WO2024007129A1 - 人源化的b7h3抗体或其抗原结合片段 - Google Patents

人源化的b7h3抗体或其抗原结合片段 Download PDF

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WO2024007129A1
WO2024007129A1 PCT/CN2022/103735 CN2022103735W WO2024007129A1 WO 2024007129 A1 WO2024007129 A1 WO 2024007129A1 CN 2022103735 W CN2022103735 W CN 2022103735W WO 2024007129 A1 WO2024007129 A1 WO 2024007129A1
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antibody
antigen
binding fragment
seq
heavy chain
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PCT/CN2022/103735
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French (fr)
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邱均专
王振生
孙楷
陈均勇
孙键
李忠良
区日山
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英诺湖医药(杭州)有限公司
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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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—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
    • 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
    • C12N15/64—General methods for preparing the vector, for introducing it into the cell or for selecting the vector-containing host
    • 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
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the present invention relates to the field of biomedical technology, specifically to humanized B7H3 antibodies or antigen-binding fragments thereof.
  • B7H3 (CD276) is a type I transmembrane protein (Chapoval A.I., et al., (2001) Nat. Immunol. 2:269). B7H3 is present in some non-immune fibroblasts, endothelial cells and osteoblasts, as well as some immune cells such as B cells, T cells, monocytes, dendritic cells (DC) or natural killer cells (NK). In many malignant tumors, B7H3 expression levels are quite high. At the same time, B7H3 has a wide range of functions in regulating the immune system.
  • the publication number is CN113402610A, and the publication date is 2021.09.17.
  • the Chinese patent discloses a group of humanized B7H3 antibodies, but its humanization level needs to be further improved.
  • the invention provides a B7H3 antibody or an antigen-binding fragment thereof, which is selected from a) or b);
  • the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 are shown in SEQ ID NO: 1 to 3, and the light chain CDR1, light chain CDR2, and light chain CDR3 are shown in SEQ ID NO: 4 to 6;
  • the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 are shown in SEQ ID NO: 7 to 9 in order, and the light chain CDR1, light chain CDR2, and light chain CDR3 are shown in SEQ ID NO: 10 to 12 in order.
  • the present invention also provides nucleic acids, vectors, host cells and corresponding preparation methods related to the above-mentioned antibodies or antigen-binding fragments thereof.
  • the present invention also relates to a conjugate, which is the B7H3 antibody or antigen-binding fragment thereof as described above combined with a therapeutic agent or a detection agent.
  • the present invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a B7H3 antibody or an antigen-binding fragment thereof as described above, or a conjugate as described above.
  • the present invention also relates to the use of the B7H3 antibody or antigen-binding fragment thereof as described above, or the conjugate as described above in the preparation of medicaments for treating B7H3-positive cancers or for modulating the immune system.
  • the present invention deeply humanizes two antibodies, hu9C8 and hu5B6, and enhances the ADCC effect; the modified ADCC-enhanced antibody shows significant ADCC activity and tumor inhibitory effect.
  • Figure 2 shows that in one embodiment of the present invention, ADCC-enhanced hu9C8 (DE) and hu5B6 (DE) antibodies exhibited significant tumor inhibitory effects in mouse tumor models; the dosage was 10 mg/kg.
  • the technical solution of "A, and/or, B, and/or, C, and/or, D” includes any one of A, B, C, and D (that is, they are all connected with "logical OR” technical solution), also includes any and all combinations of A, B, C, and D, that is, including combinations of any two or any three of A, B, C, and D, and also includes A, B, C , four combinations of D (that is, technical solutions that are all connected by "logical AND").
  • the present invention refers to concentration values, and their meaning includes fluctuations within a certain range. For example, it can fluctuate within the corresponding accuracy range. For example, 2% can allow fluctuation within the range of ⁇ 0.1%. For values that are large or do not require too fine control, the meaning is also allowed to include larger fluctuations. For example, 100mM can allow fluctuations within the range of ⁇ 1%, ⁇ 2%, ⁇ 5%, etc. Referring to molecular weight, fluctuations of ⁇ 10% are allowed.
  • the technical features described in open terms include closed technical solutions composed of the listed features, and also include open technical solutions including the listed features.
  • antibody refers to a protein that binds to a specific antigen, which generally refers to all proteins and protein fragments including complementarity determining regions (CDR regions), especially full-length antibodies or antibody functional fragments.
  • CDR regions complementarity determining regions
  • full-length antibody includes polyclonal antibodies as well as monoclonal antibodies.
  • antibody functional fragment is a substance containing part or all of the CDR of an antibody that lacks at least some of the amino acids present in the full-length chain but is still capable of specificity. Binds to antigen. Such fragments are biologically active because they bind to the target antigen and can compete with other antigen-binding molecules, including intact antibodies, for binding to a given epitope.
  • Framework or “framework sequences” are the remaining sequences of the variable regions other than those defined as the antigen-binding site. Because an antigen binding site can be defined by different terms as described above, the precise amino acid sequence of the framework depends on how the antigen binding site is defined.
  • the term "humanization” or “humanization treatment” refers to replacing a mouse antibody sequence with a human antibody sequence, thereby reducing or eliminating the human anti-mouse antibody (HAMA) reaction.
  • substitutions may be framework substitutions, such as replacement of FR sequences in the variable regions with human origin, and/or replacement of the constant region of the antibody (if present) with human origin.
  • This replacement can also be done by converting the mouse monoclonal antibody into a fully human antibody (i.e., the CDRs have also been replaced) through chain replacement, using available means such as phage antibody library technology.
  • the replaced human sequence may include the substitution or addition or deletion of some amino acids, so that the replaced sequence may not be an exact copy of the expressed human immunoglobulin sequence or germline gene sequence.
  • the antibodies produced in this way can be called human-mouse chimeric antibodies, humanized antibodies or fully human antibodies.
  • CDR complementarity determining region
  • Kabat et al. Kabat et al.
  • CDR complementarity determining region
  • CDR and “CDRs” are used Refers to a region containing one or more or even all of the major amino acid residues that contribute to the binding affinity of an antibody to the antigen or epitope it recognizes.
  • a CDR region or CDR refers to a region as defined by IMGT The highly variable regions of the heavy and light chains of immunoglobulins.
  • therapeutic agent generally refers to any agent that elicits a desired pharmacological effect when administered to an organism.
  • an agent is considered a therapeutic when it exhibits a statistically significant effect in an appropriate population.
  • a suitable population may be a population of model organisms.
  • appropriate populations may be defined by various criteria, such as a certain age group, gender, genetic background, pre-existing clinical conditions, etc.
  • therapeutic agents are useful for alleviating, ameliorating, alleviating, inhibiting, preventing, delaying onset, reducing severity, and/or reducing incidence of one or more symptoms or characteristics of a disease, disorder, and/or condition. of any substance.
  • a “therapeutic agent” is an agent that has been or needs to be approved by a governmental agency before it can be marketed for administration to humans. In some embodiments, a “therapeutic agent” is an agent required for medical prescription for administration to humans.
  • the term "detector” refers to any detectable component, molecule, functional group, compound, fragment or moiety.
  • the detection entity is provided or used separately.
  • the detection entity is provided and/or used in combination (eg, conjugated) with another agent.
  • detection entities include (but are not limited to): various ligands, radionuclides (such as 3 H, 14 C, 18 F, 19 F, 32 P, 35 S, 135 I, 125 I , 123 I, 64 Cu, 187 Re, 111 In, 90 Y, 99 mTc, 177 Lu, 89 Zr, etc.), fluorescent dyes (see below for specific exemplary fluorescent dyes), chemiluminescent agents (such as acridinium esters, stabilized dioxins cyclobutane, etc.), bioluminescent agents, spectrally resolvable inorganic fluorescent semiconductor nanocrystals (i.e., quantum dots), metal nanoparticles (such as gold, silver, copper, platinum, etc.), nanoclusters, paramagnetic metal ions, enzymes (See below for specific examples of enzymes), colorimetric labels (eg dyes, colloidal gold, etc.), biotin, digoxigenin, haptens and antisera or monoclonal
  • the first aspect of the invention relates to a B7H3 antibody or antigen-binding fragment thereof, which is selected from a) or b);
  • the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 are shown in SEQ ID NO: 1 to 3, and the light chain CDR1, light chain CDR2, and light chain CDR3 are shown in SEQ ID NO: 4 to 6;
  • the heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3 are shown in SEQ ID NO: 7 to 9 in order, and the light chain CDR1, light chain CDR2, and light chain CDR3 are shown in SEQ ID NO: 10 to 12 in order.
