-4 of Panel E are PCR amplified Anti-TNF- α VH region genes, and lanes 5-8 are PCR amplified Anti-TNF- α constant region genes; panel F is an overlap PCR ligation of Anti-TNF α -VH domain genes and constant domain genes; panel G shows the pBudCE4.1 vector fragment from PCR amplification of recombinant TNF-alpha light chain.

FIG. 2 is a forward sequencing map of the TNF- α light chain gene of Anti-TNF- α/PD-1 CrossMab.

FIG. 3 is a forward sequencing map of the TNF- α heavy chain gene of Anti-TNF- α/PD-1 CrossMab.

FIG. 4 is a reverse sequencing map of the Anti-TNF- α heavy chain gene of the bispecific antibody.

FIG. 5 is an agarose gel electrophoresis of Anti-PD-1 plasmid construction for bispecific antibodies; in the figure, lanes 1-4 of panel A are PCR amplified pBudCE4.1 vector fragment, lanes 5-8 are PCR amplified Anti-PD-1 light chain gene fragment; lanes 1-4 of panel B are PCR amplified Anti-PD-1 heavy chain gene fragments, lanes 5-8 are PCR amplified recombinant PD-1 light chain pBudCE4.1 vector fragments; lanes 1-4 of panel C are bacterial liquid PCR identification of Anti-PD-1 heavy chain gene, and lanes 5-8 are bacterial liquid PCR identification of Anti-PD-1 light chain gene.

FIG. 6 is a forward sequencing map of the Anti-PD-1 light chain gene of the bispecific antibody.

FIG. 7 is a forward sequencing map of the Anti-PD-1 heavy chain gene of the bispecific antibody.

FIG. 8 is a reverse sequencing map of the Anti-PD-1 heavy chain gene of the bispecific antibody.

Figure 9 shows the results of bispecific antibody expression purification; in the figure, A is an antibody purification chromatogram; panel B is an antibody SDS-PAGE, lane M: a protein molecular weight marker; lane 1: antibody bands under non-reducing conditions, the molecular weight is about 150 KD; lane 2: antibody bands under reducing conditions.

FIG. 10 is a graph showing the affinity assay of the final product for TNF- α and PD-L1 antigens after purification of bispecific antibody; in the figure, A is the result of the affinity determination of Anti-TNF-alpha/PD-1 CrossMab and TNF-alpha; panel B shows the SPR affinity measurement of Anti-TNF-alpha/PD-1 CrossMab and PD-1 antigen.

FIG. 11 is a graph showing the results of the inhibition of TNF-. alpha.induced cytotoxicity by bispecific antibodies.

FIG. 12 shows the morphology of L929 cells from each group in the inhibition of TNF-. alpha.induced cytotoxicity by bispecific antibodies.

FIG. 13 shows the results of dendritic cell induction experiments; in the figure, A is a mononuclear cell aggregated in a cluster under the objective lens 10 x; b is the form of the mature dendritic cells with 40 x objective lens; c, flow-type identification of the expression rate of CD86 in dendritic cells; d picture is flow identification of dendritic cell PD-L1 expression rate.

FIG. 14 shows CD4⁺(ii) T cell sorting results; in the figure, panel A: PBMC under microscope 10 × objective; and B, drawing: flow assay CD4⁺T cell sorting purity.

FIG. 15 is a graph showing the results of the mixed lymphocyte reaction using the bispecific antibody.

Figure 16 is a schematic of the structure of a bispecific antibody.

FIG. 17 shows the result of identifying the expression of PD-L1 on the surface of five tumor cells, wherein the A-E pictures in the figure are MDA-MB-231, A549, HT-29, TE-13 and H157 cells in sequence.

FIG. 18 shows the effect of TNF- α on the proliferation of three tumor cells, in which A-C are shown in the sequence of TNF- α on the proliferation of three cells, A549, HT-29 and MDA-MB-231.

FIG. 19 is a MDA-MB-231 tumor sphere, in which panels A-C show live cell staining, dead cell staining and Merge, respectively, in the tumor sphere.

FIG. 20 shows the results of the viability assay of the MDA-MB-231 tumor cells of each group.

Detailed Description

The inventors have conducted extensive and intensive studies to obtain an anti-TNF-. alpha./PD-1 bispecific antibody comprising an anti-PD-1 antibody and an anti-TNF-. alpha.antibody. Experiments show that the bispecific antibody has good binding activity to PD-1 and TNF-alpha molecules, can block the interaction of PD-1 and PD-L1 and neutralize TNF-alpha in a tumor microenvironment, and has good antitumor activity.

In order that the invention may be more readily understood, certain terms are first defined. As used in this application, each of the following terms shall have the meaning given below, unless explicitly specified otherwise herein.

As used herein, the terms "bispecific antibody of the invention", "diabody of the invention", "Anti-TNF- α/PD-1 bispecific antibody" and "Anti-TNF- α/PD-1 CrossMab" have the same meaning and all refer to bispecific antibodies that specifically recognize and bind to PD-1 and TNF- α.

As used herein, the term "antibody" or "immunoglobulin" is an approximately 150kDa heterotetrameric glycan protein with the same structural features, consisting of two identical Light Chains (LC) and two identical Heavy Chains (HC). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. There are two types of light chains, λ and κ. There are five major heavy chain types that determine the functional activity of an antibody molecule: IgM, IgD, IgG, IgA, and IgE. Each chain comprises a different sequence domain. The light chain comprises two domains, a variable domain (VL) and a constant domain (CL). The heavy chain comprises four domains, a heavy chain variable region (VH) and three constant regions (CH1, CH2, and CH3, collectively referred to as CH). The variable regions of both the light and heavy chains determine the binding recognition and specificity for the antigen. The constant domain of the light Chain (CL) and the constant region of the heavy Chain (CH) confer important biological properties such as antibody chain binding, secretion, complement binding and binding to Fc receptors (FcR). The Fv fragment is the N-terminal portion of an immunoglobulin Fab fragment, consisting of the variable portions of one light and one heavy chain. The specificity of an antibody depends on the structural complementarity of the antibody binding site and the epitope. The antibody binding site consists of residues derived primarily from the hypervariable region or Complementarity Determining Region (CDR). Occasionally, residues from non-highly variable or Framework Regions (FR) affect the overall domain structure and thus the binding site. Complementarity determining regions or CDRs refer to amino acid sequences that together define the binding affinity and specificity of the native Fv region of the native immunoglobulin binding site. The light and heavy chains of immunoglobulins each have three CDRs, designated as CDR1-L, CDR2-L, CDR3-L and CDR1-H, CDR2-H, CDR 3-H. Conventional antibody antigen binding sites therefore include six CDRs, comprising a collection of CDRs from each of the heavy and light chain variable regions (v-regions).

As used herein, the term "variable" means that certain portions of the variable regions in an antibody differ in sequence, which results in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the antibody variable region, it is concentrated in three segments called Complementarity Determining Regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The more conserved portions of the variable regions are called Framework Regions (FR). The variable regions of native heavy and light chains each comprise four FR regions, in a substantially-folded configuration, connected by three CDRs that form a connecting loop, and in some cases may form a partially-folded structure. The CDRs in each chain are held close together by the FR region and form the antigen binding site of an antibody together with the CDRs of the other chain (see Kabat et al, NIH Publ. No.91-3242, Vol. I, 647, page 669 (1991). the constant regions are not directly involved in binding of an antibody to an antigen, but they exhibit different effector functions, such as participation in antibody dependence and antibody cytotoxicity.

As used herein, the term "affinity" is theoretically defined by an equilibrium association between an intact antibody and an antigen. The affinity of the diabodies of the invention can be assessed or determined by KD values (dissociation constants), such as Surface Plasmon Resonance (SPR), determined using Biacore instrument measurements.

As used herein, the terms "heavy chain variable region" and "VH" are used interchangeably.

As used herein, the term "variable region" is used interchangeably with "Complementary Determining Region (CDR)".

The invention not only comprises the complete antibody, but also comprises antibody fragments with immunological activity or fusion proteins formed by the antibody and other sequences. Accordingly, the invention also includes fragments, derivatives and analogs of the antibodies.

