WO2024001470A1 - 靶向b7-h3的抗体及其用途 - Google Patents

靶向b7-h3的抗体及其用途 Download PDF

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WO2024001470A1
WO2024001470A1 PCT/CN2023/090397 CN2023090397W WO2024001470A1 WO 2024001470 A1 WO2024001470 A1 WO 2024001470A1 CN 2023090397 W CN2023090397 W CN 2023090397W WO 2024001470 A1 WO2024001470 A1 WO 2024001470A1
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seq
antibody
cells
cdr
cancer
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French (fr)
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李国坤
张静
孙慧芳
任江涛
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南京北恒生物科技有限公司
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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
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    • A61K39/0005—Vertebrate antigens
    • A61K39/0011—Cancer antigens
    • A61K39/001102—Receptors, cell surface antigens or cell surface determinants
    • A61K39/001111—Immunoglobulin superfamily
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    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
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    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
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    • C12N15/62—DNA sequences coding for fusion proteins
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    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • 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
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/515—Animal cells
    • A61K2039/5158—Antigen-pulsed cells, e.g. T-cells
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    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • 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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    • C07—ORGANIC CHEMISTRY
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    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
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    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
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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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    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
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    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
    • G01N2333/70503—Immunoglobulin superfamily, e.g. VCAMs, PECAM, LFA-3
    • G01N2333/70532—B7 molecules, e.g. CD80, CD86

Definitions

  • the invention belongs to the field of immunotherapy. More specifically, the present invention relates to antibodies targeting B7-H3 and their use in the prevention and/or treatment and/or diagnosis of disease.
  • B7-H3 (B7homolog 3protein), also known as CD276, is an important immune checkpoint molecule of the B7-CD28 family. As a T cell co-inhibitory molecule, B7-H3 can effectively inhibit the functions of T cells and NK cells; however, B7-H3 also has some costimulatory functions.
  • B7-H3 is expressed in a variety of malignant tumors and is closely related to the growth, metastasis, recurrence, and poor prognosis of malignant tumors.
  • B7-H3 can downregulate the immune response mediated by T helper type 1, inhibit CD4 + T cell activation, and inhibit the production of cytokines, and thus may play a role in promoting immune escape of cancer cells.
  • B7-H3 is abnormally highly expressed in a variety of cancer cells or tissues, including gastric cancer, lung cancer, prostate cancer, kidney cancer, pancreatic cancer, ovarian cancer, breast cancer, endometrial cancer, liver cancer, colorectal cancer, oral cancer, and bladder cancer Cancer, osteosarcoma and hematological malignant diseases.
  • the present invention aims to provide an antibody targeting B7-H3 and its use in disease prevention and/or treatment and/or diagnosis.
  • the invention provides an antibody or antigen-binding fragment thereof targeting B7-H3, comprising:
  • CDR-L1 as shown in SEQ ID NO: 1, CDR-L2 as shown in SEQ ID NO: 2, CDR-L3 as shown in SEQ ID NO: 3, and CDR-L3 as shown in SEQ ID NO: 4 CDR-H1, CDR-H2 as shown in SEQ ID NO: 5 and CDR-H3 as shown in SEQ ID NO: 6;
  • CDR-L1 as shown in SEQ ID NO: 10 as shown in SEQ ID CDR-L2 shown in NO: 11, CDR-L3 shown in SEQ ID NO: 12, CDR-H1 shown in SEQ ID NO: 13, CDR-H2 shown in SEQ ID NO: 14 and CDR-H2 shown in SEQ ID NO: 14.
  • CDR-L1 as shown in SEQ ID NO: 37 CDR-L2 as shown in SEQ ID NO: 38, CDR-L3 as shown in SEQ ID NO: 39, and CDR-L3 as shown in SEQ ID NO: 40
  • CDR-H1 as shown in SEQ ID NO: 41, CDR-H2 as shown in SEQ ID NO: 41 and CDR-H3 as shown in SEQ ID NO: 42 CDR-H1 as shown in SEQ ID NO: 41, CDR-H2 as shown in SEQ ID NO: 41 and CDR-H3 as shown in SEQ ID NO: 42.
  • the antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region is identical to one selected from the group consisting of SEQ ID NO: 8, 17, 26, 35,
  • the amino acid sequences of 44, 47, 50, 53, 56, 59, 62 and 65 have at least 90% identity or are selected from the group consisting of SEQ ID NO: 8, 17, 26, 35, 44, 47, 50, 53, 56 , 59, 62 and 65 have one or several amino acid modifications (for example, up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids) compared to the amino acid sequences of 59, 62 and 65; the light chain may
  • the variable region has at least 90% identity with an amino acid sequence selected from SEQ ID NO: 7, 16, 25, 34, 43, 46, 49, 52, 55, 58, 61 and 64, or with an amino acid sequence selected from SEQ ID NO: 7, 16, 25, 34, 43, 46, 49, 52, 55, 58, 61 and 64 have one or more
  • the antibody of the invention or an antigen-binding fragment thereof comprises a heavy chain selected from the group consisting of SEQ ID NO: 8, 17, 26, 35, 44, 47, 50, 53, 56, 59, 62 and 65
  • the antibody or antigen-binding fragment thereof of the invention is a murine antibody, a chimeric antibody, a humanized antibody or a human antibody, preferably a humanized antibody.
  • the antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region and a light chain variable region selected from:
  • the heavy chain variable region and the light chain variable region have at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity;
  • the heavy chain variable region and light chain variable region have one or several amino acid modifications compared to the heavy chain variable region and light chain variable region of any group of (a)-(l) , such as modifications of up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids; preferably, the modifications are conservative modifications, such as conservative substitutions, additions and deletions of amino acids.
  • the antibody or antigen-binding fragment thereof of the invention has at least an amino acid sequence selected from the group consisting of SEQ ID NO: 9, 18, 27, 36, 45, 48, 51, 54, 57, 60, 63 and 66. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity, or selected from SEQ ID NO: 9, 18, 27, 36 , 45, 48, 51, 54, 57, 60, 63 and 66 have one or several amino acids (e.g. up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10). amino acid) modification.
  • the modifications are conservative modifications, such as conservative substitutions, additions and deletions of amino acids.
  • the amino acid sequence of the antibody or antigen-binding fragment thereof of the invention is selected from SEQ ID NO: 9, 18, 27, 36, 45, 48, 51, 54, 57, 60, 63 and 66.
  • the invention also provides a multispecific antibody (preferably a bispecific antibody or a trispecific antibody), which comprises the anti-B7-H3 antibody or antigen-binding fragment thereof as described above, and one or more A second antibody or antigen-binding fragment thereof that specifically binds to other antigens.
  • a multispecific antibody preferably a bispecific antibody or a trispecific antibody
  • the antibodies or antigen-binding fragments thereof described in the invention can be in any antibody or antibody fragment form, such as full-length antibodies, Fab, Fab', (Fab') 2 , Fv, scFv, scFv-scFv , microantibodies, diabodies or sdAb.
  • the present invention also provides nucleic acid molecules encoding the above-mentioned antibodies or antigen-binding fragments thereof, or the above-mentioned multispecific antibodies.
  • the present invention also provides vectors comprising nucleic acid molecules encoding the above-mentioned antibodies or antigen-binding fragments thereof or multispecific antibodies, and host cells expressing the anti-B7-H3 antibodies or antigen-binding fragments thereof or multispecific antibodies.
  • the present invention also provides a chimeric polypeptide, which includes the anti-B7-H3 antibody of the present invention or an antigen-binding fragment thereof or a multispecific antibody, and the chimeric polypeptide is selected from the group consisting of CAR, TCR, TRuC, TAC or ImmTAC.
  • the chimeric polypeptide further comprises a transmembrane domain and an intracellular signaling domain; the intracellular signaling domain includes a primary signaling domain and/or a costimulatory domain.
  • the transmembrane domain is selected from the transmembrane domain of the following proteins: TCR ⁇ chain, TCR ⁇ chain, TCR ⁇ chain, TCR ⁇ chain, CD3 ⁇ subunit, CD3 ⁇ subunit, CD3 ⁇ subunit, CD3 ⁇ subunit, CD45, CD4, CD5 , CD8 ⁇ , CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137 and CD154;
  • the primary signaling domain is selected from the intracellular region of the following proteins: FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD22, CD79a, CD79b and CD66d;
  • the costimulatory domain is selected from the costimulatory signaling domain of the following proteins: TLR1, TLR1, T
  • the invention also provides nucleic acid molecules encoding chimeric antigen receptors targeting B7-H3 as defined above, as well as vectors comprising said nucleic acid molecules, and vectors comprising the B7-H3-targeting chimeric antigen receptors as defined above.
  • Chimeric antigen receptor cells preferably, the cells are selected from immune cells, such as T cells, NK cells, NKT cells, macrophages, dendritic cells, more preferably, the cells are selected from CD4 + CD8 + T cells, CD4 + T cells, CD8 + T cells, CD4 ⁇ CD8 ⁇ T cells, tumor infiltrating cells, memory T cells, naive T cells, ⁇ -T cells, and ⁇ -T cells.
  • the engineered immune cells further comprise a second chimeric antigen receptor targeting other tumor antigens.
  • the present invention also provides an antibody conjugate, which comprises an anti-B7-H3 antibody or an antigen-binding fragment thereof as defined in the present invention and a second functional structure, wherein the second functional structure is selected from From Fc, radioisotopes, half-life extending moieties, detectable markers and drugs.
  • the half-life extending moiety is selected from: the half-life extending moiety is selected from the binding structure of albumin, the binding structure of transferrin, polyethylene glycol molecules, recombinant polyethylene glycol molecules, Human serum albumin, fragments of human serum albumin and white polypeptides (including antibodies) that bind human serum albumin.
  • the detectable label is selected from the group consisting of fluorophores, chemiluminescent compounds, bioluminescent compounds, enzymes, antibiotic resistance genes, and contrast agents.
  • the drug is selected from the group consisting of cytotoxic and immunomodulatory agents.
  • the present invention also provides a detection kit, which contains the anti-B7-H3 antibody or antigen-binding fragment thereof, multispecific antibody, antibody conjugate, chimeric antigen receptor or Engineered immune cells.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the anti-B7-H3 antibody or antigen-binding fragment thereof, chimeric antigen receptor, multispecific antibody, engineered immune cell or An antibody conjugate, and one or more pharmaceutically acceptable excipients.
  • the present invention also provides a method for treating and/or preventing and/or diagnosing diseases related to B7-H3 expression, comprising administering to a subject an anti-B7-H3 antibody or an antigen thereof as described above. Binding fragments, chimeric antigen receptors, multispecific antibodies, antibody conjugates, engineered immune cells or pharmaceutical compositions.
  • antibody has the broadest meaning as understood by those skilled in the art and includes monoclonal antibodies (including intact antibodies), polyclonal antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies ), and an antibody fragment or synthetic polypeptide carrying one or more CDR sequences capable of exhibiting the desired biological activity.
  • the antibodies of the present invention can be of any type (such as IgG, IgE, IgM, IgD, IgA, etc.) or subclass (such as IgG1, IgG2, IgG2a, IgG3, IgG4, IgA1, IgA2, etc.).
  • antigen-binding fragment or "antibody fragment” refers to one or more fragments of an antibody that retain the ability to specifically bind an antigen. It has been shown that the antigen-binding function of antibodies can be achieved by fragments of full-length antibodies.
  • antibody fragments in the present invention include, but are not limited to: Fab, Fab', F(ab')2, Fd fragment, Fd', Fv fragment, single chain antibody (scFv), disulfide-linked Fv (sdFv ), the heavy chain variable region (VH) of an antibody Or light chain variable region (VL), linear antibodies, "dibodies” with two antigen-binding sites, single-domain antibodies, nanobodies, natural ligands of the antigen or functional fragments thereof, etc. Therefore, unless the context clearly dictates otherwise, an "antibody” of the present invention encompasses an antibody fragment or antigen-binding fragment as defined above.
  • the antibodies of the invention are selected from the group consisting of IgG, Fab, Fab', F(ab') 2 , Fd, Fd', Fv, scFv, sdFv, linear antibodies and diabodies.
  • a complete antibody consists of two heavy chains and two light chains linked together by disulfide bonds, with each light chain linked to its respective heavy chain by a disulfide bond in a "Y"-shaped structure.
  • Each heavy chain contains a heavy chain variable region (VH) and a heavy chain constant region.
  • the heavy chain variable region contains three complementarity determining regions (CDR): CDR-H1, CDR-H2 and CDR-H3.
  • the heavy chain constant region The region contains three constant domains: CH1, CH2 and CH3.
  • Each light chain contains a light chain variable region (VL) and a light chain constant region.
  • the light chain variable region contains three CDRs: CDR-L1, CDR-L2 and CDR-L3.
  • the light chain constant region contains a constant structure.
  • the CDRs are separated by more conserved framework regions (FR).
  • FR conserved framework regions
  • the variable region of the heavy chain/light chain is responsible for the recognition and binding of the antigen, while the constant region can mediate the binding of the antibody to host tissues or factors, including various cells of the immune system (such as effector cells) and the third component of the classical complement system.
  • the boundaries of a given CDR or FR may differ depending on the protocol used for identification.
  • the Kabat scheme is based on structural alignment
  • the Chothia scheme is based on structural information.
  • the numbering in both the Kabat and Chothia schemes is based on the sequence length of the most common antibody regions, where insertions are provided by intervening letters (e.g. "30a") and deletions occur in some antibodies.
  • the two schemes place certain insertions and deletions (indels) in different locations, resulting in different numbering.
  • the Contact scheme is based on the analysis of complex crystal structures and is similar in many ways to the Chothia numbering scheme.
  • the AbM scheme is a compromise between the Kabat and Chothia definitions and is based on the scheme used by Oxford Molecular's AbM antibody modeling software.
  • CDR of a given antibody or region thereof (such as a variable region thereof) encompasses CDRs defined by any of the above schemes or other known schemes.
  • CDR CDR3
  • FRs of a given antibody or region thereof encompass FRs defined by any of the above schemes or other known schemes.
  • the numbering scheme used to define the boundaries of CDRs and FRs in this article adopts the Kabat scheme.
  • Single chain antibody and “scFv” are used interchangeably herein and refer to an antibody composed of an antibody heavy chain variable region (VH) and a light chain variable region (VL) connected through a linker.
  • the optimal length and/or amino acid composition of the linker can be selected.
  • the length of the linker will significantly affect the folding and interaction of the scFv variable region. In fact, if shorter linkers are used (e.g. between 5-10 amino acids), intrachain folding can be prevented.
  • linker size and composition see, e.g., Hollinger et al., 1993 Proc Natl Acad. Sci. U.S.A. 90:6444-6448; U.S. Patent Application Publication Nos.
  • scFv can contain VH and VL linked in any order, such as VH-linker-VL or VL-linker-VH.
  • the antibody or antigen-binding fragment thereof of the invention is a murine antibody, a chimeric antibody, a humanized antibody or a human antibody, preferably a humanized antibody.
  • chimeric antibody refers to an antibody in which a portion of each heavy and light chain amino acid sequence is homologous to the corresponding sequence in an antibody from a specific species or belonging to a specific class, and the remaining segments of the chain It is homologous to the corresponding sequence of another species or belonging to another category.
  • variable regions of both the light and heavy chains are derived from the variable regions of an antibody from one species, while the constant regions are homologous to sequences of an antibody from another species.
  • a clear advantage of this chimeric form is that the variable regions can be conveniently generated from currently known sources using readily available B cells or hybridomas from non-human hosts, with the combined constant regions coming from, for example, human cells.
  • variable region has the advantage of being easy to prepare and the specificity is not affected by the source, and because the constant region is from a human, the antibody will be more likely to elicit a human immune response when injected than if the constant region is from a non-human source. Low.
  • a “humanized” antibody refers to an antibody in which all or substantially all CDR amino acid residues are derived from non-human CDRs and all or substantially all FR amino acid residues are derived from human FR.
  • a "humanized form" of a non-human antibody refers to a variant of the non-human antibody that has undergone humanization to generally reduce immunogenicity in humans while retaining the specificity and affinity of the parent non-human antibody.
  • some FR residues in a humanized antibody are replaced with corresponding residues from a non-human antibody (eg, an antibody from which CDR residues are derived), eg, to restore or improve antibody specificity or affinity.
  • Humanized antibodies and methods for their preparation are well known to those skilled in the art, see, for example, Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008).
  • Human framework regions that may be used for humanization include, but are not limited to: framework regions selected using a "best fit" approach; framework regions derived from consensus sequences of human antibodies of a specific subgroup of light or heavy chain variable regions ; Human mature (somatic mutation) framework region or human germline framework region; and the framework region obtained by screening the FR library.
  • human antibody is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences.
  • Human antibodies of the invention may contain amino acid residues that are not encoded by human germline immunoglobulin sequences (eg, mutations introduced by random or site-directed mutagenesis in vitro or by somatic mutation in vivo).