  • the heavy chain variable region of the B7H3 antibody or antigen-binding fragment thereof is as shown in SEQ ID NO: 13, and the light chain variable region is as shown in SEQ ID NO: 14;
  • its heavy chain variable region is as shown in SEQ ID NO:15
  • its light chain variable region is as shown in SEQ ID NO:16.
  • the antigen-binding fragment is one of F(ab') 2 , Fab, and scFv.
  • F(ab') 2 is derived from pepsin digestion of the entire full-length antibody, which removes most of the Fc region while leaving some of the hinge region intact.
  • the F(ab') 2 fragment has two antigen-binding Fab parts connected together by a disulfide bond, so the F(ab') 2 fragment is a bivalent antibody.
  • F(ab') 2 prepared from an IgG antibody as an example.
  • the molecular weight is about 110kDa.
  • Fab is an antibody structure that can still bind to an antigen, is monovalent and does not contain an Fc portion. After papain digestion of the full-length antibody, two Fab fragments and one Fc fragment are obtained. Each Fab fragment is approximately 50kDa.
  • scFv means a molecule comprising an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) joined by a linker.
  • Such scFv molecules may have the general structure: NH2 -VL-linker-VH-COOH or NH2 -VH-linker-VL-COOH.
  • linking peptide may be a flexible or rigid peptide, for example consisting of repeated GGGGS amino acid sequences or variants thereof, for example using variants with 1 to 4 repeats (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448).
  • Other linking peptides useful in the present invention are described by Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Eur. J. Immunol. 31:94-106, Hu et al. (1996) , Cancer Res. 56:3055-3061, described by Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56, and Roovers et al. (2001), Cancer Immunol.
  • the number of amino acids of the connecting peptide is 1 to 30; it can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 , 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30; preferably 5 to 20.
  • the amino acids of the connecting peptide are nonsense polypeptides that do not have additional functions other than connecting (such as protein localization, enzyme cleavage sites, etc.).
  • the linker peptide is a flexible linker peptide
  • the amino acid sequence of the connecting peptide is selected from one or more of Gly, Ser, Pro, Ala and Glu.
  • the amino acid sequence of the connecting peptide is selected from (GGGGS)n, (GGGS)n, (GGS)n, (GS)n or (G)n, where n is selected from 1, 2, 3, 4, 5 or 6.
  • Linking peptides are usually flexible and can reduce steric hindrance between proteins or polypeptides to be linked, thereby facilitating the correct folding of the protein.
  • the B7H3 antibody or antigen-binding fragment thereof has a constant region.
  • the heavy chain constant region can be selected from the constant region sequence of any one of IgG, IgA, IgM, IgE, and IgD, and IgG can be further selected from IgG1, IgG2, IgG3, or IgG4, with IgG1 being preferred.
  • the light chain constant region can be a kappa or lambda chain.
  • the constant region is preferably of human origin.
  • the heavy chain constant region sequence is as shown in any one of SEQ ID NO: 17-19, and the light chain constant region sequence is as shown in SEQ ID NO: 20.
  • variants of the above-mentioned amino acid sequences are also within the scope of the present invention.
  • the corresponding variants respectively contain at most 3 amino acids occurring in at least one CDR region.
  • the variant may contain less than 3 or more mutations relative to the overall sequence of SEQ ID NO:13 ⁇ SEQ ID NO:20, for example, any one of SEQ ID NO:13 ⁇ SEQ ID NO:20 Sequences that are at least 80%, 85%, 90%, 93%, 95%, 97% or 99% identical to each other between polypeptides.
  • the mutation may be substitution, deletion or addition of amino acids or any combination thereof; preferably, the mutation is a conservative substitution.
  • a “conservative substitution” refers to the substitution of an amino acid in a protein with another amino acid with similar characteristics (e.g., charge, side chain size, hydrophobicity/hydrophilicity, backbone conformation, rigidity, etc.) such that changes can be made frequently without altering the protein's properties. biological activity.
  • substitutions generally regarded as conservative substitutions are the substitution of the aliphatic amino acids Ala, Val, Leu and Ile for each other, the interchange of the hydroxyl residues Ser and Thr, the exchange of the acidic residues Asp and Glu, the exchange of the amide residues Asn and Gln. substitution between, the substitution between basic residues Lys and Arg, and the substitution between aromatic residues Phe and Tyr.
  • Those skilled in the art are aware that, in general, single amino acid substitutions in non-essential regions of polypeptides do not substantially alter biological activity (see, e.g., Watson et al. (1987) Molecular Biology of the Gene, The Benjamin/Cummings Pub. Co., Page 224, (4th ed.)).
  • substitution of amino acids with similar structure or function is unlikely to destroy biological activity.
  • a second aspect of the invention relates to an isolated nucleic acid encoding a B7H3 antibody or antigen-binding fragment thereof as described above.
  • nucleic acids include variants thereof with conservative substitutions (eg, substitutions of degenerate codons) and complementary sequences, as well as variants optimized by codons for more efficient expression in a desired host cell.
  • Nucleic acids are typically RNA or DNA, including genes, cDNA molecules, mRNA molecules, and fragments thereof such as oligonucleotides.
  • Nucleic acid molecules may be single-stranded or double-stranded, but are preferably double-stranded DNA.
  • a nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the coding sequence.
  • DNA nucleic acids are preferably used when ligated into vectors.
  • nucleic acids since antibodies are membrane proteins, nucleic acids often carry signal peptide sequences.
  • a third aspect of the invention relates to a vector comprising a nucleic acid as described above.
  • vector refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted.
  • the vector can express the protein encoded by the inserted polynucleotide, the vector is called an expression vector.
  • the vector can be introduced into the host cell through transformation, transduction or transfection, so that the genetic material elements it carries can be expressed in the host cell.
  • Vectors are well known to those skilled in the art, including but not limited to: plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC) or P1-derived artificial chromosomes (PAC) ; Phages such as lambda phage or M13 phage and animal viruses, etc.
  • Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses, Polyomavacuolating viruses (such as SV40).
  • the vector of the present invention contains regulatory elements commonly used in genetic engineering, such as enhancers, promoters, internal ribosome entry sites (IRES) and other expression control elements (such as transcription termination signals, or multiple adenylation signal and polyU sequence, etc.).
  • the vector can be a composition, for example, a mixture of multiple plasmids, with different plasmids carrying part of the antibody or its antigen-binding fragment.
  • a fourth aspect of the invention relates to a host cell comprising a nucleic acid as described above, or transformed with a vector as described above.
  • the host cell can be a prokaryotic cell or a eukaryotic cell.
  • exemplary eukaryotic cells include mammalian cells, such as primate or non-primate cells; fungal cells, such as yeast; plant cells; and insect cells.
  • Non-limiting exemplary mammalian cells include, but are not limited to, CHO cells, HEK-293 cells, BHK cells, or PER-C6 cells, and derivatives thereof, such as 293-6E, CHO-DG44, CHO-K1, CHO- S and CHO-DS cells.
  • the antibodies or antigen-binding fragments thereof provided by the invention are secreted from mammalian cells.
  • Host cells include the progeny of a single host cell, and the progeny may not necessarily be identical (in terms of morphology or genomic DNA complementation) to the original parent cell due to natural, accidental, or deliberate mutations.
  • Host cells can be isolated cells or cell lines, and also include cells transfected in vivo with the nucleic acid molecules or expression vectors provided herein. It can be shown that bacterial cells can be used as host cells. However, since proteins expressed in bacterial cells tend to be in unfolded or incorrectly folded or non-glycosylated forms, any recombinant antibodies produced in bacterial cells must be screened to retain antigen-binding ability.
  • a bacterial cell will be the desired host if the molecule it expresses is produced in a suitably folded form, or, in alternative embodiments, the molecule can be expressed in a bacterial host and subsequently refolded.
  • E. coli strains used for expression are well-known host cells in the field of biotechnology.
  • Various strains of Bacillus subtilis, Streptomyces, other Bacillus species, etc. may also be used in this method.
  • yeast cell strains known to those skilled in the art as well as insect cells, such as Drosophila and Lepidoptera, and viral expression systems can also be used as host cells.
  • the nucleic acid is inserted into the genome of the host cell and can be stably expressed.
  • the insertion method can use the vector as mentioned above, or the nucleic acid can be directly transferred into the cells without being connected to the vector (for example, liposome-mediated transfection technology).
  • the fifth aspect of the present invention relates to a method for preparing the B7H3 antibody or antigen-binding fragment thereof as described above, comprising culturing the host cell as described above under appropriate conditions, and recovering the target product from the host cell culture.
