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WO2025124463A1 - Anticorps bispécifique anti-her2 et son utilisation - Google Patents

Anticorps bispécifique anti-her2 et son utilisation Download PDF

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Publication number
WO2025124463A1
WO2025124463A1 PCT/CN2024/138695 CN2024138695W WO2025124463A1 WO 2025124463 A1 WO2025124463 A1 WO 2025124463A1 CN 2024138695 W CN2024138695 W CN 2024138695W WO 2025124463 A1 WO2025124463 A1 WO 2025124463A1
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Prior art keywords
heavy chain
bispecific antibody
her2
antibody
light chain
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Chinese (zh)
Inventor
刘彦君
王征
徐云霞
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Shanghai Bao Pharmaceuticals Co Ltd
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Shanghai Bao Pharmaceuticals Co Ltd
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/32—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against translation products of oncogenes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins

Definitions

  • the present disclosure relates to the field of biomedicine, and in particular to an anti-HER2 bispecific antibody and applications thereof.
  • Trastuzumab and Pertuzumab are anti-HER2 antibodies and are widely known for their use in the treatment of HER2-positive breast cancer and other cancers.
  • the combination of Pertuzumab and Trastuzumab is more effective than single use.
  • the response rate of Pertuzumab alone is only 3.4%, but the response rate can reach 21.4% when used in combination with Trastuzumab.
  • both Trastuzumab and Pertuzumab are only suitable for the treatment of breast cancer with strong HER2 positive (IHC test result is 3+).
  • the treatment effects of Trastuzumab and Pertuzumab are not ideal.
  • Bispecific antibodies against different HER2 epitopes are monoclonal antibody drugs used to identify two different antigenic epitopes on the surface of HER2.
  • HER2 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 surface of HER2.
  • the purpose is to provide a bispecific antibody with both trastuzumab activity and pertuzumab activity, replacing the current clinical method of trastuzumab plus pertuzumab combined therapy.
  • the existing antibodies have poor antigen binding activity, or poor stability and easy precipitation, and stability is a necessary condition for the drugability of antibodies.
  • how to improve the overall efficacy of drugs remains an important requirement for drug research.
  • the purpose of the present disclosure is to provide an anti-HER2 bispecific antibody and its application.
  • the inventors of the present disclosure have found that by using the heavy chains of pertuzumab and trastuzumab respectively and optimizing the natural trastuzumab light chain as a common light chain, a bispecific monoclonal antibody with affinity for the second and fourth domains of the HER2 target comparable to that of wild-type pertuzumab and trastuzumab can be obtained.
  • the antibody KJ015-H disclosed herein is more outstanding in stability, and shows significantly improved stability after thermal stability testing, and has enhanced proliferation inhibition effect and ADCC activity for a variety of HER2-expressing tumor cells.
  • the antibody of the present disclosure combined with PD1 antibody showed significantly better tumor inhibitory activity than the combination of trastuzumab and pertuzumab.
  • 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, the bispecific antibody is still stable after being stored in a PBS buffer at 37°C ⁇ 0.5°C for 10, 20, 22, 24, 26, 28 or 30 days, and optionally, after the bispecific antibody is stored in a PBS buffer at 37°C for 10, 20, 22, 24, 26, 28 or 30 days, the acidic peak ratio increases by less than 10%, less than 9%, less than 8%, less than 7%, less than 6% or less than 5%.
  • 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.
  • 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.
  • the bispecific antibody of the present invention 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 comprising a first heavy chain and a common light chain, the second protein functional region comprising a second heavy chain and the common light chain, wherein the common light chain comprises 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 comprises the first heavy chain variable region or a variant thereof with an amino acid sequence as shown in SEQ ID NO:2, and the second heavy chain comprises the second heavy chain variable region or a variant thereof with an amino acid sequence as shown in SEQ ID NO:5.
  • the variants of the bispecific antibodies of the present disclosure enable the bispecific antibodies to obtain stronger stability or tumor suppressor activity.
