CN117247457A - Bispecific antibody targeting HER2 and PD-L1 as well as preparation method and application thereof - Google Patents

Bispecific antibody targeting HER2 and PD-L1 as well as preparation method and application thereof Download PDF

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CN117247457A
CN117247457A CN202210657725.2A CN202210657725A CN117247457A CN 117247457 A CN117247457 A CN 117247457A CN 202210657725 A CN202210657725 A CN 202210657725A CN 117247457 A CN117247457 A CN 117247457A
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刘婵娟
郎国竣
符智祥
许彩云
司远青
曹静丽
张震
闫闰
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Sanyou Biopharmaceuticals Co Ltd
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Sanyou Biopharmaceuticals Co Ltd
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Priority to PCT/CN2023/097835 priority patent/WO2023236844A1/en
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Abstract

The invention discloses a bispecific antibody targeting HER2 and PD-L1, and a preparation method and application thereof. The bispecific antibody comprises a first protein functional region that targets HER2 and a second protein functional region that targets PD-L1; the first protein functional region comprises a VL comprising three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the sequence shown as SEQ ID NO. 3 and a VH comprising three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the sequence shown as SEQ ID NO. 4; the second protein functional region comprises a VHH comprising three complementarity determining regions CDR1, CDR2 and CDR3 comprised in the sequence as shown in SEQ ID No. 2. The bispecific antibody has better purity and thermal stability; binding activities to both targets are close to corresponding monoclonal antibodies, the activity of blocking PD-1/PD-L1 is equivalent to that of the corresponding monoclonal antibodies, and the activity of inhibiting SK-BR-3 cell proliferation is equivalent to that of the corresponding monoclonal antibodies; and simultaneously, the compound has good ADCC activity and in-vivo tumor inhibition activity.

Description

Bispecific antibody targeting HER2 and PD-L1 as well as preparation method and application thereof
Technical Field
The invention belongs to the field of biological medicine, and in particular relates to a bispecific antibody targeting HER2 and PD-L1, and a preparation method and application thereof.
Background
Human epidermal growth factor receptor 2 (human epidermal growth factor receptor, HER 2) belongs to the family of epidermal growth factor receptor (Epidermal growth factor receptor, EGFR) proteins, and through heterodimerization with other members of the EGFR family or homodimerization due to overexpression of itself, tyrosine residues on the cytoplasmic domain can autophosphorylate and initiate a range of signaling pathways for cell proliferation and tumorigenesis (Oh, D.Y. and Y.J. Bang. (2020.) "HER2-targeted therapies-a role beyond breast cancer." Nat Rev Clin Oncol 17 (1): 33-48.). HER2 is only expressed at low levels in a very small number of normal tissues. However, studies have shown that HER2 is overexpressed in cancer types such as breast, gastric, ovarian and endometrial cancers, and that its overexpression is correlated with tumor malignancy and poor prognosis (Normanno, N., et al (2005), "The ErbB receptors and their ligands in cancer: an overview.," Curr Drug Targets (3): 243-257.). Based on HER2 expression profile and biological function in tumorigenesis development, it has become a widely validated tumor therapeutic target, and a number of HER 2-targeting monoclonal antibody drugs have been marketed in bulk. However, less than 30% of patients respond to it, and most of the initially responding patients develop treatment tolerance or relapse within one year (Gu, c.l., et al (2021), "Bispecific antibody simultaneously targeting PD and HER2 inhibits tumor growth via direct tumor cell killing in combination with PD1/PDL1 block and HER2 inhibition.," Acta Pharmacol sin.).
PD-L1 is an immunoglobulin superfamily member, a type I transmembrane glycoprotein expressed in macrophages, certain activated T and B cells, DCs, and certain epithelial cells (Sharpe, A.H., et al (2007), "The function of programmed cell death 1and its ligands in regulating autoimmunity and infection.," Nat Immunol 8 (3): 239-245.). In addition, PD-L1 is also highly expressed by tumor cells to escape an adaptive anti-tumor immune response (Ohaegbulam, K.C., et al (2015), "Human cancer immunotherapy with antibodies to the PD-1and PD-L1 pathway.," Trends Mol Med 21 (1): 24-33.). In tumor microenvironment, up-regulation of PD-L1 expression can directly inhibit anti-tumor response of T cells through PD-1 signaling pathway, and mediate immune escape of tumor cells. In recent years, development of blocking-type antibody drugs against the PD-1/PD-L1 signaling pathway has attracted a great deal of attention, and a number of PD-1 or PD-L1 targeting monoclonal antibody drugs have been marketed in batches and have achieved clinically exciting therapeutic effects. Nevertheless, most patients do not respond to PD-1/PD-L1 blocked immunotherapy, and some initially responding patients also develop treatment tolerance.
Bispecific antibodies are recombinant antibodies that can bind two antigens or different epitopes of the same antigen simultaneously, not only can redirect immune effector cells to tumor tissue to achieve specific killing of the tumor by immune cells, but also can simultaneously inhibit different tumor-promoting signaling pathways on the same tumor cell to exert synergistic tumor-inhibiting functions (Labrijn, a.f., et al (2019), "Bispecific antibodies: amechanistic review of the pipeline.," Nat Rev Drug Discov (8): 585-608 "). Therefore, in order to increase the therapeutic response rate of HER2 positive tumors and overcome tumor therapeutic tolerance, there is a need to develop bispecific antibodies targeting HER2 and PD-L1.
Disclosure of Invention
In order to solve the technical problems, the invention provides a bispecific antibody targeting HER2 and PD-L1, and a preparation method and application thereof. The bispecific antibody of the invention has better physicochemical properties, better purity and thermal stability; the functional activity aiming at the double targets can be reserved, the binding activities of the two targets are close to that of the corresponding monoclonal antibodies (namely, the binding capacity of the monoclonal antibodies to PD-L1 and HER2 is equal to or better than that of the monoclonal antibodies alone), the activity of blocking the PD-1/PD-L1 is equal to that of the corresponding monoclonal antibodies, and the proliferation inhibition activity of SK-BR-3 cells is equal to that of the corresponding monoclonal antibodies; meanwhile, the antibody shows good ADCC activity, which is obviously superior to the corresponding monoclonal antibody.
In one aspect, the invention provides a bispecific antibody comprising a first protein functional region that targets HER2 and a second protein functional region that targets PD-L1; wherein the first protein functional region comprises a light chain variable region (VL) comprising three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the sequence set forth in SEQ ID NO. 3 and a heavy chain variable region (VH) comprising three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the sequence set forth in SEQ ID NO. 4;
the second protein functional region comprises a VHH, wherein the VHH comprises three complementarity determining regions CDR1, CDR2 and CDR3 comprised in the sequence as shown in SEQ ID NO. 2.
In a preferred embodiment, the VL comprises the sequence set forth in SEQ ID NO. 3, or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% sequence identity thereto; and said VH comprises an amino acid sequence as set forth in SEQ ID No. 4, or having at least 90%, 95%, 96%, 97%, 98%, 99% sequence identity thereto.
In a preferred embodiment, the VHH comprises the sequence shown as SEQ ID NO. 2, or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% sequence identity thereto.
In the bispecific antibody as described above, the first protein functional region preferably comprises a Fab;
wherein the Fab is operably linked to the VHH, e.g. the VHH is linked to the N-terminus of the light chain variable region, or the N-terminus of the heavy chain variable region, or the C-terminus of the light chain constant region in the Fab, or the Fab and the VHH are respectively operably linked to the N-terminus of the two hinge regions of the Fc region.
In the bispecific antibodies described above, the first protein functional region is preferably an IgG, and in a preferred embodiment, the VHH is operably linked to the C-terminus of the IgG heavy chain constant region.
Bispecific antibodies as described above preferably contain two first polypeptide chains and two second polypeptide chains.
In a certain preferred embodiment of the present invention,
the first polypeptide chain is represented by the formula:
the VH-CH 1-hinge region-CH 2-CH3 preferably comprises a sequence as shown in SEQ ID NO:9,
and the second polypeptide chain is of the formula:
the VHH-linker-VL-CL preferably comprises the sequence shown in SEQ ID NO. 10.
In a certain preferred embodiment of the present invention,
the first polypeptide chain is represented by the formula:
the VH-CH 1-hinge region-CH 2-CH 3-linker-VHH preferably comprises a sequence as shown in SEQ ID NO. 6,
and the second polypeptide chain is of the formula:
VL-CL preferably comprises the sequence shown in SEQ ID NO. 7.
In a certain preferred embodiment of the present invention,
the first polypeptide chain is represented by the formula:
VHH-linker-VH-CH 1-hinge region-CH 2-CH3, preferably comprising a sequence as shown in SEQ ID NO. 8,
and the second polypeptide chain is of the formula:
VL-CL preferably comprises the sequence shown in SEQ ID NO. 7.