  • the culture method of the present invention is usually a serum-free culture method, and cells are usually cultured in serum-free suspension.
  • the antibodies of the invention can be purified from cell culture contents according to standard procedures in the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis, and the like. Such technologies are within the technical scope of the art and do not limit the present invention.
  • Another method of expressing antibodies can utilize expression in animals (especially transgenic animals or nude mice). This involves an expression system utilizing an animal casein promoter that, when transgenically incorporated into a mammal, allows the female animal to produce the desired recombinant protein in its milk. Culture media secreting antibodies can be purified using conventional techniques.
  • Antibodies can be filtered and concentrated using conventional methods. Soluble mixtures and polymers can also be removed by conventional methods, such as molecular sieves and ion exchange. The obtained product needs to be frozen immediately, such as -70°C, or freeze-dried.
  • the present invention also relates to conjugates, which are the above B7H3 antibodies or antigen-binding fragments thereof combined with therapeutic agents or detection agents.
  • Therapeutic agents may be or contain any class of chemical entities, including, for example, but not limited to, proteins, carbohydrates, lipids, nucleic acids, small organic molecules, non-biological polymers, metals, ions, radioactive isotopes, and the like.
  • therapeutic agents used in accordance with the present invention may have biological activity associated with treating one or more symptoms or causes of cancer.
  • therapeutic agents used in accordance with the present invention may have biological activities associated with modulating the immune system and/or promoting T cell-mediated cytotoxicity and/or inhibiting the inhibitory effects of B7H3 on T cell proliferation and function.
  • therapeutic agents used in accordance with the invention have one or more additional activities.
  • the conjugated therapeutic agent is a radioisotope, drug conjugate, nanoparticle, immunotoxin, or any other therapeutic load.
  • a detection agent includes any moiety that can be detected using analysis, for example due to its specific functional properties and/or chemical characteristics.
  • Non-limiting examples of such agents include enzymes, radioactive labels, haptens, fluorescent labels, phosphorescent molecules, chemiluminescent molecules, chromophores, luminescent molecules, photoaffinity molecules, colored particles or ligands (such as biotin ).
  • the combined detection agent is a diagnostic or imaging agent.
  • the present invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a B7H3 antibody or an antigen-binding fragment thereof as described above, or a conjugate as described above.
  • the B7H3 antibody or antigen-binding fragment or conjugate thereof of the present invention can be used to prepare pharmaceutical compositions or sterile compositions, for example, any of them and a pharmaceutically acceptable carrier, excipient or stabilizer mix.
  • composition means that the ingredients are mixed together and may be in the form of a solid (such as a lyophilized powder) or a solution.
  • pharmaceutically acceptable means that the molecule itself, molecule fragments or compositions do not produce adverse, allergic or other adverse reactions when properly administered to animals or humans.
  • pharmaceutically acceptable carriers or components thereof include phosphoric acid, citric acid, and other organic acids; antioxidants (for example, ascorbic acid and methionine); antibacterial agents (for example, octadecane Dimethyl benzene chloride, hexahydrocarbon quaternary ammonium chloride, benzalkonium chloride, phenol, butanol or benzyl alcohol, alkylparaben, catechol, resorcinol, cyclohexanol, 3- amyl alcohol, or m-cresol); low molecular weight (less than about 10 kDa) polypeptides; proteins, e.g., serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, e.g., polyvinylpyrrolidone; amino
  • the activator containing the antibody or its functional fragment in the pharmaceutical composition can be contained in microcapsules or in a colloidal drug delivery system (such as liposomes, albumin globules, microemulsions, nanoparticles and nanocapsules) , or contained in macroemulsions, the microcapsules can be prepared by techniques such as coacervation or interfacial polymerization. Examples are hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) respectively. )Microcapsules.
  • compositions of the present invention may be administered by any route, as will be understood by those skilled in the art.
  • the pharmaceutical compositions of the present invention are administered orally (PO), intravenously (IV), intramuscularly (IM), intraarterially, intramedullary, intrathecally, subcutaneously (SQ), intraventricularly, transdermally, Intradermal, intradermal, transrectal (PR), transvaginal, intraperitoneal (IP), intragastric (IG), topical (e.g.
  • the present invention also relates to the use of the B7H3 antibody or antigen-binding fragment thereof as described above, or the conjugate as described above in the preparation of medicaments for treating B7H3-positive cancers or for modulating the immune system.
  • B7H3 is widely expressed in a variety of solid tumor types, including, for example, melanoma (Wang J., et al., (2013) J. Invest. Dermatol. 133:2050), leukemia (Hu Y., et al., (2015) )Hematology 20:187; Sun J., et al., (2014) Onco Targets Ther.7:1979), prostate cancer (Zang X., et al., (2007) Proc.Natl.Acad.Sci.104: 19458), ovarian cancer (Zang ), renal cell carcinoma, urothelial cell carcinoma (Crispen., et al., (2008), Clin Cancer Res.
  • the B7H3-positive cancer is selected from neuroblastoma and cervical cancer.
  • B7H3 can significantly inhibit the activation of T cells by CD3 antibodies or allogeneic DC cells, and B7H3 blocking antibodies can effectively reverse this inhibitory effect (Prasad D.V.R., et al., (2004) J.Immunol.173:2500) .
  • B7H3 binds to the receptor on NK cells, it can inhibit the killing effect of NK cells on neuroblastoma (Castriconi R., et al., (2004) Proc.Natl.Acad.Sci.101:12640).
  • B7H3 The inhibitory effect of B7H3 on T cells, NK and DC cells can significantly promote the immune evasion of tumor cells; in addition, B7H3 also has an important impact on tumor cell proliferation, migration, invasion, angiogenesis, and tumor cell drug resistance.
  • Transplanting tumor cells into B7H3 knockout mice or treating tumor-bearing mice with B7H3 antibodies can significantly inhibit tumor growth (Cai D., et al., (2020) Cell. Mol. Immunol. 17:227; Lee Y.H., et al., (2017) Cell Res. 27:1034), indicating that blocking B7H3 signaling can be used for tumor treatment.
  • tumor cells Due to the significant difference in B7H3 expression levels between normal tissues and tumor tissues, tumor cells can be effectively killed through the ADCC effect or toxin coupling of B7H3 antibodies without causing too many side effects on normal tissues (Koenig S., et al. al., (2014) Medicographia 36:285).
  • cancer or tumor in the present invention refers to solid tumors and/or blood tumors, which can be bones, bone connections, muscles, lungs, trachea, heart, spleen, arteries, veins, blood, capillaries, lymph nodes, lymphatic vessels, lymph fluid, oral cavity, pharynx, esophagus, stomach, duodenum, small intestine, colon, rectum, anus, appendix, liver, gallbladder, pancreas, parotid gland, sublingual gland, urinary kidney, ureter, bladder, urethra, ovary, fallopian tube, Uterus, vagina, vulva, scrotum, testicles, vas deferens, penis, eyes, ears, nose, tongue, skin, brain, brainstem, medulla oblongata, spinal cord, cerebrospinal fluid, nerves, thyroid, parathyroid glands, adrenal glands, pituitary gland, pineal gland Tumors
  • leukemia include leukemia, bladder cancer, colorectal cancer, prostate cancer, gastric cancer, pancreatic cancer, liver cancer, head and neck cancer, kidney cancer, breast cancer, ovarian cancer, desmoplastic small round cell tumor, non-small cell lung cancer, melanoma alveolar rhabdomyosarcoma, esophageal cancer, lymphoma, embryonal rhabdomyosarcoma, neuroblastoma, cervical cancer, Ewing sarcoma, Wilms tumor, neuroblastoma, diffuse pontine glioma, ganglioneuroma, Medulloblastoma, ganglioneuroblastoma, high-grade glioma, embryonal tumors with multilayered rosettes, preferably B7H3-expressing cancers.
  • the invention also relates to a method of treating, preventing, alleviating and/or diagnosing a medical condition in a subject, comprising administering a safe and effective amount of a B7H3 antibody or antigen-binding fragment as described above, or a conjugate as described above The steps, and wherein the medical condition is characterized by expression of the B7H3 antigen.
  • the medical condition is B7H3 positive cancer or an immune system related disease.
  • safe and effective amount means an amount of a compound or composition that is large enough to be significantly effective in alleviating the symptom or condition being treated, but small enough to avoid serious side effects (with a reasonable benefit/risk ratio) within reasonable pharmaceutical adjustments. .