  • the bispecific antibody of the present invention also includes a light chain constant region, and the light chain constant region is preferably a human light chain constant region ⁇ chain or ⁇ chain; preferably, the amino acid sequence of the light chain constant region is as shown in SEQ ID NO:6.
  • the first heavy chain and the second heavy chain of the bispecific antibody of the present invention further include a heavy chain constant region, and the heavy chain constant region is preferably a human heavy chain constant region; preferably, a heterodimer is formed between the first heavy chain and the second heavy chain of the bispecific antibody, 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 NO: 7 and 8, respectively.
  • the bispecific antibodies of the present disclosure have reduced fucose modification; alternatively, the antibodies are afucose-free.
  • nucleic acid molecule encoding a bispecific antibody as described herein.
  • embodiments include providing an expression vector comprising a nucleotide sequence of a nucleic acid molecule described in the present disclosure.
  • embodiments include providing a prokaryotic or eukaryotic cell comprising an expression vector described herein.
  • 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.
  • Embodiments in some aspects include providing a pharmaceutical composition comprising the bispecific antibody of the present disclosure, or a drug conjugate of the bispecific antibody of the present disclosure; optionally, the pharmaceutical composition further comprises hyaluronidase and/or anti-PD1 antibody; optionally, the hyaluronidase is rHuPH20 or modified rHuPH20; optionally, the anti-PD1 antibody comprises pembrolizumab; optionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.
  • an embodiment includes providing a method for preparing a bispecific antibody as described in the present disclosure, comprising the following steps:
  • 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
  • Embodiments in some aspects include providing use of the anti-HER2 bispecific antibody as described herein, the anti-HER2 bispecific antibody-drug conjugate as described herein, and/or the pharmaceutical composition as 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.
  • kit combination which includes kit A and kit B;
  • the kit A contains the anti-HER2 bispecific antibody, anti-HER2 bispecific antibody drug conjugate or pharmaceutical composition including bispecific antibodies disclosed in the present invention;
  • the kit B contains other antibodies, bispecific antibodies, genetically modified cells or pharmaceutical compositions targeting HER2 or other targets, such as PD1.
  • kit A and kit B is not in order, for example, kit A is used first and then kit B, or kit B is used first and then kit A, or kit A and kit B are used at the same time.
  • the anti-HER2 bispecific antibody, the immunoconjugate, the pharmaceutical composition or the kit combination disclosed in the present invention can be administered to a patient for treating related tumors.
  • 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, and thus the compositions of the present disclosure may also contain chemotherapeutic drugs and/or other antibodies.
  • 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.
  • Figure 1 Detection results of the proliferation inhibition effect of different anti-HER2 bispecific antibodies in three different tumor cells.
  • 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 activity of KJ015-D and KJ015-H.
  • Figure 2a evaluates the activity of the antibody before heated storage
  • Figure 2b evaluates the activity of the antibody after heated storage.
  • 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
  • Figure 3b shows the stability of KJ015-H stored at 37°C for 30 days using SEC-HPLC.
  • Figure 4 KJ015-H can reduce the viability of HER2-expressing cancer cell lines and inhibit HER2 downstream signal transduction.
  • Figure 4a-d CCK-8 was used to measure the viability of Calu-3 (a), NCI-N87 (b), MDA-MB-176 (c) and BT474 (d) cells, respectively.
  • Figure 5 shows the results of the test of the ability of each group of HER2 antibody samples to inhibit tumor growth in the human tumor cell line xenograft (CDX) model.
  • 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.
  • 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 bile duct cancer (GBC) patients, where T+P refers to the combined use of trastuzumab + pertuzumab.
  • PDX human tissue xenograft
  • GBC bile duct cancer
  • Figure 7 shows the results of the effects of each group of HER2 antibody samples in the PBMC humanized NCI-N87 xenograft model.
  • Figure 7a shows the tumor growth of the PBMC-humanized NCI-N87 CDX model after different treatments;
  • Figure 7b shows the comparison of tumor size in each group at d18 after administration.
