WO2023179392A1 - B7h3 antibody and bifunctional antibody comprising same - Google Patents

B7h3 antibody and bifunctional antibody comprising same Download PDF

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WO2023179392A1
WO2023179392A1 PCT/CN2023/081037 CN2023081037W WO2023179392A1 WO 2023179392 A1 WO2023179392 A1 WO 2023179392A1 CN 2023081037 W CN2023081037 W CN 2023081037W WO 2023179392 A1 WO2023179392 A1 WO 2023179392A1
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antibody
antigen
binding fragment
fusion protein
seq
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French (fr)
Chinese (zh)
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孙锴
王振生
邱均专
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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    • 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
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • C07K16/2827—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily against B7 molecules, e.g. CD80, CD86
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • 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
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
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    • 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
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    • 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
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • C—CHEMISTRY; METALLURGY
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    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
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    • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
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    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
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    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
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    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
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    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/75—Agonist effect on antigen
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    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
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    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention relates to the field of biomedicine technology, specifically to B7H3 antibodies and bifunctional antibodies containing them.
  • 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 8H9 clone (Omburtamab) in patent US20020102264 is a mouse IgG1 monoclonal antibody directed against B7H3.
  • 8H9 can bind to a variety of human solid tumors (Modak S., et al., (2001) Cancer Res. 61:4048-54), and can promote the killing effect of NK cells on B7H3-positive tumor cells ( Cheung N, et al., (2003) Hybrid Hybridomics 22:209-218).
  • Animal experiments show that 8H9 can inhibit the growth of sarcomas and brain tumors (Modak S., et al., (2005) Cancer Biother. Radiopharm.
  • bifunctional antibodies In recent years, the construction of bifunctional antibodies has created a new way for the development of new antibody drugs. Many new bifunctional antibody drugs have entered clinical trials one after another, showing stronger therapeutic effects than corresponding monoclonal antibodies. Therefore, using humanized 8H9 antibody and other antibodies or binding fragments [such as 4-1BB (CD137) antibody, VEGF-Trap protein or SIPR ⁇ protein] to construct bifunctional antibodies will further promote the function of immune cells and improve the resistance of antibodies. tumor effects.
  • 4-1BB (CD137) antibody CD137
  • VEGF-Trap protein or SIPR ⁇ protein vascular endothelial growth factor
  • the present invention relates to a B7H3 antibody or an antigen-binding fragment thereof, which includes a heavy chain complementary determining region with an amino acid sequence shown in SEQ ID NO: 1 to 3 and a light chain complement with an amino acid sequence shown in SEQ ID NO: 4 to 6. decision zone.
  • the present invention also relates to fusion proteins containing the B7H3 antibody or antigen-binding fragment thereof as described above.
  • the present invention also relates to nucleic acids, vectors and host cells related to the B7H3 antibody or its antigen-binding fragment and fusion protein as described above.
  • the present invention also relates to the preparation method of the B7H3 antibody or its antigen-binding fragment and fusion protein as described above.
  • the present invention also relates to a pharmaceutical composition
  • a pharmaceutical composition comprising the B7H3 antibody or antigen-binding fragment thereof as described above, or the fusion protein as described above, or the conjugate as described above.
  • the present invention also relates to the B7H3 antibody or antigen-binding fragment thereof as described above, or the fusion protein as described above, or the conjugate as described above, which is prepared for the treatment of B7H3-positive cancer or for regulating Applications in immune system medicines.
  • the present invention humanizes both the CDR region and the FR region of the B7H3 monoclonal antibody 8H9, and the humanized antibody can effectively block the immunosuppressive effect caused by B7H3.
  • the humanized B7H3 antibody or its antigen-binding fragment was used to construct a fusion protein with 4-1BB monoclonal antibody, VEGF-Trap protein and SIPR ⁇ protein, which further improved the activation effect of the antibody on immune cells.
  • the antibody has broad application prospects in the preparation of related drugs that can be used to inhibit cancer cells, regulate the function and level of B7H3, and enhance the body's immunity, especially drugs related to the treatment of cancer.
  • Figure 1 Determination of the binding properties of 8H9 humanized antibody and B7H3-his protein using ELISA method
  • Figure 2 The promotion effect of 8H9 humanized antibody on IFN- ⁇ secretion in human PBMC cells
  • Figure 3 Determination of the binding properties of B7H3/4-1BB bifunctional antibody and B7H3-his protein using ELISA method
  • Figure 4 The activation effect of cross-linking B7H3/4-1BB bifunctional antibody and Jurkat-B7H3 cells on the NF- ⁇ B signaling pathway;
  • FIG. 5 B7H3/4-1BB bifunctional antibody promotes IFN- ⁇ secretion in human PBMC cells
  • Figure 7 Inhibitory effect of B7H3/VEGF-Trap bifunctional antibody on VEGF-NFAT signaling pathway
  • FIG. 8 B7H3/VEGF-Trap bifunctional antibody promotes IFN- ⁇ secretion in human PBMC cells
  • Figure 9 Determination of the binding properties of B7H3/SIPR ⁇ bifunctional antibody and CD47-mFc protein using ELISA method
  • Figure 10 Using ELISA method to measure the blocking effect of B7H3/SIPR ⁇ bifunctional antibody on the binding of SIPR ⁇ and CD47;
  • Figure 11 Promoting effect of B7H3/SIPR ⁇ bifunctional antibody on IFN- ⁇ secretion in human PBMC cells.
  • 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 that contains part or all of the CDRs of an antibody, lacking at least some of the CDRs present in the full-length chain. of amino acids but still able to specifically bind to the 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 a mouse monoclonal antibody into a fully human antibody through chain replacement, using methods 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 groups 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 antibodies
  • the present invention relates to a B7H3 antibody or an antigen-binding fragment thereof, which includes a heavy chain complementary determining region whose amino acid sequence is shown in SEQ ID NO: 1 to 3 and a light chain whose amino acid sequence is shown in SEQ ID NO: 4 to 6. Chain complementarity determining region.
  • it includes a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 7 or 8, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 9 or 10; the combination thereof can Be any of the following: SEQ ID NO:7, SEQ ID NO:9; SEQ ID NO:7, SEQ ID NO:10; SEQ ID NO:8, SEQ ID NO:9; SEQ ID NO:8, SEQ ID NO:10.
  • a at position 102 of the heavy chain variable region is mutated to G.
  • the antigen-binding fragment is one of F(ab') 2 , Fab, scFv, and bispecific antibodies.
  • 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.
  • the linking peptide is usually flexible and can reduce the steric hindrance between the fusion protein and the target protein, which is more conducive to the correct folding of the protein.
  • the B7H3 antibody or antigen-binding fragment thereof further comprises an antibody constant region sequence.
  • the heavy chain constant region sequence is selected from any one of IgG, IgA, IgM, IgE, and IgD constant region sequences.
  • the heavy chain constant region sequence can be selected from human heavy chain constant regions, where IgG can be further divided into subclasses, such as IgG1, IgG2, IgG3, or IgG4.
  • the light chain constant region is a kappa or lambda chain.
  • the constant region is preferably of human origin.
  • the present invention also relates to a fusion protein containing the B7H3 antibody or antigen-binding fragment thereof as described above.
  • the fusion protein includes a first protein functional region targeting B7H3 and a second protein functional region targeting a second antigen;
  • the first functional region has the B7H3 antibody or antigen-binding fragment thereof as described above.
  • the second protein functional region is selected from the group consisting of 4-1BB antibody or antigen-binding fragment thereof, VEGF-Trap full length or fragment thereof, and SIRP ⁇ full length or fragment thereof.
  • 4-1BB belongs to the tumor necrosis factor receptor superfamily (TNFRSF9). 4-1BB on the surface of T cells can promote T cell proliferation and activation after binding to its ligand 4-1BBL (CD137L). Activating antibodies to 4-1BB have shown good anti-tumor effects in various tumor models (Vinay DS, et al., (2012) Mol. Cancer Ther. 11:1062–70). In clinical trials, the 4-1BB antibody has shown certain efficacy in the treatment of patients with melanoma, kidney cancer, and ovarian cancer, but dose-dependent liver toxicity has occurred in some patients (Sznol, et al., (2008)J .Clin.Oncol.26(supp15):3007).
  • TNFRSF9 tumor necrosis factor receptor superfamily
  • B7H3 is highly expressed in tumor tissues but at low levels in normal cells
  • constructing a bifunctional antibody with the 4-1BB antibody and the B7H3 antibody can limit the activation of T cells by the 4-1BB antibody to the tumor site. , reducing the toxic side effects on normal tissues; in addition, due to the dual effects on 4-1BB (activation) and B7H3 (blocking), the killing function of T cells against tumors has been further enhanced.
  • VEGF is a vascular endothelial growth factor that promotes blood vessel growth through interaction with its endothelial cell surface receptor (VEGFR).
  • An effective method for clinical cancer treatment is to use antibodies to block the binding of VEGF/VEGFR to inhibit the formation of new blood vessels in tumors.
  • VEGF-Trap is a fusion protein composed of the active functional regions of VEGFR1 and VEGFR2 connected in series. It has a stronger blocking effect than VEGFR monoclonal antibodies (Jocelyn H., et al., (2002) PNAS 99:11393-98 ).
  • constructing VEGF-Trap and B7H3 antibodies into bifunctional antibodies can It specifically inhibits the growth of blood vessels in tumor sites and reduces the impact on blood vessel growth in normal tissues. At the same time, by blocking B7H3, it enhances the cytotoxic activity of T cells against tumor cells.
  • CD47 also known as integrin-associated protein, is expressed in a variety of tumor cells and some normal cells.
  • SIPR ⁇ CD172 ⁇
  • CD47 is the receptor for CD47 and is mainly expressed on bone marrow cells, including monocytes, macrophages, neutrophils, dendritic cells, etc. (Adams S., et al., (1998) J Immunol 161 :1853-59).
  • the phagocytic activity of macrophages is regulated by the SIPR ⁇ /CD47 signaling pathway.
  • Tumor cells bind to SIPR ⁇ on the surface of macrophages through CD47, inhibiting the phagocytosis of tumor cells by macrophages.
  • constructing a bifunctional antibody between SIPR ⁇ and B7H3 antibodies can specifically block the combination of CD47 of tumor cells and SIPR ⁇ of macrophages, and promote the phagocytosis of tumor cells by macrophages (Chao MP., et al., ( 2010) Cell 142:699-713); on the other hand, due to the blocking effect of B7H3, the activity of T cells is enhanced, exerting another dimension of killing effect on tumor cells.
  • the 4-1BB antibody or antigen-binding fragment thereof includes the heavy chain complementarity determining regions H-CDR1, H-CDR2, H-CDR3 whose amino acid sequences are shown in SEQ ID NO: 11 to 13, and The amino acid sequence is the light chain complementarity determining region L-CDR1, L-CDR2, and L-CDR3 shown in SEQ ID NO: 14-16.
  • the 4-1BB antibody or antigen-binding fragment thereof comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 17, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 18. Change area.
  • the second protein functional region is a scFv of 4-1BB, preferably it is the scFv shown in SEQ ID NO: 19.
  • the amino acid sequence of the VEGF-Trap fragment is shown in SEQ ID NO: 20.
  • amino acid sequence of the SIRP ⁇ fragment is as shown in SEQ ID NO: 21.
  • 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.
  • the first protein functional region has an antibody heavy chain (for example, a heavy chain comprising an IgG1 constant region), and the C-terminus of the heavy chain and the N-terminus of the second protein functional region are connected through a connecting peptide .
  • an antibody heavy chain for example, a heavy chain comprising an IgG1 constant region
  • the invention also relates to an isolated nucleic acid encoding a B7H3 antibody or an antigen-binding fragment thereof as described above, or a fusion protein as described above.
  • isolated nucleic acid refers to a deoxyribonucleic acid or ribonucleic acid polymer present in single- or double-stranded form.
  • isolated nucleic acid includes RNA genomic sequences, DNA (gDNA and cDNA) or RNA sequences transcribed from DNA, and, unless otherwise specified, the polypeptide also Includes analogs of natural polynucleotides, sugars, or base changes.
  • the polynucleotide is a light chain polynucleotide.
  • the isolated nucleic acid includes a nucleotide sequence encoding the amino acid sequence of the protein complex, and also includes a nucleotide sequence complementary thereto.