As used herein, the terms "fragment," "derivative," and "analog" refer to a polypeptide that retains substantially the same biological function or activity as an antibody of the invention. A polypeptide fragment, derivative or analogue of the invention may be (i) a polypeptide in which one or more conserved or non-conserved amino acid residues, preferably conserved amino acid residues, are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a polypeptide in which the mature polypeptide is fused to another compound, such as a compound that extends the half-life of the polypeptide, e.g. polyethylene glycol, or (iv) a polypeptide in which an additional amino acid sequence is fused to the sequence of the polypeptide (e.g. a leader or secretory sequence or a sequence used to purify the polypeptide or a proprotein sequence, or a fusion protein with a 6His tag). Such fragments, derivatives and analogs are within the purview of those skilled in the art in view of the teachings herein.

The invention also provides polynucleotide molecules encoding the above antibodies or fragments or fusion proteins thereof. The polynucleotide of the present invention may be in the form of DNA or RNA. The form of DNA includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand.

The full-length nucleotide sequence of the antibody of the present invention or a fragment thereof can be obtained by a PCR amplification method, a recombinant method, or an artificial synthesis method. One possibility is to use synthetic methods to synthesize the sequence of interest, especially when the fragment length is short. Generally, fragments with long sequences are obtained by first synthesizing a plurality of small fragments and then ligating them.

Once the sequence of interest has been obtained, it can be obtained in large quantities by recombinant methods. Usually, it is cloned into a vector, transferred into a cell, and then isolated from the propagated host cell by a conventional method to obtain the relevant sequence.

At present, the DNA sequence encoding the protein of the invention (or a fragment thereof, or a derivative thereof) can be obtained completely by chemical synthesis and can then be introduced into various existing DNA molecules (or vectors, for example) and cells known in the art. Furthermore, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.

The invention also relates to a vector comprising a suitable DNA sequence as described above and a suitable promoter or control sequence. These vectors may be used to transform an appropriate host cell so that it can express the protein.

The host cell may be a prokaryotic cell, such as a bacterial cell; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples are: escherichia coli, streptomyces; bacterial cells of salmonella typhimurium; fungal cells such as yeast; insect cells of drosophila S2 or SF9 (insect ovarian cells); CHO (Chinese hamster ovary), COS7 (African green monkey SV40 transformed kidney cell), 293 (human kidney epithelial cell line) animal cells, etc.

Transformation of a host cell with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art. When the host is prokaryotic, e.g., E.coli, competent cells capable of DNA uptake can be harvested after exponential growth phase using CaCl₂Methods, the steps used are well known in the art. Another method is to use MgCl₂If desired, transformation can also be carried out by electroporation. When the host is a eukaryote, the following DNA transfection methods may be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome encapsulation, etc.

The obtained transformant can be cultured by a conventional method to express the polypeptide encoded by the gene of the present invention. The medium used in the culture may be selected from various conventional media depending on the host cell used, and the culture is performed under conditions suitable for the growth of the host cell. After the host cells have been grown to an appropriate cell density, the selected promoter is induced by suitable means (e.g., temperature shift or chemical induction) and the cells are cultured for an additional period of time.

The antibody may be expressed intracellularly or on the cell membrane, or secreted extracellularly. If desired, antibodies can be isolated and purified by various separation methods using their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of such methods include, but are not limited to: conventional renaturation treatment, treatment with a protein precipitant (such as salt precipitation), centrifugation, cell lysis by osmosis, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, High Performance Liquid Chromatography (HPLC), and other various liquid chromatography techniques, and combinations thereof.

The antibodies of the invention may be used alone or in combination or conjugated with detectable labels (for diagnostic purposes), therapeutic agents, PK (protein kinase) modifying moieties or combinations of any of the above.

Therapeutic agents that may be conjugated or conjugated to the antibodies of the invention include, but are not limited to: 1. a radionuclide; 2. biological toxicity; 3. cytokines such as IL-2, etc.; 4. gold nanoparticles/nanorods; 5. a viral particle; 6. a liposome; 7. nano magnetic particles; 8. prodrug activating enzymes, for example, DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL); 9. chemotherapeutic agents (e.g., cisplatin) or nanoparticles in any form, and the like.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight.

Example 1: Anti-TNF-alpha plasmid construction of bispecific antibody

(1) Anti-TNF-alpha VL region gene and Anti-TNF-alpha Kappa region gene amplification and connection

The Anti-TNF-alpha VL region gene is amplified by using P1 and P2, the Anti-TNF-alpha Kappa region gene is amplified by using P3 and P4, and the primer sequences of P1, P2, P3 and P4 are as follows:

P1：5’-GACCCAAGCTTGCATTCCTGCGCCATGGCTCCCGTGCAGCT-3’(SEQ ID NO:17)

P2：5’-GAGCGGCCACGGTCCGTTTGATTTCCACCTTGGTCCC-3’(SEQ ID NO:18)

P3：5’-ACCAAGG TGGAAATCAA ACGGACCGTGGCCGCTC-3’(SEQ ID NO:19)

P4：5’-AAGTACTGCTTAAGATCGATGTCGATCAGCACTCGCCCCTGTT-3’(SEQ ID NO:20)

1) Anti-TNF-alpha VL region gene amplification. The reaction system is shown in table 1; the amplification reaction conditions are shown in Table 2.

TABLE 1 Anti-TNF-alpha VL region Gene amplification reaction System

TABLE 2 Anti-TNF-alpha VL region Gene amplification reaction conditions

2) And amplifying Anti-TNF-alpha Kappa region genes. The reaction system is shown in table 3; the amplification reaction conditions are as in Table 2.

TABLE 3 Anti-TNF-alpha Kappa region Gene amplification reaction System

Preparing 1.5% (w/V) agarose gel, detecting PCR products by electrophoresis, and carrying out electrophoresis for 30min at 100V. The DNA electrophoresis bands were observed in a gel imager, and the agarose gel of the target band was recovered, as shown in FIG. 1, lane A1-4 and lane 5-8 in FIG. 1 are Anti-TNF-. alpha.VL region gene and Kappa gene fragment, respectively, with correct fragment size.

3) The Anti-TNF-alpha VL region gene and the Anti-TNF-alpha Kappa region gene are connected by the Overlap PCR. The reaction system is shown in table 4; the amplification reaction conditions are as in Table 2.

TABLE 4 Overlap PCR reaction System

The amplified products were subjected to agarose gel electrophoresis and gel recovery, and the results are shown in panel B of FIG. 1, which shows that a band appeared at 600bp, as expected.

(2) Anti-TNF-alpha light chain vector construction

The pBudCE4.1 vector fragment was amplified using P5, P6. The sequences of the primers P5 and P6 are as follows:

P5：5’-GCAGGAATGCAAGCTTGGGTC-3’(SEQ ID NO:21)

P6：5’-TCGACATCGATCTTAAGCAGTACTT-3’(SEQ ID NO:22)

1) the pBudCE4.1 vector fragment was PCR amplified. The reaction system is shown in table 5; the amplification reaction conditions are shown in Table 6.

TABLE 5 pBudCE4.1 vector fragment Gene amplification reaction System

TABLE 6 pBudCE4.1 vector fragment Gene amplification reaction conditions

The amplified products were subjected to agarose gel electrophoresis and gel recovery, and the results are shown in FIG. 1, panel C, which shows the presence of a band at 4000bp, as expected.

2) The Anti-TNF-alpha light chain gene is recombined with the vector in a homologous way. The reaction system is shown in Table 7.

TABLE 7 homologous recombination reaction System

The reaction was carried out at 37 ℃ for 30 min.