  • the safe and effective amount of the active ingredients in the pharmaceutical composition used in the method of the present invention depends on the specific symptoms to be treated, the age and physical condition of the patient being treated, the severity of the disease, treatment time, concurrent treatment conditions, and the specific active ingredients used. , the specific pharmaceutically acceptable excipients used and such factors including the knowledge and skill of the treating physician involved.
  • contemplated treatment methods will also include the administration of other immunotherapeutic entities, particularly preferably immunotherapeutic entities, including viral cancer vaccines (e.g., adenoviral vectors encoding cancer-specific antigens), bacterial cancer vaccines (e.g., expression of coli), yeast cancer vaccines, N-803 (also known as ALT-803, ALTOR Biosciences), chemotherapy drugs, antibodies (e.g., tumor-associated antigen or patient-specific tumor neoantigen binding), stem cell grafts (eg, allogeneic or autologous), and tumor-targeting cytokines (eg, NHS-IL12, IL-12 conjugated to tumor-targeting antibodies or fragments thereof).
  • contemplated methods of treatment also include subjecting said patient to radiation therapy.
  • contemplated methods of treatment also include performing surgery on the patient, such as tumor removal surgery.
  • the method of administration may be intravenous administration and/or intrabody administration (preferably intraperitoneal administration or intravesical administration).
  • Subjects or patients with the above diseases may be selected from the group consisting of humans, dogs, cats, chimpanzees, orangutans, gibbons, macaques, marmosets, pigs, horses, pandas and elephants.
  • the measurement parameters of raw material components are involved. Unless otherwise specified, there may be slight deviations within the range of weighing accuracy. Temperature and time parameters are involved, allowing for acceptable deviations due to instrument testing accuracy or operating accuracy.
  • the humanized 9C8 and 5B6 antibodies were modified to enhance the ADCC effect or increase humanization and other characteristics (the sequences are shown in Table 1).
  • reporter gene reporter gene
  • PC9 tumor cells were used as target cells, Jurkat-mCD16.2-NF-kB or Jurkat-hCD16-NF-kB cells were used as effector cells, and the activation signal of the transcription factor NF-kB mediated by mCD16.2 or hCD16 was used to screen ADCC. Positive clones.
  • each group of hu5B6 (wt, DE, DLE, MG-1) are all the same, which are the modified light chains of hu5B6, and the heavy chains are as shown in the corresponding heavy chains in Table 1;
  • each group of hu9C8 (wt, DE, The light chains of DLE and MG-1 are all the same, which are the light chains modified by hu9C8, and the heavy chains are as shown in the corresponding heavy chains in Table 1.
  • B7H3 antibody enhanced by ADCC has significant tumor inhibitory effect.
  • the anti-tumor activity of ADCC-enhanced B7H3 antibodies was tested using mouse CDX tumor model.
  • the experiment was implemented by BioDuro Technology Company (BioDuro, Shanghai).
  • human MDA-MB-231 breast cancer cells were transplanted subcutaneously into nude mice (BALB/c nude mice); when the tumors grew to about 100mm 3 , the tumor mice were tested weekly with antibody samples including Two doses of 10 mg/kg were administered intraperitoneally. Six mice in each group were injected with the same antibody, and the tumors were measured before administration.
  • Figure 2 shows the tumor growth curve.
  • ADCC-enhanced B7H3 antibodies hu9C8(DE) and hu5B6(DE) demonstrated significant tumor inhibitory effects.

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Abstract

本发明涉及生物医药技术领域，具体而言，涉及人源化的B7H3抗体或其抗原结合片段。本发明对hu9C8和hu5B6二个抗体进行了深度人源化改造，并增强ADCC作用；改造后的ADCC增强型抗体显示了显著的ADCC活性以及肿瘤抑制作用。

