Bispecific antibodies against her2 and uses thereof

Abstract

本公开公开了一种稳定的抗HER2的双特异性抗体，其包括分别用于识别HER2的胞外结构域II和胞外结构域IV的第一蛋白功能区和第二蛋白功能区，所述双特异性抗体在PBS中约37℃储存30天后仍是稳定的。本公开还公开了包括所述双特异性抗体与PD1抗体的药物组合物和它们的用途。本公开的双特异性抗体和含其的药物组合物可用于癌症的治疗。

Description

Anti-HER2 bispecific antibodies and their applications

[0001] Priority Declaration

[0002] This disclosure claims priority to Chinese patent application No. 202311705319.X, filed on December 12, 2023, and to Chinese patent application No. 2024114014713, filed on October 8, 2024. The entire text of the aforementioned Chinese patent application is incorporated herein by reference. Technical Field

[0003] The present disclosure relates to the field of biomedicine, and in particular to an anti-HER2 bispecific antibody and its application. Background Art

[0004] Trastuzumab and pertuzumab, anti-HER2 antibodies, are widely used to treat HER2-positive breast cancer and other cancers. The combination of pertuzumab and trastuzumab is more effective than either agent alone. The response rate for pertuzumab alone is only 3.4%, but when combined with trastuzumab, it reaches 21.4%. Despite this, both trastuzumab and pertuzumab are only suitable for the treatment of breast cancer with strong HER2 positivity (IHC 3+). For breast cancer with weak HER2 positivity, trastuzumab and pertuzumab are less effective. In recent years, cell therapy has achieved remarkable success in the treatment of hematological malignancies, leading scientists to develop a trastuzumab-based treatment for HER2-positive breast cancer. However, the patient died five days after CAR-T cell infusion due to severe side effects (Morgan et al., The American Society of Gene & Cell Therapy 2010;4:843-851). Bispecific antibodies against different HER2 epitopes are monoclonal antibody drugs used to recognize two different antigenic epitopes on the HER2 surface. They contain two heavy chain variable regions of different antibodies and two identical light chain variable regions (CN105829347A; CN105820251A; CN105980409A), which can specifically bind to the second and fourth domains on the HER2 surface. The purpose is to provide a bispecific antibody with both trastuzumab activity and pertuzumab activity, replacing the current clinical method of combining trastuzumab and pertuzumab for treatment. However, existing antibodies have the characteristics of poor antigen binding activity or poor stability and easy precipitation, and stability is a necessary condition for the drugability of antibodies. At the same time, how to improve the overall efficacy of drugs remains an important requirement for drug research. Summary of the Invention

[0005] In view of the technical defects of the prior art such as low affinity, poor stability, easy precipitation and easy degradation, the purpose of the present disclosure is to provide an anti-HER2 bispecific antibody and its application.

[0006] The inventors of the present disclosure discovered that by using the heavy chains of pertuzumab and trastuzumab, respectively, and optimizing the natural trastuzumab light chain as a common light chain, they were able to generate a bispecific monoclonal antibody with affinities for the second and fourth domains of the HER2 target comparable to those of wild-type pertuzumab and trastuzumab, respectively. Furthermore, they unexpectedly discovered that, compared to the anti-HER2 bispecific antibody molecule KJ015-C, while its stability had been significantly improved compared to the unmutated sequence, further studies revealed that the antibody KJ015-H disclosed herein exhibited even greater stability, demonstrating significantly improved stability after thermal stability testing and exhibiting enhanced proliferation inhibition and ADCC activity against a variety of HER2-expressing tumor cells.

[0007] In combination with PD1 antibodies, the antibodies of the present disclosure showed significantly better tumor inhibitory activity than the combination of trastuzumab and pertuzumab.

[0008] To solve the above technical problems, some aspects of the present disclosure include providing an anti-HER2 bispecific antibody, wherein the bispecific antibody comprises a first protein functional region and a second protein functional region for respectively recognizing the extracellular domain II and the extracellular domain IV of HER2, the first protein functional region comprises a first heavy chain and a common light chain, the second protein functional region comprises a second heavy chain and the common light chain, and the bispecific antibody is stable after storage in PBS buffer at 37°C ± 0.5°C for 10, 20, 22, 24, 26, 28 or 30 days. Optionally, after storage in PBS buffer at 37°C for 10, 20, 22, 24, 26, 28 or 30 days, the acidic peak ratio of the bispecific antibody increases by less than 10%, less than 9%, less than 8%, less than 7%, less than 6% or less than 5%.

[0009] In some embodiments, the bispecific antibodies of the present disclosure are placed in PBS buffer for 10, 20, 22, 24, 26, 28, or 30 days, and the acidic peak ratio increases by less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% as detected by IEC-HPLC.

[0010] In some embodiments, the bispecific antibodies of the present disclosure are placed in PBS buffer for 10, 20, 22, 24, 26, 28, or 30 days, and the main peak ratio decreases by less than 5%, less than 4%, less than 3%, less than 2.5%, or less than 2% as detected by IEC-HPLC.

[0011] In some embodiments, the bispecific antibodies of the present disclosure include a first protein functional region and a second protein functional region for recognizing the extracellular domain II and extracellular domain IV of HER2, respectively, wherein the first protein functional region includes a first heavy chain and a common light chain, and the second protein functional region includes a second heavy chain and the common light chain, wherein the common light chain includes a light chain variable region, the amino acid sequence of the light chain variable region is as shown in SEQ ID NO: 1, the first heavy chain includes the first heavy chain variable region as shown in SEQ ID NO: 2 or a variant thereof, and the second heavy chain includes the second heavy chain variable region as shown in SEQ ID NO: 5 or a variant thereof.