  • the invention provides an antibody or antigen-binding fragment thereof targeting B7-H3, comprising:
  • CDR-L1 as shown in SEQ ID NO: 1
  • CDR-L2 as shown in SEQ ID NO: 2
  • CDR-L3 as shown in SEQ ID NO: 3
  • CDR-H1 as shown in SEQ ID NO: 4
  • CDR-H2 as shown in SEQ ID NO: 5
  • CDR-H3 as shown in SEQ ID NO: 6;
  • CDR-L1 as shown in SEQ ID NO: 37 CDR-L2 as shown in SEQ ID NO: 38, CDR-L3 as shown in SEQ ID NO: 39, and CDR-L3 as shown in SEQ ID NO: 40
  • CDR-H1 as shown in SEQ ID NO: 41, CDR-H2 as shown in SEQ ID NO: 41 and CDR-H3 as shown in SEQ ID NO: 42 CDR-H1 as shown in SEQ ID NO: 41, CDR-H2 as shown in SEQ ID NO: 41 and CDR-H3 as shown in SEQ ID NO: 42.
  • the antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region and a light chain variable region, and the heavy chain variable region is identical to one selected from the group consisting of SEQ ID NO: 8, 17, 26, 35,
  • the amino acid sequences of 44, 47, 50, 53, 56, 59, 62 and 65 have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97% , at least 98%, at least 99% or 100% identical, or compared to an amino acid sequence selected from the group consisting of SEQ ID NO: 8, 17, 26, 35, 44, 47, 50, 53, 56, 59, 62 and 65 Having one or several (e.g.
  • amino acid sequences of 16, 25, 34, 43, 46, 49, 52, 55, 58, 61 and 64 have at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical, or identical to SEQ ID NOs: 7, 16, 25, 34, 43, 46, 49, 52, 55, 58, 61 and
  • the amino acid sequence of 64 has a modification of one or several amino acids (eg, up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids).
  • the modifications are conservative modifications, such as conservative substitutions, additions and deletions of amino acids.
  • the antibody of the invention or an antigen-binding fragment thereof comprises a heavy chain selected from the group consisting of SEQ ID NO: 8, 17, 26, 35, 44, 47, 50, 53, 56, 59, 62 and 65
  • the antibody or antigen-binding fragment thereof of the invention comprises a heavy chain variable region and a light chain variable region selected from:
  • the heavy chain variable region and light chain variable region have at least 90%, at least 91%, compared to the heavy chain variable region and light chain variable region of any group of (a) to (l). , at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical;
  • the heavy chain variable region and light chain variable region have one or several amino acid modifications to the heavy chain variable region and light chain variable region of any group of (a) to (l), such as Modifications of up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids; preferably, the modifications are conservative modifications, such as conservative substitutions, additions and deletions of amino acids.
  • the antibody or antigen-binding fragment thereof of the invention has at least an amino acid sequence selected from the group consisting of SEQ ID NO: 9, 18, 27, 36, 45, 48, 51, 54, 57, 60, 63 and 66. 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% identity, or selected from SEQ ID NO: 9, 18, 27, 36 , 45, 48, 51, 54, 57, 60, 63 and 66 have one or several amino acid modifications, such as up to 1, 2, 3, 4, 5, 6, 7, 8, 9 or Modification of 10 amino acids.
  • the modifications are conservative modifications, such as conservative substitutions, additions and deletions of amino acids.
  • the amino acid sequence of the antibody or antigen-binding fragment thereof of the invention is selected from SEQ ID NO: 9, 18, 27, 36, 45, 48, 51, 54, 57, 60, 63 and 66.
  • conservative modification refers to an amino acid modification that does not significantly affect or alter the binding characteristics of an antibody or antibody fragment containing the amino acid sequence. These conservative modifications include conservative substitutions, additions and deletions of amino acids. Modifications can be introduced into the chimeric antigen receptors of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. Conservative amino acid substitutions are substitutions in which an amino acid residue is replaced by an amino acid residue with a similar side chain.
  • Families of amino acid residues with similar side chains have been defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid ), uncharged polar side chains (such as glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (such as alanine, valine acid, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), ⁇ -branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).
  • Conservative modifications may be selected, for example, based on similarities in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or amphipath
  • sequence identity means the extent to which two (nucleotide or amino acid) sequences have identical residues at the same positions in an alignment, and is usually expressed as a percentage. Preferably, identity is determined over the entire length of the sequences being compared. Therefore, two with exactly the same sequence The copies are 100% identical.
  • sequence identity can be determined using several algorithms, such as Blast (Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402), Blast2 (Altschul et al. (1990) J. Mol. Biol. 215: 403-410), Smith-Waterman (Smith et al. (1981) J. Mol. Biol. 147:195-197) and ClustalW.
  • the invention also provides a multispecific antibody (preferably a bispecific antibody or a trispecific antibody) comprising an anti-B7-H3 antibody or an antigen-binding fragment thereof as described above, which further comprises one or more Secondary antibodies that specifically bind to other antigens.
  • a multispecific antibody preferably a bispecific antibody or a trispecific antibody
  • an anti-B7-H3 antibody or an antigen-binding fragment thereof as described above which further comprises one or more Secondary antibodies that specifically bind to other antigens.
  • multispecific refers to an antigen-binding protein that has multi-epitope specificity (i.e., is capable of specifically binding to two, three, or more different epitopes on a biological molecule or is capable of specifically Binds epitopes on two, three or more different biomolecules).
  • bispecific means that an antigen-binding protein has two different antigen-binding specificities.
  • the second antibody can be in any antibody or antibody fragment form, such as full-length antibody, Fab, Fab', (Fab') 2 , Fv, scFv, scFv-scFv, minibody, diabody, or sdAb.
  • the second antibody targets an antigen selected from: BCMA, CD4, CD5, CD7, CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD33, CD37 , CD38, CD40, CD40L, CD46, CD52, CD54, CD80, CD126, CD138, B7, MUC-1, Ia, HM1.24, HLA-DR, tenascin, angiogenic factor, VEGF, PIGF, ED-B Fibronectin, oncogene, oncogene product, CD66a-d, necrosis antigen, Ii, IL-2, T101, TAC, IL-6, DR4, DR5, tEGFR, Her2, L1-CAM, mesothelin, CEA, Hepatitis B surface antigen, antifolate receptor, CD24, CD30, CD44, EGFR, EGP-2, EGP-4, EPHa2, ErbB2, ErbB3, ErbB
  • MART-1 gp100, carcinoembryonic antigen, VEGF-R2, CEA, prostate-specific antigen, PSMA, Her2/neu, estrogen receptor, progesterone receptor, ephrin B2, CD123, c-Met, GD-2, OGD2, CE7, WT-1, cyclin A2, CCL-1, hTERT, MDM2, CYP1B, WT1, activin, AFP, p53, cyclin (D1), CS-1, BAFF-R , TACI, CD56, TIM-3, CD123, L1-cell adhesion molecules, cell cycle proteins such as cyclin A1 (CCNA1) and/or pathogen-specific antigens, biotinylated molecules, produced by HIV, HCV, HBV and/or molecules expressed by other pathogens; and/or neoepitopes or neoantigens.
  • CCNA1 cyclin A1
  • pathogen-specific antigens biotinylated
  • the invention in another aspect, relates to a nucleic acid molecule encoding an anti-B7-H3 antibody or multispecific antibody of the invention.
  • the nucleic acid of the invention can be RNA, DNA or cDNA.
  • the nucleic acid of the invention is a substantially isolated nucleic acid.
  • the nucleic acid of the invention may also be in the form of a vector, may be present in a vector and/or may be part of a vector, such as a plasmid, a cohesive plasmid or a YAC.
  • the vector may in particular be an expression vector, ie a vector providing for expression of the B7-H3 antibody in vitro and/or in vivo (ie in a suitable host cell, host organism and/or expression system).
  • the expression vector typically contains at least one nucleic acid molecule of the invention operably linked to one or more suitable expression control elements (eg, promoter, enhancer, terminator, etc.). Selection of such regulatory elements and their sequences for expression in a particular host is well known to those skilled in the art. Specific examples of regulatory elements and other elements useful or necessary for the expression of the B7-H3 antibody of the present invention include, but are not limited to, promoters, enhancers, terminators, integrators, selection markers, leader sequences, and reporter genes.
  • the invention also provides host cells expressing the B7-H3 antibodies, multispecific antibodies of the invention and/or containing the nucleic acids or vectors of the invention.
  • Preferred host cells of the present invention are bacterial cells, fungal cells or mammalian cells.
  • Suitable bacterial cells include Gram-negative bacterial strains (such as Escherichia coli strains, Proteus strains and Pseudomonas strains) and Gram-positive bacterial strains (such as Bacillus Bacillus strains, Streptomyces strains, Staphylococcus strains and Lactococcus strains) cells.
  • Gram-negative bacterial strains such as Escherichia coli strains, Proteus strains and Pseudomonas strains
  • Gram-positive bacterial strains such as Bacillus Bacillus strains, Streptomyces strains, Staphylococcus strains and Lactococcus strains
  • Suitable fungal cells include cells of species of the genera Trichoderma, Neurospora and Aspergillus; or include cells of the genera Saccharomyces (e.g. Saccharomyces cerevisiae), Schizosaccharomyces cerevisiae Schizosaccharomyces (such as Schizosaccharomyces pombe), Pichia (such as Pichia pastoris and Pichia methanolica) and Hansen Cells of species of the genus Hansenula.
  • Saccharomyces e.g. Saccharomyces cerevisiae
  • Schizosaccharomyces cerevisiae Schizosaccharomyces such as Schizosaccharomyces pombe
  • Pichia such as Pichia pastoris and Pichia methanolica
  • Suitable mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.
  • amphibian cells insect cells, plant cells, and any other cells known in the art for expressing heterologous proteins may also be used in the present invention.
  • the invention also provides chimeric polypeptides comprising an anti-B7-H3 antibody as described above, such as a CAR, TCR, TRuC, TAC or ImmTAC.
  • an anti-B7-H3 antibody such as a CAR, TCR, TRuC, TAC or ImmTAC.
  • the present invention also provides chimeric antigen receptors comprising anti-B7-H3 antibodies as described above.
  • chimeric antigen receptor refers to an artificially constructed hybrid polypeptide that generally includes a ligand-binding domain (such as an antigen-binding fragment of an antibody), a transmembrane domain, Optional co-stimulatory domain and intracellular signaling domain, each domain is connected through a linker.
  • CARs are able to exploit the antigen-binding properties of antibodies to redirect the specificity and reactivity of T cells and other immune cells to the target of choice in a non-MHC-restricted manner.
  • the invention provides a chimeric antigen receptor comprising an anti-B7-H3 antibody or an antigen-binding fragment thereof as described above or a multispecific antibody, a transmembrane antibody containing the anti-B7-H3 antibody, domain and intracellular signaling domain.
  • transmembrane domain refers to a polypeptide capable of expressing a chimeric antigen receptor on the surface of an immune cell (such as a lymphocyte, NK cell, or NKT cell) and directing the immune cell's cellular response against the target cell. structure.
  • the transmembrane domain may be natural or synthetic and may be derived from any membrane-bound or transmembrane protein. The transmembrane domain enables signaling when the chimeric antigen receptor binds to the target antigen.
  • Transmembrane domains particularly suitable for use in the present invention may be derived from, for example, TCR ⁇ chain, TCR ⁇ chain, TCR ⁇ chain, TCR ⁇ chain, CD3 ⁇ subunit, CD3 ⁇ subunit, CD3 ⁇ subunit, CD3 ⁇ subunit, CD45, CD4, CD5, CD8 ⁇ , CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154 and their functional fragments.
  • the transmembrane domain may be synthetic and may contain predominantly hydrophobic Residues such as leucine and valine.
  • the transmembrane domain is derived from the CD8 alpha chain, which shares at least 70%, preferably at least 80%, and more preferably at least 90%, 95%, 97% or 99% with the amino acid sequence shown in SEQ ID NO: 67 Or 100% sequence identity, or its coding sequence has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% with the nucleic acid molecule shown in SEQ ID NO: 68 sequence identity.
  • intracellular signaling domain refers to the portion of a protein that transduces effector function signals and directs the cell to perform a specified function.
  • the intracellular signaling domain comprised by the chimeric antigen receptor of the present invention can be the intracellular region sequence of a T cell receptor and a co-receptor, which function together to trigger a trigger upon binding of the antigen receptor. Signaling, as well as any derivatives or variants of these sequences and any synthetic sequences having the same or similar function.
  • the intracellular signaling domain can contain many immunoreceptor Tyrosine-based Activation Motifs (ITAM).
  • Non-limiting examples of intracellular signaling domains of the invention include, but are not limited to, intracellular regions of FcR ⁇ , FcR ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD22, CD79a, CD79b, CD66d, and the like.
  • the signaling domain of the CAR of the present invention may comprise a CD3 ⁇ intracellular region, which has at least 70%, preferably at least 80%, and more preferably at least 90%, the amino acid sequence shown in SEQ ID NO: 69. 95%, 97% or 99% or 100% sequence identity, or its coding sequence has at least 70%, preferably at least 80%, and more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity.
  • the chimeric antigen receptor of the invention may further comprise a hinge region located between the antibody and the transmembrane domain.
  • the term "hinge region” generally refers to any oligopeptide or polypeptide that functions to connect a transmembrane domain to an antibody. Specifically, the hinge region is used to provide greater flexibility and accessibility to the antibody.
  • the hinge region may comprise up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids.
  • the hinge region may be derived in whole or in part from a natural molecule, such as in whole or in part from the extracellular region of CD8, CD4 or CD28, or in whole or in part from an antibody constant region.
  • the hinge region may be a synthetic sequence corresponding to a naturally occurring hinge sequence, or may be a completely synthetic hinge sequence.
  • the hinge region includes a hinge region part of CD8 ⁇ , CD28, Fc ⁇ RIII ⁇ receptor, IgG4 or IgG1, more preferably a CD8 ⁇ , CD28 or IgG4 hinge, which has the same amino acid sequence as shown in SEQ ID NO:71 At least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity, or its coding sequence has at least 100% sequence identity with the nucleotide sequence shown in SEQ ID NO:72 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity.
  • the chimeric antigen receptor may also comprise one or more costimulatory domains.
  • a costimulatory domain may be an intracellular functional signaling domain from a costimulatory molecule, comprising the entire intracellular portion of the costimulatory molecule, or a functional fragment thereof.
  • a "costimulatory molecule” refers to a cognate binding partner that specifically binds to a costimulatory ligand on a T cell, thereby mediating a costimulatory response (eg, proliferation) of the T cell.
  • Costimulatory molecules include, but are not limited to, MHC class 1 molecules, BTLA, and Toll ligand receptors.
  • Non-limiting examples of costimulatory domains of the invention include, but are not limited to, costimulatory signaling domains derived from the following proteins: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11 , CD2, B7-H3, CD8, CD18(LFA-1), CD27, CD28, CD30, CD40, CD54(ICAM), CD83, CD134(OX40), CD137(4-1BB), CD270(HVEM), CD272( BTLA), CD276(B7-H3), CD278(ICOS), CD357(GITR), DAP10, LAT, NKG2C, SLP76, PD-1, LIGHT, TRIM and ZAP70.
  • costimulatory signaling domains derived from the following proteins: TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11 , CD2, B
  • the co-stimulatory domain of the CAR of the invention is from 4-1BB, CD28 or 4-1BB+CD28.
  • the 4-1BB costimulatory domain has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% of the amino acid sequence shown in SEQ ID NO:73 % sequence identity, or the coding sequence has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity with the nucleic acid molecule shown in SEQ ID NO:74 Identity.
  • the CAR of the invention may also comprise a signal peptide such that when expressed in a cell such as a T cell, the nascent protein is directed to the endoplasmic reticulum and subsequently to the cell surface.
  • the core of the signal peptide may contain long stretches of hydrophobic amino acids, which have a tendency to form a single ⁇ -helix.
  • At the end of the signal peptide there is usually a segment of amino acids that is recognized and cleaved by the signal peptidase.
  • Signal peptidases can cleave during or after translocation to produce free signal peptide and mature protein. The free signal peptide is then digested by specific proteases.
  • Signal peptides useful in the present invention are well known to those skilled in the art, such as signal peptides derived from B2M, CD8 ⁇ , IgGl, GM-CSFRa, etc.
  • the signal peptide useful in the present invention is from CD8 ⁇ , which has at least 70%, preferably at least 80%, and more preferably at least 90%, 95%, 97% or more of the amino acid sequence shown in SEQ ID NO:75. 99% or 100% sequence identity, or its coding sequence has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% with the nucleic acid molecule shown in SEQ ID NO:76 or 100% sequence identity.
  • the CAR contains an anti-B7-H3 antibody or an antigen-binding fragment thereof as provided herein or a multispecific antibody containing the anti-B7-H3 antibody, a CD8 ⁇ or CD28 transmembrane region, CD28 and/or or 4-1BB costimulatory domain, and CD3 ⁇ intracellular signaling domain.