  • Figure 8 Stochastic optical reconstruction microscopy determined the localization coordinates of HER2 molecules on the surface of antibody-treated SK-BR-3 cells.
  • FIG. 9 shows the results of immunoblotting experiments in Example 12.
  • the term “comprising” or “including” means including the stated elements, integers or steps, but does not exclude any other elements, integers or steps.
  • the term “comprising” or “including” when used, unless otherwise indicated, it also covers the situation consisting of the stated elements, integers or steps.
  • an antibody variable region “comprising” a specific sequence when referring to an antibody variable region “comprising” a specific sequence, it is also intended to cover the antibody variable region consisting of the specific sequence.
  • 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 or chimeric antibodies or humanized antibodies, full-length antibodies, and antibody fragments, as long as they exhibit the desired antigen-binding activity.
  • antigen-binding fragment of an antibody (interchangeably used 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.
  • the antigen-binding portion of an antibody generally 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, single domain antibody (sdAb).
  • Fab fragment antigen-binding fragment antigen-binding fragment antigen-binding fragment antigen-binding fragment antigen-binding fragments
  • sdAb single domain antibody
  • variable region 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, wherein each domain contains four conserved framework regions (FRs) and three complementarity determining regions (see, e.g., Kindt et al. Kuby Immunology, 6th edition, W.H. Freeman and Co. 91 pages (2007)).
  • a single VH or VL domain may be sufficient to confer antigen binding specificity.
  • antibodies that bind to a specific antigen can be isolated using VH or VL domains from 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).
  • Variant refers to a new peptide or fragment obtained by adding, reducing or replacing amino acid residues on the basis of the amino acid sequence of a certain peptide or fragment.
  • variable region usually exhibits the same general structure of relatively conservative framework regions (FRs) connected by three hypervariable regions, which are also called complementarity determining regions or CDRs.
  • FRs framework regions
  • CDRs from the two chains of each pair are usually aligned by the framework regions, which allow binding to specific epitopes.
  • the two light chain and heavy chain variable regions usually comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4 from N-terminus to C-terminus.
  • 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 a structurally determined loop ("hypervariable loop") and/or contains antigen contact residues ("antigen contact points").
  • CDR is primarily responsible for binding to antigen epitopes.
  • the CDRs of the heavy and light chains are generally 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.
  • the precise amino acid sequence boundaries of each CDR can be determined using any one or a combination of many well-known antibody CDR assignment systems, 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), except for HCDR1 and HCDR2, the Chothia CDR definition is the same as the Kabat CDR definition.
  • the North CDR definition is based on neighbor propagation clustering using a large number of crystal structures.
  • preventing includes the inhibition or delay of 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 before signs or symptoms occur in an at-risk individual.
  • the term "antibody stability” refers to the change in activity of an antibody after storage at a specific temperature and time.
  • Available stability detection methods include evaluating the distribution of charge variants by cation exchange chromatography using the IEC-HPLC method (other commonly used antibody stability detection methods may be supplemented as appropriate).
  • the HER2 bispecific antibody can be detected by referring to the molecular exclusion chromatography method (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 pH7.4 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 alkaline peak.
  • the increase ratio of the acid/alkaline peak (the percentage of the acid/alkaline peak after 30 days of placement-the percentage of the acidic peak before placement) ⁇ 100%/the percentage of the acid/alkaline peak before placement
  • the decrease ratio of the main peak (the percentage of the main peak before placement-the percentage of the main peak after 30 days of placement) ⁇ 100%/the percentage of the main peak before placement.
  • “Stable” means that 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 acidic peak ratio 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% as detected by IEC-HPLC; 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%.
  • “Pharmaceutical composition” refers to a preparation or combination of preparations containing one, two or more active ingredients, which allows the active ingredients contained therein to exist in a biologically effective form.