[0012] In some embodiments, variants of the bispecific antibodies disclosed herein enable the bispecific antibodies to obtain enhanced stability or tumor inhibitory activity.

[0013] In some embodiments, the bispecific antibody of the present disclosure further comprises a light chain constant region, wherein the light chain constant region is preferably a human light chain constant region λ chain or κ chain; preferably, the amino acid sequence of the light chain constant region is as shown in SEQ ID NO:6.

[0014] In some embodiments, the first heavy chain and the second heavy chain of the bispecific antibody of the present disclosure further include a heavy chain constant region, and the heavy chain constant region is preferably a human heavy chain constant region; preferably, the first heavy chain and the second heavy chain of the bispecific antibody form a heterodimer, and the first heavy chain and the second heavy chain are preferably connected by a Knob-in-Hole structure; more preferably, the heavy chain constant regions of the first heavy chain and the second heavy chain are as shown in SEQ ID NOs: 7 and 8, respectively.

[0015] In some embodiments, the bispecific antibodies of the present disclosure have reduced fucose modification; alternatively, the antibodies are afucose-free.

[0016] Other aspects of the present invention include providing a nucleic acid molecule encoding a bispecific antibody according to the present disclosure.

[0017] In some aspects, embodiments include providing an expression vector comprising the nucleotide sequence of the nucleic acid molecule described herein.

[0018] In some aspects, embodiments include providing a prokaryotic or eukaryotic cell comprising an expression vector as described herein.

[0019] In some aspects, the present invention provides an anti-HER2 bispecific antibody-drug conjugate, which comprises the bispecific antibody as described above covalently coupled to a cytotoxic drug.

[0020] In some aspects, embodiments include providing a pharmaceutical composition comprising a bispecific antibody of the present disclosure, or a drug conjugate of a bispecific antibody of the present disclosure; optionally, the pharmaceutical composition further comprises a hyaluronidase and / or an anti-PD1 antibody; optionally, the hyaluronidase is rHuPH20 or modified rHuPH20; and optionally, the anti-PD1 antibody is selected from pembrolizumab; optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

[0021] In some aspects, an embodiment includes providing a method for preparing a bispecific antibody as described herein, comprising the following steps:

[0022] (1) constructing the nucleotide sequence encoding the first heavy chain and the second heavy chain and the nucleotide sequence encoding the common light chain into the same vector or different vectors;

[0023] (2) Expressing the vector constructed in the above steps in different host cells or the same host cell.

[0024] Embodiments in some aspects include providing use of the anti-HER2 bispecific antibody, the anti-HER2 bispecific antibody-drug conjugate, and / or the pharmaceutical composition described herein in the preparation of a drug for treating HER2-positive tumors; optionally, the HER2-positive tumor is selected from breast cancer, bile duct cancer, gastric cancer, and lung cancer.

[0025] In addition, to solve the above technical problems, another technical solution is to provide a drug kit combination, which includes drug kit A and drug kit B; drug kit A contains the anti-HER2 bispecific antibody, anti-HER2 bispecific antibody drug conjugate, or pharmaceutical composition containing a bispecific antibody disclosed herein; and drug kit B contains other antibodies, bispecific antibodies, genetically modified cells, or pharmaceutical compositions targeting HER2 or other targets, such as PD1. The use of drug kits A and B is not limited to a specific order, for example, drug kit A can be used first and then drug kit B, or drug kit B can be used first and then drug kit A, or drug kit A and drug kit B can be used simultaneously.

[0026] The anti-HER2 bispecific antibodies, immunoconjugates, pharmaceutical compositions, or kits disclosed herein can be administered to patients for the treatment of related tumors.

[0027] In the present disclosure, anti-HER2 bispecific antibodies or their antigen-binding portions or mixtures thereof, or therapeutic immune cells containing their antigen-binding portions may also be used in combination with chemotherapeutic drugs and / or other antibodies. Therefore, the compositions of the present disclosure may also contain chemotherapeutic drugs and / or other antibodies.

[0028] In the present disclosure, the chemotherapy drugs include but are not limited to: Adriamycin, cyclophosphamide and taxanes [paclitaxel and docetaxel], capecitabine, gemcitabine, vinorelbine, tamoxifen, aromatase inhibitors [anastrozole, letrozole, exemestane), 5-fluorouracil (5-FU) + folinic acid, irinotecan, oxaliplatin, cisplatin, carboplatin, estramustine, mitoxantrone, prednisone, vincristine, etc., or combinations thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1. Detection results of the proliferation inhibition effects of different anti-HER2 bispecific antibodies on three different tumor cells.

[0030] Figure 2 shows the ADCC activity test results of different anti-HER2 bispecific antibodies. The ADCC activity of the samples was tested before and after storage at 37°C for 30 days. The ADCC activity of KJ015-C was taken as 100% to evaluate the activities of KJ015-D and KJ015-H. Figure 2a evaluates the activity of the antibodies before heated storage, and Figure 2b evaluates the activity of the antibodies after heated storage.

[0031] Figure 3 shows the results of different methods for testing the stability of KJ015-H. Figure 3a shows the stability of KJ015-H stored at 37°C for 30 days using IEC-HPLC, and Figure 3b shows the stability of KJ015-H stored at 37°C for 30 days using SEC-HPLC.

