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WO2023179392A1 - Anticorps b7h3 et anticorps bifonctionnel le comprenant - Google Patents

Anticorps b7h3 et anticorps bifonctionnel le comprenant 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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PCT/CN2023/081037
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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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    • C07K16/00—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
    • 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
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • C07K—PEPTIDES
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • 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
    • 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
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/46—Hybrid immunoglobulins
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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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    • C07K2317/00—Immunoglobulins specific features
    • 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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    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • 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
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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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    • C07K2317/00—Immunoglobulins specific features
    • 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)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • 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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    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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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    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
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    • C07K—PEPTIDES
    • 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

L'invention concerne un anticorps B7H3 et un anticorps bifonctionnel le comprenant. L'anticorps monoclonal 8H9 de B7H3 est humanisé, et l'anticorps humanisé peut bloquer de manière efficace l'effet immunosuppresseur provoqué par B7H3. De plus, une protéine de fusion est construite à l'aide de l'anticorps B7H3 après que celle-ci a été soumise à un traitement d'humanisation ou le fragment de liaison à l'antigène de celui-ci conjointement avec un anticorps monoclonal de 4-1BB, une protéine piège de VEGF et une protéine SIPR α, de telle sorte que l'effet d'activation de l'anticorps sur des cellules immunitaires est en outre amélioré. L'invention concerne l'utilisation de l'anticorps dans la préparation de médicaments pertinents utilisés pour inhiber des cellules cancéreuses, réguler l'effet et le niveau de B7H3 et améliorer l'immunité du corps. L'anticorps a de larges perspectives d'application en termes de médicaments associés au traitement de cancers.
PCT/CN2023/081037 2022-03-25 2023-03-13 Anticorps b7h3 et anticorps bifonctionnel le comprenant WO2023179392A1 (fr)

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