Description

人源化的B7H3抗体或其抗原结合片段 技术领域
本发明涉及生物医药技术领域，具体而言，涉及人源化的B7H3抗体或其抗原结合片段。
背景技术
B7H3(CD276)，是一种I型跨膜蛋白(Chapoval A.I.,et al.,(2001)Nat.Immunol.2:269)。B7H3存在于一些非免疫性成纤维细胞、内皮细胞和成骨细胞，以及一些免疫细胞，如B细胞，T细胞，单核细胞，树突状细胞(DC)或天然杀伤细胞(NK)。在许多恶性肿瘤中，B7H3的表达水平相当高。同时，B7H3具有较为广泛的调节免疫系统的功能。
提高B7H3抗体的人源化程度可以减少临床使用过程中产生的免疫副反应，更好地增加临床疗效。公开号为CN113402610A，公开日为2021.09.17中国专利公开了一组经人源化的B7H3抗体，但其人源化水平还有待进一步提高。
发明内容
本发明提供了一种B7H3抗体或其抗原结合片段，其选自a)或b)；
a)重链CDR1、重链CDR2、重链CDR3依次如SEQ ID NO:1～3所示，轻链CDR1、轻链CDR2、轻链CDR3依次如SEQ ID NO:4～6所示；
b)重链CDR1、重链CDR2、重链CDR3依次如SEQ ID NO:7～9所示，轻链CDR1、轻链CDR2、轻链CDR3依次如SEQ ID NO:10～12所示。
本发明还提供了如上所述抗体或其抗原结合片段相关的核酸、载体、 宿主细胞以及相应的制备方法。
本发明还涉及偶联物，其为与治疗剂或检测剂所结合的如上所述的B7H3抗体或其抗原结合片段。
本发明还涉及药物组合物，其包含如上所述的B7H3抗体或其抗原结合片段，或如上所述的偶联物。
本发明还涉及如上所述的B7H3抗体或其抗原结合片段，或如上所述的偶联物在制备用以治疗B7H3阳性癌症或用以调节免疫系统的药物中的应用。
本发明对hu9C8和hu5B6二个抗体进行了深度人源化改造，并增强ADCC作用；改造后的ADCC增强型抗体显示了显著的ADCC活性以及肿瘤抑制作用。
附图说明
为了更清楚地说明本发明具体实施方式或现有技术中的技术方案，下面将对具体实施方式或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图是本发明的一些实施方式，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为本发明一个实施例中在使用报告基因的检测(E:T＝2:1)中，经ADCC增强改造的hu9C8及hu5B6的相关抗体展现了显著的ADCC活性；
图2为本发明一个实施例中经ADCC增强的hu9C8(DE)和hu5B6(DE)抗体在小鼠肿瘤模型中展现了显著的肿瘤抑制作用；给药剂量为10mg/kg。
具体实施方式
现将详细地提供本发明实施方式的参考，其一个或多个实例描述于下文。提供每一实例作为解释而非限制本发明。实际上，对本领域技术人员而言，显而易见的是，可以对本发明进行多种修改和变化而不背离本发明的范围或精神。例如，作为一个实施方式的部分而说明或描述的特征可以用于另一实施方式中，来产生更进一步的实施方式。
除非另有说明，用于披露本发明的所有术语(包括技术和科学术语)的意义与本发明所属领域普通技术人员所通常理解的相同。通过进一步的指导，随后的定义用于更好地理解本发明的教导。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的，不是旨在于限制本发明。
本文所使用的术语“和/或”、“或/和”、“及/或”的选择范围包括两个或两个以上相关所列项目中任一个项目，也包括相关所列项目的任意的和所有的组合，所述任意的和所有的组合包括任意的两个相关所列项目、任意的更多个相关所列项目、或者全部相关所列项目的组合。需要说明的是，当用至少两个选自“和/或”、“或/和”、“及/或”的连词组合连接至少三个项目时，应当理解，在本申请中，该技术方案毫无疑问地包括均用“逻辑与”连接的技术方案，还毫无疑问地包括均用“逻辑或”连接的技术方案。比如，“A及/或B”包括A、B和A+B三种并列方案。又比如，“A，及/或，B，及/或，C，及/或，D”的技术方案，包括A、B、C、D中任一项(也即均用“逻辑或”连接的技术方案)，也包括A、B、C、D的任意的和所有的组合，也即包括A、B、C、D中任两项或任三项的组合，还包括A、B、C、D的四项组合(也即均用“逻辑与”连接的技术方案)。
本发明中所使用的术语“含有”、“包含”和“包括”是同义词，其是包容性或开放式的，不排除额外的、未被引述的成员、元素或方法步骤。
本发明中用端点表示的数值范围包括该范围内所包含的所有数值及分数，以及所引述的端点。
本发明中涉及浓度数值，其含义包括在一定范围内的波动。比如，可 以在相应的精度范围内波动。比如2％，可以允许±0.1％范围内波动。对于数值较大或无需过于精细控制的数值，还允许其含义包括更大波动。比如100mM，可以允许±1％、±2％、±5％等范围内的波动。涉及分子量，允许其含义包括±10％的波动。
本发明中，涉及“多个”、“多种”等描述，如无特别限定，指在数量上指大于等于2。
本发明中，以开放式描述的技术特征中，包括所列举特征组成的封闭式技术方案，也包括包含所列举特征的开放式技术方案。
本发明中，“优选”、“更好”、“更佳”、“为宜”仅为描述效果更好的实施方式或实施例，应当理解，并不构成对本发明保护范围的限制。本发明中，“可选地”、“可选的”、“可选”，指可有可无，也即指选自“有”或“无”两种并列方案中的任一种。如果一个技术方案中出现多处“可选”，如无特别说明，且无矛盾之处或相互制约关系，则每项“可选”各自独立。
本发明中，“抗体”此技术术语是结合特定抗原的蛋白，其泛指包含互补决定区(CDR区)的一切蛋白及蛋白片段，特别是全长抗体或抗体功能片段。“全长抗体”此用语包括多克隆抗体及单克隆抗体，术语“抗体功能片段”是包含抗体CDR的一部分或全部的物质，其缺乏至少一些存在于全长链中的氨基酸但仍能够特异性结合至抗原。此类片段具生物活性，因为其结合至靶抗原，且可与其他抗原结合分子(包括完整抗体)竞争结合至给定表位。
“框架”或“框架序列”是可变区的除了被定义为抗原结合位点的那些序列之外的其余序列。因为抗原结合位点可以由如上所述的不同术语定义，所以框架的精确氨基酸序列取决于如何定义抗原结合位点。
本发明中，术语“人源化”或“人源化处理”，是指将鼠源抗体序列替换为人源抗体序列，从而减轻或消除人抗鼠抗体(HAMA)反应。这种替换可以是框架替换，例如将可变区中的FR序列替换为人源的，和/或将抗体的恒定区(如果具有的话)替换为人源的。这种替换也可以是通过链更替将 鼠单抗转换成完全人源性抗体(即CDR也经过替换)，可用的手段例如噬菌体抗体库技术。需要注意的是，人源化的过程中，替换的人源序列可包含部分氨基酸的置换或增减，使得该替换的序列可能不是表达的人免疫球蛋白序列或种系基因序列的精确拷贝。如此产生的抗体可称为人鼠嵌合抗体(human-mouse chimeric antibody)、人源化抗体(humanized antibody)或全人源抗体(human antibody)。
本发明中，术语“互补性决定区”或“CDR”是指免疫球蛋白的重链和轻链的高度可变区，如Kabat等人所定义(Kabat等人，Sequences of proteins of immunological interest,5th Ed"US Department of Health and Human Services,NIH,1991,和后来的版本)。有三种重链CDR和三种轻链CDR。此处，取决于情况，术语“CDR”和“CDRs”用于指包含一种或多种或者甚至全部的对抗体与其识别的抗原或表位的结合亲和力起作用的主要氨基酸残基的区域。在另一具体实施方式中，CDR区或CDR是指IMGT定义的免疫球蛋白的重链和轻链的高度可变区。
如本文中所使用，短语“治疗剂”通常是指在投与生物体时引发所需药理学作用的任何药剂。在一些实施例中，药剂在其在适当人群中展示出统计学上显著的作用时被视为治疗剂。在一些实施例中，适当群体可以是模型生物体群体。在一些实施例中，适当群体可由各种准则界定，如某一年龄组、性别、遗传背景、先前存在的临床病状等。在一些实施例中，治疗剂是可用于对疾病、病症和/或病状的一或多种症状或特征进行缓解、改善、减轻、抑制、预防、延缓发作、降低严重度和/或降低发病率的任何物质。在一些实施例中，“治疗剂”是在其可市售用于向人类投药之前已经或需要由政府机构批准的药剂。在一些实施例中，“治疗剂”是用于向人类投药的医学处方所需要的药剂。