[0032] Figure 4: KJ015-H reduces the viability of HER2-expressing cancer cell lines and inhibits HER2 downstream signaling. Figures 4a-d show the viability of Calu-3 (a), NCI-N87 (b), MDA-MB-176 (c), and BT474 (d) cells measured using CCK-8, respectively.

[0033] Figure 5 shows the results of the test of the ability of each group of HER2 antibody samples to inhibit tumor growth in human tumor cell line xenograft (CDX) models. Figures 5a-5d show the tumor growth curves of xenografts derived from BT474 (Her2 3+, breast cancer), NCI-N87 (Her2 3+, gastric cancer), MDA-MB-175 (Her2 1+, breast cancer), and Calu-3 (Her2 3+, lung adenocarcinoma) cell lines in mice, respectively.

[0034] Figure 6 shows the test results of the ability of each group of HER2 antibody samples to inhibit tumor growth in a human tissue xenograft (PDX) model of biliary carcinoma (GBC) patients, where T+P refers to the combined use of trastuzumab + pertuzumab.

[0035] Figure 7 shows the effects of various HER2 antibody samples in the PBMC humanized NCI-N87 xenograft model. Figure 7a shows tumor growth in the PBMC-humanized NCI-N87 CDX model after different treatments; Figure 7b shows the comparison of tumor size among the groups on day 18 after drug administration.

[0036] Figure 8. Stochastic optical reconstruction microscopy determined the localization coordinates of HER2 molecules on the surface of antibody-treated SK-BR-3 cells.

[0037] Figure 9 shows the results of immunoblotting experiments in Example 12.

[0038] Detailed Description of the Invention

[0039] definition

[0040] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. For the purposes of this disclosure, the following terms are defined below.

[0041] The term "about" when used in conjunction with a numerical value is meant to encompass the numerical value within a range having a lower limit that is 5% less than the specified numerical value and an upper limit that is 5% greater than the specified numerical value.

[0042] The term "and / or" should be understood to mean any one of the options or a combination of any two or more of the options.

[0043] As used herein, the terms "comprising" or "including" are intended to include the recited elements, integers, or steps, but do not exclude any other elements, integers, or steps. In this document, when the terms "comprising" or "including" are used, unless otherwise indicated, the context of consisting of the recited elements, integers, or steps is also encompassed. For example, when reference is made to an antibody variable region "comprising" a specific sequence, it is intended to encompass an antibody variable region consisting of that specific sequence.

[0044] As used herein, the term "antibody" refers to a polypeptide comprising at least a light chain or heavy chain immunoglobulin variable region that specifically recognizes and binds to an antigen. The term encompasses various antibody structures, including but not limited to monoclonal antibodies, polyclonal antibodies, single-chain antibodies or multi-chain antibodies, monospecific or multispecific antibodies (e.g., bispecific antibodies), fully human antibodies, chimeric antibodies, or humanized antibodies, full-length antibodies, and antibody fragments, as long as they exhibit the desired antigen-binding activity.

[0045] The term "antigen-binding fragment" of an antibody (used interchangeably herein with "antibody fragment" and "antigen-binding portion") refers to a molecule that is not a complete antibody, which comprises a portion of a complete antibody that binds to the antigen to which the complete antibody binds. As will be appreciated by those skilled in the art, the antigen-binding portion of an antibody typically comprises amino acid residues from a "complementarity determining region" or "CDR". Antigen-binding fragments can be prepared by recombinant DNA techniques, or by enzymatic or chemical cleavage of complete antibodies. Antigen-binding fragments include, but are not limited to, Fab, scFab, Fab', F(ab')2, Fab'-SH, Fv, single-chain Fv, diabody, triabody, tetrabody, minibody, and single-domain antibody (sdAb). For a more detailed description of antibody fragments, see: Fundamental Immunology, ed. W.E. Paul, Raven Press, NY (1993); Shao Rongguang et al. (eds.), Antibody Drug Research and Application, People's Medical Publishing House (2013); Hollinger et al., PNAS USA 90: 6444-6448 (1993); Hudson et al., Nat. Med. 9: 129-134 (2003).

[0046] The term "variable region" or "variable domain" refers to the domain of an antibody heavy or light chain that is involved in binding the antibody to an antigen. The variable domains of the heavy and light chains of natural antibodies generally have similar structures, with each domain comprising four conserved framework regions (FRs) and three complementarity determining regions (see, e.g., Kindt et al., Kuby Immunology, 6th ed., WH Freeman and Co., p. 91 (2007)). A single VH or VL domain may be sufficient to confer antigen binding specificity. In addition, VH or VL domains from antibodies that bind to a particular antigen can be used to isolate antibodies that bind to the antigen to screen libraries of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150: 880-887 (1993); Clarkson et al., Nature 352: 624-628 (1991).

[0047] A "variant" refers to a new peptide or fragment obtained by adding, subtracting, or replacing other amino acid residues on the basis of the amino acid sequence of a certain peptide or fragment.

[0048] The variable regions typically exhibit the same general structure of relatively conserved framework regions (FRs) connected by three hypervariable regions, also known as complementarity determining regions or CDRs. The CDRs from the two chains of each pair are typically aligned by the framework regions, which allow for binding to specific epitopes. The two light and heavy chain variable regions typically comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4 from N-terminus to C-terminus.