  • a “pharmaceutical composition” exists in the form of a combination of separate preparations containing different, two or more active ingredients, it can be administered simultaneously, sequentially, separately or at intervals, with the purpose of exerting the biological activities of multiple active ingredients and being used together to treat diseases.
  • Effective amount is generally enough 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.
  • the effective amount is a therapeutically effective amount or a preventive effective amount.
  • “Therapeutically effective amount” is enough to treat a disease state or symptom, especially 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.
  • Preventive 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 onset (or recurrence) possibility of the disease state or related symptoms.
  • a complete treatment or preventive effect may not occur due to the administration of one dose, and may only occur after a series of doses are administered.
  • a therapeutically or preventive effective amount can be administered in one or more administrations.
  • host cell refers to cells into which exogenous nucleic acid is introduced, including 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 to the parent cell in nucleic acid content, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected in the initially transformed cell are included herein.
  • 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.
  • Nucleic acid molecule encoding refers 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.
  • tumor refers to a disease characterized by pathological proliferation of cells or tissues, and their subsequent migration or invasion of other tissues or organs. Tumor growth is usually uncontrolled and progressive, and does not induce or inhibit normal cell proliferation. Tumors include “cancer” and refer generally to all malignant tumors.
  • Treatment refers to clinical intervention in an attempt to change the disease process caused by an individual or cell, which can be either preventive or intervention in the clinical pathological process.
  • Therapeutic effects include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, slowing the progression of the disease, improving or alleviating the condition, and alleviating or improving the prognosis.
  • “Combination” involves a treatment regimen that provides at least two or more different therapies to achieve a specified therapeutic effect.
  • the therapy can be physical, such as radiotherapy, or chemical, such as administering drugs to a subject, and the drugs also include combination drugs.
  • “Combination drug” refers to a combination of two or more pharmaceutical preparations that each have an active ingredient and need to be used in combination when administered to a subject.
  • the active ingredients can be mixed together to form a single dosing unit, or they can be independently administered as dosing units and used separately; when administered, different pharmaceutical preparations can be administered essentially synchronously, simultaneously or sequentially.
  • pharmaceutically acceptable refers to those compounds, materials, compositions and/or dosage forms that are suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit/risk ratio.
  • anti-PD1 antibody refers to an antibody or antigen-binding fragment thereof that targets the immune checkpoint protein PD1 or PD-1 and can block or affect the binding of PD1 to its ligand.
  • the range of anti-PD1 antibodies available includes but is not limited to Pembrolizumab.
  • PD1 programmed death receptor 1
  • CD279 belongs to the immunoglobulin superfamily.
  • the positive progressive effects of the present disclosure are: by forming a bispecific antibody through specified amino acid mutations in the light chain variable region of trastuz, the heavy chain variable region of pertuzumab, and the heavy chain variable region of trastuz, a bispecific monoclonal antibody with high affinity, good stability, and non-degradability, strong tumor cell killing activity, and high ADCC activity is obtained, as well as an anti-PD1 antibody and/or hyaluronidase used in combination with the bispecific antibody.
  • the gene synthesis of the antibody light and heavy chains was performed 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 sequence of the first heavy chain, the second heavy chain and the common light chain was co-transfected into the ExpiCHO-S cell line (Gibco) at a certain ratio (1:1:2) using the transient transfection kit of The transfection kit instructions were followed. Finally, various bispecific antibody-expressing cell lines were obtained. Each cell line was expanded to a volume of 100 ml using CD2 basal medium and fed-batch culture was performed.
  • the Her2 antibody common light chain is formed by directly connecting 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 connecting 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 connecting 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 connecting 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 the others are similar (see Table 2 for specific sequences).
  • the distribution of charge variants was evaluated by cation exchange chromatography using IEC-HPLC.
  • the HER2 bispecific antibodies KJ015-C, KJ015-D, and KJ015-H were tested by size exclusion chromatography (Appendix IIIB of the 2010 edition of the Chinese Pharmacopoeia), and the thermal stability test data of each antibody before and after storage in a 37°C pH 7.4 PBS system for 30 days were compared.