In a certain preferred embodiment of the present invention,
the first polypeptide chain is represented by the formula:
the VH-CH 1-hinge region-CH 2-CH3 preferably comprises a sequence as shown in SEQ ID NO:9,
and the second polypeptide chain is of the formula:
VL-CL-linker-VHH preferably comprises the sequence shown as SEQ ID NO. 11.
Bispecific antibodies as described above preferably contain a first polypeptide chain, a second polypeptide chain, and a third polypeptide chain, wherein the first polypeptide chain is of the formula:
VHH-hinge region-CH 2-CH3;
the second polypeptide chain is represented by the formula:
VH-CH 1-hinge region-CH 2-CH3;
the third polypeptide chain is represented by the formula:
VL-CL。
in a preferred embodiment, the first polypeptide chain comprises the sequence shown in SEQ ID NO. 12 and the second polypeptide chain comprises the sequence shown in SEQ ID NO. 13 and the third polypeptide chain comprises the sequence shown in SEQ ID NO. 7.
In a preferred embodiment, the first polypeptide chain comprises the sequence shown as SEQ ID NO. 14 and the second polypeptide chain comprises the sequence shown as SEQ ID NO. 15 and the third polypeptide chain comprises the sequence shown as SEQ ID NO. 7.
The invention also provides an isolated nucleic acid encoding a bispecific antibody according to the invention.
The invention also provides a recombinant expression vector comprising an isolated nucleic acid according to the invention.
The invention also provides a transformant comprising an isolated nucleic acid according to the invention or a recombinant expression vector according to the invention.
The host cell of the transformant is preferably a prokaryotic cell, preferably an E.coli cell such as TG1, BL21, or a eukaryotic cell, preferably a HEK293 cell or a CHO cell, as described above.
Once the expression vector or DNA sequence has been prepared for expression, the expression vector may be transfected or introduced into a suitable host cell. Various techniques may be used to achieve this, such as protoplast fusion, calcium phosphate precipitation, electroporation, retroviral transduction, viral transfection, gene gun, lipid-based transfection, or other conventional techniques. In the case of protoplast fusion, the cells are incubated in medium and screened for appropriate activity. Methods and conditions for culturing the resulting transfected cells and for recovering the resulting antibody molecules are known to those skilled in the art and may be varied or optimized depending on the particular expression vector and mammalian host cell used, based on the present specification and methods known in the art. Alternatively, cells that have stably incorporated DNA into their chromosomes can be selected by introducing one or more markers that allow selection of transfected host cells. The marker may, for example, provide prototrophy, biocidal resistance (e.g., antibiotics) or heavy metal (e.g., copper) resistance to an auxotrophic host, and the like. The selectable marker gene may be directly linked to the DNA sequence to be expressed or introduced into the same cell by co-transformation. Additional elements may also be required for optimal synthesis of mRNA. These elements may include splicing signals, transcriptional promoters, enhancers, and termination signals.
The invention also provides a method for preparing a bispecific antibody, which comprises culturing a transformant according to the present invention, and obtaining the bispecific antibody from the culture.
The invention also provides a pharmaceutical composition comprising a bispecific antibody according to the invention, and a pharmaceutically acceptable carrier.
In a preferred embodiment, the pharmaceutical composition further comprises as active ingredient other anti-tumor antibodies and/or one or more of the group consisting of hormonal preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic agents, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules and vaccines.
In some embodiments, the pharmaceutical compositions or pharmaceutical formulations of the present invention comprise a suitable pharmaceutically acceptable carrier, e.g., a pharmaceutical excipient, such as pharmaceutically acceptable carriers, pharmaceutically acceptable excipients, including buffers, as known in the art. As used herein, "pharmaceutically acceptable carrier" or "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, isotonic and absorption delaying agents, and the like that are physiologically compatible. Pharmaceutically acceptable carriers suitable for use in the present invention may be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. When the pharmaceutical composition is administered intravenously, water is a preferred carrier. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. See also "Handbook of Pharmaceutical Excipients", fifth edition, r.c. rowe, p.j. Seskey and s.c. owen, pharmaceutical Press, london, chicago, for the use of excipients and their use. The composition may also contain minor amounts of wetting or emulsifying agents, or pH buffering agents, if desired. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained release formulations and the like. The pharmaceutical preparations or compositions comprising the antibodies of the invention or antigen binding fragments thereof of the invention of the desired purity may be prepared by mixing the antibodies with one or more optional pharmaceutical excipients (Remington's Pharmaceutical Sciences, 16 th edition, osol, code a. (1980)), preferably in the form of a lyophilized preparation or an aqueous solution. The pharmaceutical compositions or formulations of the present invention may also contain more than one active ingredient which is required for the particular indication being treated, preferably those active ingredients having complementary activities which do not adversely affect each other. For example, it may be desirable to also provide other anti-infective active ingredients, such as other antibodies, anti-infective active agents, small molecule drugs or immunomodulators, and the like. The active ingredients are suitably present in combination in an amount effective for the intended use. Sustained release formulations can be prepared. Suitable examples of sustained-release formulations include semipermeable matrices of solid hydrophobic polymers containing the antibody of the invention, or antigen-binding fragments thereof, which matrices are in the form of shaped articles, e.g., films, or microcapsules.
The invention also provides an antibody drug conjugate comprising a cytotoxic agent or tag, and a bispecific antibody of the invention; the cytotoxic agent is preferably MMAF or MMAE; the label is preferably a fluorescent agent.
The invention also provides a kit comprising a bispecific antibody according to the invention, a pharmaceutical composition according to the invention, or an antibody drug conjugate according to the invention.
The kit preferably further comprises (i) a device for administering a bispecific antibody or antibody drug conjugate or pharmaceutical composition; and/or (ii) instructions for use.
The invention also provides a kit comprising a kit a and a kit B, wherein:
the kit a contains a bispecific antibody according to the invention, a pharmaceutical composition according to the invention, or an antibody drug conjugate according to the invention.
The kit B contains other anti-tumor antibodies or pharmaceutical compositions comprising the other anti-tumor antibodies, and/or one or more of the group consisting of hormonal preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic agents, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules, and vaccines.
The invention also provides a method for detecting a non-diagnostic purpose of a specific antigen comprising detection using the bispecific antibody of the invention.
The context of use for such non-diagnostic purposes may be routine in the art, such as detecting antigens in a laboratory or studying competitive binding of antibodies.
The invention also provides the application of the bispecific antibody, the nucleic acid, the recombinant expression vector, the transformant, the pharmaceutical composition, the antibody drug conjugate, the kit or the kit in preparation of medicines for diagnosing, preventing and/or treating tumors.
The tumor is preferably a tumor associated with HER2 and/or PD-L1.
Such as breast cancer, gastric cancer, osteosarcoma, squamous cell carcinoma of the head and neck cancer, ovarian cancer, prostate cancer, pancreatic cancer, glioblastoma multiforme, gastric junction adenocarcinoma, gastroesophageal junction adenocarcinoma, cervical cancer, salivary gland carcinoma, soft tissue sarcoma, leukemia, melanoma, ewing's sarcoma, rhabdomyosarcoma, neuroblastoma, or small cell lung cancer.
The invention also provides a method of diagnosing, treating and/or preventing HER2 and/or PD-L1 mediated tumors, said method comprising administering to a patient in need thereof a therapeutically effective amount of a bispecific antibody according to the invention, a nucleic acid according to the invention, a recombinant expression vector according to the invention, a transformant according to the invention, a pharmaceutical composition according to the invention or an antibody drug conjugate according to the invention for the treatment of a patient in need thereof.
The tumor is preferably breast cancer, gastric cancer, osteosarcoma, pro-fibroproliferative small round cell carcinoma, squamous cell carcinoma of the head and neck cancer, ovarian cancer, prostate cancer, pancreatic cancer, glioblastoma multiforme, gastric junction adenocarcinoma, gastroesophageal junction adenocarcinoma, cervical cancer, salivary gland carcinoma, soft tissue sarcoma, leukemia, melanoma, ewing's sarcoma, rhabdomyosarcoma, neuroblastoma, or small cell lung cancer.
Compared with monotherapy, the bispecific antibody provided by the invention has multiple advantages of the bispecific antibody targeting HER2 and PD-L1: on one hand, the double anti-tumor activities of ADCC and PD-1/PD-L1 can be synergistically exerted, and the tumor treatment effect is improved; on the other hand, the targeting of tumor treatment can be improved, and the toxic and side effects on HER2 or PD-L1 positive normal tissues are reduced.
The invention has the positive progress effects that: the bispecific antibody of the invention has better physicochemical properties, better purity and thermal stability; the functional activity aiming at the double targets can be reserved, the binding activities of the two targets are close to that of the corresponding monoclonal antibodies (namely, the binding capacity of the monoclonal antibodies to PD-L1 and HER2 is equal to or better than that of the monoclonal antibodies alone), the activity of blocking the PD-1/PD-L1 is equal to that of the corresponding monoclonal antibodies, and the proliferation inhibition activity of SK-BR-3 cells is equal to that of the corresponding monoclonal antibodies; and simultaneously, the compound has good ADCC activity and in-vivo tumor inhibition activity.