[0049] "Complementarity determining region" or "CDR region" or "CDR" or "hypervariable region" (interchangeably used herein with hypervariable region "HVR"), is a region in an antibody variable domain that is highly variable in sequence and forms structurally determined loops ("hypervariable loops") and / or contains antigen contact residues ("antigen contact points"). CDRs are primarily responsible for binding to antigenic epitopes. The CDRs of the heavy and light chains are typically referred to as CDR1, CDR2, and CDR3, and are numbered sequentially from the N-terminus. The CDRs located within the antibody heavy chain variable domain are referred to as HCDR1, HCDR2, and HCDR3, while the CDRs located within the antibody light chain variable domain are referred to as LCDR1, LCDR2, and LCDR3. In a given light chain variable region or heavy chain variable region amino acid sequence, the precise amino acid sequence boundaries of each CDR can be determined using any one of a number of well-known antibody CDR assignment systems or a combination thereof, including, for example, Chothia based on the three-dimensional structure of the antibody and the topology of the CDR loops, Kabat based on antibody sequence variability, AbM (University of Bath), Contact (University College London), the international ImMunoGeneTics database (IMGT), with the exception of HCDR1 and HCDR2, where the Chothia CDR definition is identical to the Kabat CDR definition. The North CDR definition is based on neighbor propagation clustering using a large number of crystal structures.

[0050] The term "preventing" includes inhibiting or delaying the onset or frequency of a disease or disorder or its symptoms and generally refers to the administration of a drug before signs or symptoms occur, particularly in an at-risk individual.

[0051] As used herein, the term "antibody stability" refers to the change in activity of an antibody after storage at a specific temperature and time. Available stability testing methods include evaluating the distribution of charge variants by cation exchange chromatography using the IEC-HPLC method (other commonly used antibody stability testing methods may be supplemented as appropriate). Preferably, the HER2 bispecific antibody can be tested separately with reference to molecular exclusion chromatography (Appendix IIIB of Part III of the 2010 edition of the Chinese Pharmacopoeia), and the thermal stability test data of the antibody before and after storage in a 37°C pH 7.4 PBS system for 30 days are compared. The stability of the bispecific antibody is expressed as an indicator of the change (increase or decrease) ratio of the acidic peak, main peak, or basic peak. The increase ratio of the acidic / basic peak = (the acidic / basic peak percentage after 30 days of storage - the acidic peak percentage before storage) × 100% / the acidic / basic peak percentage before storage, and the decrease ratio of the main peak = (the main peak percentage before storage - the main peak percentage after 30 days of storage) × 100% / the main peak percentage before storage. "Stable" means that, as determined by IEC-HPLC, after the bispecific antibody is placed in PBS buffer at 37°C ± 0.5°C for 10, 20, 22, 24, 26, 28 or 30 days, the proportion of the acidic peak does not increase or increases by less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, or less than 0.5%; and / or the main peak does not decrease or decreases by less than 5%, less than 4%, less than 3%, less than 2.5% or less than 2%.

[0052] A "pharmaceutical composition" refers to a preparation or combination of preparations containing one, two, or more active ingredients that allows the active ingredients contained therein to be in a biologically effective form. When a "pharmaceutical composition" is present as a combination of separate preparations containing two or more different active ingredients, the preparations may be administered simultaneously, sequentially, separately, or at intervals, with the goal of leveraging the biological activities of the multiple active ingredients for combined treatment of a disease.

[0053] "Effective amount" is generally an amount sufficient to reduce the severity and / or frequency of symptoms, eliminate these symptoms and / or potential causes, prevent symptoms and / or their potential causes from occurring and / or improve or ameliorate the damage (e.g., lung disease) caused by or associated with the disease state. In some embodiments, an effective amount is a therapeutically effective amount or a prophylactic effective amount. A "therapeutically effective amount" is an amount sufficient to treat a disease state or symptom, particularly a state or symptom associated with the disease state, or otherwise prevent, hinder, delay, or reverse the disease state or any other undesirable symptom associated with the disease in any way. A "prophylactic effective amount" is an amount that will have a predetermined preventive effect when administered to a subject, such as preventing or delaying the onset (or recurrence) of the disease state, or reducing the likelihood of the onset (or recurrence) of the disease state or associated symptoms. A complete therapeutic or prophylactic effect may not occur with the administration of one dose, and may only occur after a series of doses are administered. Thus, a therapeutic or prophylactic effective amount can be administered in one or more administrations.

[0054] The terms "host cell," "host cell line," and "host cell culture" are used interchangeably and refer to cells into which exogenous nucleic acid has been introduced, including the progeny of such cells. Host cells include "transformants" and "transformed cells," which include the primary transformed cell and progeny derived therefrom, without regard to the number of passages. Progeny may not be completely identical in nucleic acid content to the parent cell, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included herein.

[0055] "Transfection" refers to the introduction of exogenous nucleic acid into eukaryotic cells. Transfection can be achieved by various means known in the art, including electroporation, microinjection, liposome fusion, etc.

[0056] The terms "nucleic acid molecule code," "coding DNA sequence," and "coding DNA" refer to the order of deoxyribonucleotides along a deoxyribonucleic acid chain. The order of these deoxyribonucleotides determines the order of amino acids along a polypeptide (protein) chain. Thus, a nucleic acid sequence encodes an amino acid sequence.

[0057] As used herein, the term "tumor" refers to a disease characterized by the pathological proliferation of cells or tissues, and their subsequent migration or invasion of other tissues or organs. Tumor growth is typically uncontrolled and progressive, without inducing or inhibiting normal cell proliferation. Tumor includes "cancer" and refers broadly to all malignant tumors.

