WO2019190359A1 - Anticorps bispécifique qui se lie de manière spécifique aux sous-domaines iv et ii du domaine extracellulaire her2 de l'humain - Google Patents

Anticorps bispécifique qui se lie de manière spécifique aux sous-domaines iv et ii du domaine extracellulaire her2 de l'humain Download PDF

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WO2019190359A1
WO2019190359A1 PCT/RU2019/050037 RU2019050037W WO2019190359A1 WO 2019190359 A1 WO2019190359 A1 WO 2019190359A1 RU 2019050037 W RU2019050037 W RU 2019050037W WO 2019190359 A1 WO2019190359 A1 WO 2019190359A1
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cancer
antibody
her2
human
antibodies
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PCT/RU2019/050037
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Russian (ru)
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Александр Владимирович ПРОКОФЬЕВ
Нина Грачьяевна ХАРАТЯН
Елена Андреевна КРЕНДЕЛЕВА
Виктория Олеговна ШИТИКОВА
Мария Александровна ЩЕМЕЛЕВА
Арина Витальевна АНИКИНА
Ольга Викторовна НАЗАРЕНКО
Анна Валентиновна ЕВСТРАТЬЕВА
Александра Александровна СОЗОНОВА
Алексей Владимирович ЧЕРКАСОВ
Алексей Константинович МИСОРИН
Яков Юрьевич УСТЮГОВ
Алексей Александрович АЛЕКСАНДРОВ
Павел Андреевич ЯКОВЛЕВ
Мария Игоревна ЛОМОВСКАЯ
Дмитрий Валентинович МОРОЗОВ
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Закрытое Акционерное Общество "Биокад"
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Publication of WO2019190359A1 publication Critical patent/WO2019190359A1/fr

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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
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression

Definitions

  • the present invention relates to the field of biotechnology, namely to antibodies, as well as their use. More specifically, the present invention relates to a bispecific antibody that specifically binds to the fourth subdomain of the extracellular domain (ECD4) of HER2 (human epidermal growth factor receptor 2) and the second subdomain of the extracellular domain (ECD2) of human HER2.
  • ECD4 extracellular domain
  • ECD2 human epidermal growth factor receptor 2
  • ECD2 extracellular domain 2
  • the invention also relates to a nucleic acid encoding a given antibody, an expression vector, a method for producing an antibody, and the use of an antibody for treating diseases or disorders associated with HER2 overexpression.
  • the group of human epidermal factor receptor (HER) receptors also called ErbB receptors, belongs to the family of transmembrane receptor tyrosine kinases. This family includes the epidermal growth factor receptor (EGFR), also called ErbB-1 (or HER1), and the homologous receptors ErbB-2 (HER2), ErbB-3 (HER3) and ErbB-4 (HER4 ) These receptors are expressed on the surface of most normal cells; they are involved in the regulation of the most important cellular processes, controlling proliferation, differentiation, migration, and apoptosis. Increased expression of HER receptors or their ligands, such as heregulin (HRG) or epidermal growth factor (EGF), is often observed in cancer patients (Wilson, Fridlyand et al. 2012).
  • HRG heregulin
  • EGF epidermal growth factor
  • ERBB receptor The structure of the ERBB receptor (HER) is represented by three domains: extracellular, with 4 subdomains, transmembrane and intracellular, represented by juxtamembrane, tyrosine kinase subdomains and carboxyl end (for autophosphorylation processes).
  • the binding of the ligand to the extracellular domain of tyrosine kinases causes a receptor dimerization process, which can take place between two identical receptors (homodimerization) or between different receptors within the same family (heterodimerization). Dimerization activates the intracellular domains of tyrosine kinases and causes their autophosphorylation. This, in turn, triggers a series of downstream proliferative signaling cascades, including those mediated by mitogen-activated protein kinases, as well as the AKT signaling pathway aimed at cell survival (described in Yarden and Pines, 2012).
  • ligands include, among others, neuregulin (NRG) or heregulin (HRG).
  • Ligand-dependent dimerization is characteristic of HER1 / 3/4; it is mediated by binding of the ligand to the extracellular domain of the receptor (1 ligand molecule is sufficient to maintain stable conformation of the dimer; dissociation of a single ligand molecule with a dimer leads to instantaneous dissociation of the dimer).
  • Ligand-independent dimerization is characteristic when a critical point of the HER2 level is reached, the process of spontaneous dimerization begins, i.e. hyperexpression of HER2 itself leads to a shift in the receptor equilibrium from a monomeric to an aggregated state.
  • All three receptor domains are usually involved in the dimerization process, however, both the extracellular and intracellular domains, individually, are sufficient for the dimerization process and kinase activity (in particular, the constitutive activity of the intracellular domain).
  • the allosteric interactions underlying the dimerization process begin in the region of the extracellular domain.
  • the extracellular receptor domains are responsible both for ligand binding (between I and III subdomains), and for binding of receptors to each other via II subdomains.
  • the “bent” conformation of subdomain II is characteristic of ligand-independent, inactive extracellular domains of EGFR, while the “direct” one is characteristic of ligand-dependent active dimers.
  • EbB2 receptors HER2
  • HER3B3 receptor HER3
  • the most unfavorable prognosis of breast cancer is associated with the presence of a large number of HER2-HER3 heterodimers on the surface of tumor cells, as well as with coexpression of IGFR and c-Met (associated with invasive tumor phenotype, intense metastasis, and resistance to monospecific anti-HEN2 targeted therapy).
  • trastuzumab antibody that blocks the IV subdomain of the extracellular domain of the HER2 receptor (patent EP0590058 (B1), W09222653);
  • Pertuzumab antibody which blocks the second subdomain of the extracellular domain of the HER2 receptor (patent RU2270029, W00100245).
  • the main mechanisms of resistance formation are:
  • the Beodaim drug was developed - a combination of trastuzumab and pertuzumab, used to treat metastatic breast cancer, in the absence of previously conducted anti-HE2 therapy, and also as a drug for neoadjuvant breast cancer therapy (patent RU 2430739, WO 01/00245) .
  • This drug has an inconvenient route of administration, since Beiodime is administered to the patient with two separate continuous intravenous infusions of trastuzumab and pertuzumab.
  • the above problem is solved by developing a bispecific antibody that specifically binds to different subdomains of the human extracellular domain of HER2 and is administered to the patient in a single infusion, thereby reducing the patient’s hospital stay.
  • Bispecific antibodies are known in the art that specifically bind to different subdomains of the extracellular domain of human HER2, for example, the antibodies described in the following patent documents.
  • the bispecific antibody of the invention (BCD-147-02-020) does not exhibit complement dependent cytotoxicity (CDC) at all.
  • WO2015157592 describes a bispecific antibody specifically binding to HER2 comprising a first immunoglobulin antigen binding domain, where (i) the first and second immunoglobulin antigen binding domains specifically bind to different H2 immunoglobulin binding sites binds to the first HER2 antibody binding site that contains an epitope in HER2 domain II, and (iii) the first antibody H binding site ER2 differs from the pertuzumab antibody binding site.
  • This antibody effectively inhibits HER2-mediated cell signaling, which can be used to treat tumors expressing HER2, including cancers that have a low level of HER2 presentation (p. 3 descriptions, penultimate paragraph, WO2015157592).
  • the toxic properties of the antibody are presented in the international application WO2015157592. This assumption is based on the properties of the antibody indicated in the application — enhanced internalization of the HER2 receptor and enhanced ADCC properties against HER2-expressing cells, as well as safety data for other anti-HE2 antibodies.
  • the HER2 receptor is an integral structural component of cardiomyocytes, endothelial cells, and small intestine epithelium.
  • the antibody indicated in WO2015157592 is likely to affect all healthy body cells expressing the HER2 receptor. This is confirmed by safety data obtained during phase 1 CI, where the incidence of diarrhea was 44%, which indirectly indicates toxicity to the intestinal epithelium.
  • BCD-147-02-02020 bispecific anti-HE2 antibody of this invention developed by us on healthy (non-malignant) HER2-expressing cells
  • the antibody activity against this cell line namely, antibody-dependent cell cytotoxicity
  • the antibody activity against this cell line is minimal, which suggests a potentially low toxicity of the BCD-147-02-02 020 preparation and high selectivity for HER2 tumor-expressing cells.
  • This antibody exhibits enhanced effector functions, including complement dependent cytotoxicity (CDC), compared to each corresponding monospecific divalent antigen binding construct (i.e., compared to a monospecific divalent antigen binding construct that binds to ECD2, or a monospecific bivalent antigen binding construct binds to ECD4, and / or in comparison with a combination of two monospecific divalent antigen-binding constructs (paragraph [00147 ], p. 36 of the description of W02015077891).
  • the bispecific antibody of the invention (BCD-147-02-020) does not exhibit complement dependent cytotoxicity (CDC) at all.
  • the BCD-147-02-020 bispecific antibody binds to the 4th and 2nd subdomains of the extracellular domain of HER2, so sterically, this antibody tends to effectively block both ligand-dependent and ligand-independent dimerization.
  • the BCD-147-02-020 bispecific antibody is capable of preventing not only HER2 / HER3 dimerization, but also dimerization of HER2 with EGFR and HER4.
  • the bispecific antibody BCD-147-02-020 exhibits the following properties:
  • ADCC antibody-dependent cell-mediated cytotoxicity
  • the HER2 antibody component becomes highly plastic, especially in the domain I and III domains, and this contributes to its enhanced association with the anti-HEB2 (II) component of the antibody.
  • the present invention relates to a bispecific antibody BCD-147-02-020, which specifically binds to the IV and II subdomains of the extracellular domain of HER2, and provides enhanced blockade of HER2-mediated signaling pathways.
  • Such an antibody can be used to treat a disease or disorder mediated by HER2.
  • the present invention relates to a bispecific antibody that specifically binds to the fourth subdomain of the extracellular domain (ECD4) of HER2 (human epidermal growth factor receptor 2) and the second subdomain of the extracellular domain (ECD2) of human HER2, and includes:
  • the first antigen-binding part which specifically binds to the IV subdomain of the extracellular domain (ECD4) of HER2, and is a single-chain variable fragment (scFv) trastuzumab with the amino acid sequence represented by SEQ ID NO: 1;
  • the second antigen-binding part which specifically binds to the II subdomain of the extracellular domain (ECD2) of HER2, and is an antigen binding site (Fab), comprising the variable domain of the heavy chain (VH) with the amino acid sequence represented by SEQ ID NO: 2, and variable light chain domain (VL) with the amino acid sequence represented by SEQ ID NO: 6;
  • the bispecific antibody is an IgG antibody.
  • the bispecific IgG antibody is of the human IgGl, IgG2, IgG3 or IgG4 isotype.
  • the bispecific antibody is of the human IgGl isotype.
  • the bispecific antibody comprises a crystallizing immunoglobulin fragment (Fc fragment) that contains two amino acid sequences of the second and third constant domains (CH2-CH3) represented by SEQ ID NOs: 10-11, respectively.
  • Fc fragment crystallizing immunoglobulin fragment
  • the bispecific antibody comprises a second antigen binding portion that specifically binds to the II subdomain of the extracellular domain (ECD2) of HER2, and is an antigen binding site (Fab), comprising:
  • VH variable domain of the heavy chain
  • CHI first constant domain of the heavy chain
  • VL variable domain of the light chain
  • SC constant domain of the light chain
  • the present invention relates to a bispecific antibody that specifically binds to the fourth subdomain of the extracellular domain (ECD4) of HER2 (human epidermal growth factor receptor 2) and the second subdomain of the extracellular domain (ECD2) of human HER2, and consists of:
  • the present invention relates to a nucleic acid that encodes the above antibody.
  • the nucleic acid is DNA.
  • the present invention relates to an expression vector comprising the aforementioned nucleic acid.
  • the present invention relates to a method for producing a host cell for producing the aforementioned antibody, which comprises transforming the cell with the aforementioned vector.
  • the present invention relates to a host cell for preparing the aforementioned antibody comprising the aforementioned nucleic acid.
  • the present invention relates to a method for producing the aforementioned antibody, which comprises culturing the aforementioned host cell in a culture medium under conditions sufficient to produce the aforementioned antibody, if necessary, followed by isolation and purification of the obtained antibody.
  • the present invention relates to a pharmaceutical composition for the treatment of a disease or disorder mediated by HER2, comprising the aforementioned antibody or antigen binding fragment thereof in a therapeutically effective amount, in combination with one or more pharmaceutically acceptable excipients.
  • the pharmaceutical composition is intended to treat a disease or disorder mediated by HER2, which is selected from the group: breast cancer (BC), malignant neoplasm of the stomach, non-small cell lung cancer, malignant neoplasm of the head and / or neck, squamous cell carcinoma of the head and neck (PRGS) ), colorectal cancer (CRC), esophageal cancer, ovarian cancer, pancreatic cancer, gastrointestinal cancer, kidney cancer, cervical cancer, endometrial cancer, uterine cancer, malignant melanoma, pharyngeal cancer, and oral cancer and skin cancer.
  • BC breast cancer
  • non-small cell lung cancer malignant neoplasm of the head and / or neck
  • PRGS squamous cell carcinoma of the head and neck
  • CRC colorectal cancer
  • esophageal cancer esophageal cancer
  • ovarian cancer pancreatic cancer
  • gastrointestinal cancer kidney cancer
  • cervical cancer endometrial cancer
  • the present invention relates to a pharmaceutical composition for treating a disease or disorder mediated by HER2, comprising the aforementioned antibody or antigen binding fragment thereof in a therapeutically effective amount and at least one therapeutically active antitumor compound in a therapeutically effective amount.
  • the pharmaceutical composition is intended to treat a disease or disorder mediated by HER2, which selected from the group: breast cancer (BC), malignant neoplasm of the stomach, non-small cell lung cancer, malignant neoplasm of the head and / or neck, squamous cell carcinoma of the head and neck (PRGS), colorectal cancer (CRC), esophageal cancer, ovarian cancer, pancreatic cancer glands, gastrointestinal cancer, kidney cancer, cervical cancer, endometrial cancer, uterine cancer, malignant melanoma, pharyngeal cancer, oral cancer or skin cancer.
  • HER2 selected from the group: breast cancer (BC), malignant neoplasm of the stomach, non-small cell lung cancer, malignant neoplasm of the head and / or neck, squamous cell carcinoma of the head and neck (PRGS), colorectal cancer (CRC), esophageal cancer, ovarian cancer, pancreatic cancer glands, gastrointestinal cancer, kidney cancer, cervical cancer
  • the pharmaceutical composition comprises a therapeutically active antitumor compound that is selected from a cytotoxic agent, chemotherapeutic agent, antibody, or anti-hormonal agent.
  • the present invention relates to a method for inhibiting the biological activity of HER2 in a subject that needs such inhibition, comprising administering to the subject an effective amount of the above antibody.
  • the present invention relates to a method for treating a disease or disorder mediated by HER2, comprising administering to a subject in need of such treatment the above antibody or the above pharmaceutical composition in a therapeutically effective amount.
  • the disease or disorder is selected from the group: breast cancer (BC), malignant neoplasm of the stomach, non-small cell lung cancer, malignant neoplasm of the head and / or neck, squamous cell carcinoma of the head and neck (PRGS), colorectal cancer (CRC) , esophageal cancer, ovarian cancer, pancreatic cancer, gastrointestinal cancer, kidney cancer, cervical cancer, endometrial cancer, uterine cancer, malignant melanoma, pharyngeal cancer, oral cancer or skin cancer.
  • BC breast cancer
  • non-small cell lung cancer malignant neoplasm of the head and / or neck
  • PRGS squamous cell carcinoma of the head and neck
  • CRC colorectal cancer
  • esophageal cancer esophageal cancer
  • ovarian cancer pancreatic cancer
  • gastrointestinal cancer kidney cancer
  • cervical cancer endometrial cancer
  • uterine cancer malignant melanoma
  • pharyngeal cancer
  • the present invention relates to the use of the above antibody or the above pharmaceutical composition for treating in a subject in need of such treatment a disease or disorder mediated by HER2.
  • the disease or disorder is selected from the group: breast cancer (BC), malignant neoplasm of the stomach, non-small cell lung cancer, malignant neoplasm of the head and / or neck, squamous cell carcinoma of the head and neck (PRGS), colorectal cancer (CRC), esophageal cancer, ovarian cancer, pancreatic cancer, gastrointestinal cancer, kidney cancer, cervical cancer, endometrial cancer, uterine cancer, malignant melanoma, pharyngeal cancer, oral cancer or skin cancer.
  • FIG. 1 The ring diagram of the plasmid pEE-Fc, designed to produce the extracellular domain of the human Ig2 protein in mammalian cells.