  • Different tumor cells were subcultured, diluted to different concentrations and incubated with antibodies.
  • the sample was added as 0 day.
  • the test plate was taken out of the incubator, 20 ⁇ l CCK-8 staining solution was added to each well, and the cells were stained in a 37°C incubator for 6 hours.
  • the microplate reader was read at 450nm/630nm.
  • SoftMax Pro data processing software was used for automatic analysis.
  • the concentration of the reference and test samples was used as the horizontal axis, and the OD value was used as the vertical axis.
  • the four-parameter method was selected to fit the dose-effect curve of the reference and test samples.
  • the KJ015-C sample was used as the standard, recorded as 100%, and the relative activity of other samples was calculated.
  • KJ015-H had stronger proliferation inhibition activity than KJ015-C in different tumor cells, and the proliferation inhibition of BT474, MDA-MB-175, and Calu-3 cells was 1.25 times, 1.7 times, and 1.1 times higher than that of KJ015-C, respectively ( Figure 1).
  • ADCC ADCC assay
  • tumor cells in the logarithmic growth phase were resuspended in culture medium according to the cell growth rate and inoculated into 96-well plates.
  • 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 determined using the CytoTox96 non-radioactive cytotoxicity assay, with KJ015-C and KJ015-C-heated samples as standards, recorded as 100%, and the relative activities of other samples were calculated.
  • the binding affinities of KJ015-H to Her2 domain II and domain IV were 5.24 nM and 0.63 nM, respectively (see 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.
  • Tm The thermal melting point of each sample was determined using an Uncle instrument (Unchained Labs, Pleasanton, CA, USA).
  • KJ015-H 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 greatly improved the stability. Even after being stored in a PBS system at 37°C for 30 days, the IEC purity and SEC purity did not change significantly (see Figure 3a and Figure 3b).
  • trastuzumab will be severely degraded and its antigen binding activity and biological activity will be seriously reduced after being stored for 12 days under the same conditions (Schmid et al., 2018 Commun Biol 1, 28).
  • the viability of various cell lines was detected using the CCK-8 method.
  • 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), Zanidatamab (Zani)) for 120 hours.
  • CCK-8 solution was added to each well and incubated for 1 hour. Afterwards, the absorbance of each well was measured, and the results were statistically analyzed.
  • Example 8 Anti-tumor effects of HER2 antibody samples in each group in the CDX model
  • 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. Then 1 ⁇ 10 7 cells were injected subcutaneously into the right anterior dorsal area of female nude mice to establish cancer xenograft models.
  • NCI-N87, Calu-3, and BT474 xenograft models when the average tumor volume of hormone mice reached approximately 80-150 mm 3 , the mice were randomly divided into 4 groups of 6 mice in each group according to the experimental design.
  • MDA-MB-175 xenograft model when the average tumor volume of hormone mice reached approximately 250 mm 3 , the mice were randomly divided into 5 groups of 6 mice in each group according to the experimental design.
  • 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.
  • the MDA-MB-175 xenograft groups were treated with vehicle, trastuzumab + pertuzumab (1.5 + 1.5 mg/kg, 5 + 5 mg/kg), and KJ015-H (3 mg/kg, 10 mg/kg), which were administered twice a week via tail vein injection.
  • KJ015-H was able to effectively reduce tumor growth and tended to be stronger than trastuzumab + pertuzumab ( Figure 5a-5d).
  • the low-concentration trastuzumab + pertuzumab combination (molar concentration 1:1, 0.94 mg/kg/monoclonal antibody) had no effect on tumor size, but the same concentration of KJ015-H (1.88 mg/kg) still caused tumor regression.
  • Example 9 Detection of anti-tumor effects of HER2 antibody samples in each group in PDX model
  • KJ015-H subcutaneous injection (containing hyaluronidase preparation) is a compound preparation of KJ015-H and hyaluronidase (Shanghai Baoji Pharmaceutical Co., Ltd.) for subcutaneous injection.