3) Transformation of recombinant product

1. Melting competent DH5 alpha cells in ice bath for 5min, adding 5 μ l recombinant product into 50 μ l competent cells, mixing gently with pipette, and standing on ice for 30 min;

2. thermally shocking at 42 deg.C for 90s, immediately taking out, and incubating in ice water bath for 2 min;

3. adding 450 μ l LB culture medium without antibiotic, incubating at 37 deg.C for 10min for sufficient recovery, and shaking at 37 deg.C and 150rpm for 60 min;

4. centrifuging the culture bacterial liquid at 3000rpm for 3min, absorbing part of the culture liquid, re-suspending the bacteria with the rest 100 mul LB culture medium, then uniformly coating the bacterial liquid on an LB plate containing bleomycin, standing the plate at 37 ℃ for 10min, and performing inverted culture at 37 ℃ in the dark until bacterial colonies appear;

5. single colonies were picked and cultured in LB medium containing bleomycin under magnification (37 ℃ C., 150rpm) until the medium became turbid.

4) And (5) PCR identification of the bacterial liquid. The reaction system is shown in Table 8.

TABLE 8 bacterial liquid PCR reaction system

And (3) carrying out agarose gel electrophoresis and gel recovery on the amplification product, wherein the result is shown as a diagram D in figure 1, lanes 1-4 are bacterial liquid PCR identification Anti-TNF-alpha light chain genes, lanes 1-4 in the diagram D in figure 1 show that a band appears at a position of 600bp, and the plasmid is subjected to small extraction according to the specification steps of the plasmid small extraction kit.

(3) Amplification and connection of Anti-TNF-alpha VH region gene and Anti-TNF-alpha constant region gene

The Anti-TNF-alpha VH region gene is amplified by using P7 and P8, the Anti-TNF-alpha constant region gene is amplified by using P9 and P10, and the primer sequences of P7, P8, P9 and P10 are as follows:

P7：5’-GGTACCAGCACAGTGGACTCGAGAGCCATGGCCGTGCTGGGACTG-3’(SEQ ID NO:23)

P8：5’-AGGGCCCTTTGTAGAGGCACTCGAGACGGTGACCAGGGTA-3’(SEQ ID NO:24)

P9：5’-CTGGTCACCGTCTCGAGTGCCTCTACAAAGGGCCCTTC-3’(SEQ ID NO:25)

P10：5’-AGGGTTAGGGATAGGCTTACCTTCTCACTTGCCGGGGGAC-3’(SEQ ID NO:26)

1) Anti-TNF-alpha VH region gene amplification. The reaction system is shown in table 9; the amplification reaction conditions are as in Table 2.

TABLE 9 Anti-TNF-alpha VH Gene amplification reaction System

2) Anti-TNF-alpha constant region gene amplification. The reaction system is shown in table 10; the amplification reaction conditions are as in Table 2.

TABLE 10 Anti-TNF-alpha constant region Gene amplification reaction System

Preparing 1.5% (w/V) agarose gel, detecting PCR products by electrophoresis, and carrying out electrophoresis for 30min at 100V. The DNA electrophoresis bands were observed in a gel imager, and the agarose gel of the target band was recovered, as shown in FIG. 1, lane E, lanes 1-4 and lanes 5-8 in FIG. 1, which are Anti-TNF-. alpha.VH region gene and constant region gene fragments, respectively, with the correct fragment size.

3) The Anti-TNF-alpha VH region gene and the Anti-TNF-alpha constant region gene were connected by the Overlap PCR. The reaction system is shown in table 11; the amplification reaction conditions are as in Table 2.

TABLE 11 Overlap PCR reaction System

The amplified products were subjected to agarose gel electrophoresis and gel recovery, and the results are shown in FIG. 1, panel F, which shows that a band of about 1500bp appeared in FIG. 1, and are in line with expectations.

(4) Anti-TNF-alpha vector construction

The pBudCE4.1 vector fragment was amplified using P11, P12. The sequences of the primers P11 and P12 are as follows:

P11：5’-TCTCGAGTCCACTGTGCTGGTACC-3’(SEQ ID NO:27)

P12：5’-GAAGGTAAGCCTATCCCTAACCCT-3’(SEQ ID NO:28)

1) the pBudCE4.1 vector fragment was PCR amplified. The reaction system is shown in table 12; the amplification reaction conditions are as in Table 6.

TABLE 12 pBudCE4.1 vector fragment Gene amplification reaction System

The amplified products were subjected to agarose gel electrophoresis and gel recovery, and the results are shown in FIG. 1, panel G, which shows that a band of about 5000bp appeared in FIG. 1, and are in line with expectations.

2) The Anti-TNF-alpha heavy chain gene and the Anti-TNF-alpha light chain recombinant pBudCE4.1 plasmid are subjected to homologous recombination. The reaction system is shown in Table 13.

TABLE 13 homologous recombination reaction System

The reaction was carried out at 37 ℃ for 30 min.

3) Transformation of recombinant product

The experimental procedure was as above.

4) And (5) PCR identification of the bacterial liquid. The reaction system is shown in Table 14.

TABLE 14 PCR reaction system for bacterial liquid

The amplified products were subjected to agarose gel electrophoresis and gel recovery, and the results are shown in lanes 5-9 of D in FIG. 1, and lanes 5-9 of D in FIG. 1 show that a band appeared at 1500bp, which is expected, and plasmid minification was performed according to the procedures of the plasmid minification kit.

5) Sequencing identification

The Anti-TNF-alpha heavy chain gene and Anti-TNF-alpha light chain gene sequences are consistent with the target gene sequences. The sequencing results are shown in FIG. 2, FIG. 3 and FIG. 4. FIG. 2 is a forward sequencing map of the TNF- α light chain gene of Anti-TNF- α/PD-1Crossmab, as shown in SEQ ID NO:12, the sequence of the TNF- α light chain gene after being linked to a signal peptide is shown in SEQ ID NO:15, the amino acid sequence of the Anti-human TNF- α light chain variable region of the Anti-TNF- α/PD-1 bispecific antibody is shown in SEQ ID NO:4, and the nucleotide sequence encoding the amino acid sequence of SEQ ID NO:4 is shown in SEQ ID NO: 8; FIG. 3 is a forward sequencing map of the TNF-alpha heavy chain gene of Anti-TNF-alpha/PD-1 Crossmab, FIG. 4 is a reverse sequencing map of the Anti-TNF-alpha heavy chain gene of the bispecific antibody, as shown in SEQ ID NO. 11, the sequence of the TNF-alpha heavy chain gene after being connected with the signal peptide is shown in SEQ ID NO. 16, the amino acid sequence of the Anti-human TNF-alpha heavy chain variable region of the Anti-TNF-alpha/PD-1 bispecific antibody is shown in SEQ ID NO. 3, and the nucleotide sequence of the amino acid sequence of the coding SEQ ID NO. 3 is shown in SEQ ID NO. 7.

Example 2: Anti-PD-1 plasmid construction of bispecific antibody

(1) Anti-PD-1 light chain vector construction

The Anti-PD-1 light chain gene is amplified by using P13 and P14, a pBudCE4.1 vector fragment is amplified by using P5 and P6, and the primer sequences of P13 and P14 are as follows:

P13：5’-GACCCAAGCTTGCATTCCTGCGCCACCATGGCTCCCGTG-3’(SEQ ID NO:29)

P14：5’-AAGTACTGCTTAAGATCGATGTCGACTCGAGTCAGCAGGACTTGGGC-3’(SEQ ID NO:30)

1) and (3) amplifying an Anti-PD-1 light chain gene. The reaction system is shown in Table 15; the amplification reaction conditions are shown in Table 2.

TABLE 15 Anti-TNF-alpha VL region Gene amplification reaction System

2) The pBudCE4.1 vector fragment was PCR amplified. The reaction system is shown in table 5; the amplification reaction conditions are shown in Table 6.

The amplified products were subjected to agarose gel electrophoresis and gel recovery, the results are shown in FIG. 5, lane A, lane 1-4, lane 5-8, lane A, shows that the PCR amplified pBudCE4.1 vector fragment, lane 5-8, lane A, shows that band appeared around 600bp, and it is expected.

3) The Anti-PD-1 light chain gene is subjected to homologous recombination with a vector. The reaction system is shown in Table 16.

TABLE 16 homologous recombination reaction System

The reaction was carried out at 37 ℃ for 30 min.