  • AmpR - beta-lactamase gene that provides ampicillin resistance
  • CMV promoter - promoter of early cytomegalovirus genes oriP - origin of replication
  • START - Start codon Leader - Leader mouse IgGk peptide
  • hHer2-Synthetic sequence of human Neg2 antigen Fc - Fc- fragment of human IgGl
  • His - polyhistidine tag His - polyhistidine tag
  • STOP - stop codons The ring diagram of the plasmid pEE-Fc, designed to produce the extracellular domain of the human Ig2 protein in mammalian cells.
  • FIG. 2 The ring diagram of the plasmid pEE-SK, designed to produce the light chain of antibodies in mammalian cells.
  • AmpR - beta-lactamase gene that provides ampicillin resistance
  • CMV promotor - promoter of early cytomegalovirus genes oriP - origin of replication
  • START - Start codon Leader - mouse IgGk leader peptide
  • VL - antibody light chain variable domain sequence SK
  • polyA is the polyadenylation site.
  • FIG. 3 The ring diagram of plasmids pEE-NS, designed to produce the heavy chain of antibodies in mammalian cells.
  • AmpR - beta-lactamase gene that provides ampicillin resistance
  • CMV promotor - promoter of early cytomegalovirus genes oriP - origin of replication
  • START - Start codon Leader - IgGk leader peptide
  • VH - sequence of the antibody heavy chain variable domain VH - sequence of the antibody heavy chain variable domain
  • NS - constant domains CHI, CH2, CHZ
  • STOP is the stop codon
  • polyA is the polyadenylation site.
  • FIG. 4 The ring diagram of plasmids pEE-HChole, designed to generate the heavy chain of antibodies in mammalian cells.
  • AmpR - beta-lactamase gene that provides ampicillin resistance
  • CMV promotor - promoter of early cytomegalovirus genes oriP - origin of replication
  • START - Start codon Leader - IgGk leader peptide
  • VH - sequence of the antibody heavy chain variable domain VH - sequence of the antibody heavy chain variable domain
  • NS - constant domains CHI, CH2, CHZ
  • STOP is the stop codon
  • polyA is the polyadenylation site.
  • FIG. 5 Library design with selection for stability. Design of light chain sequences.
  • FIG. b Design library with stability selection Design of heavy chain sequences.
  • FIG. 7 Library design with affinity selection. Light chain sequence design.
  • FIG. 8. Library design with affinity selection. Design sequences of heavy chains.
  • FIG. 9. The ring diagram of the plasmid pBL, designed for expression of proteins in E. coli cells.
  • AmpR is the beta-lactamase gene that provides resistance to ampicillin
  • pBR322_ori is the origin of replication from plasmid pBR322
  • lad is the lactose operon repressor gene
  • KmR is the aminoglycoside phosphotransferase gene, which provides resistance to kanamycin
  • Leader is the leader peptide that expresses periplase E.
  • VL is the sequence of the variable domain of the light chain of the antibody
  • SC is the constant domain of the light chain of the isotype of human IgGl
  • VH is the sequence of the variable domain of the heavy chain of the antibody
  • CH1 is the constant domain of human IgGl
  • Myc-tag His-tag is AGI.
  • FIG. 10 Results of kinetic analysis for 8 Fab fragments of a synthetic library (association-dissociation curve) that demonstrated binding in an enzyme-linked immunosorbent assay.
  • FIG. 11 Schematic representation of the asymmetric format.
  • a bispecific asymmetric antibody that specifically binds to the IV and II subdomains of the extracellular domain of human HER2 consists of 1) Fc-knob - scFv-Trastuzumab, 2) HC-hole - aNEB2-candidate 020-VH, 3) Ck - aNEB2-candidate 020 VL.
  • FIG. 12 The ring diagram of the plasmid pEE-scFvTrast-Fc_knob.
  • AmpR is the beta-lactamase gene that provides ampicillin resistance
  • CMV promoter is the promoter of early cytomegalovirus genes
  • oriP is the origin of replication
  • START is the start codon
  • Leader is the IgGk leader peptide
  • scFv-Trastuzumab is the sequence of variable domains of the control antibody in scFv format, S-linker - ASGDKTHT linker, Fcknob - CH2, SNZ human IgGl domains with introduced knob mutations, His-tag - polyhistidine tag, STOP - stop codon, polyA - polyadenylation site.
  • FIG. 13 Electrophoresis of antibody candidates in polyacrylamide gel under non-reducing conditions.
  • FIG. 14 Electrophoresis of antibody candidates in a polyacrylamide gel under non-reducing conditions.
  • FIG. 15 Electrophoresis of polyacrylamide gel antibody candidates under non-reducing conditions.
  • FIG. 16 Electrophoresis of antibody candidates in polyacrylamide gel under non-reducing conditions.
  • FIG. 17 Results of kinetic analysis for full-length antibodies in IgGl format (candidates 001-017) and asymmetries (candidates 019
  • FIG. 18 Curves of association-dissociation of competition of candidates 017 035 with Trastuzumab in the format “Ih-tandem”.
  • the curve labels show the loading sequence of the antigen, control antibody, and sensor candidates. Curves for different candidates overlap.
  • FIG. 19 Curves of association-dissociation of competition of candidates 017 035 with Pertuzumab in the format "Ih-tandem".
  • the curve labels show the loading sequence of the antigen, control antibody, and sensor candidates. Curves for different candidates overlap.
  • FIG. 20 Curves of association-dissociation of competition of candidates 017
  • FIG. 21 The ring diagram of the vectors pSX. p-CMVe / EFlalpha - a synthetic promoter, co-consisting of a CMV enhancer and an EF- promoter la, START - Start codon, Leader - IgGk leader peptide, INSERT - insertion sequence (antibody heavy or light chain), STOP - stop codon, polyA - polyadenylation site, Enhancer SV-40 - monkey virus enhancer SV-40, beta globin MAR - MAR (matrix attachment region) of the human b-globin gene, Rep origin 1 - pUC Origin of replication, AmpR - beta-lactamase gene providing resistance to ampicillin, F1 origin - allows a plasmid with such an origin of replication to be packaged into phage particles upon cotransformation with helper phages VCSM13 and M13K07, SV40 promoter - eukaryotic promoter of the monkey virus SV-40,
  • FIG. 22 The dependence of the fluorescence intensity on the logarithm of the concentration of antibodies in the antiproliferative test on the cell line VT-474.
  • the graph shows 11-fold insertion antibody concentrations.
  • FIG. 23 Dependence of the fluorescence intensity on the logarithm of the concentration of antibodies in the antiproliferative test on the cell line BT-474 with the introduction of hrEGF.
  • the graph shows 11-fold insertion antibody concentrations.
  • FIG. 24 The dependence of the fluorescence intensity on the logarithm of the concentration of antibodies in the antiproliferative test on the cell line BT-474 Clone 5 resistant to trastuzumab.
  • the graph shows 11-fold insertion antibody concentrations.
  • FIG. 25 Analysis of ADCC on the BT-474 cell line using a high affinity FcyRIIIa receptor reporter line.
  • the graph shows 2-fold injected antibody concentrations.
  • FIG. 26 Analysis of ADCC on the BT-474 cell line using a low affinity FcyRIIIa receptor reporter line.
  • the graph shows 2-fold injected antibody concentrations.
  • FIG. 27 CDC analysis on cell lines BT-474 and SK-BR-3. Antibodies are presented at a concentration of 50 ⁇ g / ml (3-fold injected antibody concentration), “0 point” - the point without introduction of antibodies, “PC” - positive control of CDC (rituximab and WIL2-S).
  • FIG. 28 Analysis of ADCC on a HUVEC cell line using a high affinity FcyRIIIa receptor reporter line.
  • the graph shows 4-fold injected antibody concentrations.
  • FIG. 29 Dynamics of the growth index of the tumor (tumor line ZR-75-1).
  • FIG. 30 The value of the indicator of inhibition of tumor growth (TPO) (tumor line ZR-75-1).
  • FIG. 31 The dynamics of the growth index of the tumor (tumor line SKBR3).
  • FIG. 32 The value of the inhibition of tumor growth (TPO) (tumor line SKBR3).
  • FIG. 33 The dynamics of the growth index of the tumor (tumor line BT-474).
  • FIG. 34 The value of the inhibition of tumor growth (SRW) (tumor line VT-474).
  • the terms in the singular include the terms in the plural, and the terms in the plural include the terms in the singular.
  • the classification and methods used for cell cultivation, molecular biology, immunology, microbiology, genetics, analytical chemistry, organic synthesis chemistry, medical and pharmaceutical chemistry, as well as hybridization and protein and nucleic acid chemistry described in this document are well known to specialists and widely used in this field. Enzymatic reactions and purification methods are carried out in accordance with the manufacturer's instructions, as is usually done in the art, or as described herein.
  • HER receptor is a tyrosine protein kinase receptor that belongs to the HER receptor family and includes the EGFR, HER2, HER3 and HER4 receptors and other members of this family that will be identified in the future.
  • the HER receptor may contain an extracellular domain that can bind the HER ligand; lipophilic transmembrane domain; conserved intracellular tyrosine kinase domain; and a signal transduction domain located at the carboxyl end that carries several tyrosine residues that can be phosphorylated.
  • the HER receptor is a human HER receptor with a native sequence.
  • EbB2 and "HER2” are used interchangeably herein and refer to the human HER2 protein described, for example, in Semba et al. PNAS (USA) 82: 6497-6501 (1985) and Yamamoto et al. Nature 319: 230-234 (1986) (access number in Genebank X03363).
  • eBB2 refers to a gene encoding human EbB2.
  • Preferred HER2 is human HER2 with a native sequence.
  • the extracellular domain of HER2 contains four domains: domain I (amino acid residues from about 1 to 195), domain II (amino acid residues from about 196 to 319), domain III (amino acid residues from about 320 to 488) and domain IV (amino acid residues from about 489 to 630) (residue numbering without signal peptide).
  • domain I amino acid residues from about 1 to 195
  • domain II amino acid residues from about 196 to 319
  • domain III amino acid residues from about 320 to 488
  • domain IV amino acid residues from about 489 to 630
  • HER ligand is meant a polypeptide that binds and / or activates the HER receptor.
  • binding to human HER2 or “specifically binding to human HER2” or “anti-HEU2 antibody” are used interchangeably and refer to an antibody specifically binding to a human HER2 antigen with binding affinity that matters KD 1 * 10 8 mol / L or lower at 25 ° C, in one embodiment, has a value of KD 1 * 10 9 mol / L or lower at 25 ° C.
  • Binding affinity is determined in a standard binding assay at 25 ° C, such as a surface plasma resonance technique (BIAcore®, GE-Healthcare, Uppsala, Sweden). A method for determining the KD binding affinity value is described in Example 2b).
  • antibody binding to human HER2 refers to an antibody specifically binding to a human HEy2 antigen with binding affinity with a KD value of 1 * 10 8 mol / L or lower (preferably 1 c 10 8 mol / L - 1, 0x10 12 mol / L) at 25 ° ⁇ .
  • Amplification of this gene and / or overexpression of its protein was found in many cancers, including breast cancer, breast cancer, non-small cell lung cancer, malignant neoplasms of the head and / or neck, squamous cell carcinoma of the head and neck (PRGSh ), colorectal cancer (CRC), esophageal cancer, ovarian cancer, pancreatic cancer, gastrointestinal cancer, kidney cancer, cervical cancer, endometrial cancer, uterine cancer, malignant melanoma, pharyngeal cancer, oral cancer or skin cancer.
  • binding molecule includes antibodies and immunoglobulins.
  • antibody or “immunoglobulin” (Ig), as used herein, includes whole antibodies and any antigen binding fragment (ie, “antigen binding part”) or its individual chains.
  • antibody refers to a glycoprotein containing at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or its antigen-binding part. Each heavy chain contains a variable region of the heavy chain (abbreviated herein as VH) and the constant region of the heavy chain.
  • VH variable region of the heavy chain
  • Five types of mammalian antibody heavy chains are known which are denoted by Greek letters: a, d, e, g and m.
  • the type of heavy chain present determines the class of antibody; these chains are found in antibodies such as IgA, IgD, IgE, IgG and IgM, respectively.
  • Different heavy chains differ in size and composition; and g contain approximately 450 amino acids, and m and e consist of approximately 550 amino acids.
  • Each heavy chain contains two regions, i.e. constant region and variable region. The constant region is identical in all antibodies of the same isotype, but differs in antibodies of a different isotype.
  • the heavy chains g, and d contain a constant region, which consists of three constant domains CHI, CH2 and CHZ (aligned) and a hinge region, which gives flexibility (Woof J., Burton D., Nat Rev Immunol 4, 2004, cc .89-99); heavy chains m and e contain a constant region, which consists of four constant domains CHI, CH2, CH3 and CH4.
  • heavy chains m and e contain a constant region, which consists of four constant domains CHI, CH2, CH3 and CH4.
  • lambda (l) and kappa (k) In mammals, only two types of light chains are known, which are designated as lambda (l) and kappa (k).
  • Each light chain consists of a variable region of the light chain (abbreviated herein as VL) and a constant region of the light chain.
  • the approximate length of the light chain is 211-217 amino acids.
  • the light chain is a light kappa (k) chain
  • Antibodies can be antibodies of any class (eg, IgA, IgD, IgE, IgG and IgM, preferably IgG) or a subclass (eg, IgGl, IgG2, IgG3, IgG4, IgAl and IgA2, preferably IgGl).
  • class e.g, IgA, IgD, IgE, IgG and IgM, preferably IgG
  • subclass eg, IgGl, IgG2, IgG3, IgG4, IgAl and IgA2, preferably IgGl.
  • VH and VL regions can be further subdivided into regions of hypervariability, called complementarity determining regions (CDRs), scattered between regions that are more conservative, called framework regions (FR).
  • CDRs complementarity determining regions
  • FR framework regions
  • Each VH and VL consists of three CDRs and four FRs located from the amino end to the carboxy end in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • the variable regions of the heavy and light chains contain a binding domain that interacts with the antigen.
  • the constant regions of antibodies can mediate the binding of immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells), and the first component (Clq) of the classical complement system.
  • antibody portion of an antibody or “antigen binding fragment” (or simply “antibody portion” or “antibody fragment”), as used herein, refers to one or more antibody fragments that retain the ability to specifically bind to an antigen. It has been shown that the antigen-binding function of an antibody can be performed by fragments of a full-sized antibody.
  • binding fragments included in the term “antigen binding portion” of an antibody include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) F (ab f ) 2 fragment, a divalent fragment containing two Fab fragment linked by a disulfide bridge in the hinge region; (iii) an Fd fragment consisting of the VH and CHI domains; (iv) an Fv fragment consisting of the VL and VH domains in a single arm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature 341: 544-546), which consists of a VH / VHH domain; and (vi) a designated complementarity determining region (CDR).
  • a Fab fragment a monovalent fragment consisting of the VL, VH, CL and CHI domains
  • F (ab f ) 2 fragment a divalent fragment containing two Fab fragment linked by a disul
  • two regions of the Fv fragment, VL and VH are encoded by different genes, they can be connected using recombinant methods using a synthetic linker, which makes it possible to obtain them as a single protein chain, in which the VL and VH regions mate to form monovalent molecules (known as single chain Fv (scFv); see, for example, Bird et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85: 5879 - 5883). It is contemplated that such single chain molecules are also included in the term “antigen binding portion” of an antibody. Such antibody fragments are prepared using conventional methods known to those skilled in the art, and these fragments are screened in the same manner as intact antibodies.
  • the CDR of the antigen binding site or the entire antigen binding site of the antibodies of the invention is from a mouse, llama, or human donor library, or essentially human, with certain amino acid residues altered, for example, substituted by different amino acid residues, in order to optimize specific properties of the antibody, for example KD , koff, IC50, EC50, ED50.
  • the antibody fragments of the invention are of human origin or essentially human origin (at least 80, 85, 90, 95, 96, 97, 98, or 99% human origin).
  • the antigen binding site of an antibody of the invention may be derived from, but not limited to, other non-human species, including, but not limited to, a mouse, llama, rabbit, rat, or hamster.
  • the antigen binding site may be derived from human species.
  • variable refers to the fact that certain segments of the variable domains vary widely in sequence among antibodies. Domain V mediates antigen binding and determines the specificity of a particular antibody to its specific antigen. However, the variability is not evenly distributed across the 110-amino acid variable domain region. In contrast, V regions are composed of invariant fragments called framework regions (FR) of 15-30 amino acids, separated by shorter regions of extreme variability, called “hypervariable regions” or CDRs.
  • FR framework regions
  • Each variable domain of the native heavy and light chains contains four FRs, mainly taking the configuration of beta sheets connected by three hypervariable regions that form loops that bind, and in some cases are part of the beta-folded structure.