如本文所使用，术语“检测剂”是指任何可检测的成份、分子、官能团、化合物、片段或部分。在一些实施例中，单独提供或使用检测实体。在一些实施例中，检测实体与另一试剂结合(例如接合)提供和/或使用。检测实 体的实例包括(但不限于)：各种配位体、放射性核素(例如 3H、 14C、 18F、 19F、 32P、 35S、 135I、 125I、 123I、 64Cu、 187Re、 111In、 90Y、 99mTc、 177Lu、 89Zr等)、荧光染料(关于特定例示性荧光染料，参见下文)、化学发光剂(如吖啶酯、稳定化二氧杂环丁烷等)、生物发光剂、光谱可分辨的无机荧光半导体纳米晶体(即，量子点)、金属纳米粒子(例如金、银、铜、铂等)、纳米簇、顺磁金属离子、酶(关于酶的特定实例，参见下文)、比色标记(如染料、胶态金等)、生物素、地高辛(digoxigenin)、半抗原和抗血清或单克隆抗体可适用的蛋白质。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。除非和本申请的发明目的和/或技术方案相冲突，否则，本发明涉及的引用文献以全部内容、全部目的被引用。本发明中涉及引用文献时，相关技术特征、术语、名词、短语等在引用文献中的定义也一并被引用。本发明中涉及引用文献时，被引用的相关技术特征的举例、优选方式也可作为参考纳入本申请中，但以能够实施本发明为限。应当理解，当引用内容与本申请中的描述相冲突时，以本申请为准或者适应性地根据本申请的描述进行修正。
本发明的第一方面涉及一种B7H3抗体或其抗原结合片段，其选自a)或b)；
a)重链CDR1、重链CDR2、重链CDR3依次如SEQ ID NO:1～3所示，轻链CDR1、轻链CDR2、轻链CDR3依次如SEQ ID NO:4～6所示；
b)重链CDR1、重链CDR2、重链CDR3依次如SEQ ID NO:7～9所示，轻链CDR1、轻链CDR2、轻链CDR3依次如SEQ ID NO:10～12所示。
在一些实施方式中，所述B7H3抗体或其抗原结合片段的重链可变区如SEQ ID NO:13所示，轻链可变区如SEQ ID NO:14所示；
或者，其重链可变区如SEQ ID NO:15所示，轻链可变区如SEQ ID NO:16所示。
在一些实施方式中，所述抗原结合片段为F(ab') 2、Fab以及scFv中的一种。
术语“F(ab') 2”是由胃蛋白酶消化整个全长抗体去除大部分Fc区同时完整保留一些铰链区后得到的。F(ab') 2片段具有通过二硫键连接在一起的两个抗原结合Fab部分，因此F(ab') 2片段为双价抗体，以IgG抗体制备得到的F(ab') 2为例，分子量为约110kDa。
术语“Fab”是仍可与抗原结合的抗体结构，其为单价并且不含Fc部分。木瓜蛋白酶消化全长抗体后得到两个Fab片段以及一个Fc片段，每个Fab片段均为约50kDa。
术语“scFv”意指包含通过接头连接的抗体重链可变结构域(或区域；VH)和抗体轻链可变结构域(或区域；VL)的分子。此类scFv分子可具有一般结构：NH 2-VL-连接肽(linker)-VH-COOH或NH 2-VH-连接肽(linker)-VL-COOH。
在本发明中，术语“连接肽”可以是为柔性或刚性的肽，例如由重复的GGGGS氨基酸序列或其变体组成，例如使用1～4个重复的变体(Holliger等人(1993)，Proc.Natl.Acad.Sci.USA 90:6444-6448)。可用于本发明的其他连接肽由Alfthan等人(1995)，Protein Eng.8:725-731，Choi等人(2001)，Eur.J.Immunol.31:94-106，Hu等人(1996)，Cancer Res.56:3055-3061，Kipriyanov等人(1999)，J.Mol.Biol.293:41-56和Roovers等人(2001)，Cancer Immunol.描述。
在一些实施方式中，所述连接肽的氨基酸数目为1～30个；可以是1，2，3，4，5、6、7、8、9、10、11、12、13、14、15、16、17、18、19、20、21、22、23、24、25、26、27、28、29或30个；优选5～20个。
在一些实施方式中，所述连接肽的氨基酸是不具有除连接以外的额外功能(例如蛋白定位、酶切位点等)的无意义多肽。
在一些实施方式中，所述连接肽为柔性连接肽；
在一些实施方式中，所述连接肽的氨基酸序列选自Gly、Ser、Pro、Ala以及Glu中的一种或多种。
在一些实施方式中，所述连接肽的氨基酸序列选自(GGGGS)n、(GGGS)n、(GGS)n、(GS)n或(G)n，其中n选自1，2，3，4，5或6。
连接肽通常是柔性的，可以减少待连接蛋白或多肽之间的空间位阻，从而更有利于蛋白正确折叠。
在一些实施方式中，所述的B7H3抗体或其抗原结合片具有恒定区。重链恒定区可以选自IgG、IgA、IgM、IgE、IgD任意一种的恒定区序列，IgG可进一步选自IgG1、IgG2、IgG3或IgG4，优选IgG1。
轻链恒定区可以为κ或λ链。
所述恒定区优选是人来源的。
在一些具体的实施方式中，重链恒定区序列如SEQ ID NO:17～19任一项所示，轻链恒定区序列如SEQ ID NO:20所示。
上述氨基酸序列的变体也在本发明范围内，相应的变体与SEQ ID NO:1～SEQ ID NO:12任一多肽相比，分别包含发生在至少一个CDR区域的至多3个氨基酸的突变；或者，变体相对于SEQ ID NO:13～SEQ ID NO:20整体序列而言，可包含3个以内或更多的突变，例如与SEQ ID NO:13～SEQ ID NO:20任一多肽相比具有至少80％、85％、90％、93％、95％、97％或99％同一性的序列。突变可以为氨基酸的置换、缺失或添加或其任意组合；优选地，所述突变为保守置换。
“保守置换”是指具有类似特征(例如电荷、侧链大小、疏水性/亲水性、主链构象和刚性等)的其它氨基酸置换蛋白中的氨基酸，使得可频繁进行改变而不改变蛋白的生物学活性。
通常视为保守置换的置换是在脂肪族氨基酸Ala、Val、Leu和Ile中的彼此置换、羟基残基Ser和Thr的互换、酸性残基Asp和Glu的交换、酰胺 残基Asn和Gln之间的置换、碱性残基Lys和Arg的交换以及芳香残基Phe、Tyr间的置换。本领域技术人员知晓，一般而言，多肽的非必需区域中的单个氨基酸置换基本上不改变生物学活性(参见例如Watson等(1987)Molecular Biology of the Gene,The Benjamin/Cummings Pub.Co.,第224页，(第4版))。另外，结构或功能类似的氨基酸的置换不大可能破环生物学活性。
本发明的第二方面涉及分离的核酸，其编码如上所述的B7H3抗体或其抗原结合片段。
在本文中，核酸包含其保守置换的变体(例如简并密码子的置换)和互补序列，亦包括通过密码子优化以在期望的宿主细胞中更高效表达的变体。核酸通常是RNA或DNA，包含基因、cDNA分子、mRNA分子以及它们的片段例如寡核苷酸。核酸分子可以是单链或双链的，但优选是双链DNA。当将核酸与另一个核酸序列置于功能关系中时，核酸是“有效连接的”。例如，如果启动子或增强子影响编码序列的转录，那么启动子或增强子有效地连接至所述编码序列。当其连入载体时优选采用DNA核酸。
此外，鉴于抗体为膜蛋白，所以核酸通常带有信号肽序列。
本发明的第三方面涉及载体，其包含如上所述的核酸。
术语“载体(vector)”是指，可将多聚核苷酸插入其中的一种核酸运载工具。当载体能使插入的多核苷酸编码的蛋白获得表达时，载体称为表达载体。载体可以通过转化，转导或者转染导入宿主细胞，使其携带的遗传物质元件在宿主细胞中获得表达。载体是本领域技术人员公知的，包括但不限于：质粒；噬菌粒；柯斯质粒；人工染色体，例如酵母人工染色体(YAC)、细菌人工染色体(BAC)或P1来源的人工染色体(PAC)；噬菌体如λ噬菌体或M13噬菌体及动物病毒等。可用作载体的动物病毒包括但不限于，逆转录酶病毒(包括慢病毒)、腺病毒、腺相关病毒、疱疹病毒(如单纯疱疹病毒)、痘病毒、杆状病毒、乳头瘤病毒、乳头多瘤空泡病毒(如SV40)。在一些实施方式中，本发明所述载体中包含基因工程中常用的调控元件，例如增强 子、启动子、内部核糖体进入位点(IRES)和其他表达控制元件(例如转录终止信号，或者多腺苷酸化信号和多聚U序列等)。
在本发明中，载体可以为组合物，例如为多种质粒的混合物，不同质粒负载抗体或其抗原结合片段的一部分。
本发明的第四方面涉及宿主细胞，其包含如上所述的核酸，或被如上所述的载体所转化。
宿主细胞可为原核细胞或真核细胞。示例性真核细胞包括哺乳动物细胞，诸如灵长类动物或非灵长类动物细胞；真菌细胞，诸如酵母；植物细胞；以及昆虫细胞。非限制性示例性哺乳动物细胞包括(但不限于)CHO细胞、HEK-293细胞、BHK细胞或PER-C6细胞，以及其衍生细胞，诸如293-6E、CHO-DG44、CHO-K1、CHO-S和CHO-DS细胞。在一些实施方案中，本发明提供的抗体或其抗原结合片段由哺乳动物细胞分泌产生。宿主细胞包括单个宿主细胞的后代，且后代可能由于自然、偶然或故意突变而不一定与原始亲代细胞完全一致(在形态或基因组DNA互补方面)。宿主细胞可以分离的细胞或细胞系，也包括在活体内经本文提供的核酸分子或表达载体转染的细胞。可以证明，细菌细胞可用作宿主细胞。但是，由于在细菌细胞中表达的蛋白倾向于未折叠的形式或不正确地折叠的形式或非糖基化形式，必须筛选在细菌细胞中产生的任何重组抗体，以保留抗原结合能力。如果细菌细胞表达的分子以适当地折叠的形式产生，该细菌细胞将是期望的宿主，或者，在可替代的实施方案中，可以在细菌宿主中表达分子，随后进行重新折叠。例如，用于表达的各种大肠杆菌菌株，是生物技术领域中众所周知的宿主细胞。枯草芽孢杆菌、链霉菌属、其他芽孢杆菌属等的各种菌株，也可以用于该方法中。