  • the CAR may further comprise a signal peptide from B2M, CD8 ⁇ , IgG1 or GM-CSFRa.
  • the present invention also provides nucleic acid molecules encoding chimeric antigen receptors targeting B7-H3 as defined above, and vectors comprising said nucleic acid molecules.
  • vector is a nucleic acid molecule used as a vehicle to transfer (exogenous) genetic material into a host cell in which the nucleic acid molecule can, for example, be replicated and/or expressed.
  • Vectors generally include targeting vectors and expression vectors.
  • a "targeting vector” is a medium that delivers isolated nucleic acid into the interior of a cell, for example, by homologous recombination or using a hybrid recombinase that specifically targets sequences at a site.
  • an "expression vector” is a vector used for the transcription of heterologous nucleic acid sequences (eg, those encoding the chimeric antigen receptor polypeptides of the invention) and the translation of their mRNA in a suitable host cell.
  • Suitable vectors useful in the present invention are known in the art and many are commercially available.
  • vectors of the invention include, but are not limited to, plasmids, viruses (e.g., retroviruses, lentiviruses, adenoviruses, vaccinia viruses, Rous sarcoma virus (RSV), polyomaviruses, and adeno-associated viruses (AAV), etc.
  • the vector itself is usually a nucleic acid molecule, usually a DNA sequence containing the insert (transgene) and a larger sequence that serves as the "backbone" of the vector.
  • Engineering The vector also typically contains an origin of autonomous replication in the host cell (if stable expression of the polynucleotide is required), a selectable marker, and a restriction enzyme cleavage site (such as a multiple cloning site, MCS).
  • the vector may additionally contain a promoter, multiple Elements such as poly(A) tail (polyA), 3'UTR, enhancer, terminator, insulator, operator, selectable marker, reporter gene, targeting sequence and/or protein purification tag.
  • polyA poly(A) tail
  • 3'UTR poly(A) tail
  • enhancer poly(A) tail
  • terminator insulator
  • operator selectable marker
  • reporter gene targeting sequence and/or protein purification tag
  • the vector is an in vitro transcribed vector.
  • the invention also provides engineered immune cells expressing chimeric polypeptides of the invention, such as recombinant TCR receptors or chimeric antigen receptors.
  • the term "immune cell” refers to any cell of the immune system that has one or more effector functions (eg, cytotoxic cell killing activity, secretion of cytokines, induction of ADCC and/or CDC).
  • the immune cells can be T cells, macrophages, dendritic cells, monocytes, NK cells and/or NKT cells.
  • the immune cells are derived from stem cells, such as adult stem cells, embryonic stem cells, umbilical cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells, or hematopoietic stem cells, and the like.
  • the immune cells are T cells.
  • the T cells can be any T cells, such as T cells cultured in vitro, such as primary T cells, or T cells from a T cell line cultured in vitro, such as Jurkat, SupT1, etc., or T cells obtained from a subject. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. T cells can be obtained from a variety of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from the site of infection, ascites, pleural effusion, spleen tissue, and tumors. T cells can also be concentrated or purified.
  • T cells can be at any stage of development, including, but not limited to, CD4+CD8+ T cells, CD4+ T cells (e.g., Th1 and Th2 cells), CD8+ T cells (e.g., cytotoxic T cells), CD4 - CD8 - T cells cells, tumor infiltrating cells, memory T cells, naive T cells, ⁇ -T cells, ⁇ -T cells, etc.
  • the immune cells are human T cells.
  • T cells can be obtained from the blood of a subject using a variety of techniques known to those skilled in the art, such as Ficoll isolation.
  • the engineered immune cells further comprise at least one gene selected from the group consisting of CD52, GR, dCK, TCR/CD3 genes whose expression is inhibited or silenced (for example, TRAC, TRBC, CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD3 ⁇ ), MHC related genes (HLA-A, HLA-B, HLA-C, B2M, HLA-DPA, HLA-DQ, HLA-DRA, TAP1, TAP2, LMP2, LMP7, RFX5, RFXAP, RFXANK, CIITA) and immune checkpoint genes such as PD1, LAG3, TIM3, CTLA4, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, HAVCR2, BTLA, CD160, TIGIT, CD96, CRTAM, TNFRSF10B, TNFRSF10A , CASP8, CASP10, CASP3, CASP6, C
  • the engineered immune cells further comprise at least one gene selected from the group consisting of TRAC, TRBC, HLA-A, HLA-B, HLA-C, B2M, RFX5, RFXAP, RFXANK, whose expression is inhibited or silenced.
  • RNA bait RNA bait
  • RNA aptamer siRNA
  • shRNA/miRNA trans dominant negative protein
  • TNP trans dominant negative protein
  • chimeric/antibody conjugates chemokine ligands
  • anti-infectious cellular proteins can be used , intracellular antibodies (sFv), nucleoside analogs (NRTI), non-nucleoside analogs (NNRTI), integrase inhibitors (oligonucleotides, dinucleotides and chemical agents) and protease inhibitors to inhibit gene Express.
  • genes can also be silenced by mediating DNA fragmentation through, for example, meganucleases, zinc finger nucleases, TALE nucleases or Cas enzymes in the CRISPR system.
  • the engineered immune cells further comprise a second chimeric antigen receptor targeting other tumor antigens.
  • Other tumor antigens targeted by the second chimeric antigen receptor may be selected from, for example, BCMA, CD4, CD5, CD7, CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD25, CD33, CD37, CD38 , CD40, CD40L, CD46, CD52, CD54, CD80, CD126, CD138, B7, MUC-1, HM1.24, angiogenic factors, VEGF, PIGF, ED-B fibronectin, CD66a-d, IL-2, T101, TAC, IL-6, DR4, DR5, tEGFR, Her2, L1-CAM, mesothelin, CEA, hepatitis B surface antigen, antifolate receptor, CD24, CD30, CD44, EGFR, EGP-2, EGP -4.
  • EPHa2, ErbB2, ErbB3, ErbB4, ErbB dimer EGFR vIII, FBP, FCRL5, FCRH5, fetal acetylcholine receptor, GD2, GD3, GPRC5D, HMW-MAA, IL-22R- ⁇ , IL-13R- ⁇ 2, kappa light chain, Lewis Y, L1-CAM, MAGE-A1, MAGE-A3, MAGE-A6, PRAME, survivin, EGP2, EGP40, TAG72, B7-H6, IL-13Ra2, CA9, CD171, G250/ CAIX, HLA-A1, HLA-A2, NY-ESO-1, PSCA, folate receptor-a, CD44v6, CD44v7/8, avb6 integrin, 8H9, NCAM, VEGF receptor, 5T4, fetal AchR, MUC1, MUC16 , NY-ESO-1, MART-1, gp100, carcinoembryonic antigen,
  • a plurality of immune cells are provided, each immune cell engineered to express one or more chimeric antigen receptors.
  • one immune cell is engineered to express a chimeric antigen receptor that binds and/or targets B7-H3 (e.g., a CAR comprising an anti-B7-H3 antibody of the invention), and another A cell engineered to express a chimeric antigen receptor that binds and/or targets other antigens.
  • immune cells may also express multispecific chimeric antigen receptors that target one or more antigens including B7-H3.
  • such a multispecific chimeric antigen receptor may comprise a multispecific antibody targeting B7-H3, or may comprise both an anti-B7-H3 antibody of the invention and antibodies targeting other antigens.
  • the plurality of engineered immune cells can be administered together or separately.
  • the plurality of immune cells can be in the same composition or in different compositions. Exemplary compositions of cells include those described in the following sections of this application.
  • the invention provides an antibody conjugate comprising an anti-B7-H3 antibody as defined in the invention and a second functional structure, wherein the second functional structure is selected from the group consisting of Fc, radioisotopes, extended half-life structural parts, detectable markers and drugs.
  • the invention provides an antibody conjugate comprising an anti-B7-H3 antibody and Fc as defined in the invention.
  • Fc is used to define the C-terminal region of an immunoglobulin heavy chain, which includes native Fc and variant Fc.
  • Native Fc refers to a molecule or sequence containing non-antigen-binding fragments, whether in monomeric or multimeric form, produced by digestion of an intact antibody.
  • the immunoglobulin source producing native Fc is preferably of human origin.
  • Native Fc fragments are composed of monomeric polypeptides that can be linked by covalent linkages (eg, disulfide bonds) and non-covalent linkages into dimer or multimer forms.
  • natural Fc molecules have 1-4 intermolecular disulfides between monomer subunits. key.
  • An example of a native Fc is the disulfide-linked dimer produced by digestion of IgG with papain (see Ellison et al. (1982), Nucleic Acids Res. 10:4071-9).
  • the term "native Fc" as used herein Generally refers to monomer, dimer and multimer forms.
  • “Variant Fc” refers to an amino acid sequence that differs from the amino acid sequence of a “native” or “wild-type” Fc due to at least one "amino acid modification” as defined herein, also referred to as an “Fc variant.”
  • “Fc” also includes single-chain Fc (scFc), ie, a single-chain Fc consisting of two Fc monomers connected by a polypeptide linker, which is capable of naturally folding into a functional dimeric Fc region.
  • the Fc is preferably an Fc of a human immunoglobulin, more preferably an Fc of a human IgG1.
  • the invention provides an antibody conjugate comprising an anti-B7-H3 antibody as defined in the invention and a radioactive isotope.
  • radioactive isotopes useful in the present invention include, but are not limited to, At 211 , I 131 , I 125 , Y 90 , Re 186 , Re 188 , Sm 153 , Bi 212 , P 32 , Pb 212 , 99m Tc, 123 I, 18 F and 68 Ga.
  • the invention provides an antibody conjugate comprising an anti-B7-H3 antibody as defined in the invention and a half-life extending moiety selected from the group consisting of the binding structure of albumin, transgene Binding structures of ferritin, polyethylene glycol molecules, recombinant polyethylene glycol molecules, human serum albumin, fragments of human serum albumin and white polypeptides (including antibodies) that bind human serum albumin.
  • the invention provides an antibody conjugate comprising an anti-B7-H3 antibody as defined in the invention and a detectable label.
  • detectable label as used herein means a compound that produces a detectable signal.
  • the detectable marker may be an MRI contrast agent, a scintigraphy contrast agent, an X-ray imaging contrast agent, an ultrasound contrast agent, an optical imaging contrast agent.
  • detectable labels examples include fluorophores (such as fluorescein, Alexa, or cyanine), chemiluminescent compounds (such as luminol), bioluminescent compounds (such as luciferase or alkaline phosphatase), enzymes (such as spicy Root peroxidase, glucose-6-phosphatase, ⁇ -galactosidase), antibiotics (such as kanamycin, ampicillin, chloramphenicol, tetracycline, etc.) resistance genes and contrast agents (such as nanoparticles or gadolinium).
  • fluorophores such as fluorescein, Alexa, or cyanine
  • chemiluminescent compounds such as luminol
  • bioluminescent compounds such as luciferase or alkaline phosphatase
  • enzymes such as spicy Root peroxidase, glucose-6-phosphatase, ⁇ -galactosidase
  • antibiotics such as kanamycin, ampicillin, chloramphenicol,
  • the invention provides an antibody conjugate comprising an anti-B7-H3 antibody as defined in the invention and a drug coupled to the anti-B7-H3 antibody, such as a cytotoxic or immunomodulator (i.e., antibody drug conjugates).
  • a drug coupled to the anti-B7-H3 antibody, such as a cytotoxic or immunomodulator (i.e., antibody drug conjugates).
  • drugs are linked to antibodies covalently, often relying on linkers.
  • the drug is a cytotoxin.
  • the drug is an immunomodulator.
  • cytotoxics include, but are not limited to, methotrexate, aminopterin, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, dacarbazine, nitrogen mustard, thiotepa, phentermine Nitrogen mustard, melphalan, carmustine (BSNU), lomustine (CCNU), 1-methylnitrosourea, cyclophosphamide, nitrogen mustard, busulfan, dibromomannitol, chain Zocin, mitomycin, cis-dichlorodiamine platinum (II) (DDP), cisplatin, carboplatin, zorubicin, doxorubicin, ditobicin, Kaminomycin, Darbicin, epirubicin, mitoxantrone, actinomycin D, bleomycin, californicin, radimycin, antrimycin (AMC), vincristine, vinblastine, Paclitaxel, ricin,
  • immunomodulators include, but are not limited to, ganciclovir, etanercept, tacrolimus, sirolimus, cyclosporine, cyclosporine, rapamycin, cyclophosphamide, azathioprine , mycophenolate mofetil, methotrexate, glucocorticoids and their analogs, cytokines, stem cell growth factors, lymphotoxin, tumor necrosis factor (TNF), hematopoietic factors, interleukins (such as IL-1, IL-2, IL-3, IL-6, IL-10, IL-12, IL-18 and IL-21), colony-stimulating factors (such as G-CSF and GM-CSF), interferons (such as interferon-alpha, interferon - ⁇ and interferon- ⁇ ), stem cell growth factor named "S1 factor”, erythropoietin and thrombopoietin, or combinations thereof.
  • TNF tumor nec
  • the present invention also provides a detection kit, which contains the humanized antibody, multispecific antibody, antibody conjugate, engineered immune cell or chimeric antigen receptor of the present invention.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the humanized antibody, chimeric antigen receptor, multispecific antibody, engineered immune cell or antibody conjugate of the present invention, and a one or more pharmaceutically acceptable excipients.
  • the term "pharmaceutically acceptable excipient” means an excipient that is pharmacologically and/or physiologically compatible with the subject and the active ingredient (i.e., capable of eliciting the desired therapeutic effect without causing any adverse effects). desired local or systemic effect), which are well known in the art (see, e.g., Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995).
  • Examples of pharmaceutically acceptable excipients include, but are not limited to, fillers, binders, disintegrants, coating agents, adsorbents, anti-adhesive agents, glidants, antioxidants, flavoring agents, colorants, Sweeteners, solvents, co-solvents, buffers, chelating agents, surfactants, diluents, wetting agents, preservatives, emulsifiers, coating agents, isotonic agents, absorption delaying agents, stabilizers and tonicity regulators . It is known to those skilled in the art to select suitable excipients to prepare the desired pharmaceutical compositions of the present invention.
  • excipients for use in pharmaceutical compositions of the invention include saline, buffered saline, dextrose and water.
  • selection of a suitable excipient depends inter alia on the active agent used, the disease to be treated and the desired dosage form of the pharmaceutical composition.
  • composition according to the present invention is suitable for administration by a variety of routes. Typically, administration is accomplished parenterally.
  • Parenteral delivery methods include topical, intraarterial, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, intrauterine, intravaginal, sublingual, or intranasal administration.
  • the pharmaceutical composition according to the present invention can also be prepared in various forms, such as solid, liquid, gaseous or freeze-dried forms, especially ointments, creams, transdermal patches, gels, powders, tablets, solutions, gases, etc. in the form of aerosols, granules, pills, suspensions, emulsions, capsules, syrups, elixirs, extracts, tinctures or liquid extracts, or in a form particularly suitable for the desired method of administration.
  • Processes known for the production of medicaments according to the invention may include, for example, conventional mixing, dissolving, granulating, drageeing, grinding, emulsifying, encapsulating, embedding or lyophilizing processes.
  • Pharmaceutical compositions containing immune cells such as those described herein are typically provided in solution, and preferably contain a pharmaceutically acceptable buffer.
  • compositions according to the invention may also be administered in combination with one or more other agents suitable for the treatment and/or prevention of the disease to be treated.
  • agents suitable for combination include known anticancer drugs such as cisplatin, maytansine derivatives, rachelmycin, calicheamicin, docetaxel, etoposide , gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer sodium photofrin II, temozolomide, topotecan, legislativeate glucuronate, or auristatin E, vincristine and doxorubicin; peptide cytotoxins such as ricin, diphtheria toxin, Pseudomonas bacterial exotoxin A, DNase and RNase; radionuclides such as iodine 131, rhenium 186, indium 111, iridium 90, bismuth 210 and 213, actin
  • the present invention also provides a method for treating and/or preventing and/or diagnosing diseases related to B7-H3 expression, comprising administering to the subject the humanized antibody, chimeric antigen receptor as described above. bodies, multispecific antibodies, antibody conjugates, engineered immune cells or pharmaceutical compositions.
  • diseases associated with B7-H3 expression include, but are not limited to, bladder cancer, breast cancer, cervical cancer, colorectal cancer, esophageal cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, Biliary tract cancer, oral squamous cell carcinoma, endometrial cancer, squamous cell carcinoma, gastric cancer, osteosarcoma, glioma, melanoma, adrenal malignancy, acute myeloid leukemia (AML), acute lymphoblastic leukemia ( ALL), lymphoblastic lymphoma (LBL), Hodgkin lymphoma (HL), non-Hodgkin lymphoma (NHL), multiple myeloma (MM).