  • the hypervariable regions in each chain are held together in close proximity with FR and with hypervariable regions of the other chain contribute to the formation of the antigen-binding site of antibodies.
  • the constant domains do not directly participate in the binding of the antibody to the antigen, but exhibit various effector functions, such as the participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC, ADCC).
  • hypervariable region refers to the amino acid residues of an antibody that are responsible for antigen binding.
  • the hypervariable region contains amino acid residues from the "complementarity determining region" or "CDR”, and / or such residues from the "hypervariable loop".
  • affinity maturation it may also be preferable to change one or more amino acid residues of the CDR regions in order to increase the binding affinity of the target epitope.
  • affinity maturation is known as “affinity maturation” and in some cases can be performed in connection with humanization, for example, in situations where humanization of an antibody leads to a decrease in binding specificity or affinity, and it is not possible to sufficiently improve binding specificity or affinity by using only reverse mutations.
  • affinity maturation methods are known in the art, for example, the in vitro scanning saturation mutagenesis method described by Burks et al. , Proc Natl Acad Sci USA, 94: 412-417 (1997), and a method for incremental in vitro affinity maturation proposed by Wu et al. , Proc Natl Acad Sci USA 95: 6037 6042 (1998).
  • FR Framework regions
  • FR Framework regions
  • FR light chain residues are localized approximately in residual regions 1-23 (LCFR1), 35-49 (LCFR2), 57-88 (LCFR3) and 98-107 (LCFR4)
  • heavy chain FR residues localized approximately in the region of residues 1-30 (HCFR1), 36-49 (HCFR2), 66-94 (HCFR3) and 103-113 (HCFR4) in the heavy chain.
  • FR light chain residues are located at approximately 1-25 (LCFR1), 33-49 (LCFR2), 53-90 (LCFR3) and 97-107 (LCFR4) in the light chain
  • a FR heavy chain residues are localized at about 1-25 (HCFRI), 33-52 (HCFR2), 56-95 (HCFR3) and 102-113 (HCFR4) in the heavy chain residues.
  • the FRs are adjusted accordingly. For example, when CDRH1 includes amino acids H26-H35, heavy chain FR1 residues are at positions 1-25, and FR2 residues are at positions 36-49.
  • a crystallizing fragment of an immunoglobulin (Eng. Fragment crystallizable region, Fc region, Fc) is the end part of an immunoglobulin molecule that interacts with the Fc receptor on the cell surface and with some proteins of the complement system. Given The property allows antibodies to activate the immune system.
  • the Fc region of IgG, IgA and IgD isotypes consists of two identical protein fragments, respectively, of the second and third constant domains of two heavy chains; in the case of IgM and IgE isotypes, Fc contains three constant heavy chain domains (CH 2-4 domains) in each polypeptide chain.
  • An antibody of the invention that “binds” the target antigen is an antibody that binds the antigen with sufficient affinity so that the antibody can be used as a diagnostic and / or therapeutic agent when targeting a protein or cell or tissue expressing the antigen, and slightly cross-reacts with other proteins.
  • analytical methods sorting of fluorescently activated cells (FACS), radioimmunoprecipitation (RIA) or ELISA (ELISA), in such embodiments of the invention, the degree of binding of the antibody to a protein that is not a “target” (with a “non-target protein”) is less than 10 % of the binding of the antibody to a specific target protein.
  • the term “specific binding” or the expression “specifically binds to” or “specific to” a particular polypeptide or epitope on a particular target polypeptide means a binding that is markedly (measurably) different from a non-specific interaction (e.g. in the case of LH1-44 or LH1-81, the non-specific interaction is binding to bovine serum albumin, casein, fetal bovine serum or neutravidin).
  • Specific binding can be quantified, for example, by determining the binding of a molecule compared to the binding of a control molecule. For example, specific binding can be determined by competitive reaction with another molecule similar to the target, for example, with an excess of unlabeled target. In this case, specific binding is indicated if the binding of the labeled target to the probe is competitively inhibited by an excess of unlabeled target.
  • the term “specific binding” or the expressions “specifically binds to” or “specific for” a particular polypeptide or epitope on a specific target polypeptide can be characterized by the example of a molecule having a Kd of at least 200 nM to the target, or at least at least about 150 nM, or at least about 100 nM, or at least about 60 nM, or at least about 50 nM, or at least about 40 nM, or at least about 30 nM, or at least about 20 nM, or at least about 10 n M, or at least about 8 nM, or at least about 6 nM, or at least about 4 nM, or at least about 2 nM, or at least about 1 nM or higher.
  • the term “specific binding” refers to a binding in which a molecule binds to a particular polypeptide or epitope on a particular polypeptide, with little or no binding to any other polypeptide or epitope on the polypeptide.
  • the term "Ka”, as used herein, refers to the rate of association of a particular antibody-antigen interaction.
  • Kd refers to the dissociation rate of a particular antibody-antigen interaction.
  • Binding affinity generally refers to the strength of the cumulative non-covalent interactions between a single binding site of a molecule (eg, an antibody) and its binding partner (eg, antigen). Unless otherwise indicated, “binding affinity” refers to the intrinsic (characteristic, true) binding affinity, which reflects a 1: 1 interaction between members of a binding pair (eg, antibody and antigen). The affinity of molecule X to its partner Y can usually be represented by constant dissociation (Kd).
  • the Kd value is about 200 nM, 150 nM, 100 nM, 60 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 8 nM, 6 nM, 4 nM, 2 nM, 1 nM or less.
  • Affinity can be measured by conventional methods known in the art, including those described herein. Low affinity antibodies usually bind slowly to the antigen and tend to readily dissociate, whereas high affinity antibodies usually bind to the antigen faster and tend to remain in the bound state for longer. Various methods for measuring binding affinity are known in the art, any of these methods can be used for the purposes of the present invention.
  • the "Kd" or “Kd value” of this invention is measured by surface plasmon resonance methods on a B1 Asoge TM -2000 or BIAcore TM -3000 instrument (BIAcore, Inc., Piscataway, NJ) at 25 ° C using immobilized chips CM5 antigen at ⁇ 10 relative units (response units, RU).
  • CM5 antigen at ⁇ 10 relative units (response units, RU).
  • carboxymethyldextran biosensor chips CM5, BIAcore Inc.
  • EDC -ethyl- '(3-dimethylaminopropyl) -carbodiimide
  • NHS N-hydroxysuccinimide
  • the antigen is diluted with a 10 mM sodium acetate solution, pH 4.8, to a concentration of 5 ⁇ g / ml ( ⁇ 0.2 ⁇ M), and then injected at a flow rate of 5 ⁇ l / min until approximately 10 relative units (RU) of the bound protein are reached.
  • a 1 M ethanolamine solution is administered to block unreacted groups.
  • two-fold serial dilutions of Fab e.g., from 0.78 nM to 500 nM
  • PBST Tween 20
  • association rate (cop) and dissociation rate (koff) are calculated using the simple Langmuir model for one-plus-one binding (BIAcore Evaluation Software version 3.2), using the simultaneous acquisition of association and dissociation sensograms.
  • the equilibrium dissociation constant (Kd) is calculated as the koff / kon ratio. See, for example, Chen, Y., et al. , (1999) J. Mol. Biol. 293: 865-881.
  • a spectrometer such as a stopped flow spectrophotometer (Aviv Instruments) or a SLM-Aminco spectrophotometer (ThermoSpectronie) 8000 series with a mixing cuvette.
  • koff refers to the dissociation rate constant of a particular interaction of a binding molecule and antigen.
  • the koff dissociation rate constant can be measured by bi-layer interferometry, for example using the Octet TM system.
  • the “oh-rate” or “cop” of this invention can also be determined by the same surface plasmon resonance method as described above on a B1Acoge TM -2000 or BIAcore TM -3000 instrument (BIAcore, Inc., Piscataway, NJ ) at 25 ° C using chips with immobilized CM5 antigen at ⁇ 10 relative units (response units, RU).
  • CM5 antigen at ⁇ 10 relative units (response units, RU).
  • carboxymethyldextran biosensor chips CM5, BIAcore Inc.
  • EDC -ethyl- '- (3-dimethylamino-propyl) -carbodiimide hydrochloride
  • NHS N-hydroxysuccinimide
  • the antigen is diluted with a 10 mM sodium acetate solution, pH 4.8, to a concentration of 5 ⁇ g / ml ( ⁇ 0.2 ⁇ M), and then injected at a flow rate of 5 ⁇ l / min until approximately 10 relative units (RU) of the bound protein are reached. After administration of the antigen, a 1 M ethanolamine solution is administered to block unreacted groups.
  • biologically active and “biological activity”, and “biological characteristics”, with respect to the polypeptide of this invention, mean having the ability to bind to a biological molecule.
  • biological molecule refers to nucleic acid, protein, carbohydrate, lipid, and combinations thereof. In one embodiment of the invention, the biological molecule exists in nature.
  • Antibody sites such as Fab and F (ab ') 2 fragments, can be obtained from whole antibodies using traditional methods, such as papain or pepsin hydrolysis of whole antibodies. Moreover, antibodies, parts of antibodies and immunoadhesion molecules can be obtained using standard recombinant DNA methods, for example, as described herein.
  • recombinant antibody means an antibody that is expressed in a cell or cell line containing a nucleotide sequence (nucleotide sequences) that encodes an antibody, wherein said nucleotide sequence (nucleotide sequences) is not associated with the cell in nature.
  • variant antibody refers to an antibody having an amino acid sequence that differs from the amino acid sequence of its "parent” antibody by adding, removing and / or replacing one or more amino acid residues relative to the sequence of the parent antibody.
  • the variant antibody comprises at least one or more (e.g., one to twelve, e.g., two, three, four, five, six, seven, eight or nine, ten, eleven or twelve; and in some embodiments one to about ten) additions, deletions and / or substitutions of amino acids relative to the parent antibody. In some embodiments, additions, deletions, and / or substitutions are made at the CDR regions of a variant antibody.
  • Identity or homology with respect to the sequence of the variant antibody is defined herein as the percentage of amino acid residues in the sequence of the variant antibody that are identical to the residues of the parent antibody, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percentage of sequence identity.
  • a variant antibody retains the ability to bind to the same antigen, and preferably the epitope, to which the parent antibody binds, and in some embodiments, the at least one property or biological activity exceeds that of the parent antibody.
  • a variant antibody may have, for example, a more pronounced binding affinity, a longer half-life, a lower IC50 value or an increased ability to suppress the biological activity of the antigen compared to the parent antibody.
  • a variant antibody showing a biological activity greater than at least 2 times (preferably at least 5 times, 10 times or 20 times) the biological activity of the parent antibody.
  • bispecific antibody means an antibody containing an antigen binding domain or antigen binding domains that are capable of specific binding to two different epitopes on one biological molecule or capable of specific binding to epitopes on two different biological molecules.
  • a bispecific antibody is also referred to herein as having "double specificity” or as being an antibody with "double specificity” or "biparatopic antibody”.
  • chimeric antibody refers in a broad sense to an antibody that contains one or more regions of one antibody, and one or more regions of one or more other antibodies, typically an antibody, partially human or partially non-human, that is, obtained partially from not a human animal, for example, a mouse, rat, or other rodent, or a camelid, such as a llama or alpaca.
  • Chimeric antibodies are preferred over non-human antibodies in order to reduce the risk of an immune response directed against antibodies in humans, for example, a response directed against murine antibodies in humans in the case of a murine antibody.
  • An example of a typical chimeric antibody is one in which the variable region sequences are murine, while the constant region sequences are human.
  • non-human parts can be further modified to humanize the antibody.
  • humanization refers to the fact that when an antibody is wholly or partially non-human, for example, a mouse or llama antibody obtained by immunizing mice or llamas with an antigen of interest, respectively, or is a chimeric antibody based on such a mouse or llama antibody , you can replace some amino acids, for example, in the framework regions and constant domains of the heavy and light chains, in order to avoid or minimize the immune response in humans.
  • the specificity of the interaction of the antibody with the target antigen is inherent mainly in amino acid residues located in six CDR regions of the heavy and light chains. Therefore, the amino acid sequences within the CDR regions are much more variable between individual antibodies, compared with sequences outside the CDR regions.
  • recombinant antibodies can be expressed that mimic the properties of a specific natural antibody, or more generally, any specific antibody with a given amino acid sequence, for example, by constructing expression vectors that express CDR sequences -particles from a specific antibody and frame sequences of another antibody.
  • a non-human antibody can be “humanized” and the binding specificity and affinity of the original antibody can be largely maintained.
  • non-human antibodies are generally more immunogenic than human antibodies.
  • Chimeric antibodies in which foreign (e.g., rodent or camel) constant regions have been replaced by sequences of human origin have shown generally lower immunogenicity than antibodies of completely foreign origin, and there is a tendency to use humanized or fully human antibodies in therapeutic antibodies.
  • Chimeric antibodies or other non-human antibodies can thus be humanized to reduce the risk of an anti-antibody immune response in humans.
  • humanization usually involves modifying the frame regions of the variable region sequences.
  • CDR regions complementarity determining regions
  • Amino acid residues that are part of the complementarity determining regions will most often not change due to humanization, although in some cases it may be desirable to alter individual amino acid residues of the CDR region, for example, to remove a glycosylation region, a deamidation site, an aspartate isomerization site, or an undesired cysteine or methionine residue.
  • N-linked glycosylation occurs by attaching an oligosaccharide chain to an asparagine residue in the tripeptide sequence of Asn-X-Ser or Asn-X-Thr, where X can be any amino acid except Pro.
  • Removal of the N-glycosylation site can be achieved by mutating the Asn or Ser / Thr residue with another residue, preferably by conservative substitution.
  • the deamidation of asparagine and glutamine residues can occur depending on factors such as pH and exposure of the surface.
  • Asparagine residues are particularly susceptible to deamidation, especially if they are present in the Asn-Gly sequence, and to a lesser extent in other dipeptide sequences, such as Asn-Ala. If there is such a deamidated region, for example, Asn-Gly in the sequence of the CDR region, it may be preferable to remove this region, usually by conservative substitution to remove one of the residues involved.
  • CDR site transplantation may be based on Rabat CDR site definitions, although a later publication (Magdelaine-Beuzelin et al., Crit Rev. Oncol Hematol. 64: 210 225 (2007)) suggests that the definition of no IMGT® (the international ImMunoGeneTics information system®, www.imgt.org) can improve the result of humanization (see Lefranc et al., Dev. Comp Immunol. 27: 55-77 (2003)).
  • transplantation of a CDR region can reduce the specificity and affinity of binding, and therefore biological activity, in a CDR transplanted non-human antibody, compared to the parent antibody from which the CDR regions are derived.
  • Reverse mutations (sometimes referred to as “frame repair”) can be applied at selected positions of the CDR of the transplanted antibody, typically in the frame regions, in order to restore the specificity and binding affinity of the parent antibody.
  • the determination of positions for possible reverse mutations can be performed using information available in the literature and in the antibody databases. Amino acid residues that are candidates for reverse mutations are typically located on the surface of an antibody molecule, while residues that are recessed or have a low degree of exposure of the surface usually will not be subject to change.
  • the humanization method an alternative to transplantation of the CDR region and reverse mutation, is a surface change in which residues of non-human origin unexposed on the surface are retained, while residues exposed on the surface are transformed into human residues.
  • naive and immune libraries are constructed using naturally reorganized genes encoding the variable domains of healthy or immune donor immunoglobulins, respectively. For this, mRNA of lymphoid cells producing antibodies is isolated. Most often these are peripheral blood lymphocytes, but in some cases splenocytes are used [Sheets MD, Amersdorfer P, Finnern R, Sargent P, Lindquist E, Schier R, et al. Efficient construction of a large nonimmune phage antibody library: the production of high-affinity human single-chain antibodies to protein antigens.
  • CDNAs are synthesized based on mRNA, and oligo-dT primers and statistical hexaoligonucleotides can be taken to prime the reaction, which allows one to obtain cDNA copies of all possible variants of genes encoding the variable domains of antibodies [Ulitin AB, Kapralova MV, Daman AG, Shepelyakovskaya AO, Bulgakov EB, Fursova KK, et al. Library of human mini-antibodies in phage display format. Creation and testing. DAN: Publishing House “Science”; 2005.].
  • one or more primers can be used that limit the set of amplifiable genes to one or more gene families of variable domains or antibody isotypes already at the cDNA level [Marks JD, Hoogenboom HR, Bonnert TP, McCafferty J, Griffiths AD, Winter G. Bypassing immunization. Human antibodies from V-gene libraries displayed on phage. J Mol Biol 1991,222: 581-597].