  • the complementary sequence includes a completely complementary sequence and a substantially complementary sequence, which refers to a sequence that hybridizes to the nucleotide sequence encoding the amino acid sequence of the protein complex under stringent conditions known in the art.
  • nucleotide sequence encoding the amino acid sequence of the protein complex may be altered or mutated. Such changes include additions, deletions, or non-conservative or conservative substitutions.
  • a polynucleotide encoding an amino acid sequence of a protein complex may be construed as including a nucleotide sequence that is substantially identical to the isolated nucleic acid. The substantial identity is achieved by aligning the nucleotide sequence with another random sequence in a manner that maximizes correspondence between them. When the aligned sequences are analyzed using algorithms common in the art, the sequence may show greater than 80 % homology, greater than 90% homology, or greater than 95% homology.
  • the invention also 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 transfection recording termination signal, or polyadenylation 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.
  • the invention also provides host cells comprising the nucleic acid as described above, or transformed by the vector as described above.
  • Suitable host cells or cell lines for expressing the antigen-binding proteins of the invention include mammalian cells such as NSO, Sp2/0, CHO, COS, HEK, fibroblasts, and myeloma cells. Human cells can be used, thus allowing molecules to be modified with human glycosylation patterns. Alternatively, other eukaryotic cell lines can be used. The selection of suitable mammalian host cells, as well as methods for transformation, culture, amplification, screening, and product production and purification, are known in the art.
  • Bacterial cells may prove useful as host cells suitable for expression of proteins or other embodiments of the invention. However, since proteins expressed in bacterial cells tend to be in unfolded or incorrectly folded or non-glycosylated forms, any protein 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.
  • various 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 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 present invention also relates to a method for preparing a B7H3 antibody or an antigen-binding fragment thereof as described above, or a fusion protein as described above, including culturing the host cell as described above under appropriate conditions, and recovering the target cell from the cell culture. product.
  • 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 a conjugate, which is the above B7H3 antibody or its antigen-binding fragment combined with a therapeutic agent or a detection agent, or a fusion protein as described above.
  • 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 present 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 the B7H3 antibody or antigen-binding fragment thereof as described above, or the fusion protein as described above, or the conjugate as described above.
  • the B7H3 antibody or its antigen-binding fragment or fusion protein or conjugate of the present invention can be used to prepare pharmaceutical compositions or sterile compositions, for example, any of them and pharmaceutically acceptable carriers, excipients or stabilizer mix.
  • 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 of the antibody or functional fragment thereof in the pharmaceutical composition can be contained in microcapsules, Or contained in colloidal drug delivery systems (such as liposomes, albumin globules, microemulsions, nanoparticles and nanocapsules), or contained in macroemulsions (macroemulsions), the microcapsules can be processed by, for example, coacervation ( coacervation) technology or interfacial polymerization, examples include hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules respectively.
  • colloidal drug delivery systems such as liposomes, albumin globules, microemulsions, nanoparticles and nanocapsules
  • macroemulsions macroemulsions
  • the microcapsules can be processed by, for example, coacervation ( coacervation) technology or interfacial polymerization, examples include hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) micro
  • 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 B7H3 antibody or antigen-binding fragment thereof as described above, or the fusion protein as described above, or the conjugate as described above in the preparation of medicaments for treating B7H3-positive cancer or for regulating the immune system. application.
  • 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.
  • B7H3 Positive cancers were 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 preferably cancers expressing B7H3.
  • 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, embryo with multilayered rosettes Tumors, preferably cancers expressing B7H3.
  • 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 fusion protein as described above, or the steps of the conjugate as described above, 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.
  • 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 present invention humanizes the mouse 8H9 antibody (Omburtamab) in patent US20020102264; while significantly improving the degree of humanization of the antibody, the activity of the antibody is maintained.
  • Antibodies among the bifunctional antibodies B7H3/4-1BBab, B7H3/VEGF-Trap and B7H3/SIPR ⁇ may be affected by All molecules retain their respective activities and functions.
  • the bifunctional antibody constructed in the present invention shows higher biological activity in promoting the production and secretion of cytokines by human immune cells.
  • the B7H3 control antibody MGA271 (Enoblituzumab) in the present invention comes from patent US20180134790; the CD47 control antibody 1F8 comes from patent CN201980001915; the PD-L1 antibody sequence in PD-L1-VEGF-Trap comes from patent CN201911023312.
  • the heavy chain variable region sequence of the 8H9 hybridoma antibody is SEQ ID NO: 22, and the light chain variable region sequence is SEQ ID NO: 23.
  • the complementary determinant grafting method was used to humanize the 8H9 hybridoma antibody.
  • the IMGT database was searched for human germline antibody (germline antibody) sequences with the highest homology to the light and heavy chain variable region sequences of mouse antibodies. IGHV1-18*01 and IGHV1-8*01 were selected for humanization of the heavy chain variable region, and IGKV1-39*01 and IGKV6-21*02 were selected for humanization of the antibody light chain variable region.
  • the CDR region of the murine antibody is retained, and the framework sequence of the murine antibody is replaced with the framework sequence of the human germline antibody.
  • Establish a structural model of the mouse antibody and compare the amino acids at each position in the framework region of the human antibody and the corresponding mouse antibody one by one. If the use of a human amino acid sequence at a certain position in the framework region does not lead to the destruction of the spatial structure of the CDR region or If the amino acid sequence is changed, the human amino acid sequence will be used at this site; otherwise, the corresponding mouse sequence will be used at this site (i.e., back mutation to the mouse sequence).
  • the Met at position 48 of the humanized antibody IGHV1-18*01 heavy chain was back mutated to Ile, the Val at position 67 was back mutated to Ala, and the Met at position 69 was back mutated to Leu.
  • the Met at position 48 of the heavy chain of the humanized antibody IGHV1-8*01 was back mutated to Ile, the Val at position 67 was back mutated to Ala, the Met at position 69 was back mutated to Leu, and the Arg at position 71 was back mutated to Thr.
  • the Tyr at position 49 of the humanized antibody IGKV1-39*01 was back mutated to Lys, and the Thr at position 69 was back mutated to Ser.
  • the Leu at position 4 of the humanized antibody IGKV6-21*02 was back mutated to Met, and the Thr at position 69 was back mutated to Ser.
  • variable region amino acid sequence number of the humanized antibody heavy chain (germline IGHV1-18*01) is SEQ ID NO: 7, and the degree of humanization is 83.7%; the humanized antibody heavy chain (germline IGHV1-8* 01)’s variable region amino acid sequence number is SEQ ID NO: 8, and the degree of humanization is 85.7%; the variable region amino acid sequence number of the humanized antibody light chain (germline IGKV1-39*01) is SEQ ID NO : 9, the degree of humanization is 87.4%; the amino acid sequence number of the variable region of the humanized antibody light chain (germline IGKV6-21*02) is SEQ ID NO: 10, and the degree of humanization is 89.5%.
  • the above humanized antibodies were constructed into IgG1 subtype.
  • the degree of humanization of the light and heavy chains of the antibodies constructed in the present invention is significantly higher than the 70.5% and 76.5% reported in the literature (Mahiuddin A, et al., (2015) J. Biol. Chem. 290, 30018-29).
  • the nucleic acid sequences of the humanized antibody heavy chain and light chain were synthesized and inserted into the expression vector pcDNA3.1. 200 mL of 293 cells (cell density: 1 ⁇ 10 6 /mL) were co-transfected with 0.1 mg of antibody light chain and 0.1 mg of antibody heavy chain expression plasmids, cultured with shaking in a shaker flask at 37°C for 6 days, and the supernatant was collected by centrifugation. The humanized antibody was purified with Protein A, and the activity of the purified humanized antibody was tested.
  • the combination of 8H9 humanized antibody heavy chain (germline IGHV1-18*01) and light chain (germline IGKV6-21*02) is hu8H9-V1 (Version1), and the combination of heavy chain (germline IGHV1-18*01) and light chain ( The combination of germline IGKV1-39*01) is hu8H9-V2 (Version2), the combination of heavy chain (germline IGHV1-8*01) and light chain (germline IGKV6-21*02) is hu8H9-V3 (Version3), the combination of heavy chain (germline The combination of IGHV1-8*01) and light chain (germline IGKV1-39*01) is hu8H9-V4 (Version4).
  • the inventor also mutated alanine at position 102 of the heavy chain variable region to glycine on the basis of hu8H9-V4 to obtain hu8H9-V4 (VH-A102G) to enhance the stability of the antibody.
  • the 4-1BB humanized antibody was designed into a single-chain scFv (see SEQ ID NO: 19 for the sequence) connected to the C terminus of the hu8H9-V4 heavy chain through Linker (G 4 S) 4 , and co-transfected with the light chain vector into 293 cells. Culture in a shake flask at 37°C for 6 days, collect the supernatant by centrifugation, purify with Protein A, and conduct activity detection after purification.
  • a reporter gene method was used to detect the activation effect of the bifunctional antibody hu8H9/4-1BBab on the 4-1BB signaling pathway.
  • the results are shown in Figure 4.
  • the bifunctional antibody hu8H9/4-1BBab can significantly activate the 4-1BB signaling pathway.
  • the effect of the bifunctional antibody hu8H9/4-1BBab on the secretion of cytokines by human PBMC cells please refer to Example 3 for the specific method. As shown in Figure 5, compared with monoclonal antibodies, the bifunctional antibody hu8H9/4-1BBab can significantly promote the secretion of IFN- ⁇ in PBMC cells.
  • the VEGF-Trap sequence (SEQ ID NO: 20) was connected to the C terminus of the hu8H9-V4 heavy chain through Linker (G 4 S) 4 , and was co-transfected with the light chain vector into 293 cells. Culture in a shake flask at 37°C for 6 days, collect the supernatant by centrifugation, purify with Protein A, and conduct activity detection after purification.
  • a reporter gene method was used to detect the inhibitory effect of the bifunctional antibody hu8H9/VEGF-Trap on the VEGF-NFAT signaling pathway.
  • 50 ⁇ l of different concentrations of antibodies were preincubated with 50 ⁇ l of 40 ng/ml VEGF protein for 1 hour. After adding 50 ⁇ l of the mixture to 50 ⁇ l (5 ⁇ 10 4 cells/well) 293T-VEGFR-NFAT-luc cells, incubate at 37°C for 4 hours. Add 25 ⁇ l Bright Glo (Promega, Cat No: E2620) and incubate at room temperature for 5 minutes, and measure the chemiluminescence signal of each sample using a Tecan Spark microplate reader. As shown in Figure 7, the bifunctional antibody hu8H9/VEGF-Trap can significantly inhibit the NFAT signaling pathway.
  • the effect of the bifunctional antibody hu8H9/VEGF-Trap on the secretion of cytokines by human PBMC cells please refer to Example 3 for the specific method. As shown in Figure 8, the bifunctional antibody hu8H9/VEGF-Trap can significantly promote the secretion of IFN- ⁇ by PBMC cells.
  • the SIPR ⁇ sequence (see SEQ ID NO: 21) was connected to the C terminus of the heavy chain of humanized antibody hu8H9-V1 through Linker (G 4 S) 4 , and co-transfected with the light chain vector into 293 cells. Culture in a shake flask at 37°C for 6 days, collect the supernatant by centrifugation, purify with Protein A, and conduct activity detection after purification.
  • the effect of the bifunctional antibody hu8H9-SIPR ⁇ on the secretion of cytokines by human PBMC cells please refer to Example 3 for the specific method. As shown in Figure 11, compared with hu8H9 monoclonal antibody, the bifunctional antibody hu8H9/SIPR ⁇ has a stronger promoting effect on the secretion of IFN- ⁇ by PBMC cells.

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Abstract

Provided are a B7H3 antibody and a bifunctional antibody comprising same. The monoclonal antibody 8H9 of B7H3 is humanized, and the humanized antibody can effectively block the immunosuppressive effect caused by B7H3. In addition, a fusion protein is constructed by using the B7H3 antibody after same has been subjected to a humanization treatment or the antigen-binding fragment thereof together with a 4-1BB monoclonal antibody, VEGF-Trap protein and an SIPR α protein, so that the activation effect of the antibody on immune cells is further improved. Provided is the use of the antibody in the preparation of relevant drugs used for inhibiting cancer cells, regulating the effect and level of B7H3 and enhancing the immunity of the body. The antibody has wide application prospects in terms of drugs related to treating cancers.