[0058] "Treatment" refers to clinical intervention aimed at altering the disease process in an individual or cell, and can be either preventative or interventional in the clinical pathological process. Therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of a disease, alleviating symptoms, reducing any direct or indirect pathological consequences of a disease, preventing metastasis, slowing the progression of a disease, improving or relieving the condition, and alleviating or improving the prognosis.

[0059] "Combination" refers to a treatment regimen that provides at least two or more different therapies to achieve a specified therapeutic effect. The therapies can be physical, such as radiotherapy, or chemical, such as administering a drug to a subject, including combination drugs. "Combination drug" refers to a combination of two or more pharmaceutical preparations, each with its own active ingredient, that are administered to a subject in combination. The active ingredients can be mixed together to form a single dosing unit, or they can be administered separately as separate dosing units; when administered, the different pharmaceutical preparations can be administered substantially synchronously, simultaneously, or sequentially.

[0060] The term "pharmaceutically acceptable" refers to compounds, materials, compositions and / or dosage forms that are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0061] As used herein, "anti-PD1 antibodies" refer to antibodies or antigen-binding fragments thereof that target the immune checkpoint protein PD1, also known as PD-1, and are capable of blocking or affecting the binding of PD1 to its ligand. Examples of anti-PD1 antibodies include, but are not limited to, pembrolizumab. "PD1 (programmed death receptor 1)," also known as CD279, belongs to the immunoglobulin superfamily.

[0062] On the basis of being in accordance with the common sense in the art, the above optional conditions can be arbitrarily combined to obtain the preferred embodiments of the present disclosure. The reagents and raw materials used in the present disclosure are all commercially available.

[0063] The positive progress of the present disclosure is: by mutating the specified amino acids in the light chain variable region of trastuzumab, the heavy chain variable region of pertuzumab, and the heavy chain variable region of trastuzumab, a bispecific antibody is formed, which has high affinity, good stability, is not easy to degrade, has strong tumor cell killing activity, and has high ADCC activity, as well as an anti-PD1 antibody and / or hyaluronidase used in combination with the bispecific antibody. DETAILED DESCRIPTION

[0064] The technical solution of the present disclosure is further described below through specific implementation methods. Those skilled in the art should understand that the implementation methods are only used to help understand the present disclosure and should not be regarded as specific limitations of the present disclosure.

[0065] In the following examples, the experimental methods without specific conditions were carried out according to conventional methods and conditions, or selected according to the product instructions.

[0066] Example 1. Antibody Construction, Expression and Preparation

[0067] According to the amino acid sequence in Table 2, the gene synthesis of the antibody light and heavy chains was carried out according to Table 1, and the corresponding common light chain plasmid, the first heavy chain plasmid and the second heavy chain plasmid were obtained by plasmid extraction. The plasmid DNA containing the coding sequences of the first heavy chain, the second heavy chain and the common light chain was co-transfected into ExpiCHO-S cell line (Gibco) at a certain ratio (1:1:2) using the transient transfection kit of The transfection kit instructions were followed. Various bispecific antibody-expressing cell lines were ultimately obtained. Each cell line was expanded to a volume of 100 ml using CD2 basal medium and cultured in a fed-batch format. Starting on day 3, 2% Feed4 was added daily for 12 days of suspension culture. The culture supernatants were collected and purified by Protein A affinity chromatography to obtain bispecific monoclonal antibody protein samples. Purified samples were sterile-filtered using a 0.22 μm membrane and stored at 2–8°C.

[0068] Table 1. HER2 bispecific antibody sequence combinations

[0069] *The Her2 antibody common light chain is formed by directly linking the Her2 antibody common light chain variable region and the HER2 antibody light chain constant region; the Her2 antibody first heavy chain is formed by directly linking the Her2 antibody first heavy chain variable region and the HER2 antibody first heavy chain constant region; the Her2 antibody second heavy chain is formed by directly linking the corresponding Her2 antibody second heavy chain variable region and the HER2 antibody second heavy chain constant region, that is, the Her2 antibody second heavy chain -H is formed by directly linking the Her2 antibody second heavy chain variable region -H (SEQ ID NO.5) and the HER2 antibody second heavy chain constant region (SEQ ID NO.8), and so on (see Table 2 for specific sequences).

[0070] Table 2. Amino acid sequences of HER2 bispecific antibody fragments

[0071] Example 2. Stability test

[0072] The distribution of charge variants was assessed by cation exchange chromatography using IEC-HPLC. The HER2 bispecific antibodies KJ015-C, KJ015-D, and KJ015-H were tested using size exclusion chromatography (Appendix IIIB, Part III, Chinese Pharmacopoeia 2010). The thermal stability data of each antibody were compared before and after storage in PBS at 37°C, pH 7.4, for 30 days.

[0073] A Dionex ProPac WCX-10 column (4 × 250 mm) was used as the chromatographic column. A mixture of 4 mM imidazole, 4 mM piperazine, 4 mM Tris, and 0.5 M NaCl was used as the mobile phase. The flow rate was 1 ml / min. The detection wavelength was 280 nm. 10 μl of the test solution was injected into the liquid chromatograph for detection.

[0074] IEC detected charge heterogeneity. After heating, the main peak of KJ015-H sample decreased the least and was more stable (Table 3).