Drawings
FIGS. 1A-1E show schematic structures of candidate bispecific antibodies.
FIGS. 2A-2F are graphs of candidate bispecific antibody SEC-HPLC monomer detection.
FIGS. 3A-3B show the binding activity of candidate bispecific antibodies to recombinant human PD-L1-His protein.
FIG. 4 shows the binding activity of candidate bispecific antibodies to huPD-L1-CHO-S cells.
FIGS. 5A-5B show the binding activity of candidate bispecific antibodies to recombinant human HER2-His protein.
FIG. 6 shows the binding activity of candidate bispecific antibodies to SK-BR-3 cells.
FIG. 7 shows the blocking activity of candidate bispecific antibodies on the PD-1/PD-L1 signaling pathway detected by FACS method.
FIGS. 8A-8C show the blocking activity of candidate bispecific antibodies on the PD-1/PD-L1 pathway detected by the luciferase reporter method.
FIG. 9 shows antibody-dependent cell-mediated cytotoxicity (ADCC) of candidate bispecific antibodies on SK-BR-3 cells.
FIG. 10 shows the proliferation inhibitory effect of candidate bispecific antibodies on SK-BR-3 cells.
FIGS. 11A-11B show the inhibition of tumor growth by candidate bispecific antibodies in a mouse subcutaneous engraftment tumor model.
Detailed Description
Definition of the definition
In the present invention, the amino acid sequences of the CDRs listed above are all shown according to the AbM definition rules. However, it is well known to those skilled in the art that CDRs of antibodies can be defined in the art by a variety of methods, such as Chothia (Chothia et al (1989) Nature 342:877-883, al-Lazikani et al (Journal of Molecular Biology,273, 927-948 (1997)), kabat (Kabat et al, U.S. device of Health and Human Services, national Institutes of Health (1987)), abM (University of Bath), contact (University College London), international ImMunoGeneTics database (IMGT) (world Wide Web IMGT. Cis. Fr /), based on the topology of the CDR loops, and North definition based on neighbor-transmitted clusters (affinity propagation clustering) that utilize a large number of crystal structures. It will be appreciated by those skilled in the art that unless otherwise specified, the terms "CDR" and "complementarity determining region" of a given antibody or region thereof (e.g., variable region) are to be understood as encompassing complementarity determining regions defined in any of the above known schemes as described by the present invention.
In the present invention, the protein functional region may be in the form of a full-length antibody, scFv, fab, VHH, etc., and may be selected according to the actual context. Wherein "full length antibody" is used interchangeably to refer to a glycoprotein comprising at least two Heavy Chains (HC) and two Light Chains (LC) interconnected by disulfide bonds. Each heavy chain consists of a heavy chain variable region (abbreviated VH in the present invention) and a heavy chain constant region. The heavy chain constant region consists of 3 domains, CH1, CH2 and CH 3. Each light chain consists of a light chain variable region (abbreviated as VL in the present invention) and a light chain constant region (abbreviated as CL in the present invention). The light chain constant region consists of one domain CL. Mammalian heavy chains are classified as α, δ, ε, γ, and μ. Mammalian light chains are classified as either lambda or kappa. Immunoglobulins comprising alpha, delta, epsilon, gamma and mu heavy chains are classified as immunoglobulins IgA, igD, igE, igG and IgM. The complete antibody forms a "Y" shape. The stem of Y consists of the second and third constant regions of the two heavy chains (and the fourth constant region for IgE and IgM) joined together, and disulfide bonds (interchain) formed in the hinge. Heavy chains gamma, alpha and delta have a constant region consisting of three tandem (in-line) Ig domains, and a hinge region for increased flexibility; heavy chains μ and ε have constant regions consisting of four immunoglobulin domains. The second and third constant regions are referred to as the "CH2 domain" and "CH3 domain", respectively. Each arm of Y includes a variable region and a first constant region of a single heavy chain joined to variable and constant regions of a single light chain. The variable regions of the light and heavy chains are responsible for antigen binding. In the present invention, the "Fab fragment" consists of a CH1 and variable region of a light chain and a heavy chain. The heavy chain of a Fab molecule cannot form disulfide bonds with another heavy chain molecule. The "Fc region" contains two heavy chain fragments comprising the CH2 and CH3 domains of an antibody. The two heavy chain fragments are held together by two or more disulfide bonds and by the hydrophobic effect of the CH3 domain. "Fab ' fragments" contain a portion of one light chain and one heavy chain comprising the VH domain and CH1 domain and the region between the CH1 and CH2 domains, whereby an inter-chain disulfide bond can be formed between the two heavy chains of two Fab ' fragments to form F (ab ') 2 A molecule. "F (ab') 2 The fragment "comprises two light chains and two heavy chains comprising portions of the constant region between the CH1 and CH2 domains, thereby forming an interchain disulfide bond between the two heavy chains. F (ab') 2 Fragments consist of two Fab' fragments held together by disulfide bonds between the two heavy chains. The single chain antibody (single chain antibody fragment, scFv) is an antibody in which a heavy chain variable region and a light chain variable region of an antibody are linked by a short peptide (linker) of 15 to 20 amino acids. The term "VHH", also known as single domain antibody, is an antibody consisting of only one heavy chain variable region, comprising only one chain FR4-CDR3-FR3-CDR2-FR2-CDR1-FR1 from the C-terminus to the N-terminus, also known as "nanobody". Single domain antibodies are the smallest unit currently known to bind an antigen of interest.
The three-letter and one-letter codes for amino acids used in the present invention are known to the person skilled in the art or are described in the literature (J.biol. Chem,243, p3558 (1968)).
As used herein, the terms "comprising" or "comprises" are intended to mean that the compositions and methods include the recited elements but do not exclude other elements, but that the terms "consisting of … …" are also included, as the context dictates otherwise.
As used herein, "vector" refers to a construct capable of delivering one or more genes or sequences of interest into a host cell and preferably expressing the genes or sequences in the host cell. Examples of vectors include, but are not limited to, viral vectors, naked DNA or RNA expression vectors, plasmids, cosmid or phage vectors, DNA or RNA expression vectors associated with cationic coagulants, DNA or RNA expression vectors encapsulated in liposomes, and certain eukaryotic cells, such as producer cells.
In the present invention the term "host cell" may include cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells" which include primary transformed cells and progeny derived therefrom, regardless of the number of passages. The progeny may not be exactly identical in nucleic acid content to the parent cell, but may contain mutations. The invention includes mutant progeny that have the same function or biological activity as the cell selected or selected in the originally transformed cell.
The following examples are intended to be illustrative of the invention only and should not be construed as limiting the invention in any way.
Example 1 preparation of raw materials
1.1 antigen preparation
The DNA coding sequences for recombinant human PD-L1 (UniProtKB-Q9 NZQ 7), PD-1 (UniProtKB-Q15116) and HER2 (UniProt NO-P04626) proteins were obtained by total gene synthesis from the general biosciences Co., ltd. Amplifying a target fragment by PCR, introducing a His tag at the C end of a coding sequence by a primer, and constructing the target fragment to a eukaryotic expression vector pcDNA3.4-TOPO (Invitrogen) by adopting a homologous recombination method. The constructed recombinant protein expression plasmid was transformed into E.coli DH 5. Alpha. Competent cells, which were cultured overnight at 37℃and the plasmid was extracted using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01).
Recombinant human PD-L1-His protein, recombinant human PD-1-His protein and recombinant human HER2-His protein are prepared by an Expi293 transient expression system (ThermoFisher, A14635), see Expi293 for transient methods TM Expression System USER GUIDE. After 7 days of transfection, the cell suspension was collected and centrifuged at 15000g for 10min, and the expression supernatant was affinity purified using Ni Smart Beads 6FF (Hemsl and Biotech Co., ltd., SA 036050), and the target protein was eluted with a gradient concentration of imidazole solution. Each protein obtained by elution was replaced by ultrafiltration concentration tube (Millipore, UFC 901096) into PBS buffer, respectively. And after being qualified by SDS-PAGE identification and activity detection, the samples are sub-packaged and frozen at-80 ℃.
1.2 control antibody preparation
In this example, the anti-human PD-L1 antibody D21-4 (VHH PD-L1 huFc, the amino acid sequence of which is shown in SEQ ID NO: 1) is derived from patent application WO2021083335A1; the anti-human PD-L1 antibody Avelumab was derived from drug bank (query number DB 11945); the anti-human HER2 antibody was Trastuzumab (Trastuzumab) derived from drug bank (query number DB 00072).
The control antibodies were all expressed using the transient system (ExpiCHO), transient methods of which are described in ExpiCHO TM Expression System Kit User Guide. After the completion of the culture, the cell suspension was centrifuged at a high speed and the supernatant was collected, and the obtained supernatant was filtered through a 0.22 μm filter membrane and purified by affinity chromatography using a Protein A/G column. The target protein is eluted by using 100mM glycine hydrochloride (pH 3.0), concentrated, replaced by buffer solution, split charging, SDS-PAGE identification and activity detection and then put into storage for freezing.