Description

B7H3抗体及包含其的双功能抗体B7H3 antibodies and bifunctional antibodies containing the same
相关申请的交叉引用Cross-references to related applications
本申请要求于2022年03月25日提交中国专利局的申请号为202210306215.0、名称为“B7H3抗体及包含其的双功能抗体”的中国专利申请的优先权，并将其全部内容通过引用结合在本申请中。This application claims priority to the Chinese patent application with application number 202210306215.0 and titled "B7H3 Antibody and Bifunctional Antibodies Containing the Same" submitted to the China Patent Office on March 25, 2022, and the entire content of which is incorporated by reference in in this application.
技术领域Technical field
本发明涉及生物医药技术领域，具体而言，涉及B7H3抗体及包含其的双功能抗体。The present invention relates to the field of biomedicine technology, specifically to B7H3 antibodies and bifunctional antibodies containing them.
背景技术Background technique
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***的功能。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.
专利US20020102264中的8H9克隆(Omburtamab)是一种针对B7H3的鼠源IgG1单克隆抗体。体外实验表明，8H9能与人的多种实体瘤结合(Modak S.,et al.,(2001)Cancer Res.61:4048-54)，并能促进NK细胞对B7H3阳性肿瘤细胞的杀伤作用(Cheung N,et al.,(2003)Hybrid Hybridomics 22:209-218)。动物实验显示，8H9能抑制肉瘤和脑瘤的生长(Modak S.,et al.,(2005)Cancer Biother.Radiopharm.20:534-546；Luther N.,(2008)Neurosurgery 63:1167-1174)。临床数据表明，8H9可以显著延长中枢神经***实体瘤高危病人的生存期(Kramer K,et al.,(2007)J.Clin.Oncol.25,5465–70)。同位素镥Lu177标记的8H9(Omburtamab)最近刚在美国批准用于成神经管细胞瘤的治疗。由于8H9是鼠源抗体，在用于人体治疗过程 中会因其免疫原性而产生抗药抗体，从而降低其疗效并可能产生负作用。Mahiuddin等对8H9进行人源化和亲和力成熟改造(Mahiuddin A,et al.,(2015)J.Biol.Chem.290,30018-29)，但其轻重链人源化程度相对较低，分别为70.5％和76.5％。因此，设计人源化程度更高的8H9抗体，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。The 8H9 clone (Omburtamab) in patent US20020102264 is a mouse IgG1 monoclonal antibody directed against B7H3. In vitro experiments have shown that 8H9 can bind to a variety of human solid tumors (Modak S., et al., (2001) Cancer Res. 61:4048-54), and can promote the killing effect of NK cells on B7H3-positive tumor cells ( Cheung N, et al., (2003) Hybrid Hybridomics 22:209-218). Animal experiments show that 8H9 can inhibit the growth of sarcomas and brain tumors (Modak S., et al., (2005) Cancer Biother. Radiopharm. 20: 534-546; Luther N., (2008) Neurosurgery 63: 1167-1174) . Clinical data show that 8H9 can significantly prolong the survival of high-risk patients with central nervous system solid tumors (Kramer K, et al., (2007) J. Clin. Oncol. 25, 5465–70). The isotope lutetium Lu177-labeled 8H9 (Omburtamab) has recently been approved in the United States for the treatment of medulloblastoma. Since 8H9 is a mouse-derived antibody, it is used in human treatment processes. Anti-drug antibodies will be produced due to its immunogenicity, thereby reducing its efficacy and possibly causing negative effects. Mahiuddin et al. carried out humanization and affinity maturation of 8H9 (Mahiuddin A, et al., (2015) J. Biol. Chem. 290, 30018-29), but the degree of humanization of its light and heavy chains is relatively low, respectively. 70.5% and 76.5%. Therefore, designing 8H9 antibodies with a higher degree of humanization can reduce immune side effects during clinical use and better increase clinical efficacy.
近年来，双功能抗体的构建为抗体新药研发创造了一个新的途径。不少双功能抗体新药已陆续进入临床试验，显示了比相应的单抗更强的治疗效果。因此，用人源化的8H9抗体与其他抗体或者结合片段【例如4-1BB(CD137)抗体、VEGF-Trap蛋白或SIPRα蛋白】构建双功能抗体，将可进一步促进免疫细胞的功能，提高抗体的抗肿瘤效果。In recent years, the construction of bifunctional antibodies has created a new way for the development of new antibody drugs. Many new bifunctional antibody drugs have entered clinical trials one after another, showing stronger therapeutic effects than corresponding monoclonal antibodies. Therefore, using humanized 8H9 antibody and other antibodies or binding fragments [such as 4-1BB (CD137) antibody, VEGF-Trap protein or SIPRα protein] to construct bifunctional antibodies will further promote the function of immune cells and improve the resistance of antibodies. tumor effects.
发明内容Contents of the invention
本发明涉及B7H3抗体或其抗原结合片段，其包含氨基酸序列依次如SEQ ID NO:1～3所示的重链互补决定区以及氨基酸序列依次如SEQ ID NO:4～6所示的轻链互补决定区。The present invention relates to a B7H3 antibody or an antigen-binding fragment thereof, which includes a heavy chain complementary determining region with an amino acid sequence shown in SEQ ID NO: 1 to 3 and a light chain complement with an amino acid sequence shown in SEQ ID NO: 4 to 6. decision zone.
本发明还涉及融合蛋白，其含有如上所述的B7H3抗体或其抗原结合片段。The present invention also relates to fusion proteins containing the B7H3 antibody or antigen-binding fragment thereof as described above.
本发明还涉及如上所述的B7H3抗体或其抗原结合片段及融合蛋白相关的核酸、载体及宿主细胞。The present invention also relates to nucleic acids, vectors and host cells related to the B7H3 antibody or its antigen-binding fragment and fusion protein as described above.
本发明还涉及如上所述的B7H3抗体或其抗原结合片段及融合蛋白的制备方法。The present invention also relates to the preparation method of the B7H3 antibody or its antigen-binding fragment and fusion protein as described above.
本发明还涉及药物组合物，其包括如上所述的B7H3抗体或其抗原结合片段，或如上所述的融合蛋白，或如上所述的偶联物。The present invention also relates to a pharmaceutical composition comprising the B7H3 antibody or antigen-binding fragment thereof as described above, or the fusion protein as described above, or the conjugate as described above.
本发明还涉及如上所述的B7H3抗体或其抗原结合片段，或如上所述的融合蛋白，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***的药物中的应用。The present invention also relates to the B7H3 antibody or antigen-binding fragment thereof as described above, or the fusion protein as described above, or the conjugate as described above, which is prepared for the treatment of B7H3-positive cancer or for regulating Applications in immune system medicines.
本发明对B7H3的单克隆抗体8H9的CDR区及FR区均进行了人源化，人源化后的抗体能有效阻断B7H3引起的免疫抑制作用。此外，用人源化处理后的B7H3抗体或其抗原结合片段与4-1BB单克隆抗体、VEGF-Trap蛋白和SIPRα蛋白构建成融合蛋白，进一步提高了抗体对免疫细胞的激活作用。所述抗体在制备应用于抑制癌细胞及调节B7H3的作用、水平以及增强机体免疫力的相关药物，尤其是治疗癌症相关药物方面具有广阔的应用前景。The present invention humanizes both the CDR region and the FR region of the B7H3 monoclonal antibody 8H9, and the humanized antibody can effectively block the immunosuppressive effect caused by B7H3. In addition, the humanized B7H3 antibody or its antigen-binding fragment was used to construct a fusion protein with 4-1BB monoclonal antibody, VEGF-Trap protein and SIPRα protein, which further improved the activation effect of the antibody on immune cells. The antibody has broad application prospects in the preparation of related drugs that can be used to inhibit cancer cells, regulate the function and level of B7H3, and enhance the body's immunity, especially drugs related to the treatment of cancer.
附图说明Description of the drawings
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。In order to more clearly explain the specific embodiments of the present invention or the technical solutions in the prior art, the accompanying drawings that need to be used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings in the following description The drawings illustrate some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without exerting any creative effort.
图1：用ELISA方法测定8H9人源化处理抗体与B7H3-his蛋白的结合特性；Figure 1: Determination of the binding properties of 8H9 humanized antibody and B7H3-his protein using ELISA method;
图2：8H9人源化处理抗体对人的PBMC细胞IFN-γ分泌的促进作用；Figure 2: The promotion effect of 8H9 humanized antibody on IFN-γ secretion in human PBMC cells;
图3：用ELISA方法测定B7H3/4-1BB双功能抗体与B7H3-his蛋白的结合特性；Figure 3: Determination of the binding properties of B7H3/4-1BB bifunctional antibody and B7H3-his protein using ELISA method;
图4：B7H3/4-1BB双功能抗体与Jurkat-B7H3细胞交联对NF-κB信号通路的激活作用；Figure 4: The activation effect of cross-linking B7H3/4-1BB bifunctional antibody and Jurkat-B7H3 cells on the NF-κB signaling pathway;
图5：B7H3/4-1BB双功能抗体对人的PBMC细胞IFN-γ分泌的促进作用；Figure 5: B7H3/4-1BB bifunctional antibody promotes IFN-γ secretion in human PBMC cells;
图6：用ELISA方法测定B7H3/VEGF-Trap双功能抗体与B7H3-his蛋 白的结合特性；Figure 6: Determination of B7H3/VEGF-Trap bifunctional antibody and B7H3-his protein using ELISA method The binding properties of white;
图7：B7H3/VEGF-Trap双功能抗体对VEGF-NFAT信号通路的抑制作用；Figure 7: Inhibitory effect of B7H3/VEGF-Trap bifunctional antibody on VEGF-NFAT signaling pathway;
图8：B7H3/VEGF-Trap双功能抗体对人的PBMC细胞IFN-γ分泌的促进作用；Figure 8: B7H3/VEGF-Trap bifunctional antibody promotes IFN-γ secretion in human PBMC cells;
图9：用ELISA方法测定B7H3/SIPRα双功能抗体与CD47-mFc蛋白的结合特性；Figure 9: Determination of the binding properties of B7H3/SIPRα bifunctional antibody and CD47-mFc protein using ELISA method;
图10：用ELISA方法测定B7H3/SIPRα双功能抗体对SIPRα和CD47结合的阻断作用；Figure 10: Using ELISA method to measure the blocking effect of B7H3/SIPRα bifunctional antibody on the binding of SIPRα and CD47;
图11：B7H3/SIPRα双功能抗体对人的PBMC细胞IFN-γ分泌的促进作用。Figure 11: Promoting effect of B7H3/SIPRα bifunctional antibody on IFN-γ secretion in human PBMC cells.
具体实施方式Detailed ways
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。Reference will now be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For example, features illustrated or described as part of one embodiment can be used in another embodiment, to yield still further embodiments.
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。Unless otherwise defined, all terms (including technical and scientific terms) used to disclose the invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. By way of further guidance, the following definitions are provided to better understand the teachings of the present invention. The terminology used herein in the description of the invention is for the purpose of describing specific embodiments only and is not intended to limit the invention.
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)。The terms "and/or", "or/and" and "and/or" used in this article include any one of two or more related listed items, and also include any of the related listed items. and all combinations, any and all combinations including any two of the related listed items, any More related listed items, or a combination of all related listed items. It should be noted that when at least three items are connected in combination with at least two conjunctions selected from "and/or", "or/and", "and/or", it should be understood that in this application, the technical solution It undoubtedly includes technical solutions that are all connected by "logical AND", and it also undoubtedly includes technical solutions that are all connected by "logical OR". For example, "A and/or B" includes three parallel solutions: A, B and A+B. For another example, 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").
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。The terms "comprising", "comprising" and "including" as used in this invention are synonymous and are inclusive or open-ended and do not exclude additional, unrecited members, elements or method steps.
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。Numerical ranges expressed by endpoints herein include all numbers and fractions included within the range, as well as the recited endpoints.
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。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.
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。In the present invention, descriptions such as "plurality" and "multiple" refer to a quantity greater than or equal to 2 unless otherwise specified.
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。In the present invention, 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.
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。In the present invention, the technical term "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. The term "full-length antibody" includes polyclonal antibodies as well as monoclonal antibodies. The term "antibody functional fragment" is a substance that contains part or all of the CDRs of an antibody, lacking at least some of the CDRs present in the full-length chain. of amino acids but still able to specifically bind to the 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.