  • bladder cancer bladder cancer
  • breast cancer cervical cancer
  • colorectal cancer esophageal cancer
  • kidney cancer liver cancer
  • lung cancer ovarian cancer
  • pancreatic cancer prostate cancer
  • Biliary tract cancer oral squamous cell carcinoma, endometri
  • Figure 1 B7-H3 expression levels in the CHO-B7H3 monoclonal cell line.
  • Figure 2 Binding of B7-H3 antibody to CHO-B7H3 cells.
  • Figure 3 scFv expression levels in CAR T cells expressing B7-H3 murine scFv.
  • FIG. 4 Expression of B7-H3 in target cells (A) and non-target cells (B).
  • Figure 5 The killing effect of CAR T cells expressing B7-H3 murine scFv on target cells NUGC4 (A), Hela (B), Huh7 (C) and non-target cells K562 (D) under different efficacy-to-target ratios.
  • Figure 6 The proportion of CD107a + CD8 + double-positive cells (A) and the proportion of CD107a + positive cells (B) in CAR T cells expressing B7-H3 murine scFv.
  • Figure 7 Release levels of IL2 (A) and IFN- ⁇ (B) after CAR T cells expressing B7-H3 murine scFv were co-cultured with target cells and non-target cells.
  • Figure 8 Changes in tumor burden in mice after injection of CAR T cells expressing B7-H3 murine scFv.
  • Figure 9 scFv expression levels in CAR T cells expressing B7-H3 humanized scFv.
  • Figure 10 The killing effect of CAR T cells expressing B7-H3 humanized scFv on target cells NUGC4 (A), Hela (B), and Huh7 (C) under different efficacy-to-target ratios.
  • Figure 11 The proportion of CD107a + CD8 + double-positive cells (A) and the proportion of CD107a + positive cells (B) in CAR T cells expressing B7-H3 humanized scFv.
  • Figure 12 Release levels of IL2 (A) and IFN- ⁇ (B) after CAR T cells expressing B7-H3 humanized scFv were co-cultured with target cells.
  • Figure 13 Changes in tumor burden in mice after injection of CAR T cells expressing B7-H3 humanized scFv.
  • Figure 14 Changes in the body weight of mice after injection of CAR T cells expressing B7-H3 humanized scFv.
  • Figure 15 Survival of mice after injection of CAR T cells expressing B7-H3 humanized scFv.
  • the complete coding sequence of human B7-H3 (NM_025240.3) was synthesized and cloned into the vector pGEM-T Easy (Promega, Cat. No. A1360) to obtain the pLV-B7H3 plasmid.
  • Lipofectamine transfection reagent (Roche, Cat. No. 06366546001) was used to transfect CHO cells with pLV-B7H3 plasmid, and monoclonal clones were isolated by limiting dilution method to obtain CHO-B7H3 monoclonal cell line.
  • the antibody APC anti-human CD276 (B7-H3) antibody (Biolegend, Cat. No.
  • the light and heavy chain variable regions of BH327, BH328, BH329, BH330, and BH331 in Table 1 were connected using (G4S)3, and the scFv-Fc antibody was constructed according to the structure of VH-(G4S)3-VL-hlgG1Fc.
  • the constructed scFv-Fc antibody was transiently transfected into 293 cells to express the antibody.
  • the obtained antibodies were used to stain CHO-B7H3 cells and detected by flow cytometry. The results are shown in Figure 2.
  • each antibody binds strongly to CHO-B7H3 cells and does not bind to CHO cells, indicating that its binding to B7-H3 protein is specific.
  • amino acid sequence of the anti-B7-H3 mouse scFv contained in BH328-CAR is shown in SEQ ID NO: 18; the amino acid sequence of the anti-B7-H3 mouse scFv contained in BH329-CAR is shown in SEQ ID NO: 27;
  • the amino acid sequence of the anti-B7-H3 mouse scFv contained in BH330-CAR is shown in SEQ ID NO: 36;
  • amino acid sequence of the anti-B7-H3 mouse scFv contained in BH331-CAR is shown in SEQ ID NO: 45.
  • Opti-MEM Gibco, Cat. No. 31985-070
  • packaging vector psPAX2 Adgene, No. 12260
  • enveloped vector pMD2.G Adgene, Cat. No. 12259
  • 120 ⁇ L of X-treme GENE HP DNA transfection reagent (Roche, Cat. No. 06366236001), mix immediately, incubate at room temperature for 15 minutes, and then add the plasmid/vector/transfection reagent mixture dropwise to the culture flask of 293T cells.
  • Viruses were collected at 24 hours and 48 hours, combined, and ultracentrifuged (25000g, 4°C, 2.5 hours) to obtain concentrated lentivirus.
  • T cells were activated with DynaBeads CD3/CD28 CTSTM (Gibco, Cat. No. 40203D) and cultured at 37°C and 5% CO for 1 day. Then, concentrated lentivirus was added, and after continuous culture for 3 days, each CAR T cell expressing anti-B7-H3 mouse scFv was obtained, numbered BH328-CAR T, BH329-CAR T, BH330-CAR T, and BH331-CAR T. . Unmodified wild-type T cells (NT) were used as controls.
  • anti-B7-H3 mouse scFv can be effectively expressed in BH328-CAR T, BH329-CAR T, BH330-CAR T, and BH331-CAR T cells.
  • K562 cells do not express B7-H3, so they are used as non-target cells; Hela cells, MDA-MB-231 cells, NUGC4 cells, A549 cells, 293T cells, and Huh7 cells all express B7-H3 to varying degrees, so they are used as as target cells.
  • Each target cell (NUGC4 cells, Hela cells, Huh7 cells) or non-target cells (K562 cells) expressing the luciferase gene was plated into a 96-well plate at a concentration of 1 ⁇ 10 cells/well, and then incubated at 8: 1.
  • an effector-target ratio of 1, 4:1, or 2:1 (i.e., the ratio of effector T cells to target cells).
  • use enzyme labeling Measure the fluorescence value with an instrument. According to the calculation formula: (mean fluorescence of target cells - mean fluorescence of sample)/mean fluorescence of target cells ⁇ 100%, the killing efficiency was calculated, and the results are shown in Figure 5.
  • the BH328-CAR T, BH329-CAR T, BH330-CAR T, and BH331-CAR T cells of the present invention show a strong killing effect on target cells (NUGC4 cells, Hela cells, and Huh7 cells), while on non- The killing of target cells (K562 cells) was weak, indicating that each CAR T cell only specifically killed cells expressing B7-H3.
  • the target cells Hela cells, MDA-MB-231 cells, NUGC4 cells, 293T cells, Huh7 cells
  • non-target cells K562 cells
  • BH328-CAR T, BH329-CAR T, BH330-CAR T, BH331-CAR T and NT cells negative control
  • 10 ⁇ L PE Mouse anti-human CD107a antibody BD, No. 555801
  • incubate in the dark at 37°C, 5% CO2 After 1 h, add 20 ⁇ L Golgi Stop (BD, Cat. No.
  • BH328-CAR T, BH329-CAR T, BH330-CAR T, and BH331-CAR T cells are more effective against target cells (Hela cells, MDA-MB-231 cells, NUGC4 cells, 293T cells, Huh7 cells) all showed significantly increased specific degranulation, while no significantly increased degranulation was observed for non-target cells (K562 cells).
  • Target cells Hela cells, MDA-MB-231 cells, NUGC4 cells, A549 cells, 293T cells, Huh7 cells
  • non-target cells K562 cells
  • BH328-CAR T, BH329-CAR T, BH330-CAR T, BH331-CAR T cells and NT cells negative control
  • G1NT group negative control
  • G2 BH329-CAR T G3 BH330-CAR T.
  • the specific method is as follows: first, search for human antibody sequences with high similarity through the IG BLAST database (https://www.ncbi.nlm.nih.gov/igblast/), and then Replace the FR region in the single-chain antibody with the corresponding human sequence; then replace individual amino acid residues according to the different physical and chemical properties of the amino acid residues, and finally obtain multiple humanized single-chain antibodies, whose amino acid sequences are shown in Table 2 Show.
  • CAR T cells using humanized antibodies according to the method described in 3.1, and obtain CAR T cells containing anti-B7-H3 humanized antibodies BH329V0-CAR T, BH329V1-CAR T, BH329V2-CAR T, BH329V3-CAR T, BH329V4- CAR T, BH329V5-CAR T, BH329V6-CAR T.
  • CAR T cells were detected by flow cytometry using FITC-Rabbit anti-mouse IgG, F(ab')specific (jackson immunoresearch, Cat. No. 315-095-006)
  • the expression levels of anti-B7-H3 humanized scFv on are shown in Figure 9.
  • Each target cell expressing luciferase gene (Hela cells, NUGC4 cells, Huh7 cells) was plated into a 96-well plate at a concentration of 1 ⁇ 10 4 cells/well, and then at a concentration of 16:1, 8:1, 4: 1, 2:1, 1:1 effector-target ratio (i.e., the ratio of effector T cells to target cells). Plate NT cells and each CAR T cell into a 96-well plate for co-culture. After 16-18 hours, use enzyme labeling Measure the fluorescence value with an instrument. According to the calculation formula: (mean fluorescence of target cells - mean fluorescence of sample)/mean fluorescence of target cells ⁇ 100%, the killing efficiency was calculated, and the results are shown in Figure 10.