4) Transformation of recombinant product

The experimental procedure was as above.

5) And (5) PCR identification of the bacterial liquid. The reaction system is shown in Table 17.

TABLE 17 PCR reaction system for bacterial liquid

The amplified products were subjected to agarose gel electrophoresis and gel recovery, and the results are shown in FIG. 5, panel C, and lanes 5-8 of FIG. 5, panel C, show that a band appeared at 600bp, which was expected, and plasmid minification was performed according to the procedures of the plasmid minification kit.

(2) Anti-PD-1 vector construction

The pBudCE4.1 vector fragment was amplified using P15, P16. The sequences of the primers P15 and P16 are as follows:

P15：5’-GGTACCAGCACAGTGGACTCGAGAGTGGAATTCGCCACCATGG-3’(SEQ ID NO:31)

P16：5’-AGGGTTAGGGATAGGCTTACCTTCTCACTTGCCGGGGGAC-3’(SEQ ID NO:32)

1) and (3) carrying out PCR amplification on the Anti-PD-1 heavy chain gene fragment. The reaction system is shown in Table 18; the amplification reaction conditions are as in Table 6.

TABLE 18 pBudCE4.1 vector fragment Gene amplification reaction System

The amplified products were subjected to agarose gel electrophoresis and gel recovery, and the results are shown in panel B of FIG. 5, and lanes 1-4 of panel B of FIG. 5 show that a band of about 1500bp appeared, which was expected.

2) And performing PCR amplification on the Anti-PD-1 light chain recombinant pBudCE4.1 vector fragment. The reaction system is shown in Table 19, and the amplification reaction conditions are the same as those in Table 6.

TABLE 19 pBudCE4.1 vector fragment Gene amplification reaction System

The amplified products were subjected to agarose gel electrophoresis and gel recovery, and the results are shown in panel B of FIG. 5, and lanes 5-8 of panel B of FIG. 5 show that a band appeared around 5000bp, which was expected.

3) The Anti-PD-1 heavy chain gene and the Anti-PD-1 light chain recombinant pBudCE4.1 plasmid are subjected to homologous recombination. The reaction system is shown in Table 20.

TABLE 20 homologous recombination reaction System

The reaction was carried out at 37 ℃ for 30 min.

4) Transformation of recombinant product

The experimental procedure was as above.

5) And (5) PCR identification of the bacterial liquid. The reaction system is shown in Table 21.

TABLE 21 PCR reaction system for bacterial liquid

The amplified products were subjected to agarose gel electrophoresis and gel recovery, the results are shown in FIG. 5, panel C, lanes 1-4 of FIG. 5, showing that a band appeared at 1500bp, and plasmid minification was performed according to the procedures of the plasmid minification kit.

6) Sequencing identification

The Anti-PD-1 heavy chain gene and Anti-PD-1 light chain gene sequences are consistent with the target gene sequence. The sequencing results are shown in fig. 6, 7 and 8. FIG. 6 is a PD-1 light chain gene forward sequencing map of Anti-TNF- α/PD-1Crossmab, as shown in SEQ ID NO:10, the sequence of the PD-1 light chain gene sequence after being connected with a signal peptide is shown in SEQ ID NO:13, the Anti-human PD-1 light chain variable region amino acid sequence of the Anti-TNF- α/PD-1 bispecific antibody is shown in SEQ ID NO:2, and the nucleotide sequence encoding the amino acid sequence of SEQ ID NO:2 is shown in SEQ ID NO: 6; FIG. 7 is a forward sequencing map of the PD-1 heavy chain gene of Anti-TNF-alpha/PD-1 Crossmab, FIG. 8 is a reverse sequencing map of the PD-1 heavy chain gene of Anti-TNF-alpha/PD-1 Crossmab, as shown in SEQ ID NO.9, the sequence of the PD-1 heavy chain gene after being connected with a signal peptide is shown in SEQ ID NO. 14, the amino acid sequence of the Anti-human PD-1 heavy chain variable region of the Anti-TNF-alpha/PD-1 bispecific antibody is shown in SEQ ID NO. 1, and the nucleotide sequence of the amino acid sequence coding the SEQ ID NO. 1 is shown in SEQ ID NO. 5.

Example 3: bispecific antibody expression purification

(1) Suspension cell culture

Recovering 293F suspension cells at 37 ℃ and 8% CO₂The cells were cultured in a shaker at 120rpm for five generations using OPM-293 CD05 Medium until the cell state was stable, and the cell viability was observed to be greater than 90% by trypan blue staining, and then 1X 10⁶Carrying out passage on the cells at a cell/ml density, and after passage for 12-16h, allowing the cell density to reach 1.5 multiplied by 10⁶Transfection was performed at individual cells/ml.

(2) Cell transfection

The dosage of Anti-PD-1 plasmid and Anti-TNF-alpha plasmid is 2 mug/ml, the dosage of PEI transfection reagent is 5 mug/ml, and the dosage of plasmid and transfection reagent is calculated according to the volume of suspension cells. Adding Anti-PD-1 plasmid and Anti-TNF-alpha plasmid into 5ml OPM-293 CD05 Medium, mixing, and standing for 20 min. The PEI transfection reagent was similarly added to 5ml of OPM-293 CD05 Medium, gently mixed, and allowed to stand for 20 min. The plasmid and the transfection reagent are mixed gently, and after standing for 5min, the mixture is added into the suspension cells, and then the suspension cells are put back into the shaking table again for culture.

(3) Protein purification

Seventh day after cell transfection, cells were collected and centrifuged at 13000rpm, and the supernatant was harvested. The method adopts ProteinA affinity chromatography for purification, and comprises the following specific steps:

1) the ProteinA column was mounted on the purification system and column equilibrated with equilibration buffer, about 10 column volumes, at a flow rate of 1 mL/min.

2) The supernatant from the filtration and impurity removal was purified by a ProteinA column at a flow rate of 0.5mL/min, and the flow-through was collected.

3) After the supernatant had passed through the ProteinA column, the column was washed with equilibration buffer, approximately 10 column volumes to baseline plateau.

4) Then, the antibody was collected by eluting with an eluent at a flow rate of 1 mL/min.

The purified anti-TNF-alpha/PD-1 bispecific antibody was identified by SDS-PAGE. The anti-TNF-alpha/PD-1 bispecific antibody has a band size of about 180kDa under non-reducing conditions, and heavy chain antibodies of about 50kDa and light chain antibodies of about 25kDa under reducing conditions, as shown in FIG. 9.

Example 4: biacore X100 analysis of bispecific antibody affinities

(1) Anti-Human IgG (Fc) antibody was immobilized on a CM5 sensor chip using 10mM coupling reagent, followed by ethanolamine blocking of surface residual activating groups, as required by the Biacore X-100 coupling kit protocol.

(2) The purified Anti-TNF-alpha/PD-1 Crossmab is used as a ligand, adalimumab and nivolumab are used as positive antibodies, and the value of the affinity constant is determined by the injection concentration of 20 mu g/ml and the contact time of 300 s.

(3) TNF-alpha or PD-1 antigen diluted by HEPES buffer solution is respectively used as an analyte, the initial concentration is 400nM or 500nM for dilution by multiple, 7-9 different concentrations are diluted in total, and the analytes with different concentrations sequentially flow through a chip (30 mu l/min), so as to respectively obtain a signal curve. 1 cycle for each concentration, 3M MgCl was used after 1 cycle₂The chip is regenerated to return to the original unbound state.

(4) The results of the experiment were analyzed using BIAcore X-100System software. The affinity and kinetic parameters of the antibody were obtained.

The results show that kinetic data of Anti-TNF-. alpha./PD-1 CrossMab binding to PD-1 antigen and TNF-. alpha.antigen are shown in Table 22. Among them, the KD of Anti-TNF- α/PD-1CrossMab to PD-1 antigen was 17.3nM, and the KD of Anti-TNF- α/PD-1CrossMab to TNF- α antigen was 0.0897nM, and the results are shown in fig. 10.