Example 2 construction of anti-HER 2& PD-L1 bispecific antibodies
This example describes exemplary anti-HER 2&Construction of PD-L1 bispecific antibody (BsAb) and expression vector. 6 constructs were designed and constructed: wherein the VHH domain (VHH) of the anti-human PD-L1 antibody PD-L1 ) The amino acid sequence is from D21-4, and the variable region amino acid sequence is shown in SEQ ID NO. 2; the amino acid sequence of the anti-HER 2 antibody is from trastuzumab, and the amino acid sequence of the light and heavy chain variable region of the anti-HER 2 antibody is shown as SEQ ID NO. 3 and SEQ ID NO. 4 respectively; the amino acid sequence of the linker is GGGGSGGGGSGGGGS (SEQ ID NO: 5). Exemplary BsAb constructs are shown in table 1, with corresponding amino acid sequences provided in table 2.
Construct BsAb1: comprising two identical first polypeptide chains comprising, from N-terminus to C-terminus, a heavy chain variable region of trastuzumab, a human IgG1 heavy chain constant region, a linker, and a VHH domain of an anti-human PD-L1 antibody; comprising two identical second polypeptide chains comprising, from N-terminus to C-terminus, a light chain variable region of trastuzumab and a kappa light chain constant region. BsAb1 has the form shown in FIG. 1A.
Construct BsAb2: comprising two identical first polypeptide chains comprising, from N-terminus to C-terminus, a VHH domain of an anti-human PD-L1 antibody, a heavy chain variable region of trastuzumab, a human IgG1 heavy chain constant region; comprising two identical second polypeptide chains comprising, from N-terminus to C-terminus, a light chain variable region of trastuzumab and a kappa light chain constant region. BsAb2 has the form shown in FIG. 1B.
Construct BsAb3: comprising two identical first polypeptide chains comprising, from N-terminus to C-terminus, a heavy chain variable region of trastuzumab and a human IgG1 heavy chain constant region domain; comprising two identical second polypeptide chains comprising, from N-terminus to C-terminus, a VHH domain of an anti-human PD-L1 antibody, a linker, a light chain variable region of trastuzumab and a kappa light chain constant region. BsAb3 has the form shown in FIG. 1C.
Construct BsAb4: comprising two identical first polypeptide chains comprising, from N-terminus to C-terminus, a heavy chain variable region of trastuzumab and a human IgG1 heavy chain constant region domain; comprising two identical second polypeptide chains comprising, from N-terminus to C-terminus, a light chain variable region of trastuzumab, a kappa light chain constant region, a linker and a VHH domain of an anti-human PD-L1 antibody. BsAb4 has the form shown in FIG. 1D.
Construct BsAb5: comprising 3 different polypeptide chains, a first polypeptide chain, a second polypeptide chain and a third polypeptide chain; wherein the first polypeptide chain comprises, from N-terminus to C-terminus, a VHH domain of an anti-human PD-L1 antibody, a human IgG1 heavy chain hinge region, and a human IgG1 heavy chain Fc (Knob); wherein the second polypeptide chain comprises, from N-terminus to C-terminus, a heavy chain variable region of trastuzumab and a human IgG1 heavy chain constant region (Hole); wherein the third polypeptide chain comprises, from N-terminus to C-terminus, a light chain variable region and a kappa light chain constant region of trastuzumab. BsAb5 has the form shown in FIG. 1E.
Construct BsAb6: comprising 3 different polypeptide chains, a first polypeptide chain, a second polypeptide chain and a third polypeptide chain; wherein the first polypeptide chain comprises, from N-terminus to C-terminus, a VHH domain of an anti-human PD-L1 antibody, a human IgG1 heavy chain hinge region, and a human IgG1 heavy chain Fc (Hole); wherein the second polypeptide chain comprises, from N-terminus to C-terminus, a heavy chain variable region of trastuzumab and a human IgG1 heavy chain constant region (Knob); the third polypeptide chain comprises, from N-terminus to C-terminus, a light chain variable region and a kappa light chain constant region of trastuzumab. BsAb6 has the form shown in FIG. 1E.
According to the structure of the construct, the fragments of the variable region and the constant region of each antibody are obtained through PCR amplification, the fragments are connected through overlap extension PCR, and then are respectively constructed on a modified eukaryotic expression vector plasmid pcDNA3.4-TOPO (Invitrogen) through homologous recombination, so that the complete construct polypeptide chain expression vector is formed. The constructed vectors containing the encoding sequences of the polypeptide chains of the constructs are respectively transformed into E.coli DH5 alpha and cultured at 37 ℃ overnight. Plasmid extraction was performed using an endotoxin-free plasmid extraction kit (OMEGA, D6950-01) to obtain an endotoxin-free construct polypeptide chain expression plasmid for eukaryotic expression.
TABLE 1 constructs of anti-HER 2& PD-L1 bispecific antibodies
TABLE 2 amino acid sequence of anti-HER 2& PD-L1 bispecific antibody constructs
Example 3 expression, purification and physicochemical Property analysis of anti-HER 2& PD-L1 bispecific antibodies
3.1 expression and purification of anti-HER 2& PD-L1 bispecific antibodies
The construct of example 2 was expressed by the expcho transient expression system (Thermo Fisher, a 29133) as follows: on the day of transfection, cell densities of approximately 7X 10 were confirmed 6 Up to 1X 10 7 Viable cells/mL, cell viability >98 at this time, the cells were adjusted to a final concentration of 6X 10 using fresh ExpiCHO expression medium pre-warmed at 37 ℃ 6 Individual cells/mL. With a temperature of 4 DEG CPrecooled OptiPRO TM SFM dilution of plasmid of interest (1. Mu.g plasmid was added to 1mL of the medium), while using OptiPRO TM SFM dilution of Expifectamine TM CHO, mixing the two materials in equal volume, and gently stirring to obtain the product TM The CHO/plasmid DNA mixture was incubated at room temperature for 5min, slowly added to the prepared cell suspension while gently shaking, and finally placed in a cell culture shaker at 37℃and 8% CO 2 Culturing under the condition. 18-22h after transfection, expiCHO was added to the culture broth TM Enhance and ExpiCHO TM Feed, shake flask placed on a shaker at 32℃and 5% CO 2 Culturing was continued under the conditions. On day 5 post-transfection, the same volume of ExpiCHO was added TM Feed, slowly add while gently mix the cell suspension. After 10 days of transfection, the cell culture supernatant expressing the protein of interest was centrifuged at 15000g for 10min at high speed, and the resulting supernatant was affinity purified with COLUMN XK16/20 (from cytova), then eluted with 100mM sodium acetate (ph 3.0), then neutralized with 1M Tris-HCl, and finally the resulting protein was replaced into PBS buffer by ultrafiltration concentrate (Millipore, UFC 901096).
3.2 determination of the concentration of anti-HER 2& PD-L1 bispecific antibodies
The bispecific antibody obtained in example 3.1 was subjected to concentration measurement using an ultra-micro spectrophotometer (Nano-300, of the company of australian Cheng Yiqi, hangzhou), the measured a280 reading value was divided by the theoretical extinction coefficient of the antibody, and the obtained value was used as the antibody concentration for subsequent study, and after quality inspection, the antibody was packaged and stored at-80 ℃.
3.3 SEC-HPLC monomer purity identification of anti-HER 2& PD-L1 bispecific antibodies
Material preparation: 1. mobile phase: 150mmol/L phosphate buffer, pH 7.4; 2. sample preparation: the candidate bispecific antibodies were each diluted to 0.5mg/mL with mobile phase solution. Agilent HPLC 1100 column (XBIridge BEH SEC 3.5 μm.8mm I.D.×30cm, waters) flow rate was set at 0.8mL/min, sample volume 20. Mu.L, VWD detector wavelengths 280nm and 214nm.
SEC-HPLC results for candidate bispecific antibodies were as follows: the percentages of high molecular weight polymer, antibody monomer and low molecular weight substance in the samples were calculated according to the area normalization method, and the results are shown in fig. 2A-2F and table 3, from which it was found that the monomer purities of the candidate bispecific antibodies BsAb1, bsAb2, bsAb3 and BsAb4 were all greater than 90%.
3.4 thermal stability Studies of anti-HER 2& PD-L1 bispecific antibodies
Differential scanning fluorescence (differential scanning fluorimetry; DSF) provides information about the structural stability of proteins based on the course of fluorescence changes in the protein profile, and detects changes in the configuration of the protein to obtain the melting temperature (Tm) of the protein. In this example, the Tm value of a trifunctional antibody was measured by the DSF method.