  • BH329V0-CAR T BH329V1-CAR T
  • BH329V2-CAR T BH329V3-CAR T
  • BH329V4-CAR T BH329V5-CAR T
  • BH329V6-CAR T cells showed significant effects on The killing effect on target cells (Hela cells, NUGC4 cells, and Huh7 cells) is strong, while the killing effect on non-target cells Huh7 cells is weak, indicating that each CAR T cell only specifically kills cells expressing B7-H3.
  • the target cells Hela cells, NUGC4 cells, Huh7 cells
  • non-target cells K562 cells
  • the target cells Hela cells, NUGC4 cells, Huh7 cells
  • non-target cells K562 cells
  • cells and NT cells negative control
  • cells and NT cells negative control
  • 10 ⁇ L PE Mouse anti-human CD107a antibody BD, Cat. No. 555801
  • BH329V0-CAR T, BH329V1-CAR T, BH329V2-CAR T, BH329V3-CAR T, BH329V4-CAR T, BH329V5-CAR T, BH329V6-CAR T cells are more sensitive to target cells (Hela cells, NUGC4 cells, Huh7 cells) all showed significantly increased specific degranulation, while no significantly increased degranulation was observed for non-target cells (K562 cells).
  • the target cells Hela cells, NUGC4 cells, Huh7 cells
  • non-target cells K562 cells
  • each CAR T cell was added at a ratio of 1:1.
  • cells and NT cells negative control
  • BH329V0-CAR T BH329V1-CAR T
  • BH329V2-CAR T BH329V3-CAR T
  • BH329V4-CAR T BH329V5-CAR T
  • BH329V6-CAR T cells were co-cultured with target cells. After treatment, the release levels of cytokines IL2 and IFN- ⁇ were significantly increased, and this cytokine release was specific.
  • G1NT group negative control
  • G2 BH329 group mimouse-derived CAR T control
  • G3 BH329V0 group G4 BH329V1 group
  • G5 BH329V2 group G6 BH329V3 group
  • G7 BH329V4 group G8 BH329V5 group
  • G9 BH329V6 group G9 BH329V6 group.
  • mice in each group The body weight changes of mice in each group are shown in Figure 14. It can be seen that after administration of CAR T cells, the body weight of mice in each group did not decrease significantly, indicating that the CAR T prepared by the present invention has no obvious toxicity.
  • mice in each group are shown in Figure 15. Compared with the NT group and the BH329 group (mouse-derived CAR T control), the survival rates of mice in each humanized CAR T group were significantly improved, especially in the BH329V0, BH329V2, BH329V5, and BH329V6 groups. Compared with the BH329V1 group, the survival rate was significantly higher. Rats have a higher survival rate.

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Abstract

本发明提供靶向B7-H3的抗体，以及包含它的多特异性抗体、嵌合多肽、抗体偶联物、药物组合物和试剂盒等，以及它们在诊断/治疗/预防与B7-H3表达相关的疾病中的用途。

Description

靶向B7-H3的抗体及其用途
相关申请的交叉引用
本发明要求于2022年06月30日提交中国专利局的申请号为CN202210766958.6、名称为“靶向B7-H3的抗体及其用途”的中国专利申请的优先权，其全部内容通过引用结合在本发明中。
技术领域
本发明属于免疫治疗领域。更具体地，本发明涉及靶向B7-H3的抗体，及其在预防和/或治疗和/或诊断疾病中的用途。
背景技术
B7-H3(B7homolog 3protein)又名CD276，是B7-CD28家族重要的免疫检查点分子。作为一种T细胞共抑制分子，B7-H3可有效抑制T细胞和NK细胞的功能；但B7-H3也有部分共刺激功能。
B7-H3在多种恶性肿瘤中均有表达，与恶性肿瘤的生长、转移、复发、预后不良等因素密切相关。B7-H3可下调T辅助1型介导的免疫反应抑制CD4+T细胞活化，并抑制细胞因子的产生，因而可能发挥促进癌细胞免疫逃逸的作用。
B7-H3异常高表达于多种癌细胞或组织中，包括胃癌、肺癌、前列腺癌、肾癌、胰腺癌、卵巢癌、乳腺癌、子宫内膜癌、肝癌、结肠直肠癌、口腔癌、膀胱癌、骨肉瘤及血液系统恶性疾病。
因此，针对B7-H3靶点进行药物开发、抗体开发具有重要价值和意义。本发明旨在提供一种靶向B7-H3的抗体，及其在疾病预防和/或治疗和/或诊断中的用途。
发明内容
在第一个方面，本发明提供一种靶向B7-H3的抗体或其抗原结合片段，其包含:
(1)如SEQ ID NO：1所示的CDR-L1、如SEQ ID NO：2所示的CDR-L2、如SEQ ID NO：3所示的CDR-L3、如SEQ ID NO：4所示的CDR-H1、如SEQ ID NO：5所示的CDR-H2和如SEQ ID NO：6所示的CDR-H3；(2)如SEQ ID NO：10所示的CDR-L1、如SEQ ID NO：11所示的CDR-L2、如SEQ ID NO：12所示的CDR-L3、如SEQ ID NO：13所示的CDR-H1、如SEQ ID NO：14所示的CDR-H2和如SEQ ID NO：15所示的CDR-H3；
(3)如SEQ ID NO：19所示的CDR-L1、如SEQ ID NO：20所示的CDR-L2、如SEQ ID NO：21所示的CDR-L3、如SEQ ID NO：22所示的CDR-H1、如SEQ ID NO：23所示的CDR-H2和如SEQ ID NO：24所示的CDR-H3；
(4)如SEQ ID NO：28所示的CDR-L1、如SEQ ID NO：29所示的CDR-L2、如SEQ ID NO：30所示的CDR-L3、如SEQ ID NO：31所示的CDR-H1、如SEQ ID NO：32所示的CDR-H2和如SEQ ID NO：33所示的CDR-H3；或
(5)如SEQ ID NO：37所示的CDR-L1、如SEQ ID NO：38所示的CDR-L2、如SEQ ID NO：39所示的CDR-L3、如SEQ ID NO：40所示的CDR-H1、如SEQ ID NO：41所示的CDR-H2和如SEQ ID NO：42所示的CDR-H3。
在一个实施方案中，本发明的抗体或其抗原结合片段包含重链可变区和轻链可变区，所述重链可变区与选自SEQ ID NO：8、17、26、35、44、47、50、53、56、59、62和65的氨基酸序列具有至少90％同一性，或与选自SEQ ID NO：8、17、26、35、44、47、50、53、56、59、62和65的氨基酸序列相比具有一个或几个氨基酸(例如最多1、2、3、4、5、6、7、8、9或10个氨基酸)的修饰；所述轻链可变区与选自SEQ ID NO：7、16、25、34、43、46、49、52、55、58、61和64的氨基酸序列具有至少90％同一性，或与选自SEQ ID NO：7、16、25、34、43、46、49、52、55、58、61和64的氨基酸序列相比具有一个或几个氨基酸(例如最多1、2、3、4、5、6、7、8、9或10个氨基酸)的修饰。优选地，所述修饰是保守性修饰，例如氨基酸的保守性取代、添加和缺失。在一个优选的实施方案中，本发明的抗体或其抗原结合片段包含选自SEQ ID NO：8、17、26、35、44、47、50、53、56、59、62和65的重链可变区和选自SEQ ID NO：：7、16、25、34、43、46、49、52、55、58、61和64的轻链可变区。
在一个实施方案中，本发明的抗体或其抗原结合片段是鼠源抗体、嵌合抗体、人源化抗体或人抗体，优选是人源化抗体。
在一个实施方案中，本发明的抗体或其抗原结合片段包含选自以下的重链可变区和轻链可变区：
(a)如SEQ ID NO：8所示的重链可变区和如SEQ ID NO：7所示的轻链可变区；
(b)如SEQ ID NO：17所示的重链可变区和如SEQ ID NO：16所示的轻链可变区；
(c)如SEQ ID NO：26所示的重链可变区和如SEQ ID NO：25所示的轻链可变区；
(d)如SEQ ID NO：35所示的重链可变区和如SEQ ID NO：34所示的轻链可变区；和
(e)如SEQ ID NO：44所示的重链可变区和如SEQ ID NO：43所示的轻链可变区；
(f)如SEQ ID NO：47所示的重链可变区和如SEQ ID NO：46所示的轻链可变区；
(g)如SEQ ID NO：50所示的重链可变区和如SEQ ID NO：49所示的轻链可变区；
(h)如SEQ ID NO：53所示的重链可变区和如SEQ ID NO：52所示的轻链可变区；
(i)如SEQ ID NO：56所示的重链可变区和如SEQ ID NO：55所示的轻链可变区；
(j)如SEQ ID NO：59所示的重链可变区和如SEQ ID NO：58所示的轻链可变区；
(k)如SEQ ID NO：62所示的重链可变区和如SEQ ID NO：61所示的轻链可变区；
(l)如SEQ ID NO：65所示的重链可变区和如SEQ ID NO：64所示的轻链可变区。
任选地，所述重链可变区和轻链可变区与(a)-(l)任一组的重链可变区和轻链可变区相比具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、100％同一性；
任选地，所述重链可变区和轻链可变区与(a)-(l)任一组的重链可变区和轻链可变区相比具有一个或几个氨基酸的修饰，例如最多1、2、3、4、5、6、7、8、9或10个氨基酸的修饰；优选地，所述修饰是保守性修饰，例如氨基酸的保守性取代、添加和缺失。
在一个实施方案中，本发明的抗体或其抗原结合片段与选自SEQ ID NO：9、18、27、36、45、48、51、54、57、60、63和66的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、100％同一性，或与选自SEQ ID NO：9、18、27、36、45、48、51、54、57、60、63和66的氨基酸序列相比具有一个或几个氨基酸(例如最多1、2、3、4、5、6、7、8、9或10个氨基酸)的修饰。优选地，所述修饰是保守性修饰，例如氨基酸的保守性取代、添加和缺失。优选地，本发明的抗体或其抗原结合片段的氨基酸序列选自SEQ ID NO：9、18、27、36、45、48、51、54、57、60、63和66。
在第二个方面，本发明还提供一种多特异性抗体(优选双特异性抗体或三特异性抗体)，其包含如上所述的抗B7-H3抗体或其抗原结合片段，和一个或多个与其他抗原特异性结合的第二抗体或其抗原结合片段。
在一个实施方案中，本发明中所述的抗体或其抗原结合片段可以具有任何抗体或抗体片段形式，例如全长抗体、Fab、Fab'、(Fab')2、Fv、scFv、scFv-scFv、微抗体、双抗体或sdAb。
在第三个方面，本发明还提供编码上述抗体或其抗原结合片段，或上述多特异性抗体的核酸分子。本发明还提供包含编码上述抗体或其抗原结合片段或多特异性抗体的核酸分子的载体，以及表达所述抗B7-H3抗体或其抗原结合片段或多特异性抗体的宿主细胞。
在第四个方面，本发明还提供一种嵌合多肽，其包含本发明所述的抗B7-H3抗体或其抗原结合片段或多特异性抗体，所述嵌合多肽选自CAR、TCR、TRuC、TAC或ImmTAC。
在一种实施方案中，所述嵌合多肽还包含跨膜结构域和胞内信号传导结构域；所述胞内信号传导结构域包括初级信号传导结构域和/或共刺激结构域。其中所述跨膜结构域选自以下蛋白质的跨膜结构域：TCRα链、TCRβ链、TCRγ链、TCRδ链、CD3ζ亚基、CD3ε亚基、CD3γ亚基、CD3δ亚基、CD45、CD4、CD5、CD8α、CD9、CD16、CD22、CD33、CD28、CD37、CD64、CD80、CD86、CD134、CD137和CD154；所述初级信号传导结构域选自以下蛋白的胞内区：FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD3ζ、CD22、CD79a、CD79b和CD66d；所述共刺激结构域选自以下蛋白质的共刺激信号传导结构域：TLR1、TLR2、TLR3、TLR4、TLR5、TLR6、TLR7、TLR8、TLR9、TLR10、CARD11、CD2、B7-H3、CD8、CD18(LFA-1)、CD27、CD28、CD30、CD40、CD54(ICAM)、CD83、CD134(OX40)、CD137(4-1BB)、CD270(HVEM)、CD272(BTLA)、CD276(B7-H3)、CD278(ICOS)、CD357(GITR)、DAP10、LAT、NKG2C、SLP76、PD-1、LIGHT、TRIM以及ZAP70。
在第五个方面，本发明还提供编码如上所定义的靶向B7-H3的嵌合抗原受体的核酸分子，以及包含所述核酸分子的载体，以及包含如上所定义的靶向B7-H3的嵌合抗原受体的细胞，优选地，所述细胞选自免疫细胞，例如T细胞、NK细胞、NKT细胞、巨噬细胞、树突细胞，更优选地，所述细胞选自CD4+CD8+T细胞、CD4+T细胞、CD8+T细胞、CD4-CD8-T细胞、肿瘤浸润细胞、记忆T细胞、幼稚T细胞、γδ-T细胞以及αβ-T细胞。在一个优选的实施方案中，所述工程化免疫细胞还包含靶向其他肿瘤抗原的第二嵌合抗原受体。
在第六个方面，本发明还提供一种抗体偶联物，其包含本发明所定义的抗B7-H3抗体或其抗原结合片段和第二功能性结构，其中所述第二功能性结构选自Fc、放射性同位素、延长半衰期的结构部分、可检测标记物和药物。
在一个实施方案中，所述延长半衰期的结构部分选自：所述延长半衰期的结构部分选自白蛋白的结合结构、转铁蛋白的结合结构、聚乙二醇分子、重组聚乙二醇分子、人血清白蛋白、人血清白蛋白的片段和结合人血清白蛋白的白多肽(包括抗体)。在一个实施方案中，可检测标记物选自荧光团、化学发光化合物、生物发光化合物、酶、抗生素抗性基因和造影剂。在一个实施方案中，所述药物选自细胞毒素和免疫调节剂。
在第七个方面，本发明还提供一种检测试剂盒，其包含本发明所述的抗B7-H3抗体或其抗原结合片段、多特异性抗体、抗体偶联物、嵌合抗原受体或工程化免疫细胞。
在第八个方面，本发明还提供一种药物组合物，其包含本发明所述的抗B7-H3抗体或其抗原结合片段、嵌合抗原受体、多特异性抗体、工程化免疫细胞或抗体偶联物，和一种或多种药学上可接受的赋形剂。
在第九个方面，本发明还提供一种治疗和/或预防和/或诊断与B7-H3表达相关的疾病的方法，包括向受试者施用如上所述的抗B7-H3抗体或其抗原结合片段、嵌合抗原受体、多特异性抗体、抗体偶联物、工程化免疫细胞或药物组合物。
发明详述
除非另有说明，否则本文中所使用的所有科学技术术语的含义与本发明所属领域的普通技术人员通常所了解的相同。
抗B7-H3抗体或其抗原结合片段
如本文所用，术语“抗体”具有本领域技术人员所理解的最广泛的含义，并且包括单克隆抗体(包含完整抗体)、多克隆抗体、多价抗体、多特异性抗体(例如双特异性抗体)、和能够表现期望的生物活性的携带一个或多个CDR序列的抗体片段或合成多肽。本发明所述抗体可为任何种类(例如IgG、IgE、IgM、IgD、IgA等)或亚类(例如IgG1、IgG2、IgG2a、IgG3、IgG4、IgA1、IgA2等)。
如本文所用，术语“抗原结合片段”或“抗体片段”指抗体中保留特异性结合抗原的能力的一个或更多个片段。已经显示，抗体的抗原结合功能可通过全长抗体的片段来实现。本发明中的抗体片段的实例包括但不限于：Fab、Fab'、F(ab')2、Fd片段、Fd′、Fv片段、单链抗体(scFv)、二硫键-连接的Fv(sdFv)、抗体的重链可变区(VH) 或轻链可变区(VL)、线性抗体、具有两个抗原结合位点的“双体”、单结构域抗体、纳米抗体、所述抗原的天然配体或其功能性片段等。因此，除非上下文明确指出，否则本发明的“抗体”涵盖如上定义的抗体片段或抗原结合片段。因此，在一个优选的实施方案中，本发明的抗体选自IgG、Fab、Fab'、F(ab')2、Fd、Fd′、Fv、scFv、sdFv、线性抗体和双体。