“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。"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.
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，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)。In the present invention, 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. Such 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 a mouse monoclonal antibody into a fully human antibody through chain replacement, using methods such as phage antibody library technology. It should be noted that during the humanization process, 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.
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。As used herein, the term "complementarity determining region" or "CDR" refers to the highly variable regions of the heavy and light chains of immunoglobulins, as defined by Kabat et al. (Kabat et al., Sequences of proteins of immunological interest, 5th Ed"US Department of Health and Human Services, NIH, 1991, and later editions). There are three heavy chain CDRs and three light chain CDRs. Here, depending on the context, the terms "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. In another specific embodiment, 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.
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。As used herein, the phrase "therapeutic agent" generally refers to any agent that elicits a desired pharmacological effect when administered to an organism. In some embodiments, an agent is considered a therapeutic when it exhibits a statistically significant effect in an appropriate population. In some embodiments, a suitable population may be a population of model organisms. In some embodiments, appropriate groups may be defined by various criteria, such as a certain age group, gender, genetic background, pre-existing clinical conditions, etc. In some embodiments, 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. In some embodiments, 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.
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。As used herein, the term "detector" refers to any detectable component, molecule, functional group, compound, fragment or moiety. In some embodiments, the detection entity is provided or used separately. In some embodiments, the detection entity is provided and/or used in combination (eg, conjugated) with another agent. Examples of 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 antibodies may be suitable proteins.
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。In the present invention, "preferable", "better", "better" and "suitable" are only used to describe implementations or examples with better effects. It should be understood that they do not limit the scope of protection of the present invention. In the present invention, "optionally", "optional" and "optional" mean that it is optional, that is, it refers to any one selected from the two parallel solutions of "with" or "without". If there are multiple "optionals" in a technical solution, each "optional" will be independent unless otherwise specified and there is no contradiction or mutual restriction.
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。All documents mentioned in this application are incorporated by reference in this application to the same extent as if each individual document was individually incorporated by reference. Unless it conflicts with the invention purpose and/or technical solution of the present application, the cited documents involved in the present invention are cited in their entirety and for all purposes. When referring to cited documents in the present invention, the definitions of relevant technical features, terms, nouns, phrases, etc. in the cited documents are also cited. When the present invention involves citing documents, the relevant technical features cited Examples and preferred modes may also be incorporated into this application as references, but are limited to the extent that the present invention can be implemented. It should be understood that when the quoted content conflicts with the description in this application, this application shall prevail or be adapted to be modified according to the description in this application.
抗体及融合蛋白Antibodies and fusion proteins
本发明涉及一种B7H3抗体或其抗原结合片段，其包含氨基酸序列依次如SEQ ID NO:1～3所示的重链互补决定区以及氨基酸序列依次如SEQ ID NO:4～6所示的轻链互补决定区。The present invention relates to a B7H3 antibody or an antigen-binding fragment thereof, which includes a heavy chain complementary determining region whose amino acid sequence is shown in SEQ ID NO: 1 to 3 and a light chain whose amino acid sequence is shown in SEQ ID NO: 4 to 6. Chain complementarity determining region.
在一些实施方式中，其包含氨基酸序列如SEQ ID NO:7或8所示的重链可变区，以及氨基酸序列如SEQ ID NO:9或10所示的轻链可变区；其组合可以为下述任一种：SEQ ID NO:7、SEQ ID NO:9；SEQ ID NO:7、SEQ ID NO:10；SEQ ID NO:8、SEQ ID NO:9；SEQ ID NO:8、SEQ ID NO:10。In some embodiments, it includes a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 7 or 8, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 9 or 10; the combination thereof can Be any of the following: SEQ ID NO:7, SEQ ID NO:9; SEQ ID NO:7, SEQ ID NO:10; SEQ ID NO:8, SEQ ID NO:9; SEQ ID NO:8, SEQ ID NO:10.
在一些实施方式中，所述重链可变区的第102位A突变为G。In some embodiments, A at position 102 of the heavy chain variable region is mutated to G.
在一些实施方式中，所述抗原结合片段为F(ab')2、Fab、scFv以及双特异抗体中的一种。In some embodiments, the antigen-binding fragment is one of F(ab') 2 , Fab, scFv, and bispecific antibodies.
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。The term "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. Take F(ab') 2 prepared from an IgG antibody as an example. , the molecular weight is about 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。The term "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.
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：NH2-VL-连接肽(linker)-VH-COOH或NH2-VH-连接肽(linker)-VL-COOH。 The term "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.
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。In the present invention, the term "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.
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。In some embodiments, 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.
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。In some embodiments, 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.).
在一些实施方式中，所述连接肽为柔性连接肽；In some embodiments, the linker peptide is a flexible linker peptide;
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。In some embodiments, the amino acid sequence of the connecting peptide is selected from one or more of Gly, Ser, Pro, Ala and Glu.
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。In some embodiments, 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.
连接肽通常是柔性的，可以减少融合蛋白与目的蛋白之间的空间位阻，从而更有利于蛋白正确折叠。The linking peptide is usually flexible and can reduce the steric hindrance between the fusion protein and the target protein, which is more conducive to the correct folding of the protein.
在一些实施方式中，所述B7H3抗体或其抗原结合片段还包含抗体恒定区序列。In some embodiments, the B7H3 antibody or antigen-binding fragment thereof further comprises an antibody constant region sequence.
在一些实施方式中，所述重链恒定区序列选自IgG、IgA、IgM、IgE、IgD任意一种的恒定区序列。在一些实施方式中，所述重链恒定区序列可以选自人的重链恒定区，其中IgG可进一步分成亚类，如IgG1、IgG2、IgG3或IgG4。 In some embodiments, the heavy chain constant region sequence is selected from any one of IgG, IgA, IgM, IgE, and IgD constant region sequences. In some embodiments, the heavy chain constant region sequence can be selected from human heavy chain constant regions, where IgG can be further divided into subclasses, such as IgG1, IgG2, IgG3, or IgG4.
在一些实施方式中，轻链恒定区为κ或λ链。In some embodiments, the light chain constant region is a kappa or lambda chain.
所述恒定区优选是人来源的。The constant region is preferably of human origin.
根据本发明的再一方案，还涉及融合蛋白，其含有如上所述的B7H3抗体或其抗原结合片段。According to yet another aspect of the present invention, it also relates to a fusion protein containing the B7H3 antibody or antigen-binding fragment thereof as described above.
在一些实施方式中，所述的融合蛋白包含靶向B7H3的第一蛋白功能区以及靶向第二抗原的第二蛋白功能区；In some embodiments, the fusion protein includes a first protein functional region targeting B7H3 and a second protein functional region targeting a second antigen;
所述第一功能区具有如上所述的B7H3抗体或其抗原结合片段。The first functional region has the B7H3 antibody or antigen-binding fragment thereof as described above.
在一些实施方式中，所述第二蛋白功能区选自4-1BB抗体或其抗原结合片段、VEGF-Trap全长或其片段以及SIRPα全长或其片段。In some embodiments, the second protein functional region is selected from the group consisting of 4-1BB antibody or antigen-binding fragment thereof, VEGF-Trap full length or fragment thereof, and SIRPα full length or fragment thereof.
4-1BB属于肿瘤坏死因子受体超家族(TNFRSF9)。T细胞表面的4-1BB与其配体4-1BBL(CD137L)结合后，能够促进T细胞的增殖和活化。4-1BB的激活抗体在多种肿瘤模型中显示了良好的抗肿瘤效果(Vinay DS,et al.,(2012)Mol.Cancer Ther.11:1062–70)。临床试验中，4-1BB抗体在黑色素瘤、肾癌和卵巢癌患者的治疗中显示出一定的疗效，但在部分病人中出现剂量依赖的肝脏毒性副作用(Sznol,et al.,(2008)J.Clin.Oncol.26(supp15):3007)。由于B7H3在肿瘤组织中高表达，而在正常细胞中表达水平较低；因此，将4-1BB抗体与B7H3抗体构建成双功能抗体，可以使4-1BB抗体对T细胞的激活作用限制在肿瘤部位，降低了对正常组织的毒副作用；另外，由于对4-1BB(激活)以及对B7H3(阻断)的双重影响，T细胞对肿瘤的杀伤功能得到了进一步增强。4-1BB belongs to the tumor necrosis factor receptor superfamily (TNFRSF9). 4-1BB on the surface of T cells can promote T cell proliferation and activation after binding to its ligand 4-1BBL (CD137L). Activating antibodies to 4-1BB have shown good anti-tumor effects in various tumor models (Vinay DS, et al., (2012) Mol. Cancer Ther. 11:1062–70). In clinical trials, the 4-1BB antibody has shown certain efficacy in the treatment of patients with melanoma, kidney cancer, and ovarian cancer, but dose-dependent liver toxicity has occurred in some patients (Sznol, et al., (2008)J .Clin.Oncol.26(supp15):3007). Since B7H3 is highly expressed in tumor tissues but at low levels in normal cells, constructing a bifunctional antibody with the 4-1BB antibody and the B7H3 antibody can limit the activation of T cells by the 4-1BB antibody to the tumor site. , reducing the toxic side effects on normal tissues; in addition, due to the dual effects on 4-1BB (activation) and B7H3 (blocking), the killing function of T cells against tumors has been further enhanced.
VEGF是血管内皮生长因子，可通过与其内皮细胞表面受体(VEGFR)的相互作用，促进血管生长。目前临床癌症治疗的一个有效方法是通过抗体阻断VEGF/VEGFR的结合而抑制肿瘤新生血管的形成。VEGF-Trap是由VEGFR1和VEGFR2的活性功能区串联组成的融合蛋白，它比VEGFR的单克隆抗体具有更强的阻断作用(Jocelyn H.,et al.,(2002)PNAS 99:11393-98)。因此，将VEGF-Trap与B7H3抗体构建成双功能抗体，可以 特异性的抑制肿瘤部位的血管生长，降低对正常组织中血管生长的影响，同时通过对B7H3的阻断，加强了T细胞对肿瘤细胞的毒杀活性。VEGF is a vascular endothelial growth factor that promotes blood vessel growth through interaction with its endothelial cell surface receptor (VEGFR). An effective method for clinical cancer treatment is to use antibodies to block the binding of VEGF/VEGFR to inhibit the formation of new blood vessels in tumors. VEGF-Trap is a fusion protein composed of the active functional regions of VEGFR1 and VEGFR2 connected in series. It has a stronger blocking effect than VEGFR monoclonal antibodies (Jocelyn H., et al., (2002) PNAS 99:11393-98 ). Therefore, constructing VEGF-Trap and B7H3 antibodies into bifunctional antibodies can It specifically inhibits the growth of blood vessels in tumor sites and reduces the impact on blood vessel growth in normal tissues. At the same time, by blocking B7H3, it enhances the cytotoxic activity of T cells against tumor cells.
CD47也称整联素关联蛋白，表达于多种肿瘤细胞及某些正常细胞中。SIPRα(CD172α)是CD47的受体，主要表达于骨髓细胞，包括单核细胞、巨噬细胞、中性粒细胞、树突状细胞等(Adams S.,et al.,(1998)J Immunol 161:1853-59)。巨噬细胞的吞噬活性受SIPRα/CD47信号通路的调节。肿瘤细胞通过CD47与巨噬细胞表面的SIPRα结合，抑制巨噬细胞对肿瘤细胞的吞噬作用。因此，将SIPRα与B7H3抗体构建成双功能抗体，可以特异性阻断肿瘤细胞的CD47与巨噬细胞的SIPRα结合，促进巨噬细胞对肿瘤细胞的吞噬作用(Chao MP.,et al.,(2010)Cell 142:699-713)；另一方面，由于对B7H3的阻断作用，T细胞的活性得以增强，对肿瘤细胞施以另一维度的杀伤作用。CD47, also known as integrin-associated protein, is expressed in a variety of tumor cells and some normal cells. SIPRα (CD172α) is the receptor for CD47 and is mainly expressed on bone marrow cells, including monocytes, macrophages, neutrophils, dendritic cells, etc. (Adams S., et al., (1998) J Immunol 161 :1853-59). The phagocytic activity of macrophages is regulated by the SIPRα/CD47 signaling pathway. Tumor cells bind to SIPRα on the surface of macrophages through CD47, inhibiting the phagocytosis of tumor cells by macrophages. Therefore, constructing a bifunctional antibody between SIPRα and B7H3 antibodies can specifically block the combination of CD47 of tumor cells and SIPRα of macrophages, and promote the phagocytosis of tumor cells by macrophages (Chao MP., et al., ( 2010) Cell 142:699-713); on the other hand, due to the blocking effect of B7H3, the activity of T cells is enhanced, exerting another dimension of killing effect on tumor cells.