如果需要，本领域技术人员已知的酵母细胞菌株以及昆虫细胞，例如果蝇和鳞翅目昆虫和病毒表达系统，也可用作宿主细胞。
在一些实施方式中，所述宿主细胞的基因组中插入有所述核酸，并且 能稳定表达。
插入的方式可选用如上所述的载体，或者核酸不连入载体直接转入细胞内(例如脂质体介导的转染技术)。
本发明的第五方面涉及制备如上所述的B7H3抗体或其抗原结合片段的方法，包括在合适的条件下培养如上所述的宿主细胞，以及从宿主细胞培养物中回收目的产物。
本发明培养方法通常是无血清培养方法，通常通过无血清悬浮培养细胞。同样地，一旦产生本发明抗体，可将其根据本领域标准程序从细胞培养内容物纯化出，所述标准程序包括硫酸铵沉淀、亲和柱、柱层析、凝胶电泳等。这类技术在本领域技术范围内，不限定本发明。表达抗体的另一种方法可以利用在动物(特别是转基因动物或者裸鼠)中的表达。这涉及利用动物酪蛋白启动子的表达系统，当其被转基因地并入哺乳动物中时，允许雌性动物在其奶中产生希望的重组蛋白。分泌了抗体的培养液可以用常规技术纯化。比如，用含调整过的缓冲液的A或G Sepharose FF柱进行纯化。洗去非特异性结合的组分。再用pH梯度法洗脱结合的抗体，用SDS-PAGE检测抗体片段，收集。抗体可用常规方法进行过滤浓缩。可溶的混合物和多聚体，也可以用常规方法去除，比如分子筛、离子交换。得到的产物需立即冷冻，如-70℃，或者冻干。
本发明还涉及偶联物，其为与治疗剂或检测剂所结合的如上B7H3抗体或其抗原结合片段。
治疗剂可以是或包含任何类别的化学实体，包括例如(但不限于)蛋白质、碳水化合物、脂质、核酸、小型有机分子、非生物聚合物、金属、离子、放射性同位素等。在一些实施例中，根据本发明使用的治疗剂可具有与治疗癌症的一或多种症状或起因相关的生物活性。在一些实施例中，根据本发明使用的治疗剂可具有与调节免疫系统和/或促进T细胞介导的细胞毒性和/或抑制B7H3对T细胞增殖和功能的抑制作用相关的生物活性。在一些 实施例中，根据本发明使用的治疗剂具有一或多种其它活性。
在本发明的一些实施例中，所结合的治疗剂是放射性同位素、药物结合物、纳米粒子、免疫毒素或任何其它治疗性负荷。
检测剂包含任何可使用分析来检测的部分，例如归因于其特定功能特性和/或化学特征。所述药剂的非限制性实例包括酶、放射性标记、半抗原、荧光标记、磷光分子、化学发光分子、发色团、发光分子、光亲和性分子、有色粒子或配位体(如生物素)。在本发明的一些实施例中，所结合的检测剂是诊断剂或成像剂。
本发明还涉及药物组合物，其包含如上所述的B7H3抗体或其抗原结合片段，或如上所述的偶联物。
本发明的B7H3抗体或其抗原结合片段或偶联物可以应用于制备药物组合物或无菌组合物，例如，将它们中的任一种与药学上可接受的载体、赋形剂或稳定剂混合。
术语“组合物”意味着各成分是混合在一起的，其状态可以为固体(如冻干粉)或溶液。
术语“药学上可接受的”指当分子本体、分子片段或组合物适当地给予动物或人时，它们不会产生不利的、过敏的或其他不良反应。可作为药学上可接受的载体或其组分的一些物质的具体示例包括磷酸，柠檬酸，和其它有机酸；抗氧化剂(例如，抗坏血酸和甲硫氨酸)；抗菌剂(例如，十八烷基二甲基苯氯化铵，氯化六烃季铵，苯扎氯铵，酚，丁醇或苯甲醇，烷基尼泊金，邻苯二酚，间苯二酚，环己醇，3-戊醇，或间甲酚)；低分子量(不到约10kDa)多肽；蛋白，例如，血清白蛋白，明胶，或免疫球蛋白；亲水性聚合物，例如，聚乙烯吡咯烷酮；氨基酸(例如，甘氨酸，谷氨酰胺，天冬酰胺，组氨酸，精氨酸，或赖氨酸)；单糖，二糖和其它碳水化合物(包括例如，葡萄糖，甘露糖，或葡聚糖)；螯合剂(例如，EDTA)；糖(例如，蔗糖，甘露醇，海藻糖，或山梨醇)；成盐反离子；金属复合物；和/或非离子型表 面活性剂(例如，包括TWEENTM，PLURONICSTM，或聚乙二醇)。此外，根据配制方法，可以由本领域普通技术人员适当选择常用的填充剂，稀释剂，结合剂，增湿剂，崩解剂，和/或表面活性剂。
药物组合物中包含抗体或其功能片段的激活剂可以容纳在微胶囊中，或容纳在胶体性质的药物运送系统(如脂质体，白蛋白小球体，微乳剂，纳米颗粒及纳米胶囊)中，或者容纳在大乳剂(macroemulsions)中，所述微胶囊可以通过诸如凝聚(coacervation)技术或界面聚合作用来制备，例子分别有羟甲基纤维素或明胶微胶囊和聚-(异丁烯酸甲酯)微胶囊。
本发明的医药组合物可通过任何途径投与，如所属领域的技术人员将了解。在一些实施例中，本发明的医药组合物通过口服(PO)、静脉内(IV)、肌肉内(IM)、动脉内、髓内、鞘内、皮下(SQ)、心室内、经皮、皮内、皮内、经直肠(PR)、经阴道、腹膜内(IP)、胃内(IG)、局部(例如利用粉末、软膏、乳膏、凝胶、洗剂和/或滴剂)、粘膜、鼻内、颊内、经肠、玻璃体、舌下；通过气管内滴入、支气管滴入和/或吸入；作为口服喷雾、经鼻喷雾和/或气溶胶和/或经由门静脉导管投与。
本发明还涉及如上所述的B7H3抗体或其抗原结合片段，或如上所述的偶联物在制备用以治疗B7H3阳性癌症或用以调节免疫系统的药物中的应用。
B7H3在多种实体肿瘤类型上广泛表达，包括例如黑色素瘤(Wang J.,et al.,(2013)J.Invest.Dermatol.133:2050)、白血病(Hu Y.,et al.,(2015)Hematology 20:187；Sun J.,et al.,(2014)Onco Targets Ther.7:1979)、前列腺癌(Zang X.,et al.,(2007)Proc.Natl.Acad.Sci.104:19458)、卵巢癌(Zang X.,et al.,(2010)Mod.Pathol.23:1104)、胰腺癌(Chen Y.,et al.,(2014)Onco.Targets Ther.7:1465-72)、肾细胞癌、尿道上皮细胞癌瘤(Crispen.,et al.,(2008)，Clin Cancer Res.14:5150-157；Boorjian.,et al.,(2008)，Clin Cancer Res.14:4800-4808)、神经胶母细胞瘤(Lemke.,et al.,(2012)，Clin Cancer Res. 18:105-117)、骨肉瘤(Wang.,et al.,(2013)，PLoS One.8:e70689)、神经母细胞瘤(Gregorio.,et al.,(2008)，Histopathology.53:73-80)、弥漫性内源性脑桥神经胶质瘤(DIPG)(Zhou.,et al.,(2013)，J.Neurooncol.111:257-264)、间皮瘤(Calabro.,et al.,(2011)，J.Cell Physiol.226:2595-600)和胰脏癌(Yamato.,et al.,(2009),Br.J.Cancer.101:1709-1716)中。在一些较为优选的实施例中，B7H3阳性癌症选自神经母细胞瘤、子宫颈癌。
B7H3可显著抑制CD3抗体或同种异体DC细胞对T细胞的激活作用，而B7H3阻断抗体可有效逆转这种抑制作用(Prasad D.V.R.，et al.,(2004)J.Immunol.173:2500)。B7H3与NK细胞上的受体结合后，可抑制NK细胞对成神经细胞瘤的杀伤作用(Castriconi R.,et al.,(2004)Proc.Natl.Acad.Sci.101:12640)。B7H3对T细胞、NK和DC细胞的抑制作用能显著促进肿瘤细胞的免疫逃逸；另外，B7H3对肿瘤细胞的增殖、迁移、侵袭、血管生成、以及肿瘤细胞耐药性等也有重要影响。将肿瘤细胞移植在敲除B7H3的小鼠中或用B7H3抗体处理荷瘤小鼠，能够显著抑制肿瘤的生长(Cai D.,et al.,(2020)Cell.Mol.Immunol.17:227；Lee Y.H.,et al.,(2017)Cell Res.27:1034)，说明阻断B7H3的信号传导可用于肿瘤治疗。由于正常组织和肿瘤组织在B7H3表达水平上的显著差异，可以通过B7H3抗体的ADCC效应或毒素偶联来有效地杀死肿瘤细胞，而对正常组织不引起太大的副作用(Koenig S.,et al.,(2014)Medicographia 36:285)。