[0075] Table 3. Charge heterogeneity detected by IEC

[0076] Example 3. Detection of proliferation inhibition effects of different HER2 bispecific antibodies

[0077] Different tumor cell lines were subcultured, diluted to various concentrations, and incubated with antibodies. Day 0 was used as the starting date. On the fourth day after sample addition, the assay plates were removed from the incubator and 20 μl of CCK-8 staining solution was added to each well. The cells were stained in a 37°C incubator for 6 hours and read at 450 nm / 630 nm using a microplate reader. SoftMax Pro data processing software was used for automated analysis, with the concentration of the reference and test samples plotted on the horizontal axis and the OD value plotted on the vertical axis. A four-parameter method was used to fit the dose-response curves for the reference and test samples. The KJ015-C sample was used as the standard, designated as 100%, and the relative activity of the other samples was calculated. The assays revealed that KJ015-H exhibited greater antiproliferative activity than KJ015-C in various tumor cell lines, with 1.25-fold, 1.7-fold, and 1.1-fold increases in antiproliferative activity in BT474, MDA-MB-175, and Calu-3 cells, respectively (Figure 1).

[0078] Example 4. Detection of ADCC effects of different HER2 bispecific antibodies

[0079] For ADCC testing, tumor cells in the logarithmic growth phase were resuspended in culture medium based on cell growth rate and seeded into 96-well plates. After adherence, antibodies KJ015-C, KJ015-D, KJ015-H, KJ015-C-heated, KJ015-D-heated, and KJ015-H-heated were added and incubated at 37°C, 5% CO2 for 30 minutes. ADCC was measured using the CytoTox96 non-radioactive cytotoxicity assay, with KJ015-C and KJ015-C-heated samples used as standards and recorded as 100%. The relative activity of the other samples was calculated. Testing on BT474 cells revealed that KJ015-H exhibited higher relative activity in ADCC than KJ015-C and KJ015-D. The ADCC activity of the heated KJ015-H sample was higher than that of the heated KJ015-C sample, indicating that KJ015-H exhibited stronger ADCC activity than KJ015-C and D, as well as the heated sample (see Figures 2a and 2b).

[0080] Example 5. Antibody affinity test

[0081] use The affinities of direct interactions between KJ015-H, trastuzumab, pertuzumab, anbenitamab, and zanidatamab and HER2 domains II and VI were determined using a Biacore 3000 instrument (Uppsala, Sweden).

[0082] Table 4. Kinetic and affinity constants of different antibodies binding to domains II and VI of HER2

[0083] The binding affinities of KJ015-H to Her2 domain II and domain IV were 5.24 nM and 0.63 nM, respectively (Table 4), while the affinity of pertuzumab to Her2 domain II was 2.26 nM, and the affinity of trastuzumab to domain IV was 0.90 nM. This indicates that KJ015-H has the high affinity of both trastuzumab and pertuzumab to Her2 domain II and IV.

[0084] Example 6. Antibody stability test

[0085] The thermal melting point (Tm) of each sample was determined using an Uncle instrument (Unchained Labs, Pleasanton, CA, USA).

[0086] SEC-HPLC analysis was performed under native conditions using a Waters Acquity ARC system, and purity was calculated using the area normalization method.

[0087] Table 5. Tm of different antibodies KJ015-H, trastuzumab and pertuzumab

[0088] The sequence of KJ015-H is derived from trastuzumab and pertuzumab, and its dissolution temperature is similar to that of pertuzumab (see Table 5). The modification of KJ015-H significantly improved its thermal stability. Even after 30 days of storage in PBS at 37°C, there was no significant change in IEC purity or SEC purity (see Figures 3a and 3b). However, earlier studies have shown that trastuzumab undergoes severe degradation after 12 days of storage under the same conditions, resulting in a significant decrease in antigen-binding activity and bioactivity (Schmid et al., 2018 Commun Biol 1, 28).

[0089] Example 7. KJ015-H can inhibit cell activity

[0090] The viability of various cell lines was assessed using the CCK-8 assay. Cells were seeded into 96-well plates. After cell attachment, the cells were treated with antibodies (human IgG1, trastuzumab (T), pertuzumab (P), trastuzumab plus pertuzumab (T+P), KJ015-H, anbenitamab (Anbe), and zanidatamab (Zani)) for 120 hours. CCK-8 solution was added to each well and incubated for 1 hour. The absorbance of each well was then measured, and the results were statistically analyzed.

[0091] As shown in Figure 4, the inhibition rate increased with increasing KJ015-H concentration in all four cell lines. KJ015-H exhibited strong cytotoxicity in all cell lines (Figures 4a-d). These results indicate that KJ015-H can reduce the viability of MDA-MB-175, BT474, Calu-3, and NCI-N87 cells and inhibit the proliferation of each tumor cell line.

[0092] Example 8. Anti-tumor effects of HER2 antibody samples in each group in the CDX model

[0093] NCI-N87, Calu-3, BT474, and MDA-MB-175 cells are estrogen-dependent in mice, so estradiol pellets were implanted into the right mammary pad of mice. 7 The cells were injected into the right anterior dorsal subcutaneous tissue of female nude mice to establish cancer xenograft models. For the NCI-N87, Calu-3, and BT474 xenograft models, when the average tumor volume of hormone-treated mice reached approximately 80-150 mm3 When the average tumor volume of hormone-treated mice reached approximately 250 mm, the mice were randomly divided into 4 groups with 6 mice in each group. 3 According to the experimental design, the mice were randomly divided into 5 groups, with 6 mice in each group.