The candidate bispecific antibody solution was prepared at 0.2mg/mL, each test sample was added to a 96 well plate (Nunc) at 19 μl/well, three parallel wells were set, PBS and trastuzumab were used as references, then 1 μl of a 100 x concentration of SYPRO orange dye was added to each well, and after mixing, the mixture was prepared for loading. The sample thermal stability test adopts an ABI 7500FAST RT-PCR instrument, the test type selects a melting curve, a continuous mode is adopted, the scanning temperature range is 25-95 ℃, the heating rate is 1%, the temperature is balanced for 5min at 25 ℃, data are collected in the heating process, a reporting group is selected as ROX, a quenching group is selected as None, the reaction volume is 20 mu L, and the melting temperature Tm of the antibody is determined by the temperature corresponding to the first peak and valley of the first derivative of the melting curve.
The experimental results show in table 3 that the Tm of the candidate bispecific antibodies are all greater than 60 ℃, thus having better thermal stability.
TABLE 3 preparation of anti-HER 2& PD-L1 bispecific antibodies, physicochemical data
Example 4 affinity Activity assay for anti-HER 2& PD-L1 bispecific antibodies
4.1ELISA method for detecting binding capability of candidate bispecific antibody to recombinant human PD-L1-His protein
Coating recombinant human PD on 96-well ELISA plateL1-His protein, overnight at 4 ℃. The next day, the well plate was blocked with 5% skim milk for 2h after 3 washes with PBST, and incubated for 1h after 3 washes with PBST, with different concentrations of candidate bispecific antibody, control antibody D21-4. After 3 washes with PBST, secondary Anti-human-IgG-Fc-HRP (abcam, ab 79225) was added and incubated for 1h. After incubation, PBST plates were washed six times and developed with TMB (SurModics, TMBS-1000-01). Based on the color development, the reaction was quenched by addition of 2M HCl and the plate was read by a microplate reader (Molecular Devices, specterMax 190) at OD 450. By PRISM TM (GraphPad Software, san Diego, calif.) analysis data and calculation of EC 50 Values.
The ELISA binding assay results are shown in FIGS. 3A-3B and Table 4, and the binding capacity of bispecific antibodies BsAb2 and BsAb3 to PD-L1 is significantly better than that of control antibody D21-4.
4.2 detection of binding Capacity of candidate bispecific antibodies to huPD-L1-CHO-S cells based on FACS method
The huPD-L1-CHO-S cells in exponential growth phase (stably transfected PD-L1 (Gene ID: 29126) to CHO-S (Thermo, A1461801) obtained stably transformed cell lines overexpressing human PD-L1) were collected, the cells were resuspended in FACS buffer (PBS containing 1% BSA), counted and the cell suspension density was adjusted to 2X 10 6 And each mL. Subsequently, huPD-L1-CHO-S cells were added to a 96-well round bottom plate at 100. Mu.L per well, and the supernatant was centrifuged off. Candidate bispecific antibodies, control antibody D21-4 dilutions and human IgG1 isotype antibodies (as isotype controls) were added to the corresponding wells at different concentrations, and the cells were resuspended and incubated at 4 ℃ for 30min. The incubated cell mixture was washed 3 times, and then added with PE-labeled anti-human-IgG-Fc flow antibody (Abcam, 98596), resuspended and incubated at 4 ℃ for 30min. After washing 3 times the incubated cell mixture was resuspended in 200. Mu.L of FACS buffer and analyzed by flow cytometry (Beckman, cytoFLEX AOO-1-1102) on-machine. By PRISM TM (GraphPad Software, san Diego, calif.) analysis data and calculation of EC 50 Values.
The results of the FACS binding assay are shown in FIG. 4 and Table 4, where the candidate bispecific antibodies all showed comparable binding capacity to the cell-expressed PD-L1 as the control antibody D21-4, and where the bispecific antibodies BsAb2 and BsAb3 were the strongest binding capacity.
4.3ELISA method for detecting binding ability of candidate bispecific antibody to recombinant human HER2-His protein
Recombinant human HER2-His protein was coated on 96-well ELISA plates overnight at 4 ℃. The next day, the well plate was blocked with 5% skim milk for 2h after 3 washes with PBST, and incubated with different concentrations of candidate bispecific antibody, control antibody trastuzumab for 1h after 3 washes with PBST. After 3 washes with PBST, secondary Anti-human-IgG-Fc-HRP (abcam, ab 97225) was added and incubated for 1h. After incubation, PBST plates were washed six times and developed with TMB (SurModics, TMBS-1000-01). Based on the color development, the reaction was quenched by addition of 2M HCl and the plate was read by a microplate reader (Molecular Devices, specterMax 190) at OD 450. By PRISM TM (GraphPad Software, san Diego, calif.) analysis data and calculation of EC 50 Values.
The ELISA binding assay results are shown in FIGS. 5A-5B and Table 4, and both candidate bispecific antibodies exhibited comparable binding capacity to HER2 to the control antibody trastuzumab.
4.4 detection of binding Capacity of candidate bispecific antibodies to SK-BR-3 cells based on FACS method
Endogenous HER 2-expressing SK-BR-3 cells in exponential growth phase (ATCC, HTB-30) were collected, the cells were resuspended in FACS buffer (PBS containing 1% BSA), counted and the cell suspension density was adjusted to 2X 10 6 And each mL. Subsequently, SK-BR-3 cells were added at 100. Mu.L per well to a 96-well round bottom plate, and the supernatant was centrifuged off. Candidate bispecific antibodies, control antibodies trastuzumab and human IgG1 isotype antibodies (as isotype controls) were added at different concentrations to the corresponding wells, and the cells were resuspended and incubated at 4 ℃ for 30min. The incubated cell mixture was washed 3 times, and then added with PE-labeled anti-human-IgG-Fc flow antibody (Abcam, 98596), resuspended and incubated at 4 ℃ for 30min. After 3 washes of the incubated cell mixture, 200. Mu.L of FACS buffer was added to resuspend the cells, which were finally analyzed by flow cytometry (Beckman, cytoFLEX AOO-1-1102) on-machine. By PRISM TM (GraphPad Software, san Diego, calif.) analysis data and countCalculation of EC 50 Values.
The FACS binding assay results are shown in fig. 6 and table 4, and bispecific antibodies BsAb1, bsAb2, and BsAb4 exhibit binding capacity to HER2 expressed on cells comparable to the control antibody trastuzumab.
TABLE 4 antigen binding data for anti-HER 2& PD-L1 bispecific antibodies
4.5 detection of affinity of candidate bispecific antibodies for HER2 and PD-L1 based on Fortebio
The affinity of the candidate bispecific antibodies for recombinant human HER2-His and PD-L1-His proteins was examined using the Fortebio BLItz instrument.
Recombinant human HER2-His or PD-L1-His protein as antigen was diluted to 10. Mu.g/mL with 10 XKB buffer (10 XPBS containing 1% BSA, 0.5% Tween 20), and candidate bispecific antibody and control antibody D21-4 or trastuzumab were diluted 2-fold with 10 XKB buffer, followed by 100, 50, 25, 0nM. Under the dark condition, a 10 XKB buffer solution is adopted to pre-wet a sensor (Anti-Penta-HIS, HIS1K, fortebio, CA), a sample plate (GreinerBio, PN 655209) is started to be tested after at least 10min, and the test is carried out according to a preset program after no error. The antigen and the sensor were first combined for 300s, after the combination was completed, the balance was continued for 30s in 10×kb buffer, the sensor combined with the antigen was transferred to different concentrations of antibody dilution for 300s, after the signal was stabilized, then transferred to 10×kb buffer, the dissociation time was 900s, and finally KD, kon and Koff were obtained by fitting the binding and dissociation data of different concentrations of antibodies, the results are shown in table 5, bispecific antibody BsAb2 exhibited an affinity for PD-L1 comparable to that of control antibody D21-4, and affinity of bispecific antibody BsAb2 for HER2 was significantly better than that of control antibody trastuzumab.
TABLE 5 affinity assay data table for anti-HER 2& PD-L1 bispecific antibodies
Example 5 detection of anti-HER 2& PD-L1 bispecific antibody blocking Activity based on FACS method
The blocking activity of the candidate bispecific antibody on PD-1/PD-L1 was detected by FACS method in this example, as follows: the cultured huPD-L1-CHO-S cells were collected, resuspended in the prepared FACS buffer, counted and the cell suspension density was adjusted to 2X 10 6 And each mL. The huPD-L1-CHO-S cells were added to 96-well round bottom plates at 100. Mu.L per well, the supernatant was centrifuged off, and different concentrations of candidate bispecific antibody, control antibody D21-4 and human IgG1 isotype antibody (isotype control) were added to the corresponding wells, and the cells were resuspended and incubated at 4℃for 30min. After washing 3 times the incubated cell mixture was added with 100. Mu.L of biotin-labeled recombinant human PD-1-His protein diluent (1. Mu.g/mL) and incubated at 4℃for 30min. PE-labeled streptavidine (eBioscience, 12-4317-87) was added after 3 washes, incubated at 4℃for 30min, the incubated cell mix was washed 3 times and 200. Mu.L of FACS buffer was added to resuspend cells, and finally detected by flow cytometry (Beckman, cytoFLEX AOO-1-1102). By PRISM TM (GraphPad Software, san Diego, calif.) analyzes the data and calculates the IC 50 Values.