通常，完整抗体包括通过二硫键连接在一起的两条重链和两条轻链，每条轻链通过二硫键被连至各自的重链，呈“Y”形结构。每条重链包含重链可变区(VH)和重链恒定区，其中重链可变区包含三个互补决定区(CDR)：CDR-H1、CDR-H2和CDR-H3，重链恒定区包含三个恒定结构域：CH1、CH2和CH3。每条轻链包含轻链可变区(VL)和轻链恒定区，其中轻链可变区包含三个CDR：CDR-L1、CDR-L2和CDR-L3，轻链恒定区包含一个恒定结构域CL。在重链/轻链可变区中，CDR被更保守的框架区(FR)隔开。重链/轻链的可变区负责与抗原的识别和结合，恒定区则可以介导抗体与宿主组织或因子的结合，包括免疫系统的各种细胞(例如效应细胞)和经典补体系统的第一组分。
可以使用许多本领域熟知的编号方案容易地确定给定CDR或FR的精确氨基酸序列边界，这些方案包括：Kabat等人(1991),“Sequences ofProteins of Immunological Interest,”第5版Public Health Service,NationalInstitutes of Health,贝塞斯达,马里兰州(“Kabat”编号方案)；Al-Lazikani等人,(1997)JMB 273,927-948(“Chothia”编号方案)；MacCallum等人,J.Mol.Biol.262:732-745(1996),“Antibody-antigen interactions:Contact analysis and binding sitetopography,”J.Mol.Biol.262,732-745”(“Contact”编号方案)；Lefranc MP等人,“IMGTunique numbering for immunoglobulin and T cell receptor variable domains andIg superfamily V-like domains,”Dev Comp Immunol,2003年1月；27(1):55-77(“IMGT”编号方案)；Honegger A和Plückthun A,“Yet another numbering scheme forimmunoglobulin variable domains:an automatic modeling and analysis tool,”JMol Biol,2001年6月8日；309(3):657-70(“Aho”编号方案)；和Martin等人,“Modeling antibody hypervariable loops:a combined algorithm,”PNAS,1989,86(23):9268-9272(“AbM”编号方案)。
给定CDR或FR的边界可能取决于用于鉴定的方案而不同。例如，Kabat方案是基于结构比对，而Chothia方案是基于结构信息。Kabat和Chothia方案的编号都是基于最常见的抗体区域序列长度，其中通过插入字母提供插入(例如“30a”)并且在一些抗体中出现缺失。这两种方案将某些插入和缺失(indel)放置在不同的位置，从而产生不同的编号。Contact方案是基于对复杂晶体结构的分析，并且在许多方面与Chothia编号方案相似。AbM方案是介于Kabat与Chothia定义之间的折衷，其基于Oxford Molecular的AbM抗体建模软件所使用的方案。
因此，除非另有规定，否则应当理解，给定抗体或其区域(如其可变区)的“CDR”涵盖由任何上述方案或其他已知方案所定义的CDR。例如，在指定特定的CDR(例如CDR3)含有给定氨基酸序列的情况下，应理解，这样的CDR还可以具有由任何上述方案或其他已知方案所定义的相应CDR(例如CDR3)的序列。同样，除非另有规定，否则应当理解给定抗体或其区域(如其可变区)的FR涵盖由任何上述方案或其他已知方案所定义的FR。除非特别指出，否则在本文中用于界定CDR和FR的边界的编号方案采用Kabat方案。
“单链抗体”和“scFv”在本文中可互换使用，是指由抗体重链可变区(VH)和轻链可变区(VL)通过接头连接而成的抗体。可以选择接头的最佳长度和/或氨基酸组成。接头的长度会明显影响scFv的可变区折叠和相互作用情况。事实上，如果使用较短的接头(例如在5-10个氨基酸之间)，则可以防止链内折叠。关于接头的大小和组成的选择，参见例如，Hollinger等人,1993Proc Natl Acad.Sci.U.S.A.90:6444-6448；美国专利申请公布号2005/0100543、2005/0175606、2007/0014794；以及PCT公布号WO2006/020258和WO2007/024715，其全文通过引用并入本文。常用的接头例如GSTSGSGKPGSGEGSTKG(SEQ ID NO：66)、GGGGSGGGGSGGGGS(SEQ ID NO：67)。scFv可以包含以任何顺序连接的VH和VL，例如VH-接头-VL或VL-接头-VH。
在一个实施方案中，本发明的抗体或其抗原结合片段是鼠源抗体、嵌合抗体、人源化抗体或人抗体，优选是人源化抗体。
如本文所用，术语“嵌合抗体”指这样的抗体，其中每个重链和轻链氨基酸序列的一部分与来自特定物种或者属于特定类别的抗体中相应序列同源，而该链的其余区段则与另一物种或属于另一类别的相应序列同源。一般地，轻链和重链的可变区均来自一个物种的抗体的可变区，而恒定区则与来自另一个物种的抗体序列同源。这种嵌合形式的一个明显优点是可使用易于获得的B细胞或来自非人宿主的杂交瘤从目前已知的来源方便地产生可变区，而与其组合的恒定区来自例如人细胞。所述可变区具有易于制备的优点，并且特异性不受来源的影响，而由于恒定区来自人，因此该抗体在注射时引发人免疫应答的可能性将比恒定区来自非人来源时更低。
如本文所用，“人源化”抗体是指其中所有或基本上所有CDR氨基酸残基源自非人CDR并且所有或基本上所有FR氨基酸残基源自人FR。非人抗体的“人源化形式”是指所述非人抗体的变体，其经历人源化以通常降低对人的免疫原性，同时保留亲本非人抗体的特异性和亲和力。在一些实施方案中，人源化抗体中的一些FR残基被来自非人抗体(例如，衍生CDR残基的抗体)的相应残基取代，例如以恢复或改善抗体特异性或亲和力。
人源化抗体及其制备方法是本领域技术人员熟知的，参见例如Almagro和Fransson,Front.Biosci.13:1619-1633(2008)。可用于人源化的人类框架区包括但不限于：使用“最佳拟合”方法选择的框架区；源自轻链或重链可变区的特定亚组的人类抗体的共有序列的框架区；人类成熟(体细胞突变)框架区或人类种系框架区；以及筛选FR文库得到的框架区。
如本文所用，术语“人抗体”旨在包括具有来自人生殖系免疫球蛋白序列的可变区和恒定区的抗体。本发明的人抗体可包含不是由人生殖系免疫球蛋白序列编码的氨基酸残基(例如，通过体外随机诱变或定点诱变或者通过体内体细胞突变引入的突变)。
在一个实施方案中，本发明提供一种靶向B7-H3的抗体或其抗原结合片段，其包含：
(1)如SEQ ID NO：1所示的CDR-L1、如SEQ ID NO：2所示的CDR-L2、如SEQ ID NO：3所示的CDR-L3、
如SEQ ID NO：4所示的CDR-H1、如SEQ ID NO：5所示的CDR-H2和如SEQ ID NO：6所示的CDR-H3；
(2)如SEQ ID NO：10所示的CDR-L1、如SEQ ID NO：11所示的CDR-L2、如SEQ ID NO：12所示的CDR-L3、如SEQ ID NO：13所示的CDR-H1、如SEQ ID NO：14所示的CDR-H2和如SEQ ID NO：15所示的CDR-H3；
(3)如SEQ ID NO：19所示的CDR-L1、如SEQ ID NO：20所示的CDR-L2、如SEQ ID NO：21所示的CDR-L3、如SEQ ID NO：22所示的CDR-H1、如SEQ ID NO：23所示的CDR-H2和如SEQ ID NO：24所示的CDR-H3；
(4)如SEQ ID NO：28所示的CDR-L1、如SEQ ID NO：29所示的CDR-L2、如SEQ ID NO：30所示的CDR-L3、如SEQ ID NO：31所示的CDR-H1、如SEQ ID NO：32所示的CDR-H2和如SEQ ID NO：33所示的CDR-H3；或
(5)如SEQ ID NO：37所示的CDR-L1、如SEQ ID NO：38所示的CDR-L2、如SEQ ID NO：39所示的CDR-L3、如SEQ ID NO：40所示的CDR-H1、如SEQ ID NO：41所示的CDR-H2和如SEQ ID NO：42所示的CDR-H3。
在一个实施方案中，本发明的抗体或其抗原结合片段包含重链可变区和轻链可变区，所述重链可变区与选自SEQ ID NO：8、17、26、35、44、47、50、53、56、59、62和65的氨基酸序列具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％同一性，或与选自SEQ ID NO：8、17、26、35、44、47、50、53、56、59、62和65的氨基酸序列相比具有一个或几个(例如最多1、2、3、4、5、6、7、8、9或10个氨基酸)氨基酸的修饰；所述轻链可变区与选自SEQ ID NO：7、16、25、34、43、46、49、52、55、58、61和64的氨基酸序列具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％同一性，或与选自SEQ ID NO：7、16、25、34、43、46、49、52、55、58、61和64的氨基酸序列相比具有一个或几个氨基酸(例如最多1、2、3、4、5、6、7、8、9或10个氨基酸)的修饰。优选地，所述修饰是保守性修饰，例如氨基酸的保守性取代、添加和缺失。在一个优选的实施方案中，本发明的抗体或其抗原结合片段包含选自SEQ ID NO：8、17、26、35、44、47、50、53、56、59、62和65的重链可变区和选自SEQ ID NO：：7、16、25、34、43、46、49、52、55、58、61和64的轻链可变区。在一个实施方案中，本发明的抗体或其抗原结合片段包含选自以下的重链可变区和轻链可变区：
(a)如SEQ ID NO：8所示的重链可变区和如SEQ ID NO：7所示的轻链可变区；
(b)如SEQ ID NO：17所示的重链可变区和如SEQ ID NO：16所示的轻链可变区；
(c)如SEQ ID NO：26所示的重链可变区和如SEQ ID NO：25所示的轻链可变区；
(d)如SEQ ID NO：35所示的重链可变区和如SEQ ID NO：34所示的轻链可变区；和
(e)如SEQ ID NO：44所示的重链可变区和如SEQ ID NO：43所示的轻链可变区；
(f)如SEQ ID NO：47所示的重链可变区和如SEQ ID NO：46所示的轻链可变区；
(g)如SEQ ID NO：50所示的重链可变区和如SEQ ID NO：49所示的轻链可变区；
(h)如SEQ ID NO：53所示的重链可变区和如SEQ ID NO：52所示的轻链可变区；
(i)如SEQ ID NO：56所示的重链可变区和如SEQ ID NO：55所示的轻链可变区；
(j)如SEQ ID NO：59所示的重链可变区和如SEQ ID NO：58所示的轻链可变区；
(k)如SEQ ID NO：62所示的重链可变区和如SEQ ID NO：61所示的轻链可变区；
(l)如SEQ ID NO：65所示的重链可变区和如SEQ ID NO：64所示的轻链可变区。
任选地，所述重链可变区和轻链可变区与(a)-(l)任一组的重链可变区和轻链可变区相比具有至少90％、至少91％、至少92％、至少93％、至少94％、至少95％、至少96％、至少97％、至少98％、至少99％或100％同一性；
任选地，所述重链可变区和轻链可变区与(a)-(l)任一组的重链可变区和轻链可变区具有一个或几个氨基酸的修饰，例如最多1、2、3、4、5、6、7、8、9或10个氨基酸的修饰；优选地，所述修饰是保守性修饰，例如氨基酸的保守性取代、添加和缺失。
在一个实施方案中，本发明的抗体或其抗原结合片段与选自SEQ ID NO：9、18、27、36、45、48、51、54、57、60、63和66的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％、100％同一性，或与选自SEQ ID NO：9、18、27、36、45、48、51、54、57、60、63和66的氨基酸序列相比具有一个或几个氨基酸的修饰，例如最多1、2、3、4、5、6、7、8、9或10个氨基酸的修饰。优选地，所述修饰是保守性修饰，例如氨基酸的保守性取代、添加和缺失。优选地，本发明的抗体或其抗原结合片段的氨基酸序列选自SEQ ID NO：9、18、27、36、45、48、51、54、57、60、63和66。
如本文所用，术语“保守性修饰”是指不会明显影响或改变含有该氨基酸序列的抗体或抗体片段的结合特征的氨基酸修饰。这些保守性修饰包括氨基酸的保守性取代、添加及缺失。修饰可以通过本领域中已知的标准技术，如定点诱变和PCR介导的诱变而引入本发明的嵌合抗原受体中。保守性氨基酸取代是氨基酸残基被具有类似侧链的氨基酸残基置换的取代。具有类似侧链的氨基酸残基家族已在本领域中有定义，包括碱性侧链(例如赖氨酸、精氨酸、组氨酸)、酸性侧链(例如天冬氨酸、谷氨酸)、不带电荷极性侧链(例如甘氨酸、天冬酰胺、谷氨酰胺、丝氨酸、苏氨酸、酪氨酸、半胱氨酸)、非极性侧链(例如丙氨酸、缬氨酸、亮氨酸、异亮氨酸、脯氨酸、苯丙氨酸、甲硫氨酸、色氨酸)、β-分支侧链(例如苏氨酸、缬氨酸、异亮氨酸)及芳香族侧链(例如酪氨酸、苯丙氨酸、色氨酸、组氨酸)。保守性修饰可以例如基于极性、电荷、溶解度、疏水性、亲水性和/或所涉及残基的两亲性质的相似性来进行选择。
如本文所用，术语序列“同一性”表示两个(核苷酸或氨基酸)序列在比对中在相同位置处具有相同残基的程度，并且通常表示为百分数。优选地，同一性在被比较的序列的整体长度上确定。因此，具有完全相同序列的两个 拷贝具有100％同一性。本领域技术人员知晓，可以使用一些算法来确定序列同一性，例如Blast(Altschul等(1997)Nucleic Acids Res.25：3389-3402)、Blast2(Altschul等(1990)J.Mol.Biol.215：403-410)、Smith-Waterman(Smith等(1981)J.Mol.Biol.147：195-197)和ClustalW。
在在一个方面，本发明还提供包含如上所述的抗B7-H3抗体或其抗原结合片段的多特异性抗体(优选双特异性抗体或三特异性抗体)，其还包含一个或多个与其他抗原特异性结合的第二抗体。
如本文所用，术语“多特异性”是指抗原结合蛋白具有多表位特异性(即，能够特异性结合一个生物分子上的两个、三个或更多个不同的表位或能够特异性结合两个、三个或更多个不同的生物分子上的表位)。如本文所用，术语“双特异性”表示抗原结合蛋白具有两种不同的抗原结合特异性。
在一个实施方案中，第二抗体可以具有任何抗体或抗体片段形式，例如全长抗体、Fab、Fab'、(Fab')2、Fv、scFv、scFv-scFv、微抗体、双抗体或sdAb。
因此，在一个实施方案中，所述第二抗体靶向选自以下的抗原：BCMA、CD4、CD5、CD7、CD8、CD14、CD15、CD19、CD20、CD21、CD22、CD23、CD25、CD33、CD37、CD38、CD40、CD40L、CD46、CD52、CD54、CD80、CD126、CD138、B7、MUC-1、Ia、HM1.24、HLA-DR、腱生蛋白、血管生成因子、VEGF、PIGF、ED-B纤连蛋白、致癌基因、致癌基因产物、CD66a-d、坏死抗原、Ii、IL-2、T101、TAC、IL-6、DR4、DR5、tEGFR、Her2、L1-CAM、间皮素、CEA、乙型肝炎表面抗原、抗叶酸受体、CD24、CD30、CD44、EGFR、EGP-2、EGP-4、EPHa2、ErbB2、ErbB3、ErbB4、ErbB二聚体、EGFR vIII、FBP、FCRL5、FCRH5、胎儿乙酰胆碱受体、GD2、GD3、GPRC5D、HMW-MAA、IL-22R-α、IL-13R-α2、kdr、κ轻链、Lewis Y、L1-CAM、MAGE-A1、MAGE-A3、MAGE-A6、PRAME、生存素、EGP2、EGP40、TAG72、B7-H6、IL-13Ra2、CA9、CD171、G250/CAIX、HLA-A1、HLA-A2、NY-ESO-1、PSCA、叶酸受体-a、CD44v6、CD44v7/8、avb6整合素、8H9、NCAM、VEGF受体、5T4、胎儿AchR、NKG2D配体、双抗原、与通用标签相关的抗原、癌症-睾丸抗原、MUC1、MUC16、NY-ESO-1、MART-1、gp100、癌胚胎抗原、VEGF-R2、CEA、前列腺特异性抗原、PSMA、Her2/neu、雌激素受体、孕酮受体、肝配蛋白B2、CD123、c-Met、GD-2、OGD2、CE7、WT-1、细胞周期蛋白A2、CCL-1、hTERT、MDM2、CYP1B、WT1、活素、AFP、p53、细胞周期蛋白(D1)、CS-1、BAFF-R、TACI、CD56、TIM-3、CD123、L1-细胞粘附分子、细胞周期蛋白(如细胞周期蛋白A1(CCNA1))和/或病原体特异性抗原、生物素化分子、由HIV、HCV、HBV和/或其他病原体表达的分子；和/或新表位或新抗原。
核酸、载体、宿主细胞
在另一方面中，本发明涉及编码本发明的抗B7-H3抗体或多特异性抗体的核酸分子。本发明的核酸可为RNA、DNA或cDNA。根据本发明的一个实施方案，本发明的核酸是基本上分离的核酸。
本发明的核酸也可呈载体形式，可存在于载体中和/或可为载体的一部分，该载体例如质粒、粘端质粒或YAC。载体可尤其为表达载体，即可提供B7-H3抗体在体外和/或体内(即在适合宿主细胞、宿主有机体和/或表达系统中)表达的载体。该表达载体通常包含至少一种本发明的核酸分子，其可操作地连接至一个或多个适合的表达调控元件(例如启动子、增强子、终止子等)。对所述调控元件及其序列进行选择以便在特定宿主中表达是本领域技术人员熟知的。对本发明的B7-H3抗体的表达有用或必需的调控元件及其他元件的具体实例包括但不限于启动子、增强子、终止子、整合因子、选择标记物、前导序列、报告基因。
在另一方面中，本发明还提供表达本发明的B7-H3抗体、多特异性抗体和/或含有本发明的核酸或载体的宿主细胞。本发明的优选宿主细胞为细菌细胞、真菌细胞或哺乳动物细胞。