  • the primers used to amplify genes encoding immunoglobulins are complementary to their most conserved regions.
  • Them sequences are selected from gene collections that are organized into databases, such as the Rabat database or V BASE.
  • the design of the primers also provides for the presence of internal restriction sites in them, allowing cloning PCR products into the corresponding vectors.
  • Phage display is the first and most widely used in vitro antibody search technology.
  • Smith discovered that foreign DNA sequences can be cloned into an M13 filamentous bacteriophage in such a way that cloned gene sequences are expressed on the surface of phage particles as fusion proteins (Smith GP: Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 1985, 228: 1315-1317.).
  • Smith GP Filamentous fusion phage: novel expression vectors that display cloned antigens on the virion surface. Science 1985, 228: 1315-1317.
  • the repertoire of phage libraries reflects the repertoire of antibodies of B-lymphocytes of each person or animal whose blood was used to create the library.
  • mice that expressed fully human antibodies, the repertoire of which could be comparable to those obtained by hybridoma technology
  • Hybridoma technology Liunstine M, Higgins KM, Schramm SR, Kuo CC, Mashayekh R, Wymore K, McCabe JG et al.: Antigen-specific human antibodies from mice containing four distinct genetic modifications. Nature 1994, 368: 856-859
  • mice express B-cell receptors, which are essentially hybrid mouse and human (human immunoglobulin, mouse Ig, Irb and other signaling molecules), their B cells normally develop and mature.
  • a clonal population may be a clonal population of immortalized cells.
  • immortalized cells in a clonal population are hybrid cells, hybridomas that are usually obtained by fusing individual B lymphocytes from immunized animals with individual cells of a lymphocytic tumor.
  • Hybridomas are a type of engineered cell and are not found in nature.
  • “Native antibodies” are usually heterotetrameric glycoproteins with a molecular weight of approximately 150,000 daltons, consisting of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to the heavy chain by one covalent disulfide bond, while the number of disulfide bonds between the heavy chains varies in different immunoglobulin isotypes. Each heavy and light chain also has evenly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at one end, followed by several constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at the other end.
  • VH variable domain
  • VL variable domain at one end
  • the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the variable domain of the light chain is aligned with the variable domain of the heavy chain.
  • Specific amino acid residues are believed to form an interface between the variable domains of the light chain and the heavy chain.
  • isolated used to describe the various antibodies described herein means an antibody identified and isolated and / or regenerated from the cell or cell culture in which it is expressed. Impurities (contaminants) from the natural environment are materials that, as a rule, interfere with the diagnostic or therapeutic use of the polypeptide, and may include enzymes, hormones, and other protein or non-protein solutes.
  • the antibody is purified (1) to a degree sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence using a rotating glass cup sequencer (Edman sequencer), or (2) until homogeneous by SDS-PAGE in non-reducing or reducing conditions using Coomassie staining with brilliant blue or, preferably, silver.
  • An isolated antibody includes antibodies in situ within recombinant cells, since at least one component of the natural environment of the polypeptide is absent. Typically, an isolated polypeptide is obtained from at least one purification step.
  • An “isolated” nucleic acid molecule is a nucleic acid molecule that is identified and separated from at least one impurity nucleic acid molecule with which it is usually associated in a natural source of an antibody nucleic acid.
  • An isolated nucleic acid molecule differs from the form or kit in which it is found in vivo. Thus, the isolated nucleic acid molecule is different from a nucleic acid molecule that exists in cells in vivo.
  • the isolated nucleic acid molecule includes a nucleic acid molecule located in cells in which expression of the antibody normally occurs, for example, if the nucleic acid molecule has a localization in the chromosome that is different from its localization in cells in vivo.
  • epitope refers to a part (determinant) of an antigen that specifically binds to a binding molecule (for example, an antibody or a related molecule, such as a bispecific binding molecule).
  • a binding molecule for example, an antibody or a related molecule, such as a bispecific binding molecule.
  • Epitope determinants usually consist of chemically active surface groups of molecules, such as amino acids or carbohydrates, or sugar side chains, and typically have specific three-dimensional structural characteristics, as well as specific charge characteristics.
  • the epitope can be "linear” or “conformational. In a linear epitope, all points of interaction between a protein (eg an antigen) and an interacting molecule (such as an antibody) occur linearly along the primary amino acid sequence of the protein.
  • a conformational epitope interaction points occur through amino acid residues on a protein that are separated from each other in the primary amino acid sequence.
  • antibodies to this epitope can be generated using techniques well known in the art.
  • the generation and characterization of antibodies or other binding molecules can shed light on information about the desired epitopes. Based on this information, binding molecules can then be competitively screened for binding to the same or similar epitopes, for example, by conducting competition studies to find binding molecules that compete for binding to antigen.
  • peptide linker in this document means any peptide with the ability to join domains with a length depending on the domains that it binds to each other, containing any amino acid sequence.
  • the peptide linker has a length of more than 5 amino acids and consists of any set of amino acids selected from G, A, S, P, E, T, D, K.
  • in vitro refers to a biological object, biological process or biological reaction outside the body, modeled in artificial conditions.
  • in vitro cell growth should be understood as cell growth in a medium outside the body, for example, in a test tube, culture bottle or microplate.
  • 1C50 50% inhibitory concentration refers to drug concentrations at which measured activity or response, for example, growth or proliferation of cells, such as tumor cells, is inhibited by 50%.
  • the IC50 value can be estimated using the corresponding response curves of the logarithm of the dose, s using special statistical programs for processing curves.
  • GI50 growth inhibition
  • ED50 effective dose / concentration
  • antiproliferative effect means stopping or inhibiting the growth of proliferating cells, such as cancer cells.
  • effector function of an antibody refers to the types of biological activity associated with the Fc region (native sequence of the Fc region or variants of the amino acid sequence of the Fc region) of the antibody, and vary depending on the isotype of the antibody.
  • antibody effector functions are: Clq binding; complement dependent cytotoxicity; Fc receptor binding; antibody dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B-cell receptor, BCR); and B-cell activation.
  • a CC antibody-dependent cell cytotoxicity
  • FcR Fc receptors
  • NK natural killer cells
  • monocytes express FcyRI, FcyRII and FcyRIII.
  • the expression of FcR on hematopoietic cells is summarized in table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991).
  • ADCC assays can be performed to evaluate the activity of the molecule of interest in ADCC, such as the assays described in US Pat. Nos. 5,500,362 or 5,821,337.
  • Applicable effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer cells (NKs).
  • PBMCs peripheral blood mononuclear cells
  • NKs natural killer cells
  • the ADCC activity of the molecule of interest can be evaluated in vivo, for example, in an animal model, such as the model described in Clynes et al. PNAS (USA) 95: 652-656 (1998).
  • Human effector cells are white blood cells that express one or more FcRs and perform effector functions. Preferably, the cells express at least FcyRIII and perform an ADCC effector function. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer cells (NKs), monocytes, cytotoxic T cells, and neutrophils; RVMS and NK cells are preferred. Effector cells can be isolated from their natural source, for example, from blood or PBMC, as described in this publication.
  • PBMCs peripheral blood mononuclear cells
  • NKs natural killer cells
  • monocytes cytotoxic T cells
  • neutrophils neutrophils
  • Effector cells can be isolated from their natural source, for example, from blood or PBMC, as described in this publication.
  • Fc receptor and “FcR” are used to describe a receptor that binds to the Fc region of an antibody.
  • a preferred FcR is a human FcR with a native sequence.
  • a preferred FcR is FcR, which binds an IgG antibody (gamma receptor), and preferred receptors include receptors of subclasses FcyRI, FcyRII and FcyRIII, including allelic variants and alternatively splicable forms of these receptors.
  • FcyRII receptors include FcyRI IA ("activating receptor") and FcyRI IB ("inhibitory receptor”), which have similar amino acid sequences that differ mainly in their cytoplasmic domains.
  • the activating receptor FcyRI IA contains a tyrosine-based immunoreceptor activation motif (ITAM) in its cytoplasmic domain.
  • ITAM immunoreceptor activation motif
  • the inhibitory receptor FcyRI IB contains in its cytoplasmic domain a tyrosine-based immunoreceptor inhibition motive (IT ⁇ M) (see review in Aegop, Annu. Rev. Immunol. 15: 203-234 (1997)).
  • IT ⁇ M tyrosine-based immunoreceptor inhibition motive
  • FcR is presented in Ravetch and Kinet, Annu. Rev. Immunol 9: 457-92 (1991).
  • Other FcRs, including FcRs, which will be identified in the future, are included in the present description in the term "FcR".
  • the term also includes a neonatal receptor, FcRn, which is responsible for the transfer of maternal IgG to the fetus.
  • complement dependent cytotoxicity and “SBS” refer to the ability of a molecule to lyse a target in the presence of complement.
  • the complement activation pathway is initiated by binding of the first component of the complement system (Clq) to a molecule (e.g., an antibody) in complex with its antigen.
  • a molecule e.g., an antibody
  • an SBS analysis can be performed, for example, as described in Gazzano-Santoro et al. , J. Immunol. Methods 202: 163 (1996).
  • identity or “homology” should be interpreted as meaning the percentage of amino acid residues in the candidate sequence that are identical to the residues of the corresponding sequence with which it is compared, after sequence comparison and introduction of “gaps” if it is necessary to achieve the maximum percentage of identity for the full sequence and not considering any conservative substitutions as part of sequence identity. Neither the N- or C-terminal extension nor insertion segments should be construed as reducing identity or homology. Methods and computer programs for comparison are well known. Sequence identity can be determined using sequence analysis software (e.g., Sequence Analysis Software Package, Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Ave., Madison, WI 53705). This software is suitable for such sequences by determining the degree of homology. for a variety of substitutions, deletions (eliminations) and other modifications.
  • sequence analysis software e.g., Sequence Analysis Software Package, Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Ave., Madison, WI 53705
  • nucleic acid sequence should be interpreted as a nucleotide sequence exhibiting at least 85%, preferably 90%, more preferably 95% and most preferably 97% sequence identity with respect to the nucleic acid sequence.
  • the proposed modification (s) of the amino acid sequences of the antibodies described in this application may be desirable to improve the binding affinity and / or other biological properties of the antibody.
  • Variants of the amino acid sequence of an antibody are obtained by introducing appropriate changes in nucleotides into the nucleic acid of the antibody or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues in the amino acid sequences of an antibody. Any combination of deletion, insertion, and substitution is performed to obtain the final construct, provided that the final construct possesses the required characteristics. Changes in amino acids can also change the post-translational processes in the antibody, such as changes in the number or position of glycosylation sites.
  • a variant of the modification of the amino acid sequences of antibodies using amino acid substitutions is the replacement of at least one amino acid residue in the antibody molecule with another residue.
  • Places of greatest interest for mutagenesis by substitutions include hypervariable regions or CDRs, but changes in the FR or Fc regions are also contemplated.
  • Conservative substitutions are shown in Table A under the heading of “preferred substitutions”. If such substitutions result in a change in biological activity, then additional substantial changes can be introduced, called “examples of substitutions" in Table A, or changes additionally described below in the description of the classes of amino acids, and products can be screened.
  • nucleic acid means a clear sequence of nucleotides, modified or not modified defining a fragment or region of a nucleic acid containing or not containing unnatural nucleotides and being either double-stranded DNA or RNA, or a single-chain stranded DNA or RNA or the transcription product of said DNA. It should also be mentioned here that the invention does not apply to nucleotide sequences in their natural chromosome environment, i.e. in a natural state.
  • sequences of this invention have been isolated and / or purified, i.e. were taken directly or indirectly, for example, by copying, while their environment was at least partially modified.
  • isolated nucleic acids obtained by genetic recombination, for example, using host cells (host cells), or obtained by chemical synthesis.
  • nucleotide sequence covers its compliment, unless otherwise indicated.
  • reference to a nucleic acid having a specific sequence should be understood as encompassing its complementary chain with its complementary sequence.
  • control sequences refers to DNA sequences necessary for the expression of a functionally linked coding sequence in a particular host organism.
  • Suitable control sequences for prokaryotes are, for example, a promoter, optionally an operator and a ribosome binding site.
  • promoters, polyadenylation signals and enhancers are present in eukaryotic cells.
  • a nucleic acid is “operably linked” if it is in functional association with another nucleotide sequence.
  • DNA of a presequence or secretory leader sequence is operably linked to the DNA of a polypeptide if it is expressed as a preprotein that is involved in the secretion of the polypeptide;
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence;
  • the ribosome binding site is functionally linked to the coding sequence, if located so that it can facilitate translation.
  • “operably linked” means that the linked DNA sequences are contiguous, and in the case of a secretory leader sequence, are contiguous and are in the reading phase. However, enhancers do not have to be contiguous.
  • vector means a nucleic acid molecule capable of transporting another nucleic acid to which it is connected.
  • the vector is a plasmid, i.e. a circular double-stranded portion of DNA into which additional DNA segments can be ligated.
  • the vector is a viral vector in which additional DNA segments can be ligated into the viral genome.
  • the vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors having a bacterial initiation site mammalian replication and episomal vectors).
  • vectors eg, non-episomal mammalian vectors
  • vectors can be integrated into the genome of the host cell when introduced into the host cell, and thus replicate together with the host gene.
  • some vectors are capable of directing the expression of the genes with which they are functionally linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply “expression vectors”).
  • recombinant host cell means a cell into which a recombinant expression vector has been introduced.
  • the present invention relates to host cells, which may include, for example, a vector in accordance with the present invention described above.
  • the present invention also relates to host cells, which include, for example, a nucleotide sequence encoding a heavy chain or its antigen binding parts, a nucleotide sequence encoding a light chain or its antigen binding parts, or both of them, a first binding domain and / or a second binding domain a binding molecule of the invention.
  • host cell and “host cell” mean not only the specific cell claimed, but also the progeny of such a cell. Since modifications can occur in subsequent generations due to mutations or environmental influences, such offspring may not, in fact, be identical to the parent cell, but such cells are still included within the scope of the term “host cell” as used herein.
  • disease or disorder mediated by HER2 means all diseases or disorders that are either directly or indirectly associated with HER2, including the etiology, development, progress, persistence or pathology of the disease or disorder.
  • Treatment refers to a method of alleviating or eliminating a biological disorder and / or at least one of its accompanying symptoms. Used in this document, the term “alleviate” a disease, disease or condition, means a decrease in the severity and / or frequency of symptoms of the disease, disorder or condition.
  • references to “treatment” contained herein include references to therapeutic, palliative, and prophylactic therapy.
  • the subject of treatment or the patient is a mammal, preferably a human subject.
  • the above subject may be male or female of any age.
  • violation means any condition that can be improved as a result of treatment according to the present invention.
  • the definition of this term includes chronic and acute disorders or diseases, including pathological conditions that cause a mammal's predisposition to the occurrence of this violation.
  • diseases to be treated include benign and malignant tumors, for example, cancer of the breast, ovary, stomach, cervix, endometrium, uterus, salivary gland, lung, kidney, colon, colon, thyroid, pancreas, prostate glands, skin, head and / or neck, pharynx, oral cavity or bladder.
  • the preferred disorder to be treated according to the invention is cancer, namely breast cancer (breast cancer), gastric cancer, non-small cell lung cancer, head and / or neck cancer, squamous cell carcinoma of the head and neck (CBC), colorectal cancer (CRC), esophageal cancer, ovarian cancer, pancreatic cancer, gastrointestinal cancer, kidney cancer, cervical cancer, endometrial cancer, uterine cancer, malignant melanoma, pharyngeal cancer, oral cancer or skin cancer.
  • breast cancer breast cancer
  • gastric cancer gastric cancer
  • non-small cell lung cancer non-small cell lung cancer
  • head and / or neck cancer squamous cell carcinoma of the head and neck
  • CRC colorectal cancer
  • esophageal cancer ovarian cancer
  • pancreatic cancer gastrointestinal cancer
  • kidney cancer cervical cancer
  • endometrial cancer uterine cancer
  • malignant melanoma malignant melanoma
  • pharyngeal cancer oral cancer or skin
  • cancer refers to a physiological state or describe the physiological state in mammals, which is usually characterized by unregulated (/ th) cell growth / proliferation. This definition covers benign and malignant cancers. Examples of cancers include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia.
  • cancers include squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, squamous cell carcinoma of the lung, peritoneal cancer, hepatic cell carcinoma, gastric cancer, including gastrointestinal cancer, pancreatic cancer, glioblastoma, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial and uterine carcinoma, salivary carcinoma, kidney cancer (renal cell carcinoma), gland cancer (prostate cancer), cancer of the external female genital organs, thyroid cancer, hepatic carcinoma, carcinoma of the anal canal, carcinoma of the penis, melanoma and various types of cancer of the head and neck.