The results of the FACS binding assay are shown in FIG. 7 and Table 6, with bispecific antibodies BsAb2 and BsAb3 exhibiting blocking activity against PD-1/PD-L1 comparable to control antibody D21-4.
TABLE 6 blocking Activity of anti-HER 2& PD-L1 bispecific antibodies
Antibody name IC 50 (nM)
BsAb1 9.17
BsAb2 6.62
BsAb3 6.76
BsAb4 11.85
BsAb5 12.18
BsAb6 16.61
D21-4 6.64
Example 6 detection of Activity of anti-HER 2& PD-L1 bispecific antibody blocking PD-1/PD-L1 based on reporter Gene method
In this example, a luciferase reporter gene system was used to detect candidate anti-HER 2&The PD-L1 bispecific antibody blocks the activity of the PD-1/PD-L1 signaling pathway by the following specific methods: taking logarithmic phase PD-1-NF-AT-Jurkat cells (Jurkat cells [. About. ]TIB-152) stably expressed PD-1 (UniProtKB-Q15116) and luciferase) and CD3L-PD-L1-CHO cells (stably expressed PD-L1 (UniProtKB-Q9 NZQ) and anti-CD3-scFv (sequence derived from OKT 3) in CHO cells), the two cells were mixed at 5:1 and brought to a density of 4×10, respectively 7 Individual/mL and 8X 10 7 And each mL. The antibody to be tested was diluted in a gradient, 50. Mu.L of the antibody dilution was added to each well of a 96-well clear bottom white plate (Corning, 3610), followed by adding the mixed cell suspension at 50. Mu.L/well, and the mixture was left to stand in a cell incubator at 37℃for 6 hours. 50 mu L Bright-Lite (Vazyme, DD 1204-03), incubated for 10min in the dark, and fluorescent signals were detected.
Blocking activity was measured as follows: as shown in FIGS. 8A-8C, both bispecific antibodies BsAb2, bsAb3 exhibited blocking PD-1/PD-L1 activity comparable to control antibody D21-4.
Example 7 ADCC Activity assay of anti-HER 2& PD-L1 bispecific antibody
In this example, a luciferase reporter gene system was used to detect candidate anti-HER 2&ADCC activity of PD-L1 bispecific antibodies is specifically described as follows: taking log phase SK-BR-3 cells and CD16a (V158) -NF-AT-Jurkat cells (stably transfected CD16a (V158) sequence (UniProtKB-P08637) and pGL4.30 plasmid containing NF-AT-re nucleic acid sequence (promega, # E8481) to Jurkat cellsTIB-152), see in particular the reference Parekh, b.s., et al (2012), "Development and validation of an antibody-dependent cell-mediated cytotoxicity-reporter gene assay," for stable transgenic cell lines obtained by TIB-152) "MAbs.4 (3): 310-318.) two cells were mixed at 10:1 and brought to a density of 4 x 10 respectively 5 Individual/mL and 4X 10 6 And each mL. The antibody to be tested was diluted in a gradient, 50. Mu.L of the antibody dilution was added to each well of a 96-well clear bottom white plate (Corning, 3610), followed by adding the mixed cell suspension at 50. Mu.L/well, and the mixture was left to stand in a cell incubator at 37℃for 6 hours. To each well 50. Mu.L of Bright-Lite (Vazyme, DD 1204-03) was added and incubated for 10min in the dark and fluorescence signals were detected.
ADCC assay results were as follows: as shown in fig. 9, the bispecific antibody BsAb2 exhibited significantly better ADCC activity than the control antibody trastuzumab.
Example 8 analysis of anti-HER 2& PD-L1 bispecific antibody inhibitory SK-BR-3 cell proliferation Activity
The SK-BR-3 cells in logarithmic growth phase were used to adjust the cell density to 2X 10 using RPMI1640 (1% FBS-containing) medium 4 Each mL was inoculated at 100. Mu.L/well into 96-well flat bottom cell culture plates and incubated overnight at 37℃in a cell incubator. Antibody to be tested was diluted in gradient with RPMI1640 (1% FBS-containing) medium at 50. Mu.L/wellTo the SK-BR-3 cultured overnight 96-well cell culture plate and placed in a 37℃incubator for 72 hours. MTS assay reagent (Promega, G3581) was previously placed at room temperature for thawing and equilibrated to room temperature. The cell culture plates were allowed to equilibrate to room temperature for 15min at room temperature, MTS assay reagent was added to the 96 well cell culture plates at 30. Mu.L/well, after shaking on a microplate reader for 1min, then incubated at 37℃for 3h in the absence of light, the cell culture plates were removed and equilibrated to room temperature, and OD492 was read.
Proliferation inhibition activity was measured as follows: as shown in fig. 10, bispecific antibody BsAb2 was shown to inhibit SK-BR-3 cell proliferation activity comparable to control antibody trastuzumab.
Example 9 in vivo tumor inhibition experiments with anti-HER 2& PD-L1 bispecific antibodies
Female NCG mice (purchased from Jiangsu Ji Cui Ji, strain T001475) of 6-8 weeks are used, and experimental mice are fed into a constant temperature and humidity independent ventilation box, and the temperature of a feeding room is 21-24 ℃ and the humidity is 30-53%. huPD-L1-NCI-N87 cells (stably transfected PD-L1 (Gene ID: 29126) to NCI-N87 cells (stably transfected cell lines overexpressing human PD-L1 obtained from the cell Bank of the national institute, catalog number: TCHu 130)) were cultured at 1X 10 7 The right back subcutaneous injection (day 0) is carried out on each mouse, and the subcutaneous tumor-bearing volume of the mice reaches 100-150 mm 3 On left and right (day 7), samples of mice with large differences in tumor volume were removed and then randomly grouped according to tumor volume (8 mice per group): the PBS treated group, trastuzumab-dosed group, avelumab-dosed group, trastuzumab+avelumab-combined-dosed group, and bispecific antibody BsAb 2-dosed group were each set with a 35nM/kg dose (corresponding to monoclonal antibody 5mpk and diabody 6.1 mpk), wherein the bispecific antibody BsAb 2-dosed group was also set with a 87.5nM/kg dose (corresponding to diabody 15.2 mpk). Each mouse was then given 5 x 10 i.v. injections 6 PBMC cells (C2106025) were given by intraperitoneal injection (i.p.) for the first time 4 hours, twice a week for a total of 7 times. Tumor length (mm) and width (mm) were observed and recorded at any time, and tumor volume (V) was calculated in the manner of v= (length x width) 2 ) Tumor inhibition ratio TGI (%) = (average tumor volume in 1-administration group/average tumor volume in PBS-treated group) ×100%.
The results are shown in FIGS. 11A-11B and Table 7.
TABLE 7 tumor rejection rates TGI (%)
As can be seen from fig. 11A, there was no significant difference between the weights of the mice in each group, and there was no significant change in the weights of the mice in each group during the treatment, indicating that the mice were well tolerated. As can be seen from fig. 11B and table 7, the PBS-treated mice developed the fastest tumor, and all antibody groups had significant tumor-inhibiting effects compared to the PBS group; wherein, the tumor volume of the mice in the high-dose bispecific antibody BsAb2 administration group is obviously lower than that of the trastuzumab+Avelumab combination administration group, and the mice show better tumor inhibition effect.