TABLE 22 SPR experimental data

Example 5: inhibition of TNF-alpha cytotoxicity by Anti-TNF-alpha/PD-1 Crossmab

(1) Cultured L929 cells were digested and centrifuged, counted in complete medium suspension and adjusted to 6X 10 concentration⁴cells/mL, seeded in 96-well plates, 6 replicates, 100. mu.L/well; the culture was continued for 12 h.

(2) Anti-TNF- α/PD-1CrossMab, positive control adalimumab and negative control human IgG diluted gradient concentrations of 0.1ng/mL, 1ng/mL, 10ng/mL, 100ng/mL, 1 μ g/mL, 10 μ g/mL, 100 μ L/well with complete medium containing TNF α at a concentration of 10ng/mL and 5 μ g/mL actinomycin D were added to the corresponding wells; meanwhile, a blank group (without cells and culture medium) and a cell control group (with cells and without TNF alpha) and a TNF-alpha control group (with cells and with TNF alpha) are arranged, and the culture is continued for 24 hours.

(3) MTT detection was performed.

The results show that both Anti-TNF-alpha/PD-1 Crossmab and Adalimumab (Adalilimumab) can effectively neutralize TNF-alpha in the culture medium, the cell status is consistent with that of a blank group, the IC50 values are 0.3864nM and 0.2826nM respectively, which indicates that the Anti-TNF-alpha/PD-1 Crossmab has the activity equivalent to that of Adalimumab, human IgG has no TNF-alpha neutralizing capacity, and the cells show a shrinking and apoptosis state, and the results are shown in FIG. 11 and FIG. 12.

Example 6: activation of T cells by Anti-TNF-alpha/PD-1 CrossMab

(1) PBMC extraction

Sucking fresh peripheral blood into a clean and sterile centrifuge tube, adding PBS (phosphate buffer solution) with the same volume into the centrifuge tube, taking 4ml of lymphocyte separation liquid placed at room temperature into a 15ml sterilized centrifuge tube, sucking 6ml of diluted blood, slowly and uniformly adding the diluted blood to the upper layer of the lymphocyte separation liquid along the wall of the centrifuge tube, and centrifuging for 30min in a 750g horizontal centrifuge. After the centrifugation, the liquid in the centrifuge tube was divided into four layers, and the cells in the cloud layer were aspirated by a 1ml pipette tip into another clean centrifuge tube. 5ml of PBS was added to the centrifuge tube containing PBMC, and after thoroughly mixing, 500g was centrifuged for 10min, and the supernatant was discarded. Adding an appropriate amount of RPMI-1640 culture medium into the PBMC to count cells, and performing subsequent experiments or freezing according to the experiment requirements.

(2) Dendritic cell induction

GM-CSF and IL-4 were added to PBMC of (1) at a concentration of 1000U/ml, and the cells were cultured at 37 ℃ in an incubator with the medium changed every other day and the cytokine (GM-CSF and IL-4) concentration was unchanged. On the sixth day, the medium containing 50ng/mL TNF-. alpha.was changed, and after 24 hours, the cells were harvested and subjected to flow assay for cell maturation.

(3) Magnetic bead sorting CD4⁺T cells

PBMC were extracted according to the same experimental procedure as in (1). Using Meitian and whirlwind CD4⁺Sorting CD4 using T cell magnetic bead sorting kit⁺T cells, cell sort purity by flow assay.

(4) Mixed lymphocyte reaction

Will CD4⁺And (3) co-culturing the T and the dendritic cells at a ratio of 10:1, respectively adding negative control human IgG, positive control nivolumab and Anti-TNF-alpha/PD-1 Crossmab with different concentrations for treatment, and detecting the content of IFN-gamma in the supernatant by using an ELISA kit after 48 hours.

The results show that the expression rates of PD-L1 and CD86 on the surface of dendritic cells were identified by flow, and are 77.5% and 87.7%, respectively, indicating that the dendritic cells have matured, as shown in fig. 13. Flow assay CD4⁺The purity of the T cells is 96.8%, the purity is higher, and the T cells meet the experimental requirements and are shown in figure 14. Dendritic cell PD-L1 vs CD4 in mixed lymphoid reactions⁺T cells generate immunosuppression, positive controls of nivolumab and Anti-TNF-alpha/PD-1 Crossmab can activate T cell activity, EC50 is 0.2117 mu g/ml and 0.1591 mu g/ml respectively, and control group human IgG has no cell activation capacity, as shown in FIG. 15.

Example 7: evaluation of Anti-tumor Activity of Anti-TNF-alpha/PD-1 CrossMab

(1) Screening of tumor cell lines with high expression of PD-L1 by flow cytometry, and screening five tumor cell lines with high expression of PD-L1, wherein the tumor cell lines comprise human breast cancer cell MDA-MB-231 cell lines, human colon cancer cell HT-29 cell lines, human non-small cell lung cancer H157 and A549 cell lines and human esophageal cancer TE-13 cell lines. Culturing the five cells, wherein the culture medium is a DMEM complete culture medium containing 10% fetal calf serum, when the cell density is paved at the bottom and 80% is subjected to trypsinization and passage, and when the cells grow to the logarithmic phase, 100 ten thousand cells are respectively taken and analyzed by flow cytometry for the expression condition of PD-L1 on the surface of the tumor cells.

(2) Screening of TNF-alpha proliferation promoting tumor cell line by MTT cell proliferation experiment

1) Culturing human breast cancer cell MDA-MB-231 cell line, human colon cancer cell HT-29 cell line and human non-small cell lung cancer A549 cell line, after the cells grow to logarithmic growth phase, pancreatin digesting and centrifuging the cells, wherein the MDA-MB-231 cell and the A549 cell are prepared with the density of 5 × 10⁴Cell suspension of individual cells/ml, HT-29 cell suspension density 1X 10⁵Each cell/ml was plated in a 96-well plate, and 100. mu.l of cell suspension per well was cultured in a 37 ℃ cell culture chamber.

2) After the cells adhere to the wall on the next day, a complete culture medium containing 200ng/ml of TNF-alpha is prepared, then 13 gradients are diluted by two-fold concentration gradients, the complete culture medium containing TNF-alpha with different concentrations is added into corresponding wells, each well is 100 mu l, and the culture is continued for 24 h.

3) After 24 hours, the 96-well plate was removed, 10. mu.l of MTT solution was added to each well, the plate was further placed in an incubator and cultured for 2 to 4 hours, the 96-well plate was removed, the solution in the well was aspirated by a syringe, 100. mu.l of DMSO was added to each well to dissolve formazan deposited at the bottom of the well, and the absorbance value was measured at a wavelength of 490 nm.

4) Finally, Graphpad software was used to process the experimental data and calculate the proliferation-promoting rate of tumor cells by TNF-alpha at different concentrations.

(3)3D tumor sphere cytotoxicity assay

1) Screening out high-expression PD-L1 and tumor cell lines with TNF-alpha proliferation promoting effect through the steps (1) and (2), culturing the cells to a logarithmic growth phase, carrying out trypsinization and centrifugation, adding complete culture medium containing 3.125ng/ml TNF-alpha to suspend the cells, and preparing the cells with the cell density of 7.5 multiplied by 10⁴Preparing 3D tumor balls (3000 cells/40 mu l of each drop) by a hanging drop method, standing in an incubator at 37 ℃ for 3 days to form balls, collecting the tumor balls after three days, adding calcein with the final concentration of 4.5 mu M and PI with the final concentration of 1.5 mu M, incubating in the incubator at 37 ℃ for 3 hours, and photographing by using a laser confocal microscope to identify the diameter size and the cell viability of the tumor balls.

2) PBMC were extracted according to the same procedure as in (1) of example 6.

3) PBMC are activated. One day before PBMC extraction, a PBS solution containing 2. mu.g/ml CD3 antibody, 2. mu.g/ml CD28 antibody and 300U/ml IL-2 was added to a T75 cell flask to a final volume of 10ml and incubated overnight at 4 ℃. The next day, the liquid was decanted, freshly extracted PBMCs were added and placed in a cell culture chamber for 24h activation.