在一些实施方式中，所述4-1BB抗体或其抗原结合片段包含氨基酸序列依次如SEQ ID NO:11～13所示的重链互补决定区H-CDR1、H-CDR2、H-CDR3，以及氨基酸序列依次如SEQ ID NO:14～16所示的轻链互补决定区L-CDR1、L-CDR2、L-CDR3。In some embodiments, the 4-1BB antibody or antigen-binding fragment thereof includes the heavy chain complementarity determining regions H-CDR1, H-CDR2, H-CDR3 whose amino acid sequences are shown in SEQ ID NO: 11 to 13, and The amino acid sequence is the light chain complementarity determining region L-CDR1, L-CDR2, and L-CDR3 shown in SEQ ID NO: 14-16.
在一些实施方式中，所述4-1BB抗体或其抗原结合片段包含氨基酸序列如SEQ ID NO:17所示的重链可变区，以及氨基酸序列如SEQ ID NO:18所示的轻链可变区。In some embodiments, the 4-1BB antibody or antigen-binding fragment thereof comprises a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 17, and a light chain variable region with an amino acid sequence as shown in SEQ ID NO: 18. Change area.
在一些实施方式中，所述第二蛋白功能区为4-1BB的scFv，优选其为SEQ ID NO:19所示的scFv。In some embodiments, the second protein functional region is a scFv of 4-1BB, preferably it is the scFv shown in SEQ ID NO: 19.
在一些实施方式中，所述VEGF-Trap片段的氨基酸序列如SEQ ID NO:20所示。In some embodiments, the amino acid sequence of the VEGF-Trap fragment is shown in SEQ ID NO: 20.
在一些实施方式中，所述SIRPα片段的氨基酸序列如SEQ ID NO:21所示。In some embodiments, the amino acid sequence of the SIRPα fragment is as shown in SEQ ID NO: 21.
上述氨基酸序列的变体也在本发明范围内，本发明的B7H3抗体或其抗 原结合片段，或4-1BB抗体或其抗原结合片段，或VEGF-Trap片段，或SIRPα片段所对应的变体，与SEQ ID NO:1～SEQ ID NO:210任一多肽相比，分别包含发生在CDR区域(如果具有)的至多3个氨基酸的突变；或者，变体相对于SEQ ID NO:1～SEQ ID NO:21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:1～SEQ ID NO:21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。Variants of the above amino acid sequence are also within the scope of the present invention. The B7H3 antibody of the present invention or its anti- Compared with any polypeptide of SEQ ID NO: 1 to SEQ ID NO: 210, the original binding fragment, or the 4-1BB antibody or its antigen-binding fragment, or the VEGF-Trap fragment, or the variant corresponding to the SIRPα fragment, respectively Contains mutations of up to 3 amino acids occurring in the CDR region (if any); alternatively, the variant may contain less than 3 or more mutations relative to the overall sequence of SEQ ID NO: 1 to SEQ ID NO: 21, For example, a sequence having at least 80%, 85%, 90%, 93%, 95%, 97% or 99% identity with any polypeptide of SEQ ID NO: 1 to SEQ ID NO: 21. The mutation may be substitution, deletion or addition of amino acids or any combination thereof; preferably, the mutation is a conservative substitution.
“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。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.
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。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.)). In addition, substitution of amino acids with similar structure or function is unlikely to destroy biological activity.
在一些实施方式中，所述第一蛋白功能区具有的抗体重链(例如包含IgG1恒定区的重链)，且重链的C端和所述第二蛋白功能区的N端通过连接肽连接。In some embodiments, the first protein functional region has an antibody heavy chain (for example, a heavy chain comprising an IgG1 constant region), and the C-terminus of the heavy chain and the N-terminus of the second protein functional region are connected through a connecting peptide .
分离的核酸isolated nucleic acid
本发明还涉及分离的核酸，其编码如上所述的B7H3抗体或其抗原结合片段，或如上所述的融合蛋白。The invention also relates to an isolated nucleic acid encoding a B7H3 antibody or an antigen-binding fragment thereof as described above, or a fusion protein as described above.
术语“分离的核酸”在本文中是指以单链或双链形式存在的脱氧核糖核酸或核糖核酸聚合物。所述分离的核酸包括RNA基因组序列，DNA(gDNA和cDNA)或从DNA转录的RNA序列，而且，除非特别指明，所述多肽还 包括天然多核苷酸、糖、或碱基改变的类似物。根据本发明一个方面，所述多核苷酸是轻链多核苷酸。The term "isolated nucleic acid" as used herein refers to a deoxyribonucleic acid or ribonucleic acid polymer present in single- or double-stranded form. The isolated nucleic acid includes RNA genomic sequences, DNA (gDNA and cDNA) or RNA sequences transcribed from DNA, and, unless otherwise specified, the polypeptide also Includes analogs of natural polynucleotides, sugars, or base changes. According to one aspect of the invention, the polynucleotide is a light chain polynucleotide.
所述分离的核酸包括编码蛋白复合物氨基酸序列的核苷酸序列，也包括与其互补的核苷酸序列。所述互补序列包括完全互补的序列和基本上互补的序列，这是指能在本领域已知的严谨条件下与编码蛋白复合物氨基酸序列的核苷酸序列杂交的序列。The isolated nucleic acid includes a nucleotide sequence encoding the amino acid sequence of the protein complex, and also includes a nucleotide sequence complementary thereto. The complementary sequence includes a completely complementary sequence and a substantially complementary sequence, which refers to a sequence that hybridizes to the nucleotide sequence encoding the amino acid sequence of the protein complex under stringent conditions known in the art.
而且，编码蛋白复合物氨基酸序列的核苷酸序列可以被改变或突变。所述改变包括添加、缺失、或非保守取代或保守取代。编码蛋白复合物氨基酸序列的多核苷酸可以被解释为，包括相对于该分离的核酸有实质性同一性的核苷酸序列。所述实质性同一性将该核苷酸序列与另外的随机序列以使得它们最大对应的方式进行比对，当用本领域常见的算法分析所比对的序列时，所述序列可显示大于80％的同源性，大于90％的同源性，或大于95％的同源性。Furthermore, the nucleotide sequence encoding the amino acid sequence of the protein complex may be altered or mutated. Such changes include additions, deletions, or non-conservative or conservative substitutions. A polynucleotide encoding an amino acid sequence of a protein complex may be construed as including a nucleotide sequence that is substantially identical to the isolated nucleic acid. The substantial identity is achieved by aligning the nucleotide sequence with another random sequence in a manner that maximizes correspondence between them. When the aligned sequences are analyzed using algorithms common in the art, the sequence may show greater than 80 % homology, greater than 90% homology, or greater than 95% homology.
载体carrier
本发明还涉及载体，其包含如上所述的核酸。The invention also relates to a vector comprising a nucleic acid as described above.
术语“载体(vector)”是指，可将多聚核苷酸***其中的一种核酸运载工具。当载体能使***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、***瘤病毒、***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)。The term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When 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). In some embodiments, 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 transfection recording termination signal, or polyadenylation signal and polyU sequence, etc.).
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。In the present invention, 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.
宿主细胞host cell
本发明还提供宿主细胞，其包含如上所述的核酸，或被如上所述的载体所转化。The invention also provides host cells comprising the nucleic acid as described above, or transformed by the vector as described above.
适用于表达本发明的抗原结合蛋白的宿主细胞或细胞系包括：哺乳动物细胞诸如NS0、Sp2/0、CHO、COS、HEK、成纤维细胞和骨髓瘤细胞。可以使用人细胞，因而允许分子用人糖基化模式来修饰。或者，可以采用其他真核细胞系。合适的哺乳动物宿主细胞的选择，以及用于转化、培养、扩增、筛选和产物产生和纯化的方法，是本领域已知的。Suitable host cells or cell lines for expressing the antigen-binding proteins of the invention include mammalian cells such as NSO, Sp2/0, CHO, COS, HEK, fibroblasts, and myeloma cells. Human cells can be used, thus allowing molecules to be modified with human glycosylation patterns. Alternatively, other eukaryotic cell lines can be used. The selection of suitable mammalian host cells, as well as methods for transformation, culture, amplification, screening, and product production and purification, are known in the art.
可以证明，细菌细胞可用作宿主细胞，其适合表达本发明的蛋白或其他实施方案。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蛋白，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。Bacterial cells may prove useful as host cells suitable for expression of proteins or other embodiments of the invention. However, since proteins expressed in bacterial cells tend to be in unfolded or incorrectly folded or non-glycosylated forms, any protein 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. For example, various 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.
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***，也可用作宿主细胞。If desired, 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.
在一些实施方式中，所述细胞基因组中***有所述核酸，并且能稳定表达。In some embodiments, the nucleic acid is inserted into the genome of the cell and can be stably expressed.
***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)。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).
制备方法Preparation
本发明还涉及一种制备如上所述B7H3抗体或其抗原结合片段，或如上所述的融合蛋白的方法，包括在合适的条件下培养如上所述的宿主细胞，以及从细胞培养物中回收目的产物。The present invention also relates to a method for preparing a B7H3 antibody or an antigen-binding fragment thereof as described above, or a fusion protein as described above, including culturing the host cell as described above under appropriate conditions, and recovering the target cell from the cell culture. product.
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***，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。The culture method of the present invention is usually a serum-free culture method, and cells are usually cultured in serum-free suspension. Likewise, once the antibodies of the invention are produced, they 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. For example, purify using an A or G Sepharose FF column containing adjusted buffer. Wash away non-specifically bound components. Then use the pH gradient method to elute the bound antibodies, and use SDS-PAGE to detect the antibody fragments and collect them. 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.
偶联物conjugate
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，或如上所述的融合蛋白。The present invention also relates to a conjugate, which is the above B7H3 antibody or its antigen-binding fragment combined with a therapeutic agent or a detection agent, or a fusion protein as described above.
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***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。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. In some embodiments, therapeutic agents used in accordance with the present invention may have biological activity associated with treating one or more symptoms or causes of cancer. In some embodiments, 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. In some embodiments, therapeutic agents used in accordance with the present invention have one or more additional activities.
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。 In some embodiments of the invention, the conjugated therapeutic agent is a radioisotope, drug conjugate, nanoparticle, immunotoxin, or any other therapeutic load.
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。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 ). In some embodiments of the invention, the combined detection agent is a diagnostic or imaging agent.
药物组合物、医药用途与治疗方法Pharmaceutical compositions, medicinal uses and treatment methods
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，或如上所述的融合蛋白，或如上所述的偶联物。The present invention also relates to a pharmaceutical composition comprising the B7H3 antibody or antigen-binding fragment thereof as described above, or the fusion protein as described above, or the conjugate as described above.
本发明的B7H3抗体或其抗原结合片段或融合蛋白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。The B7H3 antibody or its antigen-binding fragment or fusion protein or conjugate of the present invention can be used to prepare pharmaceutical compositions or sterile compositions, for example, any of them and pharmaceutically acceptable carriers, excipients or stabilizer mix.
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。The term "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. Specific examples of some substances that can serve as 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 acids (e.g., glycine, glutamine, asparagine, histidine, arginine, or lysine); monosaccharides, disaccharides, and other carbohydrates (including, for example, glucose, mannose, or dextran); chelating agents (e.g., EDTA); sugars (e.g., sucrose, mannitol, trehalose, or sorbitol); salt-forming counterions; metal complexes; and/or nonionic surfactants (e.g., including TWEENTM, PLURONICSTM, or polyethylene glycol). In addition, according to the formulation method, commonly used fillers, diluents, binding agents, humectants, disintegrants, and/or surfactants can be appropriately selected by those of ordinary skill in the art.