本发明所称癌症或肿瘤是指实体肿瘤和/或血液肿瘤，其可以是骨、骨连接、肌肉、肺、气管、心脏、脾脏、动脉、静脉、血液、毛细血管、淋巴结、淋巴管、淋巴液、口腔、咽、食管、胃、十二指肠、小肠、结肠、直肠、肛门、阑尾、肝、胆、胰腺、腮腺、舌下腺、泌尿肾、输尿管、膀胱、尿道、卵巢、输卵管、子宫、阴道、外阴部、阴囊、睾丸、输精管、阴茎、眼、耳、鼻、舌、皮肤、脑、脑干、延髓、脊髓、脑脊液、神经、甲状腺、甲状旁腺、肾上腺、垂体、松果体、胰岛、胸腺、性腺、舌下腺以及腮腺中任一处病变生成的肿瘤。具体如白血病、膀胱癌、结肠直肠癌、 前列腺癌、胃癌、胰腺癌、肝癌、头颈部癌、肾癌、乳腺癌、卵巢癌、促纤维增生性小圆细胞肿瘤、非小细胞肺癌、黑色素瘤、肺泡横纹肌肉瘤、食管癌、淋巴癌、胚胎横纹肌肉瘤、神经母细胞瘤、宫颈癌、尤文肉瘤、肾母细胞瘤、神经母细胞瘤、弥漫性脑桥脑胶质瘤、神经节神经瘤、髓母细胞瘤、神经节神经母细胞瘤、高级别胶质瘤、具有多层玫瑰花结的胚胎肿瘤，优选为表达B7H3的癌症。
本发明还涉及一种治疗、预防、减轻和/或诊断受试者的医学状况的方法，包括施用安全和有效量的如上所述的B7H3抗体或抗原结合片段，或如上所述的偶联物的步骤，并且其中所述医学状况的特征在于B7H3抗原的表达。
优选其中所述医学状况为B7H3阳性癌症或免疫系统相关疾病。
短语“安全和有效量的”。如本文所用，意指在合理的医药调节范围内化合物或组合物的量大到足以明显有效地缓解所治疗的症状或病症，但小到足以避免严重的副作用(以合理的有益/危险比率)。本发明的方法所用的药物组合物中的活性成份的安全和有效量随所治疗的特定症状、年龄和所治疗患者的身体状况，疾病的严重性、治疗时间、同期治疗情况、使用的特定活性成份、使用的特定的药物学可接受的赋形剂及包括参与治疗医师的知识和技能在内的这类因素的不同而不同。
应该理解的是，设想的治疗方法还将包括施用其他免疫治疗实体，特别优选为免疫治疗实体，包括病毒癌症疫苗(例如，编码癌症特异性抗原的腺病毒载体)、细菌癌症疫苗(例如，表达一种或多种癌症特异性抗原的非热原性大肠杆菌)、酵母癌症疫苗、N-803(也被称为ALT-803，ALTOR生物科学公司)、化疗药物、抗体(例如，与肿瘤相关抗原或患者特异性肿瘤新抗原结合)、干细胞移植物(例如，异体或自体)和肿瘤靶向细胞因子(例如，NHS-IL12，IL-12与肿瘤靶向抗体或其片段偶联)。在一些实施方式中，设想的治疗方法还包括对所述患者进行放射性治疗。在一些实施方式中，设想的治疗方法还包括对所述患者进行手术，例如肿瘤切除手术。
施用的方法可以为静脉给药和/或体腔内给药(优选腹腔给药或膀胱腔内给药)。
上述疾病的受试者或患者可选自人类、狗、猫、黑猩猩、猩猩、长臂猿、猕猴、狨猴、猪、马、熊猫和大象的群组。
下面将结合实施例对本发明的实施方案进行详细描述。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，优先参考本发明中给出的指引，还可以按照本领域的实验手册或常规条件，还可以参考本领域已知的其它实验方法，或者按照制造厂商所建议的条件。
下述的具体实施例中，涉及原料组分的量度参数，如无特别说明，可能存在称量精度范围内的细微偏差。涉及温度和时间参数，允许仪器测试精度或操作精度导致的可接受的偏差。
B7H3抗体的制备、初步人源化和基础性能检测参见公开号为CN113402610A，公开日为2021.09.17中国专利的实施例1～12。
实施例
1.对B7H3抗体进行增强ADCC作用的改造，以及ADCC活性的测定
对人源化9C8和5B6抗体进行了增强ADCC作用或者增加人源化等特性的改造(序列如表1所示)。
表1 ADCC增强型人源化B7H3抗体序列
Figure PCTCN2022103735-appb-000001
Figure PCTCN2022103735-appb-000002
Figure PCTCN2022103735-appb-000003
用报告基因(Reporter)法测试抗体的ADCC活性；具体的方法参照CN113402610A中的相应方法。以PC9肿瘤细胞为靶向细胞，Jurkat-mCD16.2-NF-kB或Jurkat-hCD16-NF-kB细胞为效应细胞，利用mCD16.2或hCD16介导的转录因子NF-kB的活化信号筛选ADCC阳性克隆。取50μL在上述ELISA和FACS检测中呈阳性的杂交瘤抗体与25μL的293T-B7H3、PC9或DLD1肿瘤细胞(7.5×10 4个/孔)混合，加至96孔板，再加入25μL Jurkat-mCD6.2-NF-kB细胞(2.5×10 4个/孔)，混匀，置于37℃培养箱中孵育4小时。每孔加入25μL预热的Bright-Glo溶液(Promega,Cat.No.:E2620)，室温避光静置3分钟，用酶标仪(Tecan Spark)测定各样品的冷光(Luminescence)信号值。
结果如图1和表2所示，人源化B7H3抗体hu5B6、hu9C8的ADCC加强型的ADCC活性得到了显著提高。
表2
  hu9C8(wt) hu9C8(DLE) hu9C8(DE) hu9C8(MG-1) MGA271
EC50 120.0 5.941 8.849 14.50 301.9
  hu5B6(wt) hu5B6(DLE) hu5B6(DE) hu5B6(MG-1) MGA271
EC50 164.2 5.533 4.989 13.51 305.4
图1中hu5B6各组(wt、DE、DLE、MG-1)轻链均相同，为hu5B6 改造后轻链，重链分别如表1中相应重链所示；hu9C8各组(wt、DE、DLE、MG-1)轻链均相同，为hu9C8改造后轻链，重链分别如表1中相应重链所示。
2.经ADCC增强的B7H3抗体具显著的肿瘤抑制效果。
使用小鼠CDX肿瘤模型检测经ADCC增强的B7H3抗体的抗肿瘤活性。实验由保诺科技公司(BioDuro，上海)实施完成。在实验过程中，人源MDA-MB-231乳腺癌细胞被移植到裸鼠(BALB/c nude mouse)皮下；等到在肿瘤长到约100mm 3，开始用抗体样品包括对肿瘤小鼠进行每周两次剂量为10mg/kg的腹腔注射。每一组6只小鼠用同一种抗体注射处理，给药前进行肿瘤的测量，第9次给药后的第四天，实验结束；肿瘤的体积由肿瘤的长度(mm)×宽度(mm)×一半的高度(mm)进行计算。图2显示了肿瘤的生长曲线，经ADCC增强的B7H3抗体hu9C8(DE)和hu5B6(DE)展现了显著的肿瘤抑制效果。
以上所述实施例仅表达了本发明的几种实施方式，其描述较为具体和详细，但并不能因此而理解为对发明专利范围的限制。应当指出的是，对于本领域的普通技术人员来说，在不脱离本发明构思的前提下，还可以做出若干变形和改进，这些都属于本发明的保护范围。因此，本发明专利的保护范围应以所附权利要求为准，说明书及附图可以用于解释权利要求的内容。

Claims (12)

  1. B7H3抗体或其抗原结合片段，其选自a)或b)；
    a)重链CDR1、重链CDR2、重链CDR3依次如SEQ ID NO:1～3所示，轻链CDR1、轻链CDR2、轻链CDR3依次如SEQ ID NO:4～6所示；
    b)重链CDR1、重链CDR2、重链CDR3依次如SEQ ID NO:7～9所示，轻链CDR1、轻链CDR2、轻链CDR3依次如SEQ ID NO:10～12所示。
  2. 根据权利要求1所述的B7H3抗体或其抗原结合片段，其重链可变区如SEQ ID NO:13所示，轻链可变区如SEQ ID NO:14所示；
    或者，其重链可变区如SEQ ID NO:15所示，轻链可变区如SEQ ID NO:16所示。
  3. 根据权利要求1或2所述的B7H3抗体或其抗原结合片段，所述抗原结合片段为F(ab') 2、Fab以及scFv中的一种。
  4. 根据权利要求1或2所述的B7H3抗体或其抗原结合片段，所述抗体具有恒定区，重链恒定区序列选自IgG、IgA、IgM、IgE、IgD任意一种的恒定区序列，优选IgG1；轻链恒定区为κ或λ链；所述恒定区优选是人来源的。
  5. 根据权利要求1或2所述的B7H3抗体或其抗原结合片段，所述抗体具有恒定区：
    重链恒定区序列如SEQ ID NO:17～19任一项所示，轻链恒定区序列如SEQ ID NO:20所示。
  6. 分离的核酸，其编码权利要求1～5任一项所述的B7H3抗体或其抗原结合片段。
  7. 载体，其包含权利要求6所述的核酸。
  8. 宿主细胞，其包含权利要求6所述的核酸，或被权利要求7所述的载体所转化。
  9. 制备权利要求1～5任一项所述的B7H3抗体或其抗原结合片段的方法，包括在合适的条件下培养权利要求8所述的宿主细胞，以及从宿主细胞培养物中回收目的产物。
  10. 偶联物，其为与治疗剂或检测剂所结合的权利要求1～5任一项所述的B7H3抗体或其抗原结合片段。
  11. 药物组合物，其包含权利要求1～5任一项所述的B7H3抗体或其抗原结合片段，或权利要求10所述的偶联物。
  12. 权利要求1～5任一项所述的B7H3抗体或其抗原结合片段，或权利要求10所述的偶联物在制备用以治疗B7H3阳性癌症或用以调节免疫系统的药物中的应用。
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