4) Tumor spheres were collected in ultra-low adsorption 6-well plates, 10 tumor spheres per well, and activated PBMC (3X 10 per well) was added⁵Individual cells) and then adding Anti-TNF-alpha/PD-1 Crossmab, PD-1 monoclonal antibody and TNF-alpha monoclonal antibody with different concentrations into different wells respectively, and incubating for 48h in a cell culture box.

5) And (5) staining tumor balls. Calcein was added to each well at a final concentration of 4.5. mu.M and PI at 1.5. mu.M, and the cells were incubated in a cell incubator for 3h and examined for viability by photographing with a fluorescence microscope.

The results showed that the expression rates of PD-L1 on the surface of HT-29, A549 and MDA-MB-231 were 18.3%, 21.2% and 22.4%, respectively, as shown in FIG. 17, the expression rates were relatively high, while the expression rates of PD-L1 on the surface of H157 and TE-13 were low, 5.52% and 6.65%, respectively. And then MTT experiments are carried out on the HT-29, A549 and MDA-MB-231 tumor cells, when the TNF-alpha concentration is in the range of 100ng/ml, the proliferation inhibiting effect is exerted on the A549 cells, no obvious proliferation influence is exerted on the HT-29 cells, but the proliferation promoting effect is exerted on the MDA-MB-231 cells, and when the TNF-alpha concentration is 3.125ng/ml, the maximum proliferation promoting rate is 113%, as shown in figure 18. Therefore, complete medium containing 3.125ng/ml TNF-alpha was used to mimic TNF-alpha in the tumor microenvironment in subsequent tumor sphere cytotoxicity experiments.

As shown in figure 19, the diameter of the tumor sphere is about 200-300 mu m and the size meets the requirements of cytotoxicity experiments through confocal laser photography, and living cells can be seen to emit green fluorescence in the tumor sphere, because non-fluorescent Calcein-AM can penetrate through the living cell membrane and is sheared by intracellular esterase to form green-emitting Calcein, and PI can directly permeate dead cells to enable the dead cells to emit red fluorescence. Tumor cells are more viable and a small number of cells inside may die due to hypoxia and lack of nutrients. In a 3D tumor cell cytotoxicity experiment, as shown in fig. 20, the activity of a single tumor cell was better, and the live cell was stained with calcein dye to show green fluorescence; in the presence of PBMC or Anti-TNF-alpha/PD-1 bispecific antibody only, some cells within the tumor sphere died and showed red fluorescence when stained with PI dye. In the presence of PBMC, Anti-TNF- α/PD-1 bispecific antibody and PD-1mAb each at a concentration of 1 μ M effectively killed MDA-MB-231 tumor cells, promoting tumor cell death. Similarly, mixed solutions of PD-1mAb and TNF- α mAb (0.5 μ M each) also showed comparable cytotoxicity in 3D tumor spheres.

Sequence listing

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cctggcaagg gcctggaatg ggtggccgtg atctggtacg acggctccaa gcggtactac 180

gctgattccg tcaagggaag gttcaccatc tcccgggata acagcaagaa caccctcttc 240

ctgcagatga acagcctgag ggccgaagat accgccgtgt actactgcgc cacaaacgac 300

gattactggg gccagggaac cctggtgaca gtgtcctccc ggaccgtggc cgctccttcc 360

gtgttcatct tccctcctag cgacgagcag ctgaagagcg gcaccgccag cgtggtgtgc 420

ctgctgaaca acttctaccc cagggaggcc aaggtgcagt ggaaggtgga caacgccctc 480

cagagcggca acagccagga gtccgtgacc gagcaggact ccaaggacag cacctactcc 540

ctgtccagca ccctgaccct gtccaaggct gactacgaga agcacaaggt gtacgcctgc 600

gaggtgaccc atcagggcct gtcctccccc gtgaccaagt ccttcaacag gggcgagtgc 660

gacaagaccc acacctgccc tccttgtcct gctcctgagc tgctgggcgg cccttctgtg 720

tttctgttcc ctcctaagcc caaggacacc ctgatgatct ccaggacccc cgaggtgacc 780

tgcgtggtgg tggacgtgtc tcacgaggac cctgaggtga agtttaactg gtacgtggat 840

ggcgtggagg tgcataacgc taagaccaag cctagggagg agcagtacaa ctccacctac 900

agggtggtgt ccgtgctgac cgtgctgcac caggactggc tgaacggcaa ggagtacaag 960

tgcaaggtgt ccaacaaggc cctgcctgct cctatcgaga agaccatctc caaggctaag 1020

ggccagccta gagagcccca ggtgtgcacc ctgcccccct ccagggagga gatgaccaag 1080

aaccaggtgt ccctgagctg cgcggtgaag ggcttctacc cctccgacat cgccgtggag 1140

tgggagtcca acggccagcc cgagaacaac tacaagacca ccccccccgt gctggactcc 1200

gacggctcct tcttcctggt ctccaagctg accgtggaca agtccaggtg gcagcagggc 1260

aacgtgttct cctgctccgt gatgcacgag gccctgcaca accactacac ccagaagtcc 1320

ctgtccctgt cccccggcaa gtga 1344

<210> 10

<211> 633

<212> DNA

<213> Intelligent (Homo sapiens)

<400> 10

gagatcgtgc tgacacagtc ccctgctaca ctgtccctgt cccccggcga gagggctaca 60

ctgagctgtc gggcctccca gtccgtgagc agctacctgg cctggtacca gcagaagcct 120

ggccaggctc ccaggctgct gatctacgac gcctccaaca gggccaccgg catccctgcc 180

aggtttagcg gaagcggcag cggcaccgac ttcaccctga ccatctcctc cctggagccc 240

gaggacttcg ccgtgtacta ctgccagcag tccagcaact ggcccaggac attcggccag 300

ggcaccaagg tggagatcaa ggcctctaca aagggccctt ctgtgttccc tctggcccct 360

tcctctaagt ctacatctgg cggaaccgct gctctgggct gtctggtgaa ggactacttc 420

cctgagcctg tgacagtgtc ttggaactct ggcgctctga cctccggcgt gcacaccttc 480

cctgctgtgc tgcagtcctc tggactgtac tctctgtctt ctgtggtgac cgtgccttct 540

tcctctctgg gcacccagac ctacatctgc aacgtgaacc ataagccttc taacacaaag 600

gtggacaaga aggtggagcc caagtcctgc tga 633

<210> 11

<211> 1356

<212> DNA

<213> Intelligent (Homo sapiens)

<400> 11

gaggtgcagc tggtggagtc tgggggaggc ttggtacagc ccggcaggtc cctgagactc 60

tcctgtgcgg cctctggatt cacctttgat gattatgcca tgcactgggt ccggcaagct 120

ccagggaagg gcctggaatg ggtctcagct atcacttgga atagtggtca catagactat 180

gcggactctg tggagggccg attcaccatc tccagagaca acgccaagaa ctccctgtat 240

ctgcaaatga acagtctgag agctgaggat acggccgtat attactgtgc gaaagtctcg 300

taccttagca ccgcgtcctc ccttgactat tggggccaag gtaccctggt caccgtctcg 360

agtgcctcta caaagggccc ttctgtgttc cctctggccc cttcctctaa gtctacatct 420

ggcggaaccg ctgctctggg ctgtctggtg aaggactact tccctgagcc tgtgacagtg 480

tcttggaact ctggcgctct gacctccggc gtgcacacct tccctgctgt gctgcagtcc 540

tctggactgt actctctgtc ttctgtggtg accgtgcctt cttcctctct gggcacccag 600

acctacatct gcaacgtgaa ccataagcct tctaacacaa aggtggacaa gaaggtggag 660

cccaagtcct gcgacaagac ccacacctgc cctccttgtc ctgctcctga gctgctgggc 720

ggcccttctg tgtttctgtt ccctcctaag cccaaggaca ccctgatgat ctccaggacc 780

cccgaggtga cctgcgtggt ggtggacgtg tctcacgagg accctgaggt gaagtttaac 840

tggtacgtgg atggcgtgga ggtgcataac gctaagacca agcctaggga ggagcagtac 900

aactccacct acagggtggt gtccgtgctg accgtgctgc accaggactg gctgaacggc 960

aaggagtaca agtgcaaggt gtccaacaag gccctgcctg ctcctatcga gaagaccatc 1020

tccaaggcta agggccagcc tagagagccc caggtgtaca ccctgccccc ctgcagggag 1080

gagatgacca agaaccaggt gtccctgtgg tgcctggtga agggcttcta cccctccgac 1140

atcgccgtgg agtgggagtc caacggccag cccgagaaca actacaagac cacccccccc 1200

gtgctggact ccgacggctc cttcttcctg tactccaagc tgaccgtgga caagtccagg 1260

tggcagcagg gcaacgtgtt ctcctgctcc gtgatgcacg aggccctgca caaccactac 1320

acccagaagt ccctgtccct gtcccccggc aagtga 1356

<210> 12

<211> 645

<212> DNA

<213> Intelligent (Homo sapiens)