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***(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。The activator of the antibody or functional fragment thereof in the pharmaceutical composition can be contained in microcapsules, Or contained in colloidal drug delivery systems (such as liposomes, albumin globules, microemulsions, nanoparticles and nanocapsules), or contained in macroemulsions (macroemulsions), the microcapsules can be processed by, for example, coacervation ( coacervation) technology or interfacial polymerization, examples include hydroxymethylcellulose or gelatin microcapsules and poly-(methyl methacrylate) microcapsules respectively.
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***、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。The pharmaceutical compositions of the present invention may be administered by any route, as will be understood by those skilled in the art. In some embodiments, 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. using powders, ointments, creams, gels, lotions and/or drops), Mucosal, intranasal, intrabuccal, enteral, vitreous, sublingual; by intratracheal instillation, bronchial instillation and/or inhalation; administered as oral spray, nasal spray and/or aerosol and/or via portal vein catheter.
本发明还涉及如上所述的B7H3抗体或其抗原结合片段，或如上所述的融合蛋白，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***的药物中的应用。The present invention also relates to the B7H3 antibody or antigen-binding fragment thereof as described above, or the fusion protein as described above, or the conjugate as described above in the preparation of medicaments for treating B7H3-positive cancer or for regulating the immune system. application.
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)、***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***。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. 14: 5150-157; Boorjian., et al., (2008), Clin Cancer Res. 14: 4800-4808), glioblastoma (Lemke., et al., (2012), Clin Cancer Res. 18:105-117), osteosarcoma (Wang., et al., (2013), PLoS One. 8:e70689), neuroblastoma (Gregorio., et al., (2008), Histopathology. 53:73-80), diffuse intrinsic pontine glioma (DIPG) (Zhou., et al. , (2013), J. Neurooncol. 111:257-264), mesothelioma (Calabro., et al., (2011), J. Cell Physiol. 226: 2595-600) and pancreatic cancer (Yamato., et al., (2009), Br.J.Cancer.101:1709-1716). In some preferred embodiments, B7H3 Positive cancers were selected from neuroblastoma and cervical cancer.
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)。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) . After 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). 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. 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).
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、***、***、淋巴液、口腔、咽、食管、胃、十二指肠、小肠、结肠、直肠、***、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、***、外***、阴囊、睾丸、输精管、***、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、***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、***、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。The term 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 arising from lesions in any of the body, pancreatic islets, thymus, gonads, sublingual glands, and parotid glands. Specific examples 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, embryo with multilayered rosettes Tumors, preferably cancers expressing B7H3.
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，或如上所述的融合蛋白，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。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 fusion protein as described above, or the steps of the conjugate as described above, and wherein the medical condition is characterized by expression of the B7H3 antigen.
优选其中所述医学状况为B7H3阳性癌症或免疫***相关疾病。Preferably the medical condition is B7H3 positive cancer or an immune system related disease.
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。The phrase "safe and effective amount." As used herein, 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.
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。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.
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。The embodiments of the present invention will be described in detail below with reference to examples. It should be understood that these examples are only used to illustrate the invention and are not intended to limit the scope of the invention. For experimental methods that do not indicate specific conditions in the following examples, priority is given to the guidelines given in the present invention. You can also follow the experimental manuals or conventional conditions in the field. You can also refer to other experimental methods known in the field, or according to the manufacturing methods. Conditions recommended by the manufacturer.
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。In the following specific examples, 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.
本发明将专利US20020102264中的鼠源8H9抗体(Omburtamab)进行了人源化改造；在显著提高抗体人源化程度的同时，保持了抗体的活性。双功能抗体B7H3/4-1BBab、B7H3/VEGF-Trap和B7H3/SIPRα中的抗体或受 体分子均保留了各自的活性和功能。与8H9单克隆抗体相比，本发明构建的双功能抗体在促进人的免疫细胞产生和分泌细胞因子方面，表现出比更高的生物活性。本发明中的B7H3对照抗体MGA271(Enoblituzumab)来自专利US20180134790；CD47的对照抗体1F8来自专利CN201980001915；PD-L1-VEGF-Trap中PD-L1抗体序列来自专利CN201911023312。The present invention humanizes the mouse 8H9 antibody (Omburtamab) in patent US20020102264; while significantly improving the degree of humanization of the antibody, the activity of the antibody is maintained. Antibodies among the bifunctional antibodies B7H3/4-1BBab, B7H3/VEGF-Trap and B7H3/SIPRα may be affected by All molecules retain their respective activities and functions. Compared with the 8H9 monoclonal antibody, the bifunctional antibody constructed in the present invention shows higher biological activity in promoting the production and secretion of cytokines by human immune cells. The B7H3 control antibody MGA271 (Enoblituzumab) in the present invention comes from patent US20180134790; the CD47 control antibody 1F8 comes from patent CN201980001915; the PD-L1 antibody sequence in PD-L1-VEGF-Trap comes from patent CN201911023312.
实施例1Example 1
8H9抗体的人源化Humanization of 8H9 Antibody
8H9杂交瘤抗体的重链可变区序列为SEQ ID NO：22，轻链可变区序列为SEQ ID NO：23。采用互补决定簇嫁接法进行8H9杂交瘤抗体的人源化改造。首先，在IMGT数据库中分别搜寻与鼠源抗体的轻、重链可变区序列同源性最高的人胚系抗体(germline antibody)序列。重链可变区人源化选取IGHV1-18*01和IGHV1-8*01，抗体轻链可变区人源化选取的胚系为IGKV1-39*01和IGKV6-21*02。保留鼠源抗体的CDR区，将鼠源抗体的框架区(framework)序列用人胚系抗体的框架区序列置换。建立鼠源抗体的结构模型，逐个对比人源抗体与相应鼠源抗体框架区中每个位点的氨基酸，如果框架区的某个位点采用人的氨基酸序列没有导致CDR区域空间结构的破坏或改变，则该位点使用人的氨基酸序列，否则在该位点使用对应的鼠源序列(即回复突变为鼠源序列)。The heavy chain variable region sequence of the 8H9 hybridoma antibody is SEQ ID NO: 22, and the light chain variable region sequence is SEQ ID NO: 23. The complementary determinant grafting method was used to humanize the 8H9 hybridoma antibody. First, the IMGT database was searched for human germline antibody (germline antibody) sequences with the highest homology to the light and heavy chain variable region sequences of mouse antibodies. IGHV1-18*01 and IGHV1-8*01 were selected for humanization of the heavy chain variable region, and IGKV1-39*01 and IGKV6-21*02 were selected for humanization of the antibody light chain variable region. The CDR region of the murine antibody is retained, and the framework sequence of the murine antibody is replaced with the framework sequence of the human germline antibody. Establish a structural model of the mouse antibody, and compare the amino acids at each position in the framework region of the human antibody and the corresponding mouse antibody one by one. If the use of a human amino acid sequence at a certain position in the framework region does not lead to the destruction of the spatial structure of the CDR region or If the amino acid sequence is changed, the human amino acid sequence will be used at this site; otherwise, the corresponding mouse sequence will be used at this site (i.e., back mutation to the mouse sequence).
根据结构模拟，将人源化处理抗体IGHV1-18*01重链的第48位Met回复突变为Ile，第67位Val回复突变为Ala，第69位Met回复突变为Leu。将人源化处理抗体IGHV1-8*01重链的第48位Met回复突变为Ile，第67位Val回复突变为Ala，第69位Met回复突变为Leu，第71位Arg回复突变为Thr。将人源化处理抗体IGKV1-39*01的第49位Tyr回复突变为Lys，第69位Thr回复突变为Ser。将人源化处理抗体IGKV6-21*02的第4位Leu回复突变为Met，第69位Thr回复突变为Ser。 According to structural simulation, the Met at position 48 of the humanized antibody IGHV1-18*01 heavy chain was back mutated to Ile, the Val at position 67 was back mutated to Ala, and the Met at position 69 was back mutated to Leu. The Met at position 48 of the heavy chain of the humanized antibody IGHV1-8*01 was back mutated to Ile, the Val at position 67 was back mutated to Ala, the Met at position 69 was back mutated to Leu, and the Arg at position 71 was back mutated to Thr. The Tyr at position 49 of the humanized antibody IGKV1-39*01 was back mutated to Lys, and the Thr at position 69 was back mutated to Ser. The Leu at position 4 of the humanized antibody IGKV6-21*02 was back mutated to Met, and the Thr at position 69 was back mutated to Ser.
人源化处理抗体重链(germline IGHV1-18*01)的可变区氨基酸序列号为SEQ ID NO：7，人源化程度为83.7％；人源化处理抗体重链(germline IGHV1-8*01)的可变区氨基酸序列号为SEQ ID NO：8，人源化程度为85.7％；人源化处理抗体轻链(germline IGKV1-39*01)的可变区氨基酸序列号为SEQ ID NO：9，人源化程度为87.4％；人源化处理抗体轻链(germline IGKV6-21*02)的可变区氨基酸序列号为SEQ ID NO：10，人源化程度为89.5％。将以上人源化处理抗体构建为IgG1亚型。本发明中构建的抗体轻、重链人源化程度显著高于文献报道的70.5％和76.5％(Mahiuddin A,et al.,(2015)J.Biol.Chem.290,30018-29)。The variable region amino acid sequence number of the humanized antibody heavy chain (germline IGHV1-18*01) is SEQ ID NO: 7, and the degree of humanization is 83.7%; the humanized antibody heavy chain (germline IGHV1-8* 01)’s variable region amino acid sequence number is SEQ ID NO: 8, and the degree of humanization is 85.7%; the variable region amino acid sequence number of the humanized antibody light chain (germline IGKV1-39*01) is SEQ ID NO : 9, the degree of humanization is 87.4%; the amino acid sequence number of the variable region of the humanized antibody light chain (germline IGKV6-21*02) is SEQ ID NO: 10, and the degree of humanization is 89.5%. The above humanized antibodies were constructed into IgG1 subtype. The degree of humanization of the light and heavy chains of the antibodies constructed in the present invention is significantly higher than the 70.5% and 76.5% reported in the literature (Mahiuddin A, et al., (2015) J. Biol. Chem. 290, 30018-29).
合成人源化处理抗体重链和轻链的核酸序列，并***到表达载体pcDNA3.1。用0.1mg抗体轻链和0.1mg抗体重链表达质粒共转染200mL的293细胞(细胞密度为1×106/mL)，在37℃摇瓶振摇培养6天，离心收集上清液，用Protein A纯化人源化处理抗体，纯化后的人源化处理抗体进行活性检测。8H9人源化处理抗体重链(germline IGHV1-18*01)与轻链(germline IGKV6-21*02)组合为hu8H9-V1(Version1)，重链(germline IGHV1-18*01)与轻链(germline IGKV1-39*01)组合为hu8H9-V2(Version2)，重链(germline IGHV1-8*01)与轻链(germline IGKV6-21*02)组合为hu8H9-V3(Version3)，重链(germline IGHV1-8*01)与轻链(germline IGKV1-39*01)组合为hu8H9-V4(Version4)。发明人还在hu8H9-V4的基础上将重链可变区的第102位的丙氨酸突变为甘氨酸，得到hu8H9-V4(VH-A102G)，以增强抗体的稳定性。The nucleic acid sequences of the humanized antibody heavy chain and light chain were synthesized and inserted into the expression vector pcDNA3.1. 200 mL of 293 cells (cell density: 1×10 6 /mL) were co-transfected with 0.1 mg of antibody light chain and 0.1 mg of antibody heavy chain expression plasmids, cultured with shaking in a shaker flask at 37°C for 6 days, and the supernatant was collected by centrifugation. The humanized antibody was purified with Protein A, and the activity of the purified humanized antibody was tested. The combination of 8H9 humanized antibody heavy chain (germline IGHV1-18*01) and light chain (germline IGKV6-21*02) is hu8H9-V1 (Version1), and the combination of heavy chain (germline IGHV1-18*01) and light chain ( The combination of germline IGKV1-39*01) is hu8H9-V2 (Version2), the combination of heavy chain (germline IGHV1-8*01) and light chain (germline IGKV6-21*02) is hu8H9-V3 (Version3), the combination of heavy chain (germline The combination of IGHV1-8*01) and light chain (germline IGKV1-39*01) is hu8H9-V4 (Version4). The inventor also mutated alanine at position 102 of the heavy chain variable region to glycine on the basis of hu8H9-V4 to obtain hu8H9-V4 (VH-A102G) to enhance the stability of the antibody.