适合的细菌细胞包括革兰氏阴性细菌菌株(例如大肠杆菌(Escherichia coli)菌株、变形杆菌属(Proteus)菌株及假单胞菌属(Pseudomonas)菌株)及革兰氏阳性细菌菌株(例如芽孢杆菌属(Bacillus)菌株、链霉菌属(Streptomyces)菌株、葡萄球菌属(Staphylococcus)菌株及乳球菌属(Lactococcus)菌株)的细胞。
适合的真菌细胞包括木霉属(Trichoderma)、脉孢菌属(Neurospora)及曲菌属(Aspergillus)的物种的细胞；或者包括酵母属(Saccharomyces)(例如酿酒酵母(Saccharomyces cerevisiae))、裂殖酵母属(Schizosaccharomyces)(例如粟酒裂殖酵母(Schizosaccharomyces pombe))、毕赤酵母属(Pichia)(例如巴斯德毕赤酵母(Pichiapastoris)及嗜甲醇毕赤酵母(Pichia methanolica))及汉森酵母属(Hansenula)的物种的细胞。
适合的哺乳动物细胞包括例如HEK293细胞、CHO细胞、BHK细胞、HeLa细胞、COS细胞等。
然而，本发明也可使用两栖类细胞、昆虫细胞、植物细胞及本领域中用于表达异源蛋白的任何其他细胞。
嵌合多肽
在另一方面，本发明还提供包含如上所述的抗B7-H3抗体的嵌合多肽，例如CAR、TCR、TRuC、TAC或ImmTAC。优选地，本发明还提供包含如上所述的抗B7-H3抗体的嵌合抗原受体。
如本文所用，术语“嵌合抗原受体”或“CAR”是指人工构建的杂合多肽，该杂合多肽一般包括配体结合结构域(例如抗体的抗原结合片段)、跨膜结构域、任选的共刺激结构域和细胞内信号传导结构域，各个结构域之间通过接头连接。CAR能够利用抗体的抗原结合特性以非MHC限制性的方式将T细胞和其它免疫细胞的特异性和反应性重定向至所选择的靶标。
在一个实施方案中，本发明提供一种嵌合抗原受体，其包含如上所述的抗B7-H3抗体或其抗原结合片段或含有所述抗B7-H3抗体的多特异性抗体、跨膜结构域和胞内信号传导结构域。
如本文所用，术语“跨膜结构域”是指能够使嵌合抗原受体在免疫细胞(例如淋巴细胞、NK细胞或NKT细胞)表面上表达，并且引导免疫细胞针对靶细胞的细胞应答的多肽结构。跨膜结构域可以是天然或合成的，也可以源自任何膜结合蛋白或跨膜蛋白。当嵌合抗原受体与靶抗原结合时，跨膜结构域能够进行信号传导。特别适用于本发明中的跨膜结构域可以源自例如TCRα链、TCRβ链、TCRγ链、TCRδ链、CD3ζ亚基、CD3ε亚基、CD3γ亚基、CD3δ亚基、CD45、CD4、CD5、CD8α、CD9、CD16、CD22、CD33、CD28、CD37、CD64、CD80、CD86、CD134、CD137、CD154及其功能性片段。或者，跨膜结构域可以是合成的并且可以主要地包含疏水性 残基如亮氨酸和缬氨酸。优选地，所述跨膜结构域源自CD8α链，其与SEQ ID NO:67所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性，或其编码序列与SEQ ID NO:68所示的核酸分子具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。
如本文所用，术语“胞内信号传导结构域”是指转导效应子功能信号并指导细胞进行指定功能的蛋白质部分。在一个实施方案中，本发明的嵌合抗原受体包含的胞内信号传导结构域可以是T细胞受体和共受体的胞内区序列，其在抗原受体结合以后一同起作用以引发信号传导，以及这些序列的任何衍生物或变体和具有相同或相似功能的任何合成序列。胞内信号传导结构域可以包含许多免疫受体酪氨酸激活基序(Immunoreceptor Tyrosine-based Activation Motifs,ITAM)。本发明的胞内信号传导结构域的非限制性施例包括但不限于FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD3ζ、CD22、CD79a、CD79b和CD66d等的胞内区。在优选的实施方式中，本发明CAR的信号传导结构域可以包含CD3ζ胞内区，其与SEQ ID NO:69所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性，或其编码序列与SEQ ID NO:70所示的核酸分子具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。
在一个实施方案中，本发明的嵌合抗原受体还可以包含位于抗体和跨膜结构域之间的铰链区。如本文所用，术语“铰链区”一般是指作用为连接跨膜结构域至抗体的任何寡肽或多肽。具体地，铰链区用来为抗体提供更大的灵活性和可及性。铰链区可以包含最多达300个氨基酸，优选10至100个氨基酸并且最优选25至50个氨基酸。铰链区可以全部或部分源自天然分子，如全部或部分源自CD8、CD4或CD28的胞外区，或全部或部分源自抗体恒定区。或者，铰链区可以是对应于天然存在的铰链序列的合成序列，或可以是完全合成的铰链序列。在优选的实施方式中，所述铰链区包含CD8α、CD28、FcγRIIIα受体、IgG4或IgG1的铰链区部分，更优选CD8α、CD28或IgG4铰链，其与SEQ ID NO:71所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性，或其编码序列与SEQ ID NO:72所示的核苷酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。
在一个实施方案中，嵌合抗原受体还可以包含一个或多个共刺激结构域。共刺激结构域可以是来自共刺激分子的细胞内功能性信号传导结构域，其包含所述共刺激分子的整个细胞内部分，或其功能片段。“共刺激分子”是指在T细胞上与共刺激配体特异性结合，由此介导T细胞的共刺激反应(例如增殖)的同源结合配偶体。共刺激分子包括但不限于1类MHC分子、BTLA和Toll配体受体。本发明的共刺激结构域的非限制性施例包括但不限于源自以下蛋白质的共刺激信号传导结构域：TLR1、TLR2、TLR3、TLR4、TLR5、TLR6、TLR7、TLR8、TLR9、TLR10、CARD11、CD2、B7-H3、CD8、CD18(LFA-1)、CD27、CD28、CD30、CD40、CD54(ICAM)、CD83、CD134(OX40)、CD137(4-1BB)、CD270(HVEM)、CD272(BTLA)、CD276(B7-H3)、CD278(ICOS)、CD357(GITR)、DAP10、LAT、NKG2C、SLP76、PD-1、LIGHT、TRIM以及ZAP70。优选地，本发明CAR的共刺激结构域来自4-1BB、CD28或4-1BB+CD28。在一个实施方案中，4-1BB共刺激结构域与SEQ ID NO:73所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性，或其编码序列与SEQ ID NO:74所示的核酸分子具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。
在一个实施方案中，本发明的CAR还可以包含信号肽，使得当其在细胞例如T细胞中表达时，新生蛋白质被引导至内质网并随后引导至细胞表面。信号肽的核心可以含有长的疏水性氨基酸区段，其具有形成单个α-螺旋的倾向。在信号肽的末端，通常有被信号肽酶识别和切割的氨基酸区段。信号肽酶可以在移位期间或完成后切割，以产生游离信号肽和成熟蛋白。然后，游离信号肽被特定蛋白酶消化。可用于本发明的信号肽是本领域技术人员熟知的，例如衍生自B2M、CD8α、IgG1、GM-CSFRα等的信号肽。在一个实施方案中，可用于本发明的信号肽来自CD8α，其与SEQ ID NO:75所示的氨基酸序列具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性，或其编码序列与SEQ ID NO:76所示的核酸分子具有至少70％，优选至少80％，更优选至少90％、95％、97％或99％或100％的序列同一性。
在一个实施方案中，所述CAR含有如本文所提供的抗B7-H3抗体或其抗原结合片段或含有所述抗B7-H3抗体的多特异性抗体、CD8α或CD28跨膜区、CD28和/或4-1BB共刺激结构域，和CD3ζ胞内信号传导结构域。在该实施方案中，所述CAR还可以进一步包含来自B2M、CD8α、IgG1或GM-CSFRα的信号肽。
本发明还提供编码如上所定义的靶向B7-H3的嵌合抗原受体的核酸分子，以及包含所述核酸分子的载体。
如本文所用，术语“载体”是用作将(外源)遗传材料转移到宿主细胞中的媒介核酸分子，在该宿主细胞中所述核酸分子可以例如复制和/或表达。载体一般包括靶向载体和表达载体。“靶向载体”是通过例如同源重组或使用特异性靶向位点处序列的杂合重组酶将分离的核酸递送至细胞内部的介质。“表达载体”是用于异源核酸序列(例如编码本发明的嵌合抗原受体多肽的那些序列)在合适的宿主细胞中的转录以及它们的mRNA的翻译的载体。可用于本发明的合适载体是本领域已知的，并且许多可商购获得。在一个实施方案中，本发明的载体包括但不限于质粒、病毒(例如逆转录病毒、慢病毒、腺病毒、牛痘病毒、劳氏肉瘤病毒(RSV、多瘤病毒和腺相关病毒(AAV)等)、噬菌体、噬菌粒、粘粒和人工染色体(包括BAC和YAC)。载体本身通常是核酸分子，通常是包含插入物(转基因)的DNA序列和作为载体“骨架”的较大序列。工程化载体通常还包含在宿主细胞中自主复制的起点(如果需要多核苷酸的稳定表达)、选择标记和限制酶切割位点(如多克隆位点，MCS)。载体可另外包含启动子、多聚腺苷酸尾(polyA)、3’UTR、增强子、终止子、绝缘子、操纵子、选择标记、报告基因、靶向序列和/或蛋白质纯化标签等元件。在一个具体的实施方案中，所述载体是体外转录的载体。
工程化免疫细胞
在一个方面，本发明还提供表达本发明所述嵌合多肽，例如重组TCR受体或嵌合抗原受体的工程化免疫细胞。
如本文所用，术语“免疫细胞”是指免疫系统的具有一种或多种效应子功能(例如，细胞毒性细胞杀伤活性、分泌细胞因子、诱导ADCC和/或CDC)的任何细胞。例如，免疫细胞可以是T细胞、巨噬细胞、树突状细胞、单核细胞、NK细胞和/或NKT细胞。在一个实施方案中，免疫细胞衍生自干细胞，例如成体干细胞、胚胎干细胞、脐带血干细胞、祖细胞、骨髓干细胞、诱导多能干细胞、全能干细胞或造血干细胞等。优选地，免疫细胞是T细胞。T细胞可以是任何T细胞，如体外培养的T细胞，例如原代T细胞，或者来自体外培养的T细胞系例如Jurkat、SupT1等的T细胞，或获得自受试者的T细胞。受试者的实例包括人、狗、猫、小鼠、大鼠及其转基因物种。T细胞可以从多种来源获得，包括外周血单核细胞、骨髓、淋巴结组织、脐血、胸腺组织、来自感染部位的组织、腹水、胸膜积液、脾组织及肿瘤。T细胞也可以被浓缩或纯化。T细胞可以处于任何发育阶段，包括但不限于，CD4+CD8+T细胞、CD4+T细胞(例如Th1和Th2细胞)、CD8+T细胞(例如，细胞毒性T细胞)、CD4-CD8-T细胞、肿瘤浸润细胞、记忆T细胞、幼稚T细胞、γδ-T细胞、αβ-T细胞等。在一个优选的实施方案中，免疫细胞是人T细胞。可以使用本领域技术人员已知的多种技术，如Ficoll分离从受试者的血液获得T细胞。
在一个实施方案中，为减少移植物抗宿主病的风险，所述工程化免疫细胞还包含至少一种选自以下的基因的表达被抑制或沉默：CD52、GR、dCK、TCR/CD3基因(例如TRAC、TRBC、CD3γ、CD3δ、CD3ε、CD3ζ)、MHC相关基因(HLA-A、HLA-B、HLA-C、B2M、HLA-DPA、HLA-DQ、HLA-DRA、TAP1、TAP2、LMP2、LMP7、RFX5、RFXAP、RFXANK、CIITA)和免疫检查点基因，如PD1、LAG3、TIM3、CTLA4、PPP2CA、PPP2CB、PTPN6、PTPN22、PDCD1、HAVCR2、BTLA、CD160、TIGIT、CD96、CRTAM、TNFRSF10B、TNFRSF10A、CASP8、CASP10、CASP3、CASP6、CASP7、FADD、FAS、TGFBRII、TGFRBRI、SMAD2、SMAD3、SMAD4、SMAD10、SKI、SKIL、TGIF1、IL10RA、IL10RB、HMOX2、IL6R、IL6ST、EIF2AK4、CSK、PAG1、SIT、FOXP3、PRDM1、BATF、GUCY1A2、GUCY1A3、GUCY1B2和GUCY1B3。优选地，所述工程化免疫细胞还包含至少一种选自以下的基因的表达被抑制或沉默：TRAC、TRBC、HLA-A、HLA-B、HLA-C、B2M、RFX5、RFXAP、RFXANK、CIITA、PD1、LAG3、TIM3、CTLA4，更优选TRAC、TRBC、HLA-A、HLA-B、HLA-C、B2M、RFX5、RFXAP、RFXANK、CIITA。
抑制基因表达或使基因沉默的方法是本领域技术人员熟知的。例如，可以使用反义RNA、RNA诱饵、RNA适体、siRNA、shRNA/miRNA、反式显性阴性蛋白(TNP)、嵌合/抗体偶联物、趋化因子配体、抗感染性细胞蛋白、细胞内抗体(sFv)、核苷类似物(NRTI)、非核苷类似物(NNRTI)、整合酶抑制剂(寡核苷酸、二核苷酸和化学剂)和蛋白酶抑制剂来抑制基因的表达。另外，也可以通过例如大范围核酸酶、锌指核酸酶、TALE核酸酶或CRISPR系统中的Cas酶介导DNA断裂，从而使基因沉默。
在一个实施方案中，所述工程化免疫细胞还包含靶向其他肿瘤抗原的第二嵌合抗原受体。所述第二嵌合抗原受体靶向的其他肿瘤抗原可以选自例如BCMA、CD4、CD5、CD7、CD8、CD14、CD15、CD19、CD20、CD21、CD22、CD23、CD25、CD33、CD37、CD38、CD40、CD40L、CD46、CD52、CD54、CD80、CD126、CD138、B7、MUC-1、HM1.24、血管生成因子、VEGF、PIGF、ED-B纤连蛋白、CD66a-d、IL-2、T101、TAC、IL-6、DR4、DR5、tEGFR、Her2、L1-CAM、间皮素、CEA、乙型肝炎表面抗原、抗叶酸受体、CD24、CD30、CD44、EGFR、EGP-2、EGP-4、EPHa2、ErbB2、ErbB3、ErbB4、ErbB二聚体、EGFR vIII、FBP、FCRL5、FCRH5、胎儿乙酰胆碱受体、GD2、GD3、GPRC5D、HMW-MAA、IL-22R-α、IL-13R-α2、κ轻链、Lewis Y、L1-CAM、MAGE-A1、MAGE-A3、MAGE-A6、PRAME、生存素、EGP2、EGP40、TAG72、B7-H6、IL-13Ra2、CA9、CD171、G250/CAIX、HLA-A1、HLA-A2、NY-ESO-1、PSCA、叶酸受体-a、CD44v6、CD44v7/8、avb6整合素、8H9、NCAM、VEGF受体、5T4、胎儿AchR、MUC1、MUC16、NY-ESO-1、MART-1、gp100、癌胚胎抗原、VEGF-R2、CEA、前列腺特异性抗原、PSMA、Her2/neu、雌激素受体、孕酮受体、肝配蛋白B2、CD123、c-Met、GD-2、O-乙酰化GD2(OGD2)、CE7、WT-1、细胞周期蛋白A2、CCL-1、hTERT、MDM2、CYP1B、WT1、活素、AFP、p53、D1、CS-1、BAFF-R、TACI、CD56、TIM-3、CD123、L1-细胞粘附分子、MAGE-A1、MAGEA3、CCNA1和/或病原体特异性抗原、生物素化分子、由HIV、HCV、HBV和/或其他病原体表达的分子。
在一个实施方案中，提供多种免疫细胞，每种免疫细胞被改造为表达一种或多种嵌合抗原受体。例如，在一些实施方案中，将一种免疫细胞改造为表达结合和/或靶向B7-H3的嵌合抗原受体(例如包含本发明所述抗B7-H3抗体的CAR)，并且将另一种细胞改造为表达结合和/或靶向其他抗原的嵌合抗原受体。在一个实施方案中，免疫细胞也可以表达多特异性嵌合抗原受体，其靶向包括B7-H3在内的一种或多种抗原。例如，这种多特异性嵌合抗原受体可以包含靶向B7-H3的多特异性抗体，或者同时包含本发明所述的抗B7-H3抗体和靶向其他抗原的抗体。在此类实施方案中，所述多种工程化免疫细胞可以一起或单独施用。在一个实施方案中，所述多种免疫细胞可以在同一组合物中或在不同组合物中。细胞的示例性组合物包括本申请以下章节中所描述的组合物。
抗体偶联物
在一个方面，本发明提供一种抗体偶联物，其包含本发明所定义的抗B7-H3抗体和第二功能性结构，其中所述第二功能性结构选自Fc、放射性同位素、延长半衰期的结构部分、可检测标记物和药物。
在一个实施方案中，本发明提供一种抗体偶联物，其包含本发明所定义的抗B7-H3抗体和Fc。如本文所用，术语“Fc”用于定义免疫球蛋白重链的C末端区，其包括天然Fc和变体Fc。“天然Fc”是指包含通过消化完整抗体产生的、无论是单体形式或是多聚体形式的非抗原结合片段的分子或序列。产生天然Fc的免疫球蛋白源优选来源于人类。天然Fc片段由可以通过共价连接(例如二硫键)和非共价连接而连接为二聚体或多聚体形式的单体多肽构成。根据类别(例如IgG、IgA、IgE、IgD、IgM)或亚型(例如IgG1、IgG2、IgG3、IgA1、IgGA2)的不同，天然Fc分子单体亚基之间具有1-4个分子间二硫键。天然Fc的一个实例是通过用木瓜蛋白酶消化IgG产生的二硫键连接的二聚体(参见Ellison等(1982)，Nucleic Acids Res.10：4071-9)。本文所用的术语“天然Fc” 一般是指单体、二聚体和多聚体形式。“变体Fc”是指由于至少一个本文定义的“氨基酸修饰”而与“天然”或“野生型”Fc的氨基酸序列不同的氨基酸序列，也称为“Fc变体”。因此，“Fc”也包括单链Fc(scFc)，即，由多肽接头连接的两个Fc单体组成的单链Fc，其能够自然折叠成功能性二聚体Fc区域。在一个实施方案中，所述Fc优选是人免疫球蛋白的Fc，更优选是人IgG1的Fc。
在一个实施方案中，本发明提供一种抗体偶联物，其包含本发明所定义的抗B7-H3抗体和放射性同位素。可用于本发明的放射性同位素的实例包括但不限于At211、I131、I125、Y90、Re186、Re188、Sm153、Bi212、P32、Pb212、99mTc、123I、18F和68Ga。
在一个实施方案中，本发明提供一种抗体偶联物，其包含本发明所定义的抗B7-H3抗体和延长半衰期的结构部分，所述延长半衰期的结构部分选自白蛋白的结合结构、转铁蛋白的结合结构、聚乙二醇分子、重组聚乙二醇分子、人血清白蛋白、人血清白蛋白的片段和结合人血清白蛋白的白多肽(包括抗体)。
在一个实施方案中，本发明提供一种抗体偶联物，其包含本发明所定义的抗B7-H3抗体和可检测标记物。术语“可检测标记物”在本文中意指产生可检测信号的化合物。例如，可检测标记物可以是MRI造影剂、闪烁扫描造影剂、X射线成像造影剂、超声造影剂、光学成像造影剂。可检测标记物的实施例包括荧光团(如荧光素、Alexa或花青)、化学发光化合物(如鲁米诺)、生物发光化合物(如荧光素酶或碱性磷酸酶)、酶(如辣根过氧化物酶、葡萄糖-6-磷酸酶、β-半乳糖苷酶)、抗生素(例如卡那霉素、氨苄霉素、氯霉素、四环素等)抗性基因和造影剂(如纳米颗粒或钆)。本领域技术人员可以根据所用的检测系统选择合适的可检测标记物。