  • gastric cancer including gastrointestinal cancer, pancreatic cancer, glioblastoma, glioma, cervical cancer, ovarian cancer, liver cancer, bladder cancer, breast cancer, colon cancer, colorectal cancer, endometrial and uterine
  • a “therapeutically effective amount” is the amount of a therapeutic agent administered during treatment that will relieve to some extent one or more of the symptoms of the disease being treated.
  • chronic use refers to the continuous (long-term) use of the agent (s) as opposed to the acute (short-term) route of administration, so as to maintain the initial therapeutic effect (activity) for a long period of time.
  • “Intermittent” use refers to a treatment that is not administered sequentially without interruption, but which is rather periodic in nature.
  • the present invention relates to a bispecific antibody that specifically binds to the fourth subdomain of the extracellular domain (ECD4) of HER2 (human epidermal growth factor receptor 2) and the second subdomain of the extracellular domain (ECD2) of human HER2.
  • ECD4 extracellular domain
  • ECD2 human epidermal growth factor receptor 2
  • the present invention relates to a bispecific antibody that specifically binds to the fourth subdomain of the extracellular domain (ECD4) of HER2 (human epidermal growth factor receptor 2) and the second subdomain of the extracellular domain (ECD2) of human HER2, and includes:
  • the first antigen-binding part that specifically binds to the IV subdomain of the extracellular domain (ECD4) of HER2, and is a single-chain variable fragment (scFv) of trastuzumab with the amino acid sequence
  • the second antigen-binding part which specifically binds to the II subdomain of the extracellular domain (ECD2) of HER2, and is an antigen binding site (Fab), including the variable domain of the heavy chain (VH) with the amino acid sequence
  • HCDR1 GFTFTDYTMD (SEQ ID NO: 3);
  • HCDR2 DVNPNSGES IYNQRFKG (SEQ ID NO: 4);
  • HCDR3 NLGPSFYFDY (SEQ ID NO: 5).
  • VL variable domain of the light chain
  • DIQMTQSPSSLSASVGDRVTITCKALQDVSRGVAWYQQKPGKAPKLLIYSAHYRYTGVPSRFSGSGSGSG TDFTLTISSLQPEDFATYYCQQYYIYPYTFGQGTKVEIK (SEQ ID NO: b: 3), includes LCD
  • LCDR1 KALQDVSRGVA (SEQ ID NO: 7);
  • LCDR2 SAHYRYT (SEQ ID NO: 8);
  • LCDR3 QQYYIYPYT (SEQ ID NO: 9).
  • the bispecific antibody is an IgG antibody.
  • the bispecific IgG antibody is of the human IgGl, IgG2, IgG3 or IgG4 isotype.
  • the bispecific antibody is of the human IgGl isotype.
  • the bispecific antibody comprises a crystallizing immunoglobulin fragment (Fc fragment) that contains two amino acid sequences of the second and third constant domains (CH2-CH3) represented by the amino acid sequence
  • the bispecific antibody comprises a second antigen binding portion that specifically binds to the II subdomain of the extracellular domain (ECD2) of HER2, and is an antigen binding site (Fab), comprising:
  • VH variable domain
  • CHI first constant domain of the heavy chain
  • amino acid sequence EVQLVESGGGLVQPGGSLRLSCAAWGFTFTDYTMDWVRQAPGKGLEWVADVNPNSGESIYNQRFKGRF TLSVDRSKNTLYLQMNSLRAEDTAVYYCARNLGPSFYFDYWGLGTLVTVSSASTKGPSVFPLAPSSKS TSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNL NHKPSNTKVDKRV (SEQ ID NO: 12);
  • the present invention relates to a bispecific antibody that specifically binds to the IV subdomain of the extracellular domain (ECD4) HER2 (epidermal receptor human growth factor 2) and II subdomain of the extracellular domain (ECD2) of human HER2, consisting of:
  • an antibody light chain that specifically binds to the II subdomain of the extracellular domain (ECD2) of HER2, including the variable domain of the light chain (VL) and the constant domain of the light chain (SC), with the amino acid sequence
  • the bispecific antibody is a BCD147-02-020 bispecific antibody that specifically binds to the IV subdomain of the extracellular domain (ECD4) HER2 (human epidermal growth factor receptor 2) and the second subdomain of the extracellular domain (ECD2) HER2.
  • the BCD147-02-020 bispecific antibody is an asymmetric antibody, that is, it includes 1) a first antigen binding portion that specifically binds to the IV subdomain of the extracellular domain (ECD4) of HER2, and is a single chain variable fragment (scFv) of trastuzumab; and 2) a second antigen binding part that specifically binds to the II subdomain of the extracellular domain (ECD2) of HER2, and is antigen binding site (Fab).
  • the BCD147-02-020 bispecific antibody is a biparatopic antibody, that is, an antibody that binds to two paratopes, IV and II subdomains of the human extracellular domain of HER2.
  • the bispecific antibody is a bispecific asymmetric antibody BCD147-02-020, which specifically binds to the IV and II subdomains of the extracellular domain of human HER2.
  • the BCD147-02-020 bispecific antibody consists of:
  • an antibody light chain that specifically binds to the II subdomain of the extracellular domain (ECD2) of HER2, including the variable domain of the light chain (VL) and the constant domain of the light chain (SC), with the amino acid sequence
  • amino acid sequence of SEQ ID NO: 14, which specifically binds to the IV subdomain of the extracellular domain (ECD4) HER2, consists of: 1) the constant domain of CH2 and CHZ, with the amino acid sequence
  • the antibody heavy chain (SEQ ID NO: 15), which specifically binds to the II subdomain of the extracellular domain (ECD2) of HER2, consists of:
  • VH heavy chain variable domain
  • CHI first constant domain of the heavy chain
  • the antibody light chain (SEQ ID NO: 13), which specifically binds to the II subdomain of the extracellular domain (ECD2) of HER2, consists of:
  • the present invention also relates to nucleic acid molecules, in particular to sequences encoding a bispecific antibody that specifically binds to the IV subdomain of the extracellular domain (ECD4) HER2 (human epidermal growth factor receptor 2) and the second subdomain of the extracellular domain (ECD2) HER2, according to this inventions as described herein, optionally including any sequence of a peptide linker connecting them.
  • ECD4 extracellular domain
  • HER2 human epidermal growth factor receptor 2
  • ECD2 extracellular domain 2
  • nucleotide sequence covers its compliment, unless otherwise indicated. Thus, a reference to a nucleic acid having a specific sequence should be understood as encompassing its complementary chain with its complementary sequence.
  • polynucleotide means a polymeric form of nucleotides of at least 10 bases in length, or ribonucleotides, or deoxynucleotides, or a modified form of any type of nucleotide. The term includes single and double chain forms.
  • the present invention also relates to nucleotide sequences encoding an amino acid sequence selected from the group consisting of SEQ ID NO: 1-15, or any combination thereof.
  • the present invention relates to a nucleic acid molecule comprising a nucleotide sequence that encodes an amino acid sequence selected from SEQ ID NO: 1-15.
  • the nucleic acid molecule may also contain any combination of these nucleotide sequences.
  • the nucleic acid molecule comprises a nucleotide sequence encoding SEQ ID NO: 1, 2, and b. In another embodiment, the nucleic acid molecule comprises nucleotide sequences encoding SEQ ID NO: 1, 2, b, 10-11. In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding SEQ ID NO: 1, 12 and 13. In one embodiment, the nucleic acid molecule comprises a nucleotide sequence encoding SEQ ID NO: 1, 10-13.
  • the present invention relates to a nucleic acid molecule that contains nucleotide sequences encoding the amino acid sequences of SEQ ID NO: 13-15.
  • the present invention relates to a nucleic acid molecule comprising any combination of the above nucleic acid sequences.
  • the nucleic acid molecules may be isolated.
  • the nucleic acid molecule of this invention can be isolated from any source that produces a bispecific antibody that specifically binds to the IV and II subdomains of the extracellular domain of human HER2.
  • a nucleic acid molecule of the invention may be synthesized rather than isolated.
  • the nucleic acid molecule of the invention may comprise a nucleotide sequence encoding a VH domain of a first or second domain of an antibody of the invention, coupled as read from a nucleotide sequence encoding a constant domain of a heavy chain from any source.
  • a nucleic acid molecule of this invention may comprise a nucleotide sequence encoding a VL domain from a first or second region of an antibody of this invention, combined as a read with a nucleotide sequence encoding a constant domain of a light chain from any source.
  • nucleic acid molecules encoding the variable domain of the heavy (VH) and / or light (VL) chains of the first or second binding domain can be "transformed” along the entire length of the antibody genes.
  • the nucleic acid molecules encoding the VH or VL domains are transformed into antibody genes along the entire length by insertion into an expression vector already encoding the constant domains of the heavy chain (CH) or light chain (CL), respectively, so that the VH segment is functional connected to the CH segment (s) in the vector and / or the VL segment is operatively connected to the CL segment in the vector.
  • nucleic acid molecules encoding the VH and / or VL domains are converted into genes along the entire length of the antibody by connecting, for example, ligation, a nucleic acid molecule encoding the VH and / or VL domains to a nucleic acid molecule encoding the CH and / or CL domains using standard molecular biological methods. Nucleic acid molecules encoding the entire length of the heavy and / or light chains can then be expressed from the cell into which they were introduced.
  • Nucleic acid molecules can be used to express a large amount of a recombinant bispecific antibody that specifically binds to the IV and II subdomains of the human extracellular domain of HER2. Nucleic acid molecules can be used to produce bispecific antibodies, as described herein.
  • Vectors a recombinant bispecific antibody that specifically binds to the IV and II subdomains of the human extracellular domain of HER2. Nucleic acid molecules can be used to produce bispecific antibodies, as described herein.
  • the present invention relates to a vector suitable for expression of any of the nucleotide sequences described herein.
  • the present invention relates to vectors containing nucleic acid molecules that encode any of the amino acid sequences of a bispecific antibody that specifically binds to the IV and II subdomains of the human extracellular domain of HER2, or parts thereof (e.g., heavy and / or heavy and / or heavy chain sequences light chain of the second binding domains) as described herein.
  • the present invention further relates to vectors containing nucleic acid molecules encoding a bispecific antibody or parts thereof.
  • a bispecific antibody that specifically binds to the IV and II subdomains of the human extracellular domain of HER2 of this invention is expressed by inserting DNA encoding partially or fully the sequence of the first and second binding domain (e.g., heavy and light chain sequences, where the binding domain contains heavy and light chain sequences) obtained as described above in expression vectors so that the genes are functionally linked with the necessary expression control sequences, such as transcriptional and translational control sequences.
  • Expression vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses such as cauliflower mosaic virus, tobacco mosaic viruses, cosmids, YAC, EBV derived episomas and the like.
  • DNA molecules can be ligated into a vector so that sequences that control transcription and translation in the vector fulfill the intended function of regulating the transcription and translation of DNA.
  • the expression vector and expression control sequences can be selected so as to be compatible with the expression host cell used.
  • DNA molecules encoding partially or along the entire length of the sequence of the first and second binding domains e.g., heavy and light chain sequences, where the binding domain contains a heavy and light chain sequence
  • any combination of the above DNA molecules is introduced into the same expression vector.
  • DNA molecules can be introduced into the expression vector by standard methods (for example, by ligation of complementary restriction sites on a fragment of an antibody and vector gene or by ligation of blunt ends if there are no restriction sites).
  • a suitable vector is one that encodes functionally complete sequences of CH or CL of human immunoglobulin with the construction of an appropriate restriction site so that any the VH or VL sequence can be easily incorporated and expressed as described above.
  • NS and LC coding of genes in such vectors may contain intron sequences, which leads to a general increase in the protein products of antibodies by stabilization of the corresponding mRNA.
  • Intron sequences are surrounded by a splice donor and a splice acceptor sites that determine where RNA splicing will occur.
  • the arrangement of intron sequences can be either in the variable or constant regions of the antibody chains, or in both variable and constant regions when several introns are used.
  • Termination of polyadenylation and transcription may occur downstream of the native chromosome site of the encoded regions.
  • the recombinant expression vector can also encode a signal peptide that facilitates the production of an antibody chain by a host cell.
  • the antibody chain gene can be cloned into a vector so that the signal peptide is connected to the reading frame of the amino terminus of the immunoglobulin chain.
  • the signal peptide may be an immunoglobulin signal peptide or a heterologous signal peptide (i.e., a signal peptide of a non-immunoglobulin nature protein).
  • the recombinant expression of the vectors of this invention can carry regulatory sequences that control the expression of antibody chain genes in the host cell.
  • regulatory sequences that control the expression of antibody chain genes in the host cell.
  • the design of the expression vector including the choice of regulatory sequences, may depend on factors such as selection of the host cell for transformation, expression level of the desired protein, etc.
  • Preferred regulatory sequences for an expressing mammalian host cell include viral elements providing a high level of protein expression in mammalian cells, such as promoters and / or enhancers derived from retroviral LTR, cytomegalovirus (CMV) (e.g., CMV promoter / enhancer), monkey virus 40 (SV40) (e.g.
  • CMV cytomegalovirus
  • SV40 monkey virus 40
  • SV40 promoter / enhancer adenovirus, (e.g. Late Adenovirus late promoter (AdMLP)), polyoma virus, as well as strong mammalian promoters such as the native promoter x immunoglobulins or actin promoter.
  • AdMLP Late Adenovirus late promoter
  • strong mammalian promoters such as the native promoter x immunoglobulins or actin promoter.
  • recombinant expression vectors of the invention may carry additional sequences, such as sequences that regulate vector replication in host cells (eg, replication origin) and selectable marker genes.
  • the selectable marker gene facilitates the selection of host cells into which the vector has been introduced (see, for example, US patents 4,399,216, 4,634,665 and 5,179,017).
  • a selectable marker gene confers resistance to drugs, such as G418, hygromycin or methotrexate, to the host cell into which the vector is introduced.
  • breeding marker genes include the dihydrofolate reductase gene (DHFR) (for use in dhfr host cells for the selection / amplification of methotrexate), the neo gene (for G418 selection), and the glutamate synthetase gene.
  • DHFR dihydrofolate reductase gene
  • neo for G418 selection
  • glutamate synthetase gene for use in dhfr host cells for the selection / amplification of methotrexate
  • glutamate synthetase gene glutamate synthetase gene
  • expression control sequence means polynucleotide sequences that are necessary to affect the expression and processing of the coding sequences to which they are ligated.
  • Expression control sequences include corresponding transcription, termination, promoter and enhancer initiation sequences; effective RNA processing signals, such as splicing and polyadenylation signals; sequences that stabilize the cytoplasmic mRNA; sequences that increase translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and, if desired, sequences that enhance protein secretion.
  • control sequences vary depending on the host organism; in prokaryotes, such control sequences typically include a promoter, a ribosome binding site, and transcription termination sequences; in eukaryotes, typically, such control sequences include promoters and transcription termination sequences.
  • control sequences includes at least all components whose presence is important for expression and processing, and may also include additional components whose presence is useful, for example, lead sequences and sequences of fused cells.
  • An additional aspect of the present invention relates to methods for producing a bispecific antibody that specifically binds to the IV and II subdomains of the extracellular domain of human HER2, according to this invention.
  • One embodiment of the invention relates to a method for producing a bispecific antibody that specifically binds to the IV and II subdomains of the human HER2 extracellular domain, as defined herein, comprising preparing a recombinant host cell capable of expressing a bispecific antibody that specifically binds to IV and II subdomains human extracellular domain HER2 cultivation the specified host cell under conditions suitable for expression of the production of a bispecific antibody that specifically binds to the IV and II subdomains of the extracellular domain of human HER2, and the selection of this antibody.
  • a bispecific antibody that specifically binds to the IV and II subdomains of the human extracellular domain of HER2 obtained by such expression in such recombinant host cells is referred to herein as a “recombinant bispecific antibody that specifically binds to the IV and II subdomains of the human extracellular domain of HER2".
  • the invention also relates to the progeny of cells of such host cells and a bispecific antibody that specifically binds to the IV and II subdomains of the extracellular domain of human HER2, obtained in a similar way.
  • Nucleic acid molecules encoding a bispecific antibody that specifically binds to the IV and II subdomains of the human extracellular domain of HER2 according to the invention and vectors containing these nucleic acid molecules can be used to transfect a suitable mammal or cell, plant or cell thereof, bacterial or yeast host cell. The conversion can occur by any known method for introducing polynucleotides into a host cell.
  • Methods for introducing heterologous polynucleotides into mammalian cells include dextran-mediated transfection, transfection with a nucleic acid complex and a positively charged polymer, transfection with a nucleic acid and calcium phosphate precipitate, polybrene-mediated transfection, protoplast fusion, and transfection of the nucleotide DNA to the nucleus.