实施例2Example 2
人源化8H9抗体(hu8H9)与B7H3抗原结合的ELISA检测。ELISA detection of humanized 8H9 antibody (hu8H9) binding to B7H3 antigen.
用50μL B7H3-his(终浓度：2μg/mL)包被96孔ELISA板(Corning,Cat.No.:9018)，室温过夜；用洗涤缓冲液(PBS+0.05％Tween20)洗涤3次后，加入封闭缓冲液(PBS+2％BSA(Sigma,Cat.No.:V90093))室温孵育1小时，用洗涤缓冲液洗涤ELISA板3次；加入50μL不同浓度的抗体， 室温孵育1小时，洗涤3次；每孔加入50μL HRP偶联羊抗鼠IgG二抗(Thermo,Cat.No.:31432)，室温避光孵育1小时，洗涤3次；每孔加入100μL TMB(北京百奥赛博，Cat.No.:ES-002)，室温孵育显色2分钟，加入100μL/孔的终止液(2N H2SO4)终止显色反应，用酶标仪(Tecan Spark)读取各孔OD450数值。结果如图1所示，与嵌合抗体相比，人源化8H9抗体及其突变体保持了对B7H3较高的亲和力。Coat a 96-well ELISA plate (Corning, Cat. No.: 9018) with 50 μL B7H3-his (final concentration: 2 μg/mL) and keep overnight at room temperature; wash 3 times with washing buffer (PBS+0.05% Tween20), and then add Incubate in blocking buffer (PBS+2% BSA (Sigma, Cat. No.: V90093)) at room temperature for 1 hour, wash the ELISA plate three times with washing buffer; add 50 μL of antibodies of different concentrations. Incubate at room temperature for 1 hour and wash 3 times; add 50 μL HRP-conjugated goat anti-mouse IgG secondary antibody (Thermo, Cat. No.: 31432) to each well, incubate at room temperature for 1 hour in the dark, and wash 3 times; add 100 μL TMB ( Beijing Biocyber, Cat. No.: ES-002), incubate at room temperature for 2 minutes to develop color, add 100 μL/well of stop solution (2N H 2 SO 4 ) to terminate the color reaction, and use a microplate reader (Tecan Spark) Read the OD 450 value of each well. The results are shown in Figure 1. Compared with chimeric antibodies, the humanized 8H9 antibody and its mutants maintained higher affinity for B7H3.
实施例3Example 3
人源化8H9抗体(hu8H9)对PBMC的激活作用Activation effect of humanized 8H9 antibody (hu8H9) on PBMC
测试hu8H9对人PBMC细胞分泌细胞因子的影响：在96孔(Corning,Cat.No.:3799)加入用完全培养基(RPMI1640+10％FCS)重悬的PBMC细胞(TPCS，Cat.No.:PB025C)，再加入40ng/mL OKT3(eBioscience,Cat.No.:16-0037-85)，置37℃孵育72小时。活化的PBMC细胞计数后，用完全培养基重悬(2.5×105细胞/mL)。在96孔板中，每孔加入100μL PBMC细胞，50μL不同浓度的hu8H9抗体(起始浓度为20μg/mL，10倍系列稀释)，将96孔细胞培养板(Corning,Cat.No.:3599)置于37℃，5％CO2培养箱中孵育48小时，收集上清液。用IFN-γELISA试剂盒(R&D Systems,Cat.No.:DY285)检测细胞因子的浓度。结果如图2所示，人源化8H9抗体及其突变体能够显著促进PBMC细胞分泌IFN-γ，优于对照抗体MGA271。Test the effect of hu8H9 on the secretion of cytokines by human PBMC cells: add PBMC cells (TPCS, Cat. No.: PB025C), then add 40ng/mL OKT3 (eBioscience, Cat. No.: 16-0037-85), and incubate at 37°C for 72 hours. After the activated PBMC cells were counted, they were resuspended in complete culture medium (2.5 × 10 5 cells/mL). In a 96-well plate, add 100 μL of PBMC cells and 50 μL of different concentrations of hu8H9 antibodies to each well (initial concentration is 20 μg/mL, 10-fold serial dilution), and place the 96-well cell culture plate (Corning, Cat. No.: 3599) Place in a 37°C, 5% CO2 incubator and incubate for 48 hours, and collect the supernatant. The concentration of cytokines was detected using IFN-γ ELISA kit (R&D Systems, Cat. No.: DY285). The results are shown in Figure 2. Humanized 8H9 antibody and its mutants can significantly promote the secretion of IFN-γ in PBMC cells, which is better than the control antibody MGA271.
实施例4Example 4
双功能抗体hu8H9/4-1BBab的构建和功能检测Construction and functional detection of bifunctional antibody hu8H9/4-1BBab
将4-1BB人源化处理抗体设计成单链形式scFv(序列见SEQ ID NO：19)通过Linker(G4S)4连接在hu8H9-V4重链C末端，与轻链载体共转染到293细胞。在37℃摇瓶振摇培养6天，离心收集上清液，用Protein A纯化，纯化后进行活性检测。The 4-1BB humanized antibody was designed into a single-chain scFv (see SEQ ID NO: 19 for the sequence) connected to the C terminus of the hu8H9-V4 heavy chain through Linker (G 4 S) 4 , and co-transfected with the light chain vector into 293 cells. Culture in a shake flask at 37°C for 6 days, collect the supernatant by centrifugation, purify with Protein A, and conduct activity detection after purification.
双功能抗体hu8H9/4-1BBab与B7H3抗原结合的ELISA检测。具体方法参考实施例2。结果如图3所示，hu8H9/4-1BBab双功能抗体对B7H3具 有较高的亲和力。ELISA detection of the binding of bifunctional antibody hu8H9/4-1BBab to B7H3 antigen. Please refer to Example 2 for specific methods. The results are shown in Figure 3. The hu8H9/4-1BBab bifunctional antibody is effective against B7H3. Have higher affinity.
采用报告基因方法检测双功能抗体hu8H9/4-1BBab对4-1BB信号通路的激活作用。在96孔板中加入50μl(5×104细胞/孔)Jurkat-4-1BB-NFkB-luc细胞以及50μl不同浓度的抗体，再加入Jurkat-B7H3细胞(2×104细胞/孔)，混合后置37℃孵育4小时。加入25μl Bright Glo(Promega,Cat No:E2620)室温孵育5分钟，用Tecan Spark酶标仪测定各样品的化学发光信号。结果如图4所示，在Jurkat-B7H3细胞存在时，双功能抗体hu8H9/4-1BBab能显著激活4-1BB信号通路。A reporter gene method was used to detect the activation effect of the bifunctional antibody hu8H9/4-1BBab on the 4-1BB signaling pathway. Add 50 μl (5×10 4 cells/well) Jurkat-4-1BB-NFkB-luc cells and 50 μl antibodies of different concentrations to the 96-well plate, then add Jurkat-B7H3 cells (2×10 4 cells/well), and mix Post-incubate at 37°C for 4 hours. Add 25 μl Bright Glo (Promega, Cat No: E2620) and incubate at room temperature for 5 minutes, and measure the chemiluminescence signal of each sample using a Tecan Spark microplate reader. The results are shown in Figure 4. In the presence of Jurkat-B7H3 cells, the bifunctional antibody hu8H9/4-1BBab can significantly activate the 4-1BB signaling pathway.
双功能抗体hu8H9/4-1BBab对人PBMC细胞分泌细胞因子的影响，具体方法参考实施例3。如图5所示，相较于单抗，双功能抗体hu8H9/4-1BBab能够显著促进PBMC细胞IFN-γ的分泌。The effect of the bifunctional antibody hu8H9/4-1BBab on the secretion of cytokines by human PBMC cells, please refer to Example 3 for the specific method. As shown in Figure 5, compared with monoclonal antibodies, the bifunctional antibody hu8H9/4-1BBab can significantly promote the secretion of IFN-γ in PBMC cells.
实施例5Example 5
双功能抗体hu8H9/VEGF-Trap的构建和功能检测Construction and functional detection of bifunctional antibody hu8H9/VEGF-Trap
将VEGF-Trap序列(SEQ ID NO：20)通过Linker(G4S)4连接在hu8H9-V4重链C末端，与轻链载体共转染到293细胞。在37℃摇瓶振摇培养6天，离心收集上清液，用Protein A纯化，纯化后进行活性检测。The VEGF-Trap sequence (SEQ ID NO: 20) was connected to the C terminus of the hu8H9-V4 heavy chain through Linker (G 4 S) 4 , and was co-transfected with the light chain vector into 293 cells. Culture in a shake flask at 37°C for 6 days, collect the supernatant by centrifugation, purify with Protein A, and conduct activity detection after purification.
双功能抗体hu8H9/VEGF-Trap与B7H3结合的ELISA检测，具体方法参考实施例2。如图6所示，hu8H9/VEGF-Trap双功能抗体对B7H3仍具有较高的亲和力。ELISA detection of the binding of bifunctional antibody hu8H9/VEGF-Trap to B7H3. Please refer to Example 2 for the specific method. As shown in Figure 6, the hu8H9/VEGF-Trap bifunctional antibody still has a high affinity for B7H3.
采用报告基因方法检测双功能抗体hu8H9/VEGF-Trap对VEGF-NFAT信号通路的抑制作用。将50μl不同浓度的抗体与50μl 40ng/ml VEGF蛋白预孵育1小时。将50μl混合物加入50μl(5×104细胞/孔)293T-VEGFR-NFAT-luc细胞后，置37℃孵育4小时。加入25μl Bright Glo(Promega,Cat No:E2620)室温孵育5分钟，用Tecan Spark酶标仪测定各样品的化学发光信号。如图7所示，双功能抗体hu8H9/VEGF-Trap能够显著抑制NFAT信号通路。 A reporter gene method was used to detect the inhibitory effect of the bifunctional antibody hu8H9/VEGF-Trap on the VEGF-NFAT signaling pathway. 50 μl of different concentrations of antibodies were preincubated with 50 μl of 40 ng/ml VEGF protein for 1 hour. After adding 50 μl of the mixture to 50 μl (5×10 4 cells/well) 293T-VEGFR-NFAT-luc cells, incubate at 37°C for 4 hours. Add 25 μl Bright Glo (Promega, Cat No: E2620) and incubate at room temperature for 5 minutes, and measure the chemiluminescence signal of each sample using a Tecan Spark microplate reader. As shown in Figure 7, the bifunctional antibody hu8H9/VEGF-Trap can significantly inhibit the NFAT signaling pathway.
双功能抗体hu8H9/VEGF-Trap对人PBMC细胞分泌细胞因子的影响，具体方法参考实施例3。如图8所示，双功能抗体hu8H9/VEGF-Trap能够显著促进PBMC细胞分泌IFN-γ。The effect of the bifunctional antibody hu8H9/VEGF-Trap on the secretion of cytokines by human PBMC cells, please refer to Example 3 for the specific method. As shown in Figure 8, the bifunctional antibody hu8H9/VEGF-Trap can significantly promote the secretion of IFN-γ by PBMC cells.
实施例6Example 6
双功能抗体hu8H9/SIPRα的构建和功能检测。Construction and functional detection of bifunctional antibody hu8H9/SIPRα.
将SIPRα序列(见SEQ ID NO：21)通过Linker(G4S)4连接在人源化处理抗体hu8H9-V1重链C末端，与轻链载体共转染到293细胞。在37℃摇瓶振摇培养6天，离心收集上清液，用Protein A纯化，纯化后进行活性检测。The SIPRα sequence (see SEQ ID NO: 21) was connected to the C terminus of the heavy chain of humanized antibody hu8H9-V1 through Linker (G 4 S) 4 , and co-transfected with the light chain vector into 293 cells. Culture in a shake flask at 37°C for 6 days, collect the supernatant by centrifugation, purify with Protein A, and conduct activity detection after purification.