[0094] The BT474 or NCI-N87 xenograft groups received vehicle, trastuzumab + pertuzumab (0.94 + 0.94 mg / kg, 1.88 + 1.88 mg / kg), or KJ015-H (1.88 mg / kg), respectively, injected twice a week via the tail vein.

[0095] The MDA-MB-175 xenograft groups received vehicle, trastuzumab + pertuzumab (1.5 + 1.5 mg / kg, 5 + 5 mg / kg), and KJ015-H (3 mg / kg, 10 mg / kg) twice a week via tail vein injection.

[0096] Calu-3 xenograft mice received vehicle, trastuzumab + pertuzumab (1.88 + 1.88 mg / kg, 3.75 + 3.75 mg / kg), or KJ015-H (3.75 mg / kg) twice weekly via tail vein injection. Tumor diameter and mouse body weight were measured twice weekly, and tumor volume was calculated based on this. Data are mean ± SEM (n = 6).

[0097] Throughout the treatment and observation period, KJ015-H potently reduced tumor growth, with a trend toward greater efficacy than trastuzumab plus pertuzumab (Figures 5a–5d). Specifically, in the BT474 breast cancer cell transplant model, a low-dose trastuzumab plus pertuzumab combination (1:1 molar ratio, 0.94 mg / kg / mAb) had no effect on tumor size compared to the control group, while the same concentration of KJ015-H (1.88 mg / kg) still caused tumor regression. These results demonstrate that, compared to trastuzumab plus pertuzumab, KJ015-H significantly inhibits tumor growth at low concentrations without producing significant toxicity.

[0098] Example 9. Anti-tumor effect detection of each group of HER2 antibody samples in PDX model

[0099] To simplify treatment options for cancer patients and reduce costs for both patients and the healthcare system, Shanghai Baoji Pharmaceutical Co., Ltd. has added hyaluronidase to its KJ015-H injection, switching the traditional intravenous administration of the antibody to a subcutaneous route. KJ015-H subcutaneous injection (containing hyaluronidase) is a compound of KJ015-H and hyaluronidase (Shanghai Baoji Pharmaceutical Co., Ltd.) for subcutaneous administration. Hyaluronidase can temporarily degrade hyaluronic acid in the human body, improving tissue permeability and promoting rapid dispersion and absorption of the injected drug, enabling subcutaneous administration.

[0100] This example studies the in vivo antitumor activity of KJ015-H in a cholangiocarcinoma patient xenograft (PDX) mouse model. 3 Tumor tissues of 130 mm in size were implanted subcutaneously into the right forelimb of 8-week-old C-NKG mice (severely immunodeficient mice generated by Cyagen by deleting the Il2rg gene from NOD-Scid genetic background mice). 3 At the time of the study, 30 mice were randomly divided into five groups and received intravenous injection of vehicle, trastuzumab + pertuzumab (12.5 + 12.5 mg / kg), or subcutaneous injection of KJ015-H (12.5, 25, 50 mg / kg). Tumor growth was measured twice a week. Throughout the study, mice were weighed and observed regularly for signs of illness or discomfort related to tumor growth and / or drug toxicity.

[0101] Compared with the control group, there were no significant differences between the experimental groups for subcutaneous injection of KJ015-H and trastuzumab plus pertuzumab. Both significantly inhibited tumor growth and did not cause significant toxicity, as measured by weight loss. The degree of tumor inhibition with subcutaneous injection of KJ015-H was dose-dependent. Consistent with the in vitro and in vivo findings, subcutaneous injection of KJ015-H exhibited the same antitumor effect as high-concentration intravenous trastuzumab plus pertuzumab at low subcutaneous concentrations (see Figure 6 and Table 6).

[0102] Table 6. Effect of subcutaneous injection of KJ015-H on tumor weight in PDX model

[0103] Example 10. Detection of the effect of KJ015-H subcutaneous injection combined with αPD-1 antibody in the treatment of HER2-overexpressing tumors

[0104] The efficacy of KJ015-H subcutaneous injection or trastuzumab combined with anti-PD-1 antibody (pembrolizumab, trade name: Keytruda, manufacturer: Merck) in treating tumors was tested using humanized NOG mice in the NCI-N87 model. 3×10 6 Human PBMCs were injected into NOG mice via the tail vein; the day of tumor cell implantation was set as d0; on the second day after tumor cell inoculation (d1), mice in groups G3 and G4 began to receive anti-PD-1 antibody treatment, 10 mg / kg, iv, BIW. When the average tumor volume reached 100 mm 3 After the experiment (4 days after tumor cell inoculation (d3)), NOG mice received control group (normal saline, iv, G1), subcutaneous injection of KJ015-H (4 mg / kg, sc, G2), trastuzumab + anti-PD-1 mAb (4+10 mg / kg, iv+iv, G3), and subcutaneous injection of KJ015-H + anti-PD-1 mAb (4+10 mg / kg, sc+iv, G4) treatment respectively. The tumor volume was measured twice a week, and the tumor was removed on the 19th day after tumor cell inoculation (d18).

[0105] The results showed that compared with the control group, the combination of αPD-1 with subcutaneous KJ015-H or trastuzumab inhibited the growth of HER2-positive NCI-N87 tumors without causing significant weight loss. Compared with trastuzumab combined with αPD-1, subcutaneous KJ015-H combined with αPD-1 demonstrated stronger synergistic anti-tumor activity (see Figures 7a and 7b).