SEQUENCE LISTING
<110> Sanyou biomedical (Shanghai) Co., ltd
<120> bispecific antibody targeting HER2 and PD-L1, and preparation method and application thereof
<130> P21016686C
<160> 15
<170> PatentIn version 3.5
<210> 1
<211> 348
<212> PRT
<213> Artificial Sequence
<220>
<223> antibody D21-4
<400> 1
Glu 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 Ala Ala Ser Arg Thr Asp Arg Asn Ile Asn
20 25 30
Thr Met His Trp Tyr Arg Gln Ala Pro Gly Lys Gly Arg Glu Trp Val
35 40 45
Gly Thr Ile Phe Ile Asp Leu Asn Thr Ile Val Thr Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Thr Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Asp Val Ser Gly Tyr Gly Arg Ala Trp Gly Gln Gly Thr Thr Val
100 105 110
Thr Val Ser Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro
115 120 125
Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
130 135 140
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
145 150 155 160
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
165 170 175
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
180 185 190
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
195 200 205
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
210 215 220
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
225 230 235 240
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg
245 250 255
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly
260 265 270
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
275 280 285
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
290 295 300
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
305 310 315 320
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
325 330 335
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
340 345
<210> 2
<211> 116
<212> PRT
<213> Artificial Sequence
<220>
<223> variable region of VHH Domain
<400> 2
Glu 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 Ala Ala Ser Arg Thr Asp Arg Asn Ile Asn
20 25 30
Thr Met His Trp Tyr Arg Gln Ala Pro Gly Lys Gly Arg Glu Trp Val
35 40 45
Gly Thr Ile Phe Ile Asp Leu Asn Thr Ile Val Thr Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Thr Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Asp Val Ser Gly Tyr Gly Arg Ala Trp Gly Gln Gly Thr Thr Val
100 105 110
Thr Val Ser Ser
115
<210> 3
<211> 107
<212> PRT
<213> Artificial Sequence
<220>
<223> light chain variable region of anti-HER 2 antibody
<400> 3
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105
<210> 4
<211> 120
<212> PRT
<213> Artificial Sequence
<220>
<223> heavy chain variable region of anti-HER 2 antibody
<400> 4
Glu 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 Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser
115 120
<210> 5
<211> 15
<212> PRT
<213> Artificial Sequence
<220>
<223> connector
<400> 5
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
1 5 10 15
<210> 6
<211> 581
<212> PRT
<213> Artificial Sequence
<220>
<223> BsAb 1-first polypeptide chain
<400> 6
Glu 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 Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 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 Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
355 360 365
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
Gly Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly
450 455 460
Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly
465 470 475 480
Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Thr Asp Arg Asn Ile
485 490 495
Asn Thr Met His Trp Tyr Arg Gln Ala Pro Gly Lys Gly Arg Glu Trp
500 505 510
Val Gly Thr Ile Phe Ile Asp Leu Asn Thr Ile Val Thr Asp Ser Val
515 520 525
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr
530 535 540
Leu Gln Met Asn Thr Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
545 550 555 560
Ala Ala Asp Val Ser Gly Tyr Gly Arg Ala Trp Gly Gln Gly Thr Thr
565 570 575
Val Thr Val Ser Ser
580
<210> 7
<211> 214
<212> PRT
<213> Artificial Sequence
<220>
<223> BsAb1/BsAb 2-second polypeptide chain, bsAb5/BsAb 6-third polypeptide chain
<400> 7
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys
210
<210> 8
<211> 581
<212> PRT
<213> Artificial Sequence
<220>
<223> BsAb 2-first polypeptide chain
<400> 8
Glu 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 Ala Ala Ser Arg Thr Asp Arg Asn Ile Asn
20 25 30
Thr Met His Trp Tyr Arg Gln Ala Pro Gly Lys Gly Arg Glu Trp Val
35 40 45
Gly Thr Ile Phe Ile Asp Leu Asn Thr Ile Val Thr Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Thr Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Asp Val Ser Gly Tyr Gly Arg Ala Trp Gly Gln Gly Thr Thr Val
100 105 110
Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
115 120 125
Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln
130 135 140
Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile
145 150 155 160
Lys Asp Thr Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
165 170 175
Glu Trp Val Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala
180 185 190
Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn
195 200 205
Thr Ala Tyr Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
210 215 220
Tyr Tyr Cys Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr
225 230 235 240
Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly
245 250 255
Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly
260 265 270
Thr Ala Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val
275 280 285
Thr Val Ser Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe
290 295 300
Pro Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val
305 310 315 320
Thr Val Pro Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val
325 330 335
Asn His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys
340 345 350
Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
355 360 365
Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr
370 375 380
Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val
385 390 395 400
Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val
405 410 415
Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
420 425 430
Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu
435 440 445
Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala
450 455 460
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
465 470 475 480
Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln
485 490 495
Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala
500 505 510
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr
515 520 525
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu
530 535 540
Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
545 550 555 560
Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser
565 570 575
Leu Ser Pro Gly Lys
580
<210> 9
<211> 450
<212> PRT
<213> Artificial Sequence
<220>
<223> BsAb3/BsAb 4-first polypeptide chain
<400> 9
Glu 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 Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 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 Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
355 360 365
Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
Gly Lys
450
<210> 10
<211> 345
<212> PRT
<213> Artificial Sequence
<220>
<223> BsAb 3-second polypeptide chain
<400> 10
Glu 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 Ala Ala Ser Arg Thr Asp Arg Asn Ile Asn
20 25 30
Thr Met His Trp Tyr Arg Gln Ala Pro Gly Lys Gly Arg Glu Trp Val
35 40 45
Gly Thr Ile Phe Ile Asp Leu Asn Thr Ile Val Thr Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Thr Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Asp Val Ser Gly Tyr Gly Arg Ala Trp Gly Gln Gly Thr Thr Val
100 105 110
Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly
115 120 125
Gly Gly Ser Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala
130 135 140
Ser Val Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val
145 150 155 160
Asn Thr Ala Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
165 170 175
Leu Leu Ile Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg
180 185 190
Phe Ser Gly Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser
195 200 205
Leu Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr
210 215 220
Thr Pro Pro Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr
225 230 235 240
Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu
245 250 255
Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro
260 265 270
Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly
275 280 285
Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr
290 295 300
Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His
305 310 315 320
Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val
325 330 335
Thr Lys Ser Phe Asn Arg Gly Glu Cys
340 345
<210> 11
<211> 345
<212> PRT
<213> Artificial Sequence
<220>
<223> BsAb 4-second polypeptide chain
<400> 11
Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly
1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala
20 25 30
Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45
Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60
Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro
65 70 75 80
Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro
85 90 95
Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala
100 105 110
Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln Leu Lys Ser Gly
115 120 125
Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala
130 135 140
Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln
145 150 155 160
Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser
165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr
180 185 190
Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205
Phe Asn Arg Gly Glu Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser
210 215 220
Gly Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
225 230 235 240
Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Thr
245 250 255
Asp Arg Asn Ile Asn Thr Met His Trp Tyr Arg Gln Ala Pro Gly Lys
260 265 270
Gly Arg Glu Trp Val Gly Thr Ile Phe Ile Asp Leu Asn Thr Ile Val
275 280 285
Thr Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
290 295 300
Asn Thr Leu Tyr Leu Gln Met Asn Thr Leu Arg Ala Glu Asp Thr Ala
305 310 315 320
Val Tyr Tyr Cys Ala Ala Asp Val Ser Gly Tyr Gly Arg Ala Trp Gly
325 330 335
Gln Gly Thr Thr Val Thr Val Ser Ser
340 345
<210> 12
<211> 348
<212> PRT
<213> Artificial Sequence
<220>
<223> BsAb 5-first polypeptide chain
<400> 12
Glu 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 Ala Ala Ser Arg Thr Asp Arg Asn Ile Asn
20 25 30
Thr Met His Trp Tyr Arg Gln Ala Pro Gly Lys Gly Arg Glu Trp Val
35 40 45
Gly Thr Ile Phe Ile Asp Leu Asn Thr Ile Val Thr Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Thr Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Asp Val Ser Gly Tyr Gly Arg Ala Trp Gly Gln Gly Thr Thr Val
100 105 110
Thr Val Ser Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro
115 120 125
Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
130 135 140
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
145 150 155 160
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
165 170 175
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
180 185 190
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
195 200 205
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
210 215 220
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
225 230 235 240
Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Cys Arg
245 250 255
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Trp Cys Leu Val Lys Gly
260 265 270
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
275 280 285
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
290 295 300
Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
305 310 315 320
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
325 330 335
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
340 345
<210> 13
<211> 450
<212> PRT
<213> Artificial Sequence
<220>
<223> BsAb 5-second polypeptide chain
<400> 13
Glu 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 Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 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 Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Cys
340 345 350
Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
355 360 365
Ser Cys Ala Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Val Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
Gly Lys
450
<210> 14
<211> 348
<212> PRT
<213> Artificial Sequence
<220>
<223> BsAb 6-first polypeptide chain
<400> 14
Glu 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 Ala Ala Ser Arg Thr Asp Arg Asn Ile Asn
20 25 30
Thr Met His Trp Tyr Arg Gln Ala Pro Gly Lys Gly Arg Glu Trp Val
35 40 45
Gly Thr Ile Phe Ile Asp Leu Asn Thr Ile Val Thr Asp Ser Val Lys
50 55 60
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu
65 70 75 80
Gln Met Asn Thr Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95
Ala Asp Val Ser Gly Tyr Gly Arg Ala Trp Gly Gln Gly Thr Thr Val
100 105 110
Thr Val Ser Ser Glu Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro
115 120 125
Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe
130 135 140
Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val
145 150 155 160
Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
165 170 175
Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro
180 185 190
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr
195 200 205
Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val
210 215 220
Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala
225 230 235 240
Lys Gly Gln Pro Arg Glu Pro Gln Val Cys Thr Leu Pro Pro Ser Arg
245 250 255
Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Ser Cys Ala Val Lys Gly
260 265 270
Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro
275 280 285
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
290 295 300
Phe Phe Leu Val Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln
305 310 315 320
Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His
325 330 335
Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys
340 345
<210> 15
<211> 450
<212> PRT
<213> Artificial Sequence
<220>
<223> BsAb 6-second polypeptide chain
<400> 15
Glu 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 Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr
20 25 30
Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45
Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val
50 55 60
Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr
65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys
85 90 95
Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110
Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val
115 120 125
Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly 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 Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205
Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp
210 215 220
Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly
225 230 235 240
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile
245 250 255
Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu
260 265 270
Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
275 280 285
Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg
290 295 300
Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys
305 310 315 320
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu
325 330 335
Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr
340 345 350
Thr Leu Pro Pro Cys Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu
355 360 365
Trp Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp
370 375 380
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val
385 390 395 400
Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp
405 410 415
Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His
420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro
435 440 445
Gly Lys
450

Claims (15)

1. A bispecific antibody comprising a first protein functional region that targets HER2 and a second protein functional region that targets PD-L1; wherein:
the first protein functional region comprises a VL comprising three complementarity determining regions LCDR1, LCDR2 and LCDR3 contained in the sequence shown as SEQ ID NO. 3 and a VH comprising three complementarity determining regions HCDR1, HCDR2 and HCDR3 contained in the sequence shown as SEQ ID NO. 4;
the second protein functional region comprises a VHH, wherein the VHH comprises three complementarity determining regions CDR1, CDR2 and CDR3 comprised in the sequence as shown in SEQ ID NO. 2.