双功能抗体hu8H9/SIPRα与CD47结合的ELISA检测。用50μL CD47-mFc(终浓度：2μg/mL)包被96孔ELISA板(Corning,Cat.No.:9018)，室温过夜；用洗涤缓冲液(PBS+0.05％Tween20)洗涤3次后，加入封闭缓冲液(PBS+2％BSA(Sigma,Cat.No.:V90093))室温孵育1小时，用洗涤缓冲液洗涤ELISA板3次；加入50μL不同浓度的抗体，室温孵育1小时，洗涤3次；每孔加入50μL HRP偶联羊抗鼠IgG二抗(Thermo,Cat.No.:31432)，室温避光孵育1小时，洗涤3次；每孔加入100μL TMB(北京百奥赛博，Cat.No.:ES-002)，室温孵育显色2分钟，加入100μL/孔的终止液(2N H2SO4)终止显色反应，用酶标仪(Tecan Spark)读取各孔OD450数值。结果如图9所示，hu8H9/SIPRα双功能抗体对CD47有较高的亲和力。ELISA detection of the binding of bifunctional antibody hu8H9/SIPRα to CD47. Coat a 96-well ELISA plate (Corning, Cat. No.: 9018) with 50 μL CD47-mFc (final concentration: 2 μg/mL) and keep overnight at room temperature; wash 3 times with washing buffer (PBS+0.05% Tween20), and then add Incubate in blocking buffer (PBS+2% BSA (Sigma, Cat. No.: V90093)) at room temperature for 1 hour, wash the ELISA plate 3 times with washing buffer; add 50 μL of antibodies of different concentrations, incubate at room temperature for 1 hour, and wash 3 times ; Add 50 μL HRP-conjugated goat anti-mouse IgG secondary antibody (Thermo, Cat. No.: 31432) to each well, incubate at room temperature for 1 hour in the dark, and wash 3 times; add 100 μL TMB (Beijing Biocyber, Cat. No.: ES-002), incubate at room temperature for 2 minutes to develop color, add 100 μL/well of stop solution (2N H 2 SO 4 ) to terminate the color reaction, and read the OD 450 value of each well with a microplate reader (Tecan Spark). The results are shown in Figure 9. The hu8H9/SIPRα bifunctional antibody has a high affinity for CD47.
双功能抗体hu8H9/SIPRα阻断SIPRα与CD47结合的ELISA检测。用50μL CD47-mFc(终浓度：2μg/mL)包被96孔ELISA板(Corning,Cat.No.:9018)，室温过夜；用洗涤缓冲液(PBS+0.05％Tween20)洗涤3次后，加入封闭缓冲液(PBS+2％BSA(Sigma,Cat.No.:V90093))室温孵育1小时，用洗涤缓冲液洗涤ELISA板3次；加入50μL不同浓度的抗体，室温孵育1小时，继续向样品孔中加入50μL SIPRα-Biotin，室温孵育1小时，洗涤3次；加入二抗Avidin HRP(Invitrogen，Cat No:18-4100-51)，孵育30分钟， 洗涤3次后加入TMB显色3分钟，用100μL/孔的终止液(2N H2SO4)终止反应，用酶标仪(Tecan Spark)读取各孔OD450数值。结果如图10所示，hu8H9/SIPRα双功能抗体能有效阻断SIPRα和CD47的结合。ELISA detection of bifunctional antibody hu8H9/SIPRα blocking the binding of SIPRα to CD47. Coat a 96-well ELISA plate (Corning, Cat. No.: 9018) with 50 μL CD47-mFc (final concentration: 2 μg/mL) and keep overnight at room temperature; wash 3 times with washing buffer (PBS+0.05% Tween20), and then add Blocking buffer (PBS+2% BSA (Sigma, Cat. No.: V90093)) was incubated at room temperature for 1 hour, and the ELISA plate was washed 3 times with washing buffer; 50 μL of antibodies of different concentrations were added, incubated at room temperature for 1 hour, and continued to the sample. Add 50 μL SIPRα-Biotin to the well, incubate at room temperature for 1 hour, and wash 3 times; add secondary antibody Avidin HRP (Invitrogen, Cat No: 18-4100-51), and incubate for 30 minutes. After washing three times, TMB was added to develop color for 3 minutes. The reaction was terminated with 100 μL/well stop solution (2N H 2 SO 4 ), and the OD 450 value of each well was read with a microplate reader (Tecan Spark). The results are shown in Figure 10. The hu8H9/SIPRα bifunctional antibody can effectively block the binding of SIPRα and CD47.
双功能抗体hu8H9-SIPRα对人PBMC细胞分泌细胞因子的影响，具体方法参考实施例3。如图11所示，与hu8H9单抗相比，双功能抗体hu8H9/SIPRα对PBMC细胞分泌IFN-γ具有更强的促进作用。The effect of the bifunctional antibody hu8H9-SIPRα on the secretion of cytokines by human PBMC cells, please refer to Example 3 for the specific method. As shown in Figure 11, compared with hu8H9 monoclonal antibody, the bifunctional antibody hu8H9/SIPRα has a stronger promoting effect on the secretion of IFN-γ by PBMC cells.
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。 The above-mentioned embodiments only express several implementation modes of the present invention, and their descriptions are relatively specific and detailed, but they should not be construed as limiting the scope of the invention. It should be noted that, for those of ordinary skill in the art, several modifications and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention should be determined by the appended claims, and the description and drawings can be used to interpret the content of the claims.

Claims (22)

  1. B7H3抗体或其抗原结合片段，其包含氨基酸序列依次如SEQ ID NO:1～3所示的重链互补决定区以及氨基酸序列依次如SEQ ID NO:4～6所示的轻链互补决定区。The B7H3 antibody or its antigen-binding fragment includes a heavy chain complementarity-determining region with an amino acid sequence shown in SEQ ID NO: 1 to 3 and a light chain complementarity-determining region with an amino acid sequence shown in SEQ ID NO: 4-6.
  2. 根据权利要求1所述的B7H3抗体或其抗原结合片段，其包含氨基酸序列如SEQ ID NO:7或8所示的重链可变区，以及氨基酸序列如SEQ ID NO:9或10所示的轻链可变区。The B7H3 antibody or antigen-binding fragment thereof according to claim 1, which includes a heavy chain variable region with an amino acid sequence as shown in SEQ ID NO: 7 or 8, and an amino acid sequence as shown in SEQ ID NO: 9 or 10 Light chain variable region.
  3. 根据权利要求2所述的B7H3抗体或其抗原结合片段，所述重链可变区的第102位A突变为G。The B7H3 antibody or antigen-binding fragment thereof according to claim 2, wherein A at position 102 of the heavy chain variable region is mutated to G.
  4. 根据权利要求1～3任一项所述的B7H3抗体或其抗原结合片段，所述抗原结合片段为F(ab')2、Fab、scFv以及双特异抗体中的一种。The B7H3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antigen-binding fragment is one of F(ab') 2 , Fab, scFv, and bispecific antibodies.
  5. 根据权利要求1～3任一项所述的B7H3抗体或其抗原结合片段，其具有恒定区，重链恒定区序列选自IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE、IgD任意一种的恒定区序列；轻链恒定区为κ或λ链；所述恒定区优选是人来源的。The B7H3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, which has a constant region, and the heavy chain constant region sequence is selected from any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD. The constant region sequence; the light chain constant region is a kappa or lambda chain; the constant region is preferably of human origin.
  6. 融合蛋白，其含有权利要求1～5任一项所述的B7H3抗体或其抗原结合片段。A fusion protein containing the B7H3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 5.
  7. 根据权利要求6所述的融合蛋白，其包含靶向B7H3的第一蛋白功能区以及靶向第二抗原的第二蛋白功能区；The fusion protein according to claim 6, which includes a first protein functional region targeting B7H3 and a second protein functional region targeting a second antigen;
    所述第一功能区具有权利要求1～5任一项所述的B7H3抗体或其抗原结合片段。The first functional region has the B7H3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 5.
  8. 根据权利要求7所述的融合蛋白，所述第二蛋白功能区选自4-1BB抗体或其抗原结合片段、VEGF-Trap全长或其片段以及SIRPα全长或其片段。According to the fusion protein of claim 7, the second protein functional region is selected from the group consisting of 4-1BB antibody or its antigen-binding fragment, VEGF-Trap full length or its fragment, and SIRPα full length or its fragment.
  9. 根据权利要求8所述的融合蛋白，所述4-1BB抗体或其抗原结合片 段包含氨基酸序列依次如SEQ ID NO:11～13所示的重链互补决定区H-CDR1、H-CDR2、H-CDR3，以及氨基酸序列依次如SEQ ID NO:14～16所示的轻链互补决定区L-CDR1、L-CDR2、L-CDR3。The fusion protein according to claim 8, the 4-1BB antibody or its antigen-binding fragment The segment includes the heavy chain complementarity determining regions H-CDR1, H-CDR2, and H-CDR3 whose amino acid sequences are shown in SEQ ID NO: 11 to 13 in sequence, and the light chain whose amino acid sequence is shown in SEQ ID NO: 14 to 16 in sequence. Complementarity determining regions L-CDR1, L-CDR2, L-CDR3.
  10. 根据权利要求9所述的融合蛋白所述4-1BB抗体或其抗原结合片段包含氨基酸序列如SEQ ID NO:17所示的重链可变区，以及氨基酸序列如SEQ ID NO:18所示的轻链可变区，优选其为SEQ ID NO:19所示的scFv。The 4-1BB antibody or antigen-binding fragment thereof of the fusion protein according to claim 9 comprises the heavy chain variable region with an amino acid sequence as shown in SEQ ID NO:17, and an amino acid sequence as shown in SEQ ID NO:18 The light chain variable region is preferably the scFv shown in SEQ ID NO: 19.
  11. 根据权利要求8所述的融合蛋白，所述VEGF-Trap片段的氨基酸序列如SEQ ID NO:20所示。According to the fusion protein of claim 8, the amino acid sequence of the VEGF-Trap fragment is shown in SEQ ID NO: 20.
  12. 根据权利要求8所述的融合蛋白，所述SIRPα片段的氨基酸序列如SEQ ID NO:21所示。According to the fusion protein of claim 8, the amino acid sequence of the SIRPα fragment is shown in SEQ ID NO: 21.
  13. 根据权利要求7～12任一项所述的融合蛋白，所述第一蛋白功能区具有的抗体重链，且重链的C端和所述第二蛋白功能区的N端通过连接肽连接。According to the fusion protein according to any one of claims 7 to 12, the first protein functional region has an antibody heavy chain, and the C-terminus of the heavy chain and the N-terminus of the second protein functional region are connected through a connecting peptide.
  14. 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，或权利要求6～13任一项所述的融合蛋白。An isolated nucleic acid encoding the B7H3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, or the fusion protein according to any one of claims 6 to 13.
  15. 载体，其包含权利要求14所述的核酸。A vector comprising the nucleic acid of claim 14.
  16. 宿主细胞，其包含权利要求14所述的核酸，或被权利要求15所述的载体所转化。A host cell comprising the nucleic acid of claim 14 or transformed with the vector of claim 15.
  17. 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，或权利要求6～13任一项所述的融合蛋白的方法，包括在合适的条件下培养权利要求16所述的宿主细胞，以及从细胞培养物中回收目的产物。The method for preparing the B7H3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, or the fusion protein according to any one of claims 6 to 13, comprising culturing the antibody according to claim 16 under appropriate conditions. host cells, and recovery of the product of interest from cell culture.
  18. 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，或权利要求6～13任一项所述的融合蛋白。A conjugate, which is the B7H3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, or the fusion protein according to any one of claims 6 to 13, bound to a therapeutic agent or a detection agent.
  19. 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6～13任一项所述的融合蛋白，或权利要求18所 述的偶联物。A pharmaceutical composition comprising the B7H3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, or the fusion protein according to any one of claims 6 to 13, or the fusion protein according to claim 18 the above-mentioned conjugates.
  20. 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6～13任一项所述的融合蛋白，或权利要求18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***的药物中的应用。The B7H3 antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, or the fusion protein according to any one of claims 6 to 13, or the conjugate according to claim 18 for use in the treatment of B7H3 positive cancer or use of drugs to modulate the immune system.
  21. 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权利要求1～5任一项所述的B7H3抗体或抗原结合片段，或权利要求6～13任一项所述的融合蛋白，或权利要求18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。A method of treating, preventing, alleviating and/or diagnosing a medical condition in a subject, comprising administering a safe and effective amount of the B7H3 antibody or antigen-binding fragment of any one of claims 1 to 5, or claims 6 to The fusion protein of any one of 13, or the steps of the conjugate of claim 18, and wherein the medical condition is characterized by expression of the B7H3 antigen.
  22. 根据权利要求21所述的方法，其中所述医学状况为B7H3阳性癌症或免疫***相关疾病。 The method of claim 21, wherein the medical condition is B7H3-positive cancer or an immune system-related disease.
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