  • nucleic acid molecules can be introduced into mammalian cells by viral vectors.
  • Cell transfection methods are well known in the art. See, for example, U.S. Patents 4,399,216, 4,912,040, 4,740,461 and 4,959,455.
  • Methods for transforming plant cells are well known in the art, including, for example, Agrobacterium-mediated transformation, biolistic transformation, direct injection, electroporation and viral transformation. Methods for transforming bacterial and yeast cells are also well known in the art.
  • Mammalian cell lines used as hosts for transformation are well known in the art and include many immortalized cell lines available. These include, for example, Chinese hamster ovary cells (CHO), NS0 cells, SP2 cells, HEK-293T cells, 293 Freestyle cells (Invitrogen), NIH-3T3 cells, HeLa cells, hamster kidney cells (BHK), African kidney cells green monkeys (COS), human hepatocellular carcinoma cells (e.g., Hep G2), A549 cells, and a number of other cell lines. Cell lines are selected by determining which cell lines have high levels of expression and provide the necessary characteristics of the protein produced. Other cell lines which can be used are insect cell lines, such as Sf9 or Sf21 cells.
  • the antibodies are produced by culturing the host cells for a time sufficient to express the antibodies in the host cells or, more preferably, isolating antibodies into the culture medium in which the host cells are grown.
  • a bispecific antibody that specifically binds to the IV and II subdomains of the human extracellular domain of HER2 can be isolated from the culture medium using standard protein purification methods.
  • Plant host cells include Nicotiana, Arabidopsis, duckweed, corn, wheat, potatoes, etc.
  • Host bacteria cells include Escherichia and Streptomyces species.
  • Yeast host cells include Schizosaccharomyces pombe, Saccharomyces cerevisiae, and Pichia pastoris.
  • the production level of a bispecific antibody that specifically binds to the IV and II subdomains of the human extracellular domain of HER2 of the present invention from a producing cell line can be enhanced using a number of known methods.
  • the glutamine synthetase gene expression system (GS system) is common enough to enhance expression under certain conditions.
  • the GS system is discussed in whole or in part in connection with the patents EP 0216846, 0256055, 0323997 and 0338841.
  • bispecific antibody that specifically binds to the IV and II subdomains of the extracellular domain of human HER2 obtained from different cell lines or transgenic animals, will differ from each other by a glycosylation profile.
  • a bispecific antibody that specifically binds to the IV and II subdomains of the human HER2 extracellular domain, encoded by the nucleic acid molecules described herein or containing the amino acid sequences provided herein is part of this invention, regardless of the glycosylation state of the binding molecules and Generally, regardless of the presence or absence of post-translational modifications.
  • the invention also relates to methods and processes for the preparation of a bispecific antibody that specifically binds to the IV and II subdomains of the extracellular domain of human HER2.
  • Monoclonal antibodies can be created, for example, using the hybridoma-based method, which was first described by Kohler et al. , Nature,
  • lymphocytes can be obtained by in vitro immunization. After immunization, lymphocytes are isolated and then fused to the myeloma cell line using an acceptable binding agent, such as polyethylene glycol, to produce a hybridoma cell.
  • an acceptable binding agent such as polyethylene glycol
  • the hybridoma cells thus obtained are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused parent myeloma cells.
  • a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused parent myeloma cells.
  • the hybridoma culture medium should typically include hypoxanthine, aminopterin and thymidine (NAT medium), i.e. substances that inhibit the growth of HGPRT-deficient cells.
  • Myeloma cells used as a component for cell fusion are preferably fusion cells, maintain a stable high level of antibody production by selected antibody producing cells, and are sensitive to the selective medium on which unbound parental cells are selected.
  • Preferred myeloma cell lines are mouse myeloma lines, such as those based on the murine tumor cell lines of the MORS-21 and MPC-11 lines, which can be obtained from the Salk Institite Cell Disrtibution Center, San Diego, pc. California, USA, and the SP-2 or HBZ-Ad8-653 lines, which can be obtained from the American Type Culture Collection, Rockville, pc. Maryland, USA
  • the use of human myeloma and mouse-human heteromyeloma cell lines for the production of monoclonal antibodies has also been described (Kozbor, J. Immunol., 133, 1984, p. 3001).
  • the binding specificity of the monoclonal antibodies obtained using hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).
  • an in vitro binding assay such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).
  • the binding affinity of a monoclonal antibody can, for example, be determined using the Scatchard analysis described by Munson et al. Anal. Biochem., 107, 1980, p. 220.
  • clones can be subcloned using the method of limiting dilutions and grown by standard methods. Suitable media for this purpose include, for example, DMEM or RPMI-1640.
  • hydriboma cells can grow in vivo as ascites tumors in animals, for example, by intraperitoneal (ip) injection of cells into mice.
  • Monoclonal antibodies secreted by subclones can be separated from the culture medium, ascites fluid or serum using conventional antibody purification methods, for example, affinity chromatography (for example, using protein A - or protein Q-Sepharose), or ion exchange chromatography, chromatography on hydroxylapatites, gel electrophoresis, dialysis, etc.
  • affinity chromatography for example, using protein A - or protein Q-Sepharose
  • ion exchange chromatography chromatography on hydroxylapatites, gel electrophoresis, dialysis, etc.
  • DNA encoding monoclonal antibodies can be easily isolated and sequenced using conventional procedures (for example, using oligonucleotide probes that have the ability to specifically bind to genes encoding the heavy and light chains of murine antibodies).
  • Hybridoma cells serve as a preferred source of such DNA.
  • DNA can be included in expression vectors, which are then transfected with host cells, such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not produce antibody proteins without transfection, resulting in the synthesis of monoclonal antibodies in recombinant host cells.
  • monoclonal antibodies or antibody fragments can be isolated from phage libraries of antibodies generated using methods described by McCafferty et al. , Nature, 348, 1990, ss. 552-554. Clackson et al. , Nature, 352, 1991, ss. 624-628 and Marks et al. , J. Mol. Biol., 222,
  • the DNA encoding the antibody can also be modified, for example, so as to obtain chimeric or fused polypeptides of antibodies, for example, by replacing the sequences of the constant regions of the heavy and light chains (Cn and CL) with homologous mouse sequences (US 4816567 and Morrison et al., Proc. Natl. Acad. Sci. USA: 81, 1984, p. 6851) or by covalently linking the immunoglobulin coding sequence to all or part of the coding sequence of a non-immunoglobulin polypeptide (heterologous polypeptide).
  • Cn and CL homologous mouse sequences
  • Non-immunoglobulins polypeptide sequences can be replaced by constant regions of the antibody or replaced by the variable regions of the antigen-binding center of the antibody, creating a chimeric bivalent an antibody that contains one antigen binding center having specificity for an antigen, and another antigen binding center having specificity for another antigen.
  • transgenic animals e.g., mice
  • mice can be obtained which, after immunization, can produce a full range of human antibodies without producing endogenous immunoglobulin.
  • Dn segment the region of the junction of the antibody heavy chain gene
  • transferring the germline set of the human immunoglobulin gene to such a mutant mouse germline results in the production of human antibodies after challenge with the antigen (US 5545806, 5569825, 5591669 (all in the name of GenPharm); 5545807; and WO 97/17852).
  • phage display technology (McCafferty et al., Nature, 348, 1990, pp. 552-553) can be used to obtain human antibodies and in vitro antibody fragments from the spectrum of immunoglobulin variable region (V) gene genes from immunized donor organisms.
  • V immunoglobulin variable region
  • the genes of the V region of the antibody are cloned into the reading frame with either the main or minor gene of the coat protein of a filamentous bacteriophage, such as M13 or fd, and present as functional fragments of the antibody on the surface of the phage particle.
  • the filamentous particle contains a copy of the single-stranded DNA of the phage genome, selection based on the functional properties of the antibody also leads to the selection of a gene encoding an antibody that has the indicated properties.
  • the phage mimics some of the properties of b cells.
  • Phage presentation can be carried out in various formats. For phage presentation, you can use various sources of segments of V-genes. Clackson et al. , Nature, 352, 1991, ss. 624-628 isolated various sets of antibodies to oxazolone from a small random combinatorial library of V genes obtained from the spleen of immunized mice.
  • V genes obtained from the body of immunized human donors can be constructed, and antibodies to a different set of antigens (including autoantigens) can be isolated in general according to the methods described by Marks et al. , J. Mol. Biol., 222, 1991, ss. 581-597.
  • human antibodies can also be produced in vitro by activated B cells (see US 5567610 and 5229275).
  • Bespecifically antibodies are antibodies that have specificity for binding to two different epitopes.
  • a bispecific antibody that specifically binds to two different protein epitopes, namely the IV and II subdomains the extracellular domain of human HER2.
  • Bespecifically antibodies can be obtained in the form of full-sized antibodies or fragments of antibodies (for example, F (ab ') 2 fragments of bespecifically antibodies).
  • bispecific antibodies Methods for creating bispecific antibodies are known in the art.
  • the generally accepted production of full-sized bispecific antibodies is based on the joint expression of two pairs of immunoglobulin heavy and light chains, where both chains have different specificities. Due to the random set of immunoglobulin heavy and light chains, these hybridomas (quadromas) can potentially produce a mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule, which is usually carried out in several stages using affinity chromatography, is quite laborious, and the product yield is low. Similar processes are described in WO 93/08829.
  • variable regions of an antibody with the desired binding specificity are fused to the sequences of the constant region of an immunoglobulin.
  • the fusion is preferably carried out with the constant region of the Ig heavy chain, which includes at least a portion of the hinge CH2 and CH3 regions.
  • at least one of the fusions contains the first constant region of the heavy chain (CH1) containing the site necessary for binding of the light chain.
  • DNAs encoding the fusion of the immunoglobulin heavy chain and, if necessary, the immunoglobulin light chain are inserted into various expression vectors and together they transfect an acceptable host organism.
  • the bispecific antibodies are an immunoglobulin heavy chain hybrid providing the first binding specificity in the first arm, and an immunoglobulin light chain-heavy chain hybrid (which provides the second binding specificity) in the second arm. It has been found that this asymmetric structure facilitates the separation of the desired bispecific molecule from undesired combinations of immunoglobulin chains, since the presence of the immunoglobulin light chain in only one half of the bispecific molecule facilitates separation.
  • This approach is described in WO 94/04690.
  • a preferred contact region includes at least a portion of the CHZ region.
  • one or more small amino acids with side chains from the contact region of the first antibody molecule are replaced with molecules with larger side chains (for example, tyrosine or tryptophan).
  • Balancing “cavities” that are identical or close in size to the large side chain (s) (s) in the contact area of the second antibody molecule are created by replacing amino acids with large side chains with amino acids with smaller side chains (for example, alanine or threonine ) This provides a mechanism to increase the yield of the heterodimer relative to other undesired end products.
  • Bespecific antibodies include crosslinked antibodies or “heteroconjugates”.
  • one of the antibodies in the heteroconjugate may be crosslinked with avidin, and the other with biotin.
  • Such antibodies can be used, for example, for targeted transfer of cells of the immune system to unwanted cells (US 4676980) and for the treatment of HIV infection (WO 91/00360, WO 92/200373 and EP 03089).
  • Heteroconjugate antibodies can be created using any of the conventional cross-linking methods. Acceptable cross-linkers are well known in the art and are described in US 4,676,980 along with various methods for introducing cross-linking.
  • bispecific antibodies can be obtained using chemical binding.
  • Brennan et al. Science, 229, 1985, p. 81 described a technique whereby intact antibodies are proteolytically cleaved to obtain F (ab ′) 2 fragments. These fragments are reduced in the presence of a complexing agent with dithiol, such as sodium arsenite, to stabilize neighboring dithiols and prevent the formation of intermolecular disulfide bonds.
  • the resulting Fab ′ fragments are then converted to a thionitrobenzoate (TNB) derivative.
  • TAB thionitrobenzoate
  • bispecific antibodies can be used as agents for the selective immobilization of enzymes.
  • bispecific antibodies were prepared using leucine zippers (Kostelny et al., J. Immunol., 148 (5), 1992, pp. 1547-1553). Leucine zipper peptides from Fos and Jun proteins were linked to Fab 'fragments of two different antibodies by gene fusion. Antibody homodimers were reduced in the hinge region to produce monomers, and then antibody heterodimers were obtained by reoxidation. This method can also be used to obtain antibody homodimers. Double Antibody Technology Described by Hollinger et al. , Proc. Natl. Acad. Sci.
  • Fragments contain a VH region linked to the VL region by a linker that is too short to allow pairing of two domains of the same chain. Thus, the VH and VL regions of one fragment must pair with the complementary VL and VH regions of another fragment, thereby forming two antigen-binding centers.
  • Another strategy for producing bispecific antibody fragments is also described, based on the use of single chain (Fv) - (sFv) dimers (see Gruber et al., J. Immunol, 152, 1994, p. 5368).
  • compositions comprising, as an active ingredient (or as a sole active ingredient), a bispecific antibody that specifically binds to the IV and II subdomains of the human extracellular domain of HER2.
  • the pharmaceutical composition may include at least one bispecific antibody that specifically binds to the IV and II subdomains of the human extracellular domain of HER2, as described herein, and one or more additional binding molecules (eg, antibodies) that target one or more of the corresponding surface receptors.
  • the compositions are intended to improve, prevent, or treat disorders that may be associated with HER2.
  • “Pharmaceutical composition” means a composition comprising a bispecific antibody that specifically binds to the IV and II subdomains of the extracellular domain of human HER2, according to the invention and at least one of the components selected from the group consisting of pharmaceutically acceptable and pharmacologically compatible excipients, such as excipients, solvents, diluents, carriers, excipients, distributors, delivery vehicles, preservatives, stabilizers, emulsifiers, suspending agents, thickeners, regulators of prolonged delivery, the choice and ratio of which depends on the nature and method of administration and dosage.
  • the pharmaceutical compositions of the present invention and methods for their manufacture will be undeniably apparent to those skilled in the art.
  • the manufacture of pharmaceutical compositions should preferably comply with GMP (Good Manufacturing Practice) requirements.
  • the composition may include a buffer composition, tonic agents, stabilizers and solubilizers.
  • the prolonged action of the composition can be achieved using agents that slow down the absorption of the active pharmaceutical ingredient, for example, aluminum monostearate and gelatin.
  • suitable carriers, solvents, diluents and delivery vehicles are water, ethanol, polyalcohols, and also mixtures thereof, oils and injectable organic esters.
  • “Medicinal product (preparation)” a substance or mixture of substances in the form of a pharmaceutical composition in the form of tablets, capsules, powders, lyophilisates, injections, infusions, ointments, etc. ready-made forms, designed to restore, correct or change the physiological functions in humans and animals, as well as for the treatment and prevention of diseases, diagnosis, anesthesia, contraception, cosmetology and other things. Any method of administering peptides, proteins, or antibodies adopted in the art can be suitably used for a bispecific antibody that specifically binds to the IV and II subdomains of the human extracellular domain of HER2 of this invention.
  • pharmaceutically acceptable means one or more compatible liquid or solid components that are suitable for administration to a mammal, preferably a human.
  • excipient or “excipient” is used herein to describe any component other than previously described in this invention. These are substances of inorganic or organic origin, used in the production process, the manufacture of drugs to give them the necessary physical and chemical properties.
  • buffer By the term “buffer”, “buffer composition”, “buffer agent” is meant a solution capable of maintaining a pH value due to the interaction of the acid and alkaline components included in its composition, which enables the preparation of a bispecific antibody that specifically binds to IV and II subdomains the extracellular domain of human HER2, show resistance to pH changes.
  • pharmaceutical pHs are preferred.
  • compositions from 4.0 to 8.0.
  • buffering agents for example, acetate, phosphate, citrate, histidine, succinate and the like can be used. buffer solutions, but not limited to them.
  • tonic agent refers to an excipient that can apply the osmotic pressure of a liquid antibody preparation.
  • An “isotonic” drug is a drug that has an osmotic pressure equivalent to that of human blood. Isotonic preparations typically have an osmotic pressure of about 250 to 350 mOsm / kg.
  • isotonic agents polyols, mono- and disaccharides, amino acids, metal salts, for example, sodium chloride, and the like, can be used, but not limited to.
  • stabilizer is meant an auxiliary substance or a mixture of two or more auxiliary substances that provide the physical and / or chemical stability of the active agent.