2. The bispecific antibody of claim 1, wherein the VL comprises an amino acid sequence as set forth in SEQ ID No. 3, or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% sequence identity thereto; and said VH comprises an amino acid sequence as set forth in SEQ ID No. 4, or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% sequence identity thereto; and/or the VHH comprises an amino acid sequence as shown in SEQ ID NO. 2, or an amino acid sequence having at least 90%, 95%, 96%, 97%, 98%, 99% sequence identity thereto.
3. The bispecific antibody of claim 2, wherein the first protein functional region comprises a Fab;
wherein the Fab is operably linked to the VHH, which is preferably linked to the N-terminus of the light chain variable region or heavy chain variable region, or the C-terminus of the light chain constant region, in the Fab;
alternatively, the Fab and the VHH are each operably linked to the N-terminus of two hinge regions of the Fc region.
4. The bispecific antibody of claim 2, wherein the first protein functional region is IgG; the VHH is operably linked to the C-terminus of the IgG heavy chain constant region.
5. The bispecific antibody of any one of claims 1-4, which satisfies any one of the following conditions:
(i) The bispecific antibody comprises two first polypeptide chains and two second polypeptide chains, wherein:
the first polypeptide chain is represented by the formula:
the VH-CH 1-hinge region-CH 2-CH3 preferably comprises a sequence as shown in SEQ ID NO:9,
and the second polypeptide chain is of the formula:
VHH-linker-VL-CL, preferably comprising the sequence shown as SEQ ID NO. 10;
or, the first polypeptide chain is of the formula:
the VH-CH 1-hinge region-CH 2-CH 3-linker-VHH preferably comprises a sequence as shown in SEQ ID NO. 6,
And the second polypeptide chain is of the formula:
VL-CL, preferably comprising a sequence as shown in SEQ ID NO. 7;
or, the first polypeptide chain is of the formula:
VHH-linker-VH-CH 1-hinge region-CH 2-CH3, preferably comprising a sequence as shown in SEQ ID NO. 8,
and the second polypeptide chain is of the formula:
VL-CL, preferably comprising a sequence as shown in SEQ ID NO. 7;
or, the first polypeptide chain is of the formula:
the VH-CH 1-hinge region-CH 2-CH3 preferably comprises a sequence as shown in SEQ ID NO:9,
and the second polypeptide chain is of the formula:
VL-CL-linker-VHH, preferably comprising the sequence shown as SEQ ID NO. 11;
(ii) The bispecific antibody comprises a first polypeptide chain, a second polypeptide chain and a third polypeptide chain, wherein the first polypeptide chain is shown in the following formula:
VHH-hinge region-CH 2-CH3;
the second polypeptide chain is represented by the formula:
VH-CH 1-hinge region-CH 2-CH3;
the third polypeptide chain is represented by the formula:
VL-CL；
preferably, the first polypeptide chain comprises the sequence shown as SEQ ID NO. 12 and the second polypeptide chain comprises the sequence shown as SEQ ID NO. 13 and the third polypeptide chain comprises the sequence shown as SEQ ID NO. 7,
or the first polypeptide chain comprises the sequence shown as SEQ ID NO. 14 and the second polypeptide chain comprises the sequence shown as SEQ ID NO. 15 and the third polypeptide chain comprises the sequence shown as SEQ ID NO. 7.
6. An isolated nucleic acid encoding the bispecific antibody of any one of claims 1-5.
7. A recombinant expression vector comprising the isolated nucleic acid of claim 6.
8. A transformant comprising the isolated nucleic acid of claim 6 or the recombinant expression vector of claim 7;
preferably, the host cell of the transformant is a prokaryotic cell, preferably an E.coli cell such as TG1, BL21, or a eukaryotic cell, preferably a HEK293 cell or a CHO cell.
9. A method of preparing a bispecific antibody comprising the steps of: culturing the transformant according to claim 8, and obtaining the bispecific antibody from the culture.
10. A pharmaceutical composition comprising the bispecific antibody of any one of claims 1-5, and a pharmaceutically acceptable carrier;
preferably, the pharmaceutical composition further comprises as active ingredient other anti-tumor antibodies and/or one or more of the group consisting of hormonal preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic agents, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules and vaccines.
11. An antibody drug conjugate comprising a cytotoxic agent or tag, and the bispecific antibody of any one of claims 1-5;
preferably, the cytotoxic agent is MMAF or MMAE and the tag is a fluorescent agent.
12. A kit comprising the bispecific antibody of any one of claims 1-5, the pharmaceutical composition of claim 10, or the antibody drug conjugate of claim 11;
preferably, the kit further comprises (i) a device for administering a bispecific antibody or antibody drug conjugate or pharmaceutical composition; and/or (ii) instructions for use.
13. A kit of parts comprising a kit a and a kit B, wherein:
the kit a contains a bispecific antibody according to any one of claims 1 to 5, a pharmaceutical composition according to claim 10 or an antibody drug conjugate according to claim 11;
the kit B contains other anti-tumor antibodies or pharmaceutical compositions comprising the other anti-tumor antibodies, and/or one or more of the group consisting of hormonal preparations, targeted small molecule preparations, proteasome inhibitors, imaging agents, diagnostic agents, chemotherapeutic agents, oncolytic agents, cytotoxic agents, cytokines, activators of co-stimulatory molecules, inhibitors of inhibitory molecules, and vaccines.
14. Use of a bispecific antibody according to any one of claims 1-5, a nucleic acid according to claim 6, a recombinant expression vector according to claim 7, a transformant according to claim 8, a pharmaceutical composition according to claim 10, an antibody drug conjugate according to claim 11, a kit according to claim 12 or a kit of parts according to claim 13 for the preparation of a medicament for the diagnosis, prevention and/or treatment of a tumor; preferably, the tumor is a tumor associated with HER2 and/or PD-L1, more preferably the tumor is breast cancer, gastric cancer, osteosarcoma, myelogenous small round cell carcinoma, squamous cell carcinoma of head and neck cancer, ovarian cancer, prostate cancer, pancreatic cancer, glioblastoma multiforme, gastric junction adenocarcinoma, gastroesophageal junction adenocarcinoma, cervical cancer, salivary gland carcinoma, soft tissue sarcoma, leukemia, melanoma, ewing's sarcoma, rhabdomyosarcoma, neuroblastoma, or small cell lung cancer.
15. A method for detecting a non-diagnostic purpose of a specific antigen comprising detecting using the bispecific antibody of any one of claims 1-5.
CN202210657725.2A 2022-06-10 2022-06-10 Bispecific antibody targeting HER2 and PD-L1 as well as preparation method and application thereof Pending CN117247457A (en)

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PCT/CN2023/097835 WO2023236844A1 (en) 2022-06-10 2023-06-01 Bispecific antibody targeting her2 and pd-l1, preparation method therefor and use thereof

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US11325982B2 (en) * 2017-04-09 2022-05-10 Xuanzhu Biopharmaceutical Co., Ltd. Biparatopic and multiparatopic antibodies with common light chain and method of use
US20200354460A1 (en) * 2017-09-29 2020-11-12 Wuxi Biologics Ireland Limited. Bispecific antibodies against EGFR and PD-1
WO2019153200A1 (en) * 2018-02-08 2019-08-15 北京韩美药品有限公司 Anti-pd-1/anti-her2 natural antibody structure-like bispecific antibody in heterodimeric form and preparation thereof
CN111196856A (en) * 2018-11-19 2020-05-26 三生国健药业（上海）股份有限公司 anti-HER 2/PD1 bispecific antibodies
CN111848800B (en) * 2020-07-31 2023-07-04 三优生物医药(上海)有限公司 PD-L1 single-domain antibody and application thereof
CN113754775A (en) * 2020-06-02 2021-12-07 三生国健药业（上海）股份有限公司 Bispecific antibody for resisting PD-L1 and HER2

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