<400> 12

gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtagggga cagagtcacc 60

atcacttgtc gggcaagtca gggcatcaga aattacttag cctggtatca gcaaaaacca 120

gggaaagccc ctaagctcct gatctatgct gcatccactt tgcaatcagg ggtcccatct 180

cggttcagtg gcagtggatc tgggacagat ttcactctca ccatcagcag cctacagcct 240

gaagatgttg caacttatta ctgtcaaagg tataaccgtg caccgtatac ttttggccag 300

gggaccaagg tggaaatcaa acggaccgtg gccgctcctt ccgtgttcat cttccctcct 360

agcgacgagc agctgaagag cggcaccgcc agcgtggtgt gcctgctgaa caacttctac 420

cccagggagg ccaaggtgca gtggaaggtg gacaacgccc tccagagcgg caacagccag 480

gagtccgtga ccgagcagga ctccaaggac agcacctact ccctgtccag caccctgacc 540

ctgtccaagg ctgactacga gaagcacaag gtgtacgcct gcgaggtgac ccatcagggc 600

ctgtcctccc ccgtgaccaa gtccttcaac aggggcgagt gctga 645

<210> 13

<211> 690

<212> DNA

<213> Intelligent (Homo sapiens)

<400> 13

atggctcccg tgcagctgct gggactgctg gtgctgttcc tgcccgccat gcggtgtgag 60

atcgtgctga cacagtcccc tgctacactg tccctgtccc ccggcgagag ggctacactg 120

agctgtcggg cctcccagtc cgtgagcagc tacctggcct ggtaccagca gaagcctggc 180

caggctccca ggctgctgat ctacgacgcc tccaacaggg ccaccggcat ccctgccagg 240

tttagcggaa gcggcagcgg caccgacttc accctgacca tctcctccct ggagcccgag 300

gacttcgccg tgtactactg ccagcagtcc agcaactggc ccaggacatt cggccagggc 360

accaaggtgg agatcaaggc ctctacaaag ggcccttctg tgttccctct ggccccttcc 420

tctaagtcta catctggcgg aaccgctgct ctgggctgtc tggtgaagga ctacttccct 480

gagcctgtga cagtgtcttg gaactctggc gctctgacct ccggcgtgca caccttccct 540

gctgtgctgc agtcctctgg actgtactct ctgtcttctg tggtgaccgt gccttcttcc 600

tctctgggca cccagaccta catctgcaac gtgaaccata agccttctaa cacaaaggtg 660

gacaagaagg tggagcccaa gtcctgctga 690

<210> 14

<211> 1401

<212> DNA

<213> Intelligent (Homo sapiens)

<400> 14

atggccgtgc tgggactgct gttctgtctg gtgacctttc ctagctgtgt gctgagccag 60

gtgcagctgg tggaaagcgg aggcggagtg gtccagcctg gaaggtccct gaggctcgac 120

tgcaaggcca gcggcatcac cttcagcaac agcggcatgc actgggtgag gcaggctcct 180

ggcaagggcc tggaatgggt ggccgtgatc tggtacgacg gctccaagcg gtactacgct 240

gattccgtca agggaaggtt caccatctcc cgggataaca gcaagaacac cctcttcctg 300

cagatgaaca gcctgagggc cgaagatacc gccgtgtact actgcgccac aaacgacgat 360

tactggggcc agggaaccct ggtgacagtg tcctcccgga ccgtggccgc tccttccgtg 420

ttcatcttcc ctcctagcga cgagcagctg aagagcggca ccgccagcgt ggtgtgcctg 480

ctgaacaact tctaccccag ggaggccaag gtgcagtgga aggtggacaa cgccctccag 540

agcggcaaca gccaggagtc cgtgaccgag caggactcca aggacagcac ctactccctg 600

tccagcaccc tgaccctgtc caaggctgac tacgagaagc acaaggtgta cgcctgcgag 660

gtgacccatc agggcctgtc ctcccccgtg accaagtcct tcaacagggg cgagtgcgac 720

aagacccaca cctgccctcc ttgtcctgct cctgagctgc tgggcggccc ttctgtgttt 780

ctgttccctc ctaagcccaa ggacaccctg atgatctcca ggacccccga ggtgacctgc 840

gtggtggtgg acgtgtctca cgaggaccct gaggtgaagt ttaactggta cgtggatggc 900

gtggaggtgc ataacgctaa gaccaagcct agggaggagc agtacaactc cacctacagg 960

gtggtgtccg tgctgaccgt gctgcaccag gactggctga acggcaagga gtacaagtgc 1020

aaggtgtcca acaaggccct gcctgctcct atcgagaaga ccatctccaa ggctaagggc 1080

cagcctagag agccccaggt gtgcaccctg cccccctcca gggaggagat gaccaagaac 1140

caggtgtccc tgagctgcgc ggtgaagggc ttctacccct ccgacatcgc cgtggagtgg 1200

gagtccaacg gccagcccga gaacaactac aagaccaccc cccccgtgct ggactccgac 1260

ggctccttct tcctggtctc caagctgacc gtggacaagt ccaggtggca gcagggcaac 1320

gtgttctcct gctccgtgat gcacgaggcc ctgcacaacc actacaccca gaagtccctg 1380

tccctgtccc ccggcaagtg a 1401

<210> 15

<211> 702

<212> DNA

<213> Intelligent (Homo sapiens)

<400> 15

atggctcccg tgcagctgct gggactgctg gtgctgttcc tgcccgccat gcggtgtgac 60

atccagatga cccagtctcc atcctccctg tctgcatctg taggggacag agtcaccatc 120

acttgtcggg caagtcaggg catcagaaat tacttagcct ggtatcagca aaaaccaggg 180

aaagccccta agctcctgat ctatgctgca tccactttgc aatcaggggt cccatctcgg 240

ttcagtggca gtggatctgg gacagatttc actctcacca tcagcagcct acagcctgaa 300

gatgttgcaa cttattactg tcaaaggtat aaccgtgcac cgtatacttt tggccagggg 360

accaaggtgg aaatcaaacg gaccgtggcc gctccttccg tgttcatctt ccctcctagc 420

gacgagcagc tgaagagcgg caccgccagc gtggtgtgcc tgctgaacaa cttctacccc 480

agggaggcca aggtgcagtg gaaggtggac aacgccctcc agagcggcaa cagccaggag 540

tccgtgaccg agcaggactc caaggacagc acctactccc tgtccagcac cctgaccctg 600

tccaaggctg actacgagaa gcacaaggtg tacgcctgcg aggtgaccca tcagggcctg 660

tcctcccccg tgaccaagtc cttcaacagg ggcgagtgct ga 702

<210> 16

<211> 1413

<212> DNA

<213> Intelligent (Homo sapiens)