  • amino acids can be used, for example, but not limited to arginine, histidine, glycine, lysine, glutamine, proline; surfactants, for example, polysorbate 20 (trade name Tween 20), polysorbate 80 (trade name Tween 80), polyethylene-polypropylene glycol and its copolymers (trade names Poloxamer, Pluronic), sodium dodecyl sulfate (SDS ), but not limited to them; antioxidants, for example, methionine, acetylcysteine, ascorbic acid, monothioglycerol, salts of seric acids, and the like, but not limited to; chelating agents, for example, ethylene diamine tetraacetic acid (EDTA), diethylene triamine pentaacetic acid (DTPA), sodium citrate, and the like
  • the pharmaceutical composition is “stable” if the active agent retains its physical stability and / or chemical stability and / or biological activity during the stated shelf life at a storage temperature, for example, at 2-8 ° C.
  • the active agent retains both physical and chemical stability as well as biological activity.
  • the storage period is selected based on the results of stability studies during accelerated and natural storage.
  • the pharmaceutical composition of this invention can be manufactured, packaged, or sold widely as a unit dosage unit or as a plurality of unit dosage units in the form of a finished dosage form.
  • unit dosage unit means a discrete amount of a pharmaceutical composition containing a predetermined amount of an active ingredient.
  • the amount of active ingredient is usually equal to the dosage of the active ingredient that will be administered to the subject, or a convenient portion of such a dosage, for example half or a third of such a dosage.
  • the pharmaceutical compositions of the present invention are suitable for parenteral administration in the form of sterile drugs intended for administration to the human body with a violation of the integrity of the skin or mucous membranes, bypassing the gastrointestinal tract by injection, infusion or implantation.
  • parenteral administration includes, but is not limited to, subcutaneous, intraperitoneal, intramuscular, intravenous, intraarterial, intrathecal, intraventricular, intraurethral, intracranial, intraarticular, transdermal injection or infusion; and renal dialysis infusion techniques.
  • Intratumoral delivery for example, intratumoral injection, may also be applicable.
  • Regional perfusion is also provided.
  • Preferred embodiments of the invention include intravenous and subcutaneous routes. Any method of administering peptides or proteins adopted in this field can be suitably used for a bispecific antibody that specifically binds to the IV and II subdomains of the human extracellular domain of HER2, according to this invention.
  • Injectable drugs can be manufactured, packaged or sold in unit dosage form, for example, but not limited to ampoules, vials, polymer containers, prefilled syringes, auto-injection devices.
  • Medicines for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous bases, pastes, and the like.
  • Another embodiment of the invention is a medicament for parenteral administration, wherein the pharmaceutical composition is provided in dry form, that is, a powder or granules for dissolution in a suitable solvent (eg, sterile pyrogen-free water) before administration.
  • a suitable solvent eg, sterile pyrogen-free water
  • Such a drug can be obtained, for example, by lyophilization, i.e. a process known in the art as freeze-drying, which includes freezing the preparation and then removing the solvent from the frozen contents.
  • a bispecific antibody that specifically binds to the IV and II subdomains of the human extracellular domain of HER2, according to this invention, can also be administered intranasally or by inhalation, alone, as a mixture with a suitable pharmaceutically acceptable excipient, such as a pressurized aerosol container, pump, spray, nebulizer) or a nebulizer in which a suitable propellant is used or not used, or in the form of nasal drops or a spray.
  • a suitable pharmaceutically acceptable excipient such as a pressurized aerosol container, pump, spray, nebulizer
  • a nebulizer in which a suitable propellant is used or not used, or in the form of nasal drops or a spray.
  • the parenteral drug may be immediate or modified release.
  • Modified release drugs include delayed, slow, pulsating, controlled, targeted and programmed release.
  • a bispecific antibody that specifically binds to the IV and II subdomains of the human extracellular domain of HER2 of the present invention is used in the treatment of diseases or disorders that are associated with HER2 activity.
  • the subject of treatment or the patient is a mammal, preferably a human subject.
  • the above subject may be male or female and of any age.
  • a therapeutically effective amount of a bispecific antibody that specifically binds to the IV and II subdomains of the human extracellular domain of HER2 can reduce the number of cancer cells; reduce the initial size of the tumor; inhibit (i.e., to some extent slow down and, preferably, stop) cancer cell infiltration of peripheral organs; inhibit (i.e., to some extent slow down and, preferably, stop) tumor metastasis; inhibit, to some extent, tumor growth; and / or alleviate, to some extent, one or more symptoms associated with the disorder.
  • An antibody or antibody fragment can, to some extent, prevent the growth and / or kill existing cancer cells, it can cause a cytostatic and / or cytotoxic effect.
  • in vivo efficacy can be determined, for example, by assessing life expectancy, time to disease progression (TTR), tumor response rate (RR), response duration and / or quality of life.
  • co-administration refers to a bispecific antibody that specifically binds to the IV and II subdomains of the human extracellular domain of HER2, with one or more other therapeutic agents, which are intended to mean are referred to or include:
  • a bispecific antibody that specifically binds to the IV and II subdomains of the human extracellular domain of HER2 according to this invention can be administered without additional therapeutic treatment, i.e. as an independent therapy.
  • treatment with a bispecific antibody that specifically binds to the IV and II subdomains of the human extracellular domain of HER2 according to the invention may include at least one additional therapeutic treatment (combination therapy).
  • a bispecific antibody that specifically binds to the IV and II subdomains of the human extracellular domain of HER2 can be co-administered or formulated with another cancer treatment / medication.
  • cytotoxic agent refers to a substance that inhibits or prevents the functioning of cells and / or causes destruction of cells.
  • the term is intended to include radioactive isotopes (e.g., At211, 1131, 1125, Y90, Rel86, Rel88, Sml53, Bi212, P32 and Lu radioactive isotopes), chemotherapeutic agents and toxins, such as low molecular weight toxins or enzymatically active toxins originating from bacteria, fungi, plants or animals, including fragments and / or variants thereof.
  • a “chemotherapeutic agent” is a chemical compound useful in the treatment of a malignant tumor.
  • chemotherapeutic agents include alkylating agents such as thiotepa and cyclophosphamide (CYTOXAN®); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquinone, meturedopa and uredopa; ethyleneimines and methylmelamines, including altretamine, triethylene melamine, triethylene phosphoramide, triethylene thiophosphoramide and trimethyl melamine; acetogenins (e.g., bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta lapachone; lapachol; colchicines; betulinic acid; camptothecin (including the synthetic analogue of topotecan (HYCAMTIN®), CPT-11 (irinotecan, CAMPTOSAR®), acety
  • cryptophycin 1 and cryptophycin 8 dolastatin; duocarmycin (including synthetic analogues KW-2189 and CB1-TM1); eleutherobin; pankratistatin; sarcodiktiin; spongistatin; nitrogen mustards, such as chlorambucil, chlornaphazine, holophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembikhine, phenesterol, prednimustine, trophosphamide, uramustine; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine and ranimnustine; antibiotics such as enediin antibiotics (e.g.
  • calicheamicin e.g. calicheamicin gamma II and calicheamicin omega II (see, for example, Agnew, Chem. Inti. Ed. Engl., 33: 183-186 (1994)); dynamin, including dynemycin A; esperamycin; as well as the neocarcinostatin chromophore and related chromophores of chromoproteins - enediin antibiotics, aclacinomisins, actinomycin, autramycin, azaserin, bleomycin, cactinomycin, carabicin-5, carcininomycinomycinomycinomycinomycinomycinomycinomycinomycinomycinomycin L-norleucine, doxorubicin (including ADRIAMICIN®, my rfolinodoksorubitsin, tsianomorfolinodoksorubitsin, 2- pyrrolino, doksorubitsin
  • LURTOTECAN® LURTOTECAN®
  • rmRH e.g., ABARELIX®
  • BAY439006 sorafenib; Bayer
  • SU-11248 Pfizer
  • perifosin a COX-2 inhibitor (e.g., celecoxib or etoricoxib), a proteosome inhibitor (e.g., PS341); bortezomib (VELCADE®); CCI-779; tipifarnib (811577); orafenib, ABT510; a Bc1-2 inhibitor such as oblimersen sodium (GENASENSE®); pixantron; EGFR inhibitors (see definition below); tyrosine kinase inhibitors (see definition below); and pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above, such as CHOP, short name for combination therapy with cyclophosphamide, doxorubicin, vin
  • anti-hormonal drugs that act to regulate or inhibit the action of hormones on tumors, such as antiestrogens with a mixed agonist / antagonist profile, including tamoxifen (NOLVADEX®), 4-hydroxytamoxifen, trioxifene, toremifene (FARESTON®); idoxifene, droloxifene, raloxifene (EVISTA®), trioxifene, keyoxifene and selective estrogen receptor modulators (SERMs) such as SERM3; pure antiestrogens without agonistic properties, such as fulvestrant (FASLODEX®) and EM800 (such drugs can block estrogen receptor dimerization (ER), inhibit DNA binding, increase ER metabolism and / or reduce ER levels); aromatase inhibitors, including steroidal aromatase inhibitors such as formestane and exemestane (AROMAS IN®), and non-steroidal aromatase inhibitors such as anastrazole
  • the bispecific antibody that specifically binds to the IV and II subdomains of the human extracellular domain of HER2 will be administered in an amount effective to treat the condition in question, i.e. in doses and for periods of time necessary to achieve the desired result.
  • a therapeutically effective amount may vary depending on factors such as the particular condition being treated, the patient’s age, gender and weight, and whether the administration of a bispecific antibody that specifically binds to the IV and II subdomains of the human extracellular domain of HER2 is independent treatment or it is performed in combination with one or more additional drugs or treatments.
  • Drug regimens can be adjusted to provide the optimum desired response. For example, a single bolus may be administered, several divided doses may be administered during some time, or the dose can be proportionally reduced or increased depending on the severity of the therapeutic situation. Particularly useful is the manufacture of parenteral compositions in unit dosage form for ease of administration and uniformity of dosage.
  • the unit dosage form refers to physically discrete units suitable as unit doses for patients / subjects to be treated; each unit contains a predetermined amount of the active compound, calculated to produce the desired therapeutic effect in combination with the desired pharmaceutical carrier.
  • unit dosage forms of the present invention is generally dictated and directly dependent on (a) the unique characteristics of the chemotherapeutic agent and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the compounding technique of such an active compound for treatment of sensitivity in subjects.
  • the dose and dosage regimen are adjusted in accordance with methods well known in the therapeutic field.
  • the maximum tolerated dose can be easily set and an effective amount can also be determined that provides a detectable therapeutic effect for the patient, as well as the time requirements for the administration of each agent to achieve a visible therapeutic effect for the patient.
  • some dosages and regimen regimens are given as examples herein, these examples do not in any way limit the dosages and regimens that may be necessary for the patient to practice the present invention.
  • dosage values may vary, depending on the type and severity of the condition, which should be alleviated, and may include one or more doses.
  • specific administration schedules should be adjusted after some time according to individual needs and at the discretion of the medical professional who administers or controls the administration of the compositions, and that the concentration ranges given in this description are only given as an example and are not intended to limit the scope or practice of the claimed compositions.
  • the dosage regimen with the compositions of this invention can be based on various factors, including the type of disease, age, weight, gender, health status of the patient, severity of the condition, route of administration, and the particular bispecific antibody used that specifically binds to the IV and II subdomains the extracellular domain of human HER2.
  • the dosage regimen can vary widely, but can be determined regularly using standard methods. For example, doses may be adjusted based on pharmacokinetic and pharmacodynamic parameters, which may include clinical effects, such as toxic effects or laboratory values.
  • the present invention encompasses an individual dose increase, which is determined by a qualified specialist. Determination of the required dose and modes are well known in the relevant field of technology and will be clear to a person skilled in the art after familiarization with the ideas disclosed herein.
  • a suitable dose of a bispecific antibody that specifically binds to the IV and II subdomains of the human extracellular domain of HER2 in the present invention will be in the range of 0.1-200 mg / kg, preferably 0.1-100 mg / kg, including about 0.5-50 mg / kg, for example, about 1-20 mg / kg.
  • a bispecific antibody that specifically binds to the IV and II subdomains of the extracellular domain of human HER2 can be administered, for example, at a dose of at least 0.25 mg / kg, for example at least 0.5 mg / kg, including not less than 1 mg / kg, for example at least 1.5 mg / kg, for example, as well as at least 2 mg / kg, for example at least 3 mg / kg, including at least 4 mg / kg, for example at least 5 mg / kg; and for example up to a maximum of 50 mg / kg, including up to a maximum of 30 mg / kg, for example, up to a maximum of 20 mg / kg, including up to a maximum of 15 mg / kg.
  • the administration will usually be repeated at suitable intervals, for example, once a week, once every two weeks, once every three weeks, or once every four weeks, and for as long as it is deemed appropriate by the responsible physician, who may in some cases increase or reduce the dose if necessary.
  • the next embodiment of the invention is a product that contains products used to treat cancer selected from the group: breast cancer (BC), malignant neoplasm of the stomach, non-small cell lung cancer, malignant neoplasm of the head and / or neck, squamous cell carcinoma of the head and neck (PRGS) ), colorectal cancer (CRC), esophageal cancer, ovarian cancer, pancreatic cancer, gastrointestinal cancer, kidney cancer, cervical cancer, endometrial cancer, uterine cancer, malignant melanoma, pharyngeal cancer, oral cancer or cancer skin.
  • the product is a container with a label and a leaflet, which can be placed in a blister and / or bundle.
  • Suitable containers are, for example, vials, ampoules, syringes, etc.
  • Containers can be made of various materials, such as glass or polymeric materials.
  • the container contains a composition effective to treat a particular condition and may have a sterile inlet channel).
  • At least one active ingredient in the composition is a bispecific antibody that specifically binds to IV and II subdomains of the extracellular domain of human HER2, proposed in the invention.
  • the label and package insert indicate that the drug is used to treat a specific condition.
  • the label and / or package leaflet must additionally contain instructions for administering the antibody composition to the patient, including information about the indications, use, dose, route of administration, contraindications and / or precautions regarding the use of such therapeutic products.
  • the package insert indicates that the composition is used to treat a disease or disorder mediated by HER2, namely a cancer selected from the group: breast cancer (BC), gastric cancer, non-small cell lung cancer, malignant neoplasm of the head and / or neck, squamous cell carcinoma of the head and neck (CBC), colorectal cancer (CRC), esophageal cancer, ovarian cancer, pancreatic cancer, gastrointestinal cancer, kidney cancer, cervical cancer, endometrial cancer, uterine cancer, zl qualitative melanoma, cancer of the pharynx, cancer of the oral cavity or skin cancer.
  • a cancer selected from the group: breast cancer (BC), gastric cancer, non-small cell lung cancer, malignant neoplasm of the head and / or neck, squamous cell carcinoma of the head and neck (CBC), colorectal cancer (CRC), esophageal cancer, ovarian cancer, pancreatic cancer, gastrointestinal cancer, kidney cancer, cervical cancer, endometrial cancer
  • the product may include other products necessary from a commercial point of view and from the point of view of the consumer, for example, but not limited to solvents, diluents, filters, needles and syringes.
  • a bispecific antibody that specifically binds to the IV and II subdomains of the human extracellular domain of HER2 of the present invention is also used for diagnostic purposes (e.g., in vitro, ex vivo).
  • this bispecific antibody can be used to detect or measure HER2 levels in samples obtained from a patient, for example, a tissue sample or a sample of body fluid, such as inflammatory exudate, blood, blood serum, intestinal fluid, saliva, or urine.
  • Suitable detection and measurement methods include immunological methods such as flow cytometry, enzyme-linked immunosorbent assay (ELISA), chemiluminescent analysis, radioimmunoassay and immunohistology.
  • the invention further includes kits, for example, diagnostic kits containing a bispecific antibody that specifically binds to the IV and II subdomains of the human HER2 extracellular domain described herein.
  • the desired gene segments were obtained from oligonucleotides created by chemical synthesis. Gene segments ranging in length from 300 to 4000 kb, which are flanked by unique restriction sites, were collected by annealing and ligation of oligonucleotides, including PCR amplification and subsequent cloning through these restriction sites. The DNA sequences of the subcloned gene fragments were confirmed by DNA sequencing.
  • DNA sequences were determined by Sanger sequencing.
  • variants of expression plasmids were used for expression in prokaryotic (E. coli) cells of short-term expression in eukaryotic cells (for example, in CHO cells).
  • the vectors contained: a replication initiation site for replication of the indicated plasmid in E. coli, genes conferring resistance in E. coli to various antibiotics (e.g., ampicillin and kanamycin).
  • antibodies with the published sequence of Pertuzumab, Trastuzumab were used.
  • the genes of the variable domains of the heavy and light chains of antibodies were synthesized and cloned into the vectors pEE-HC, pEE-CK (Figs. 2, 3, 4), intended for the production of protein in mammalian cells, at the restriction sites Sall / Nhel and Sall / BstWI, respectively .