  • Hyaluronidase can temporarily degrade hyaluronic acid in the human body, improve the permeability of tissues, promote the rapid dispersion and absorption of injected drugs, and achieve subcutaneous injection.
  • PDX tumor xenograft
  • a piece of tumor tissue of 2 ⁇ 2 ⁇ 2mm 3 size was implanted subcutaneously in the right forelimb of 8-week-old C-NKG mice (severely immunodeficient mice produced by Cyagen by deleting the Il2rg gene from NOD-Scid genetic background mice).
  • mice When the tumor reached an average of 130mm 3 , 30 mice were randomly divided into 5 groups, receiving intravenous injection of vehicle, trastuzumab + pertuzumab (12.5+12.5mg/kg) or KJ015-H subcutaneous injection (12.5, 25, 50mg/kg), respectively, and tumor growth was measured twice a week. Throughout the study, the mice were weighed and regularly observed for symptoms of disease or discomfort associated with tumor growth and/or drug toxicity.
  • Example 10 Detection of the effect of subcutaneous injection of KJ015-H combined with ⁇ PD-1 antibody in the treatment of HER2-overexpressing tumors
  • the humanized NOG mice in the NCI-N87 model were used to test the efficacy of subcutaneous injection of KJ015-H or trastuzumab combined with anti-PD-1 antibody (pembrolizumab, trade name: Keytruda, manufacturer: Merck) in treating tumors.
  • 3 ⁇ 10 6 human PBMCs were injected into NOG mice via the tail vein 3 days before tumor cell inoculation (d-3); the tumor cell implantation day 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.
  • NOG mice were treated with 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).
  • the tumor volume was measured twice a week, and the tumor was removed on day 19 (d18) after tumor cell inoculation.
  • 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 min.
  • the labeling kit (ab188288) was pre-labeled with Cy5, with an average of 2.5 AF647 molecules labeled per antibody molecule.
  • PFA 2% phosphoric acid
  • the unbound beads were removed by thorough washing with PBS and precipitated overnight at 4°C with a fiducial marker (ThermoFisher Scientific, Waltham, MA, USA), and the adhered particles were imaged by stochastic optical reconstruction microscopy (iSTORM) (Ningbo INVIEW Intelligent Technology Co., Ltd., China).
  • iSTORM stochastic optical reconstruction microscopy

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Abstract

La présente divulgation concerne un anticorps bispécifique anti-HER2 stable, qui comprend une première région fonctionnelle de protéine et une seconde région fonctionnelle de protéine utilisées pour reconnaître le domaine extracellulaire II et le domaine extracellulaire IV de HER2, respectivement. L'anticorps bispécifique reste stable après avoir été stocké dans du PBS à environ 37 °C pendant 30 jours. La présente divulgation concerne en outre une composition pharmaceutique contenant l'anticorps bispécifique et un anticorps PD1, ainsi que son utilisation. L'anticorps bispécifique et la composition pharmaceutique le contenant de la présente divulgation peuvent être utilisés pour traiter des cancers.
PCT/CN2024/138695 2023-12-12 2024-12-12 Anticorps bispécifique anti-her2 et son utilisation Pending WO2025124463A1 (fr)

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CN105017421A (zh) * 2014-04-16 2015-11-04 中国人民解放军海军总医院 一种抗her2双特异性抗体及其制备方法与应用
CN105829347A (zh) * 2013-12-20 2016-08-03 豪夫迈·罗氏有限公司 双特异性her2抗体及使用方法
CN105820251A (zh) * 2015-01-08 2016-08-03 苏州康宁杰瑞生物科技有限公司 具有共同轻链的双特异性抗体或抗体混合物
CN109715671A (zh) * 2016-07-22 2019-05-03 北京天广实生物技术股份有限公司 双特异性抗her2抗体
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CN109715671A (zh) * 2016-07-22 2019-05-03 北京天广实生物技术股份有限公司 双特异性抗her2抗体
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