<400> 16

atggccgtgc tgggactgct gttctgtctg gtgacctttc ctagctgtgt gctgagcgag 60

gtgcagctgg tggagtctgg gggaggcttg gtacagcccg gcaggtccct gagactctcc 120

tgtgcggcct ctggattcac ctttgatgat tatgccatgc actgggtccg gcaagctcca 180

gggaagggcc tggaatgggt ctcagctatc acttggaata gtggtcacat agactatgcg 240

gactctgtgg agggccgatt caccatctcc agagacaacg ccaagaactc cctgtatctg 300

caaatgaaca gtctgagagc tgaggatacg gccgtatatt actgtgcgaa agtctcgtac 360

cttagcaccg cgtcctccct tgactattgg ggccaaggta ccctggtcac cgtctcgagt 420

gcctctacaa agggcccttc tgtgttccct ctggcccctt cctctaagtc tacatctggc 480

ggaaccgctg ctctgggctg tctggtgaag gactacttcc ctgagcctgt gacagtgtct 540

tggaactctg gcgctctgac ctccggcgtg cacaccttcc ctgctgtgct gcagtcctct 600

ggactgtact ctctgtcttc tgtggtgacc gtgccttctt cctctctggg cacccagacc 660

tacatctgca acgtgaacca taagccttct aacacaaagg tggacaagaa ggtggagccc 720

aagtcctgcg acaagaccca cacctgccct ccttgtcctg ctcctgagct gctgggcggc 780

ccttctgtgt ttctgttccc tcctaagccc aaggacaccc tgatgatctc caggaccccc 840

gaggtgacct gcgtggtggt ggacgtgtct cacgaggacc ctgaggtgaa gtttaactgg 900

tacgtggatg gcgtggaggt gcataacgct aagaccaagc ctagggagga gcagtacaac 960

tccacctaca gggtggtgtc cgtgctgacc gtgctgcacc aggactggct gaacggcaag 1020

gagtacaagt gcaaggtgtc caacaaggcc ctgcctgctc ctatcgagaa gaccatctcc 1080

aaggctaagg gccagcctag agagccccag gtgtacaccc tgcccccctg cagggaggag 1140

atgaccaaga accaggtgtc cctgtggtgc ctggtgaagg gcttctaccc ctccgacatc 1200

gccgtggagt gggagtccaa cggccagccc gagaacaact acaagaccac cccccccgtg 1260

ctggactccg acggctcctt cttcctgtac tccaagctga ccgtggacaa gtccaggtgg 1320

cagcagggca acgtgttctc ctgctccgtg atgcacgagg ccctgcacaa ccactacacc 1380

cagaagtccc tgtccctgtc ccccggcaag tga 1413

<210> 17

<211> 41

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 17

gacccaagct tgcattcctg cgccatggct cccgtgcagc t 41

<210> 18

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 18

gagcggccac ggtccgtttg atttccacct tggtccc 37

<210> 19

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 19

accaaggtgg aaatcaaacg gaccgtggcc gctc 34

<210> 20

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 20

aagtactgct taagatcgat gtcgatcagc actcgcccct gtt 43

<210> 21

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 21

gcaggaatgc aagcttgggt c 21

<210> 22

<211> 25

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 22

tcgacatcga tcttaagcag tactt 25

<210> 23

<211> 45

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 23

ggtaccagca cagtggactc gagagccatg gccgtgctgg gactg 45

<210> 24

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 24

agggcccttt gtagaggcac tcgagacggt gaccagggta 40

<210> 25

<211> 38

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 25

ctggtcaccg tctcgagtgc ctctacaaag ggcccttc 38

<210> 26

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 26

agggttaggg ataggcttac cttctcactt gccgggggac 40

<210> 27

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 27

tctcgagtcc actgtgctgg tacc 24

<210> 28

<211> 24

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 28

gaaggtaagc ctatccctaa ccct 24

<210> 29

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 29

gacccaagct tgcattcctg cgccaccatg gctcccgtg 39

<210> 30

<211> 47

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 30

aagtactgct taagatcgat gtcgactcga gtcagcagga cttgggc 47

<210> 31

<211> 43

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 31

ggtaccagca cagtggactc gagagtggaa ttcgccacca tgg 43

<210> 32

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 32

agggttaggg ataggcttac cttctcactt gccgggggac 40

Claims (9)

1. A bispecific antibody characterized by: is constructed by combining an anti-human PD-1 antibody and an anti-human TNF-alpha antibody; the construction method comprises the following steps: the Fc segment of the anti-human PD-1 heavy chain and the Fc segment of the anti-human TNF-alpha heavy chain are exchanged by a CH1 structural domain of the anti-human PD-1 heavy chain and a CL structural domain of the anti-human PD-1 light chain through site-specific mutation respectively to realize the correct assembly of the antibody light chain and the antibody heavy chain; the heavy chain variable region of the anti-human PD-1 antibody is an amino acid sequence shown by SEQ ID NO. 1, or the heavy chain variable region of the anti-human PD-1 antibody is an amino acid sequence coded by a nucleotide sequence shown by a sequence SEQ ID NO. 5, or the heavy chain variable region of the anti-human PD-1 antibody is a nucleotide sequence coded by an amino acid sequence shown by the sequence SEQ ID NO. 1; the light chain variable region of the anti-human PD-1 antibody is an amino acid sequence shown by SEQ ID NO. 2, or the light chain variable region of the anti-human PD-1 antibody is an amino acid sequence coded by a nucleotide sequence shown by SEQ ID NO. 6, or the light chain variable region of the anti-human PD-1 antibody is a nucleotide sequence coded by an amino acid sequence shown by SEQ ID NO. 2; the heavy chain variable region of the anti-human TNF-alpha antibody is an amino acid sequence shown by SEQ ID NO. 3, or the heavy chain variable region of the anti-human TNF-alpha antibody is an amino acid sequence coded by a nucleotide sequence shown by SEQ ID NO. 7, or the heavy chain variable region of the anti-human TNF-alpha antibody is a nucleotide sequence coded by an amino acid sequence shown by SEQ ID NO. 3; the variable region of the light chain of the anti-human TNF-alpha antibody is an amino acid sequence shown in SEQ ID NO. 4, or the variable region of the light chain of the anti-human TNF-alpha antibody is an amino acid sequence coded by a nucleotide sequence shown in SEQ ID NO. 8, or the variable region of the light chain of the anti-human TNF-alpha antibody is a nucleotide sequence coded by an amino acid sequence shown in SEQ ID NO. 4.

2. The bispecific antibody of claim 1, characterized in that: the light chain coding sequence of the anti-human PD-1 antibody is a nucleotide sequence shown as SEQ ID NO. 10; the heavy chain coding sequence of the anti-human PD-1 antibody is a nucleotide sequence shown as SEQ ID NO. 9.

3. The bispecific antibody of claim 1, characterized in that: the light chain coding sequence of the anti-human TNF-alpha antibody is a nucleotide sequence shown in SEQ ID NO. 12; the heavy chain coding sequence of the anti-human TNF-alpha antibody is the nucleotide sequence shown in SEQ ID NO. 11.

4. A polynucleotide encoding the bispecific antibody of any one of claims 1-3.

5. An expression vector, characterized in that: the bispecific antibody of any one of claims 1 to 3 is expressed, wherein the heavy chain variable region of the anti-human PD-1 antibody is the amino acid sequence shown in SEQ ID NO. 1, the light chain variable region of the anti-human PD-1 antibody is the amino acid sequence shown in SEQ ID NO. 2, the heavy chain variable region of the anti-human TNF-alpha antibody is the amino acid sequence shown in SEQ ID NO. 3, and the light chain variable region of the anti-human TNF-alpha antibody is the amino acid sequence shown in SEQ ID NO. 4; the vector plasmid of the expression vector is pBudCE4.1 plasmid.

6. A host cell, characterized in that: the host cell comprising the polynucleotide of claim 4 or the expression vector of claim 5.

7. Use of a bispecific antibody according to any one of claims 1 to 3 for the preparation of a medicament for the treatment of tumor cells expressing PD-L1 and being pro-proliferative by TNF- α; the tumor is breast cancer.

8. Use of a bispecific antibody according to any one of claims 1 to 3 for the preparation of a PD-L1-positive tumor medicament or a medicament for the treatment of tumors that are promoted for proliferation by TNF- α; the tumor is breast cancer.

9. A pharmaceutical composition comprising a bispecific antibody according to any one of claims 1 to 3.

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