  • Plasmids were generated in the required quantities in E. coli cells and purified using the Qiagen kit.
  • Antigens and control antibodies were produced in cells of a constant cell line obtained from Chinese hamster ovary cells (CHO-T line). Suspension cultivation was carried out in flasks on an orbital shaker incubator using serum-free media manufactured by HyCell TransFx-C with the addition of 8 mM L-glutamine and 1 g / l pluronic 68. For transient cell expression at a concentration of 2-2.2 * 10 6 cells / ml were transfected using linear polyethyleneimine (PEI "MAX", the company "Polysciences”). The ratio of DNA / PEI was 1: 3/1: 10.
  • the culture fluid was separated from the cells by filtration through a depth filter with a pore size of 0.5 / 0.22 ⁇ m.
  • Target proteins were isolated from the culture fluid by affinity chromatography.
  • Recombinant proteins with Fc from the culture fluid were isolated and purified using a Protein A affinity chromatography column.
  • the clarified culture fluid was passed through a 5 ml HiTrap rProtein A Sepharose FF column (GE Healthcare), balanced with phosphate-buffered saline (FSB, pH 7, four) . Then the column was washed with 5 column volumes of FSB to wash the non-specific binding components.
  • the bound antigen was eluted using 0.1 M glycine pH 3 buffer.
  • the main protein elution peak was collected and adjusted to neutrality with 1 M Tris buffer (pH 8). All stages were carried out at a flow rate of 110 cm / h. Next, the protein was transferred to PBS (pH 7.4) using dialysis using SnakeSkin Dialysis Tubing technology, filtered (0.22 ⁇ m), transferred to test tubes and stored at -70 ° C.
  • the purity of the resulting protein solution was evaluated using SDS gel electrophoresis under reducing and non-reducing conditions.
  • the optimal poses were selected by estimating the average binding energy over a 50 nanosecond molecular dynamics interval (Desmond tool, part of the Schrodinger Suite) using the MM-GBSA method (with a force field OPLS_2005) for dynamics frames taken at different simulation intervals.
  • the visualization of the resulting structure was created using Schrodinger's PyMOL tool.
  • the library contained 335 different Fab candidates: 1) 135 candidates in the set with affinity selection (up to 10 substitutions in b CDR); 2) 211 candidates in the set with stability selection (1 mutation in each CDR from the set) combined from 196 different heavy chains and 37 light chains (Figs. 5-8).
  • Antibody Fab genes were synthesized using degenerate oligonucleotides and cloned into the pBL vector, designed to generate Fab fragments in E. coli cells, at the Ncol, Nhel sites (Fig. 9).
  • Fab was produced according to the standard method: E. coli B121Gold bacterial cells were transformed with expression vectors containing Fab genes, and the subsequent addition of an inducer triggered transcription of the 1ac operon, which, when cultured, transformed the expression of Fabs, which were exported to the periplasmic space of the cells. Then, ELISA was performed to check the Fab binding with the substrate immobilized ⁇ réelle2-Fc antigen at a concentration of 0.2 ⁇ g / ml on plates (medium binding from Greiner bio one) in 0.1 M ANCO3, pH 9.0 (the antigen was immobilized overnight at 4 ° ⁇ ) As a positive control, Fab control antibodies Pertuzumab (Roche) were used.
  • Detection of immune complexes was carried out using goat anti-Fab antibodies, peroxidase-conjugated (from Pierce-Thermo Scientific) at a dilution of 1: 7500. Staining of the substrate – chromogenic mixture was carried out by adding a substrate solution ( ⁇ 2 ⁇ 2 - 0.02% and TMB in CHsCOONa pH 5.5) in a volume of 50 ⁇ l for 15 minutes. The reaction was stopped by 25 ⁇ l of H2S04 1%. The color signal was measured at a wavelength of 450 nm using a suitable Tecan-Sunrise plate reader (from Tecan). The degree of antibody binding was proportional to the color signal intensity. Clones in which the color signal intensity exceeded the signal from the control antibody was tested in ELISA for non-specific binding.
  • ELISA was also used to analyze nonspecific binding of the studied Fab fragments to other antigens.
  • the study was carried out as described above, but Angiopoetin2-H6F, Cmet-Fc, GM-CSFl-Fc, INF 2b in 0.1 M INHCO3, pH 9.0 were used as antigens for immobilization (the antigen was immobilized overnight at 4 ° C).
  • Her2-Fc was used as a control for specific binding (the antigen was immobilized overnight at 4 ° C). All subsequent steps were carried out according to the standard ELISA protocol using a high-performance automated platform based on Genetix Qpix2xt robotic systems (from Molecular Device) and Tecan Freedom EVO 200 (from Tecan).
  • variable domains of the positive clones were sequenced according to standard protocols and analyzed.
  • FIG. 11 A schematic representation of the asymmetry format is shown in FIG. 11.
  • the bulge-containing heavy chain (knob) contains the following domains: Trastuzumab in scFv format, flexible linker, Fc-knob (Fig. 12).
  • the heavy chain containing the hole consists of domains: VH optimized candidates, CH1, hinge region IgGl, Fc-hole.
  • a light chain containing VL domains of optimized candidates and a CK domain was also developed.
  • the obtained genetic constructs transformed the CHO-T-NS cell line.
  • Proteins were isolated and purified by standard methods by affinity chromatography on a Protein A bacterial protein as described in Example 1. Electrophoresis was performed under denaturing conditions in 7.5% PAG (Figs. 13, 14, 15, 16). The productivity of candidates BCD147-02-004, -006, -011, -015, -016, -018, -025 turned out to be below the threshold level (50 mg / l), and accordingly they were not taken for isolation and purification.
  • Anti-human Fc sensors (ForteBio, AHC) were immersed in a solution of antibodies at a concentration of 30 ⁇ g / ml for 300 seconds to immobilize them. Sensors loaded with antibody were then immersed in wells with solutions of non-targeted antigens at a concentration of 30 ⁇ g / ml for 150 s. Then, dissociation of the complex was detected for 150 seconds.
  • the panel of non-specific antigens includes: Ang2, IL23, GMCSF Fe, IL6R, IL5R, IL4R, DLL4, PDL1, LAG3, FGF2, Neg3, CD3 ED, CD137, PCSK9, PD1. These antigens do not contain Fc-tag.
  • candidate BCD147-02-020 showed himself best.
  • the obtained plasmids were obtained in E. coli cells and isolated using a BenchPro device in an amount of 600-700 ⁇ g. Plasmids were linearized overnight with Pvul endonuclease, then reprecipitated with ethanol and the final concentration was adjusted to 900-1100 ng / ⁇ l.
  • the CHO-K1-S cell line was cultured in S.3.87 MM (synthetic medium developed in BIOCAD without FBS) + 6 mM Glutamine.
  • Transfection with genetic constructs containing the coding sequences of candidate BCD147-02-020 chains was performed by electroporation on a Nucleofector TM instrument (Lonza) according to the manufacturer's protocol.
  • the cell clone expressing BCD147-02-020 was selected based on the analysis of the level of the target protein and the homogeneity of its structure, taking into account the growth rate, population homogeneity and the absence of morphological changes.
  • trastuzumab Comparison of candidate antibodies against HER2-HER2 BCD147-02-020 and monoclonal antibodies trastuzumab, pertuzumab, as well as a mixture of trastuzumab and pertuzumab in the antiproliferative test on cell culture BT-474
  • a BT-474 breast cancer cell line was used with HER2 receptor overexpression (HER2 +++ line).
  • Cells were cultured in DMEM / F12 medium with 2 mM glutamine, 10% FBS (fetal bovine serum), 10 ⁇ g / ml insulin at 37 ° C, 5% CO2. Cells were removed from the surface of the culture vials using trypsin. Cell viability and cell count were evaluated using trypan blue dye. A cell suspension was prepared in DMEM / F12 medium with 2 mM glutamine, 10% FBS, 10 ⁇ g / ml gentamicin antibiotic with a density of 1 * 10 5 cells / ml. 100 ⁇ l of the obtained cell suspension were added to the wells of a 96-well plate, the plate was incubated for 1 hour at 37 ° C and 5% COg.
  • FBS fetal bovine serum
  • trastuzumab, pertuzumab, a mixture of trastuzumab and pertuzumab and BCD147-02-02 from a concentration of 100 ⁇ g / ml was prepared, introduced into a plate with cells of 10 ⁇ l per well of the tablet.
  • trastuzumab and pertuzumab were mixed in a 1: 1 ratio two-fold solutions of each of the antibodies.
  • the plate was incubated for 5 days at 37 ° C, 5% COg. After incubation time, cell viability analysis was performed using the Alamar Blue stain (alamarBlue®).
  • trastuzumab Comparison of the candidate antibody against HER2-HER2 BCD147-02-020 and monoclonal antibodies trastuzumab, pertuzumab, as well as a mixture of trastuzumab and pertuzumab in the antiproliferative test with the introduction of hrEGF on cell culture BT-474
  • hrEGF human recombinant epidermal growth factor
  • the level of inhibition of proliferation at a maximum concentration of candidate BCD147-02-020 is 1.44 times higher than the mixture of monoclonal antibodies trastuzumab and pertuzumab.
  • candidate BCD147-02-020 the ratio of the maximum level of inhibition of proliferation to point 0 was 1.5 times.
  • candidate BCD147-02-020 unlike the antibodies trastuzumab and a mixture of trastuzumab and pertuzumab, has an antiproliferative effect on the cell line resistant to trastuzumab in proliferative tests.
  • ADCC analysis was performed using Jurkat reporter lines stably expressing the FcyRIIIa (CD16a) receptor and carrying the gene encoding luciferase expressed in response to activation of the NFAT pathway, which in turn occurs after the interaction of the FcyRIIIa receptor with the Fc portion of the antibody, provided that the antibody binds to the antigen on the surface of the target cells.
  • Two Jurkat reporter cell lines stably expressing the FcyRIIIa receptor, variant V158 (with high affinity for Fc - Jurkat Reporter ADCC High) and stably expressing FcyRIIIa receptor, variant F158 (with low affinity for Fc - Jurkat Reporter ADCC Low) were used in the analysis.
  • the supernatant was taken from the wells with BT-474 cells and 40 ⁇ l of the prepared dilutions of BCD147-02-020 antibody series and a mixture of trastuzumab and pertuzumab in RPMI-1640 medium, 2 mM glutamine, 4% FBS ultra-low IgG were added. 40 ⁇ l of a cell suspension of Jurkat Reporter ADCC High or Jurkat Reporter ADCC Low with a concentration of 1.875 * 10 6 cells / ml was added. The plates were incubated at 37 ° C, 5% C0 2 for 5 hours.
  • candidate BCD147-02-020 was 99.2% of the activity of the mixture of antibodies trastuzumab and pertuzumab using the reporter line with the high affinity FcyRIIIa receptor, and 121.2% of the reporter line with the low affinity FcyRIIIa receptor.
  • the analysis was carried out in RPMI-1640 medium, 2 mM glutamine, 0.1% bovine serum albumin, 50 ⁇ g / ml gentamicin.
  • a series of dilutions of the antibodies trastuzumab, a mixture of trastuzumab and pertuzumab, BCD147-02-020 from a concentration of 50 ⁇ g / ml were prepared. Contributed to 96-well plates at 50 ⁇ l per well. 50 ⁇ l of cell suspension BT-474 or SK-BR-3 with a density of 0.4 * 10 6 cells / ml was added.
  • a cell suspension of WIL2-S with a density of 1 * 10 6 cells / ml was added. Prepared a working solution of complement dilution of the liquid preparation of human complement 4 times. A complement solution of 50 ⁇ l / well was added. The plates were incubated for 2 hours at 37 ° C, 5% COg. 15 ⁇ l of Alamar Blue dye was added to the wells of the plate, the plate was incubated at 37 ° C, 5% COg until the development of a gradient color. The fluorescence level was estimated using an Infinite M200Pgo flatbed detector at an excitation / emission wavelength of 544/590 nm.
  • HUVEC cells were used as the target line. HUVEC cells were thawed on the day of analysis, washed from DMSO, a cell suspension was prepared with a concentration of 2 * 10 5 cells / ml in Medium200 medium with lx LSGS (Gibco), 1: 1 mixed with Medium200 medium, 5% Attachment factor (Gibco), 4 ⁇ g / ml gentamicin was added at 50 ⁇ l / well into 96-well plates with white walls. Cell plates were incubated for 1.5-2 hours at 37 ° C, 5% C0 2 .
  • An antibody titer of a mixture of trastuzumab and pertuzumab, BCD147-02-020, and antibodies binding to DLL4 as a positive ADCC control on HUVEC was added at 25 ⁇ l / well.
  • An antibody titer was prepared in steps of 2, at a concentration of 400 ⁇ g / ml.
  • a suspension of Jurkat Reporter ADCC High was added at a concentration of 3.75 * 10 6 cells / ml, 25 ⁇ l / well.
  • the weight of the animals was assessed (before administration, and then twice a week), the volume of the tumor node, the following formula was used:
  • W is the width of the tumor node
  • L is the length of the tumor node
  • the criterion for the effectiveness of the studied drug is the tumor growth inhibition index (SRW) and the tumor growth index (I), which are calculated by the formulas:
  • TPO (%) (Vo-Vk) / Vk * 100,
  • Vk and Vo are the average tumor volume (mm 3 ) in the control experimental groups, respectively.
  • I is the tumor growth index
  • i-day of the experiment Vo is the tumor volume on the day treatment begins.
  • the weight of the animals was assessed (before administration, and then twice a week), the volume of the tumor node, the following formula was used:
  • W is the width of the tumor node
  • L is the length of the tumor node
  • the criterion for the effectiveness of the studied drug is the tumor growth inhibition index (SRW) and the tumor growth index (I), which are calculated by the formulas:
  • TPO (%) (Vo-Vk) / Vk * 100,
  • Vk and Vo are the average tumor volume (mm 3 ) in the control experimental groups, respectively.
  • I is the tumor growth index
  • i-day of the experiment Vo is the tumor volume on the day treatment begins.
  • the weight of the animals was assessed (before administration, and then twice a week), the volume of the tumor node, the following formula was used:
  • W is the width of the tumor node
  • L is the length of the tumor node
  • the criterion for the effectiveness of the studied drug is the tumor growth inhibition index (SRW) and the tumor growth index (I), which are calculated by the formulas:
  • TPO (%) (Vo-Vk) / Vk * 100,
  • Vk and Vo are the average tumor volume (mm 3 ) in the control experimental groups, respectively.
  • I is the tumor growth index
  • i-day of the experiment Vo is the tumor volume on the day treatment begins.
  • - a general blood test according to indicators: the number of red blood cells, the number of leukocytes, the concentration of hemoglobin, the number of lymphocytes, the number of monocytes, the number of neutrophils, the number of eosinophils, the number of basophils;
  • the local irritant effect of the drugs was also evaluated, for which they isolated and performed a histological examination of the soft tissues at the injection site. For all doses tested, the toxic effect of the drug was not shown.

Abstract

La présente invention concerne la biotechnologie et propose un anticorps bispécifique qui se lie spécifiquement au sous-domaine IV de domaine extracellulaire (ECD4) HER2 (récepteur du facteur de croissance épidermique 2) de l'humain II et au sous-domaine II du domaine extracellulaire (ECD2) HER2 de l'humain. L'invention concerne des ADN qui codent pour ledit anticorps, qui correspondent aux vecteurs d'expression, et des procédés de production ainsi que des méthodes de traitement utilisant ces anticorps.
PCT/RU2019/050037 2018-03-29 2019-03-28 Anticorps bispécifique qui se lie de manière spécifique aux sous-domaines iv et ii du domaine extracellulaire her2 de l'humain WO2019190359A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2015077891A1 (fr) * 2013-11-27 2015-06-04 Zymeworks Inc. Produits de recombinaison de liaison à l'antigène bispécifiques ciblant her2
RU2627185C1 (ru) * 2012-03-16 2017-08-03 Коваген Аг Новые связывающие молекулы с противоопухолевой активностью

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2627185C1 (ru) * 2012-03-16 2017-08-03 Коваген Аг Новые связывающие молекулы с противоопухолевой активностью
WO2015077891A1 (fr) * 2013-11-27 2015-06-04 Zymeworks Inc. Produits de recombinaison de liaison à l'antigène bispécifiques ciblant her2

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
GAIL D. LEWIS PHILLIPS ET AL.: "Targeting HER2-Positive Breast Cancer with Trastuzumab-DMl, an Antibody-Cytotoxic Drug Conjugate", CANCER RES 2008, vol. 68, no. 22, 15 November 2008 (2008-11-15), pages 9280 - 9290, XP055013498, doi:10.1158/0008-5472.CAN-08-1776 *

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