[0106] Example 11

[0107] SK-BR-3 cells were detached, suspended in complete RPMI medium, and treated with 200 nM KJ015-H or trastuzumab + pertuzumab (100 + 100 nM) at 37°C for 15 minutes. The labeling kit (ab188288) was pre-labeled with Cy5, with each antibody molecule labeled with an average of 2.5 AF647 molecules. After fixation with 2% phosphoric acid (PFA) in PBS for 10 minutes at room temperature, cells were washed with PBS, pelleted on a 0.01% poly-l-lysine cover slip, and fixed again with 2% PFA in PBS. After thorough PBS washing and pelleting with fiducial markers (ThermoFisher Scientific, Waltham, MA, USA) at 4°C overnight, unbound beads were removed, and adhered particles were imaged using stochastic optical reconstruction microscopy (iSTORM) (Ningbo INVIEW Intelligent Technology Co., Ltd., China). Data were processed using instrument software, and the average area of ​​Cy5 fluorescence signal was calculated, with distribution presented as violin plots.

[0108] When Cy5-labeled KJ015-H or T+P were studied in SK-BR-3 cells, varying degrees of high aggregation signal distribution were observed (Figure 8). The figure shows that KJ015H induced a significantly stronger aggregation effect than T+P.

[0109] Example 12

[0110] Western blotting experiments were used to detect the expression of multiple signaling pathways after different tumor cells were incubated with different antibodies (human IgG1, trastuzumab (T), pertuzumab (P), trastuzumab plus pertuzumab (T+P), KJ015-H, Anbenitamab (Anbe), and Zanidatamab (Zani)).

[0111] In immunoblotting results, HER2 and HER3 expression levels remained unchanged, while downstream MAPK signaling was reduced (Figure 9). It is reasonable to infer that aggregation reduces normal HER2 heterodimer levels, preventing downstream signaling from being activated through normal dimer signaling pathways. It can be inferred that aggregation may promote endocytosis and hinder downstream signaling pathways, thereby reducing tumor cell activity and inhibiting proliferation.

[0112] Although the specific embodiments of the present disclosure have been described above, those skilled in the art will appreciate that these are merely illustrative and that various changes or modifications may be made to these embodiments without departing from the principles and essence of the present disclosure. Therefore, the scope of protection of the present disclosure is defined by the appended claims.

Claims

1. An anti-HER2 bispecific antibody, comprising a first protein functional region and a second protein functional region for respectively recognizing the extracellular domain II and the extracellular domain IV of HER2, wherein the first protein functional region comprises a first heavy chain and a common light chain, and the second protein functional region comprises a second heavy chain and the common light chain, and the bispecific antibody is stable after storage in PBS buffer at 37±0.5°C for 30 days.

2. The anti-HER2 bispecific antibody according to claim 1, wherein The common light chain includes a light chain variable region, the amino acid sequence of which is shown in SEQ ID NO: 1, the first heavy chain includes a first heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 2 or a variant thereof, and the second heavy chain includes a second heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 5 or a variant thereof.

3. The bispecific antibody according to claim 1 or 2, further comprising a light chain constant region, wherein the light chain constant region may be a human light chain constant region λ chain or κ chain; optionally, the amino acid sequence of the light chain constant region is as shown in SEQ ID NO:

6.

4. The bispecific antibody according to any one of claims 1 to 3, wherein the first heavy chain and the second heavy chain of the bispecific antibody further comprise a heavy chain constant region, and the heavy chain constant region may be a human heavy chain constant region; optionally, the first heavy chain and the second heavy chain of the bispecific antibody form a heterodimer, and the first heavy chain and the second heavy chain may be connected by a Knob-in-Hole structure; optionally, the heavy chain constant regions of the first heavy chain and the second heavy chain are as shown in SEQ ID NOs: 7, 9, 10 and 8, 11, 12, respectively.

5. The bispecific antibody of any one of claims 1-4, wherein the bispecific antibody has reduced fucose modification; alternatively, the antibody is non-fucose modified.

6. A nucleic acid molecule encoding the bispecific antibody according to any one of claims 1 to 5.

7. An expression vector comprising the nucleotide sequence of the nucleic acid molecule according to claim 6.

8. A prokaryotic or eukaryotic cell comprising the expression vector according to claim 7. 9 . An anti-HER2 bispecific antibody-drug conjugate, which comprises the bispecific antibody according to any one of claims 1 to 5 covalently coupled to a cytotoxic drug.

10. A pharmaceutical composition comprising the bispecific antibody of any one of claims 1 to 5 or the drug conjugate of the bispecific antibody of claim 9; optionally, the pharmaceutical composition further comprises hyaluronidase and / or anti-PD1 antibody; optionally, the hyaluronidase is rHuPH20 or modified rHuPH20; optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

11. A method for preparing the bispecific antibody according to any one of claims 1 to 5, comprising the following steps: (1) constructing the nucleotide sequence encoding the first heavy chain and the second heavy chain and the nucleotide sequence encoding the common light chain in the same vector or in different vectors; (2) Expressing the vector constructed in the above steps in different host cells or the same host cell.

12. Use of the anti-HER2 bispecific antibody according to any one of claims 1 to 5, the anti-HER2 bispecific antibody-drug conjugate according to claim 9, and the pharmaceutical composition according to claim 10 in the preparation of a medicament for treating HER2-positive tumors; optionally, the HER2-positive tumor is selected from breast cancer, bile duct cancer, gastric cancer and lung cancer.

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