在一个实施方案中，本发明提供一种抗体偶联物，其包含本发明所定义的抗B7-H3抗体和与所述抗B7-H3抗体偶联的药物，例如细胞毒素或免疫调节剂(即，抗体药物偶联物)。通常药物通过共价与抗体连接，并且通常依赖于接头。在一个实施方案中，所述药物是细胞毒素。在另一个实施方案中，所述药物是免疫调节剂。细胞毒素的实例包括但不限于甲氨蝶呤、氨基蝶呤、6-巯基嘌呤、6-硫鸟嘌呤、阿糖胞苷、5-氟尿嘧啶、达卡巴嗪、氮芥、噻替派、苯丁酸氮芥、美法仑、卡莫司汀(BSNU)、洛莫司汀(CCNU)、1-甲基亚硝基脲、环磷酰胺、氮芥、白消安、二溴甘露醇、链佐星、丝裂霉素、顺-二氯二胺铂(II)(DDP)、顺铂、卡铂、佐柔比星、多柔比星、地托比星、卡米诺霉素、伊达比星、表柔比星、米托蒽醌、放线菌素D、博来霉素、刺孢霉素、光辉霉素、安曲霉素(AMC)、长春新碱、长春花碱、紫杉醇、蓖麻毒素、假单胞菌外毒素、吉西他滨、细胞松弛素B、短杆菌肽D、溴乙锭、依米丁、依托泊苷、替尼泊苷、秋水仙素、二羟基蒽二酮、1-脱氢睾酮、糖皮质激素、普鲁卡因、丁卡因、利多卡因、普萘洛尔、嘌呤霉素、丙卡巴肼、羟基脲、天冬酰胺酶、皮质类固醇、米托坦(O,P'-(DDD))、干扰素，以及它们的组合。免疫调节剂的实例包括但不限于更昔洛韦、依那西普、他克莫司、西罗莫司、伏环孢素、环孢灵、雷帕霉素、环磷酰胺、硫唑嘌呤、霉酚酸酯、甲氨蝶呤、糖皮质素及其类似物、细胞因子、干细胞生长因子、淋巴毒素、肿瘤坏死因子(TNF)、造血因子、白介素(例如IL-1、IL-2、IL-3、IL-6、IL-10、IL-12、IL-18及IL-21)、集落刺激因子(例如G-CSF及GM-CSF)、干扰素(例如干扰素-α、干扰素-β及干扰素-γ)、命名为“S1因子”的干细胞生长因子、红细胞生成素和血小板生成素，或其组合。
试剂盒和药物组合物
在另一个方面，本发明还提供一种检测试剂盒，其包含本发明所述的人源化抗体、多特异性抗体、抗体偶联物、工程化免疫细胞或嵌合抗原受体。
在另一个方面，本发明还提供一种药物组合物，其包含本发明所述的人源化抗体、嵌合抗原受体、多特异性抗体、工程化免疫细胞或抗体偶联物，和一种或多种药学上可接受的赋形剂。
如本文所用，术语“药学上可接受的赋型剂”是指在药理学和/或生理学上与受试者和活性成分相容(即，能够引发所需的治疗效果而不会引起任何不希望的局部或全身作用)的载体和/或赋形剂，其是本领域公知的(参见例如Remington's Pharmaceutical Sciences.Edited by Gennaro AR,19th ed.Pennsylvania:Mack Publishing Company,1995)。药学上可接受的赋型剂的实例包括但不限于填充剂、粘合剂、崩解剂、包衣剂、吸附剂、抗粘附剂、助流剂、抗氧化剂、调味剂、着色剂、甜味剂、溶剂、共溶剂、缓冲剂、螯合剂、表面活性剂、稀释剂、润湿剂、防腐剂、乳化剂、包覆剂、等渗剂、吸收延迟剂、稳定剂和张力调节剂。本领域技术人员已知选择合适的赋型剂以制备本发明期望的药物组合物。用于本发明的药物组合物中的示例性赋型剂包括盐水、缓冲盐水、葡萄糖和水。通常，合适的赋形剂的选择尤其取决于所使用的活性剂、待治疗的疾病和药物组合物的期望剂型。
根据本发明的药物组合物可适用于多种途径施用。通常，通过胃肠外完成施用。胃肠外递送方法包括局部、动脉内、肌内、皮下、髓内、鞘内、心室内、静脉内、腹膜内、子宫内、阴道内、舌下或鼻内施用。
根据本发明的药物组合物也可以制备成各种形式，如固态、液态、气态或冻干形式，特别可以是软膏、乳膏、透皮贴剂、凝胶、粉末、片剂、溶液、气雾剂、颗粒、丸剂、混悬剂、乳剂、胶囊、糖浆、酏剂、浸膏剂、酊剂或流浸膏提取物的形式，或者是特别适用于所需施用方法的形式。本发明已知的用于生产药物的过程可包括例如常规混合、溶解、制粒、制糖衣、研磨、乳化、包封、包埋或冻干过程。包含例如本文所述的免疫细胞的药物组合物通常以溶液形式提供，并且优选包含药学上可接受的缓冲剂。
根据本发明的药物组合物还可以与一种或多种适用于治疗和/或预防待治疗疾病的其它药剂组合施用。适用于组合的药剂的优选实例包括已知的抗癌药物，比如顺铂、美登素衍生物、雷查霉素(rachelmycin)、卡里奇霉素(calicheamicin)、多西紫杉醇、依托泊苷、吉西他滨、异环磷酰胺、伊立替康、美法仑、米托蒽醌、sorfimer卟啉钠II(sorfimer sodiumphotofrin II)、替莫唑胺、拓扑替康、葡萄糖醛酸曲美沙特(trimetreate glucuronate)、奥利斯他汀E(auristatin E)、长春新碱和阿霉素；肽细胞毒素，比如蓖麻毒素、白喉毒素、假单胞菌细菌外毒素A、DNA酶和RNA酶；放射性核素，比如碘131、铼186、铟111、铱90、铋210和213、锕225和砹213；前药，比如抗体定向的酶前药；免疫刺激剂，比如血小板因子4、黑色素瘤生长刺激蛋白等；抗体或其片段，比如抗CD3抗体或其片段，补体活化剂，异种蛋白结构域，同种蛋白结构域，病毒/细菌蛋白结构域和病毒/细菌肽。此外，本发明的药物组合物也可以与其他一种或多种治疗方法，例如化疗、放疗组合使用。
治疗/预防/诊断用途
在另一个方面，本发明还提供一种治疗和/或预防和/或诊断与B7-H3表达相关的疾病的方法，包括向受试者施用如上所述的人源化抗体、嵌合抗原受体、多特异性抗体、抗体偶联物、工程化免疫细胞或药物组合物。
在一个实施方案中，与B7-H3表达相关的疾病包括但不限于膀胱癌、乳腺癌、宫颈癌、结直肠癌、食管癌、肾癌、肝癌、肺癌、卵巢癌、胰腺癌、前列腺癌、胆道癌、口腔鳞状细胞癌、子宫内膜癌、鳞状细胞癌、胃癌、骨肉癌、胶质瘤、黑色素瘤、肾上腺恶性肿瘤、急性髓细胞样白血病(AML)、急性淋巴细胞性白血病(ALL)、淋巴母细胞淋巴瘤(LBL)、霍奇金淋巴瘤(HL)、非霍奇金淋巴瘤(NHL)、多发性骨髓瘤(MM)。
下面将参考附图并结合实例来详细说明本发明。需要说明的是，本领域的技术人员应该理解本发明的附图及其实施例仅仅是为了例举的目的，并不能对本发明构成任何限制。在不矛盾的情况下，本申请中的实施例及实施例中的特征可以相互组合。
附图说明
图1：CHO-B7H3单克隆细胞系中的B7-H3表达水平。
图2：B7-H3抗体与CHO-B7H3细胞的结合情况。
图3：表达B7-H3鼠源scFv的CAR T细胞中的scFv表达水平。
图4：靶细胞(A)和非靶细胞(B)中的B7-H3的表达情况。
图5：表达B7-H3鼠源scFv的CAR T细胞在不同效靶比下对靶细胞NUGC4(A)、Hela(B)、Huh7(C)和非靶细胞K562(D)的杀伤效果。
图6：表达B7-H3鼠源scFv的CAR T细胞中的CD107a+CD8+双阳性细胞占比(A)和CD107a+阳性细胞占比(B)。
图7：表达B7-H3鼠源scFv的CAR T细胞与靶细胞和非靶细胞共培养后的IL2(A)和IFN-γ(B)的释放水平。
图8：注射表达B7-H3鼠源scFv的CAR T细胞后，小鼠肿瘤负荷的变化情况。
图9：表达B7-H3人源化scFv的CAR T细胞中的scFv表达水平。
图10：表达B7-H3人源化scFv的CAR T细胞在不同效靶比下对靶细胞NUGC4(A)、Hela(B)、Huh7(C)的杀伤效果。
图11：表达B7-H3人源化scFv的CAR T细胞中的CD107a+CD8+双阳性细胞占比(A)和CD107a+阳性细胞占比(B)。
图12：表达B7-H3人源化scFv的CAR T细胞与靶细胞共培养后的IL2(A)和IFN-γ(B)的释放水平。
图13：注射表达B7-H3人源化scFv的CAR T细胞后，小鼠肿瘤负荷的变化情况。
图14：注射表达B7-H3人源化scFv的CAR T细胞后，小鼠的体重变化情况。
图15：注射表达B7-H3人源化scFv的CAR T细胞后，小鼠的存活情况。
具体实施方式
实施例1.构建B7-H3过表达细胞株
分别合成人B7-H3的完整编码序列(NM_025240.3)，并将其克隆入载体pGEM-T Easy(Promega，货号A1360)，得到pLV-B7H3质粒。使用脂质体转染试剂(Roche，货号06366546001)，用pLV-B7H3质粒转染CHO细胞，有限稀释法分离单克隆，获得CHO-B7H3单克隆细胞系。然后，使用抗体APC anti-human CD276(B7-H3)antibody(Biolegend，货号351005)进行染色，通过流式细胞术检测上述单克隆细胞系中的B7-H3的表达，结果如图1所示。可以看出，B7-H3过表达细胞株CHO-B7H3构建成功。
实施例2.筛选抗B7-H3抗体
将Human B7-H3/CD276 Protein,His Tag(MALS verified)(Acrobiosystems，货号B73-H52E2)蛋白免疫适龄Balb/c小鼠，随后每隔2～3周重复免疫注射一次，共计4次。然后，取小鼠脾脏淋巴细胞，与SP2/0骨髓瘤细胞混合并进行电融合，以制备杂交瘤细胞。使用B7-H3过表达细胞株(CHO-B7H3细胞)通过ELISA或流式细胞术筛选与B7-H3结合的杂交瘤克隆。经过多轮筛选，获得5个可特异性结合B7-H3的抗体克隆，相应编号分别为BH327、BH328、BH329、BH330、BH331。
分别对各抗体进行测序，得到的5个抗B7-H3鼠源抗体的氨基酸序列如表1所示。
表1.抗B7-H3鼠源抗体的氨基酸序列
分别将表1中BH327、BH328、BH329、BH330、BH331的轻重链可变区，使用(G4S)3连接，按照VH-(G4S)3-VL-hIgG1Fc的结构，构建scFv-Fc抗体。将构建好的scFv-Fc抗体瞬时转染293细胞，表达抗体。 用所得抗体对CHO-B7H3细胞进行染色，并通过流式细胞术进行检测，结果如图2所示。
可以看出，各抗体与CHO-B7H3细胞都存在强烈结合，同时不结合CHO细胞，说明其对B7-H3蛋白的结合是特异性的。
实施例3.制备表达抗B7-H3鼠源scFv的CAR T细胞并验证其功能
3.1制备CAR T细胞
分别合成编码以下蛋白的序列，并将其克隆至pLVX载体(Public Protein/Plasmid Library(PPL)，货号PPL00157-4a)：CD8α信号肽(SEQ ID NO:75)、抗B7-H3鼠源scFv、CD8a铰链区(SEQ ID NO:71)、CD8α跨膜区(SEQ ID NO:67)、4-1BB胞内区(SEQ ID NO:73)和CD3ζ胞内区(SEQ ID No:69)，并通过测序确认目标序列的正确插入。其中，BH328-CAR包含的抗B7-H3鼠源scFv的氨基酸序列如SEQ ID NO：18所示；BH329-CAR包含的抗B7-H3鼠源scFv的氨基酸序列如SEQ ID NO：27所示；BH330-CAR包含的抗B7-H3鼠源scFv的氨基酸序列如SEQ ID NO：36所示；BH331-CAR包含的抗B7-H3鼠源scFv的氨基酸序列如SEQ ID NO：45所示。
在无菌管中加入3mL Opti-MEM(Gibco，货号31985-070)稀释上述质粒后，再根据质粒：病毒包装载体：病毒包膜载体＝4:2:1的比例加入包装载体psPAX2(Addgene，货号12260)和包膜载体pMD2.G(Addgene，货号12259)。然后，加入120μL X-treme GENE HP DNA转染试剂(Roche，货号06366236001)，立即混匀，于室温下孵育15min，然后将质粒/载体/转染试剂混合物逐滴加入到293T细胞的培养瓶中。在24小时和48小时收集病毒，将其合并后，超速离心(25000g，4℃，2.5小时)获得浓缩的慢病毒。
用DynaBeads CD3/CD28 CTSTM(Gibco,货号40203D)激活T细胞，并在37℃和5％CO2下培养1天。然后，加入浓缩的慢病毒，持续培养3天后，获得表达抗B7-H3鼠源scFv的各CAR T细胞，编号分别为BH328-CAR T、BH329-CAR T、BH330-CAR T、BH331-CAR T。未经修饰的野生型T细胞(NT)用作对照。
在37℃和5％CO2下培养7天之后，使用Biotin-Goat anti-mouse IgG,F(ab’)specific(jackson immunoresearch，货号115-065-072)作为一抗，PE Streptavidin(Biolegend，货号405204)，通过流式细胞仪检测CAR T细胞上的抗B7-H3鼠源scFv的表达水平，结果如图3所示。
可以看出，BH328-CAR T、BH329-CAR T、BH330-CAR T、BH331-CAR T细胞中的抗B7-H3鼠源scFv可以有效表达。
3.2检测CAR T细胞对靶细胞的杀伤效果
取K562细胞、Hela细胞、MDA-MB-231细胞、NUGC4细胞、A549细胞、293T细胞、Huh7细胞，使用抗体APC anti-human CD276(B7-H3)antibody(Biolegend，货号351005)进行染色，通过流式细胞术检测各细胞系中的B7-H3的表达情况，结果如图4所示。
可以看出，K562细胞不表达B7-H3，因此用作非靶细胞；Hela细胞、MDA-MB-231细胞、NUGC4细胞、A549细胞、293T细胞、Huh7细胞均不同程度表达B7-H3，因此用作靶细胞。
以1×104个细胞/孔的浓度将表达荧光素酶基因的各靶细胞(NUGC4细胞、Hela细胞、Huh7细胞)或非靶细胞(K562细胞)铺入96孔板中，然后以8:1、4:1、2:1的效靶比(即效应T细胞与靶细胞之比)将NT细胞和各CAR T细胞铺入到96孔板进行共培养，16-18小时后利用酶标仪测定荧光值。根据计算公式：(靶细胞荧光均值-样品荧光均值)/靶细胞荧光均值×100％，计算得到杀伤效率，结果如图5所示。
可以看出，本发明的BH328-CAR T、BH329-CAR T、BH330-CAR T、BH331-CAR T细胞显示出对靶细胞(NUGC4细胞、Hela细胞、Huh7细胞)的强烈杀伤作用，而对非靶细胞(K562细胞)的杀伤则较弱，表明各CAR T细胞仅对表达B7-H3的细胞呈现特异性杀伤。
3.3检测CAR T细胞的脱颗粒作用
以1×105个细胞/孔的浓度将靶细胞(Hela细胞、MDA-MB-231细胞、NUGC4细胞、293T细胞、Huh7细胞)和非靶细胞(K562细胞)分别铺于96孔板中，按1:1的比例加入BH328-CAR T、BH329-CAR T、BH330-CAR T、BH331-CAR T和NT细胞(阴性对照)，然后向各孔加入10μL PE Mouse anti-human CD107a antibody(BD,货号555801)，并于37℃、5％CO2条件下避光孵育。1h后，向各孔加入20μL Golgi Stop(BD,货号51-2092K2)，并于37℃、5％CO2条件下避光孵育2.5h。然后向各孔加入10μL APC anti-human CD8(BD,货号555369)，并于37℃、5％CO2条件下避光孵育0.5h。通过流式细胞术检测各孔细胞样品，并分析CD107a、CD8双阳性细胞占T细胞的比例，结果图6所示。
可以看出，与NT细胞相比，BH328-CAR T、BH329-CAR T、BH330-CAR T、BH331-CAR T细胞对靶细胞(Hela细胞、MDA-MB-231细胞、NUGC4细胞、293T细胞、Huh7细胞)均显示出显著升高的特异性的脱颗粒作用，而对非靶细胞(K562细胞)则没有观察到显著升高的脱颗粒作用。
3.4检测CAR T细胞的细胞因子释放水平
以1×105个细胞/孔的浓度将靶细胞(Hela细胞、MDA-MB-231细胞、NUGC4细胞、A549细胞、293T细胞、Huh7细胞)和非靶细胞(K562细胞)铺于96孔板中，按1:1的比例分别加入BH328-CAR T、BH329-CAR T、BH330-CAR T、BH331-CAR T细胞和NT细胞(阴性对照)，共培养18-24小时后收集细胞共培养上清液。
分别使用Human IL-2DuoSet ELISA Kit(R&D systems,货号DY202)、Human IFN-gamma DuoSet ELISA Kit(R&D systems,货号DY285)检测共培养上清液中IL2和IFN-γ的含量，结果如图7所示。
可以看出，与NT细胞相比，BH328-CAR T、BH329-CAR T、BH330-CAR T、BH331-CAR T与各靶细胞共培养后，细胞因子IL2和IFN-γ的释放水平均显著升高，并且这种细胞因子释放是特异性的。
实施例4.验证CAR T细胞的肿瘤抑制效果
将28只约7周龄的健康雌性NPI小鼠分成3组：G1NT组(阴性对照)、G2 BH329-CAR T、G3 BH330-CAR  T。
在第0天(D0)，向每只小鼠皮下注射4×106个NUGC4细胞。6天后(D6)，根据分组情况向每只小鼠尾静脉注射4×106个NT细胞或相应CAR T细胞。每周评估小鼠肿瘤负荷的变化，结果如图8所示。
可以看出，与NT细胞相比，注射BH329-CAR T、BH330-CAR T后，小鼠肿瘤负荷显著降低。值得注意的是，注射BH329-CAR T细胞25天后，小鼠肿瘤几乎完全消失。
实施例5.鼠抗人B7-H3抗体的人源化改造
对鼠源抗体BH329进行人源化改造，具体方法如下：首先通过IG BLAST数据库(https://www.ncbi.nlm.nih.gov/igblast/)检索相似度较高的人源抗体序列，然后将单链抗体中的FR区替换为相应的人源序列；再根据氨基酸残基的不同理化性质对个别氨基酸残基进行替换，最终获得多个人源化单链抗体，其氨基酸序列如表2所示。
表2.B7-H3人源化抗体的序列。
实施例6.制备人源化CAR T细胞并验证其功能
6.1制备CAR T细胞
根据3.1所述方法用人源化抗体制备CAR T细胞，获得包含抗B7-H3人源化抗体的CAR T细胞BH329V0-CAR T、BH329V1-CAR T、BH329V2-CAR T、BH329V3-CAR T、BH329V4-CAR T、BH329V5-CAR T、BH329V6-CAR T。
在37℃和5％CO2下培养10天之后，使用FITC-Rabbit anti-mouse IgG,F(ab’)specific(jackson immunoresearch，货号315-095-006)，通过流式细胞仪检测CAR T细胞上的抗B7-H3人源化scFv的表达水平，结果如图9所示。
可以看出，本发明制备的各人源化CAR T细胞中的抗B7-H3人源化scFv可以有效表达。
6.2检测CAR T细胞对靶细胞的杀伤效果
以1×104个细胞/孔的浓度将表达荧光素酶基因的各靶细胞(Hela细胞、NUGC4细胞、Huh7细胞)铺入96孔板中，然后以16:1、8:1、4:1、2:1、1:1的效靶比(即效应T细胞与靶细胞之比)将NT细胞和各CAR T细胞铺入到96孔板进行共培养，16-18小时后利用酶标仪测定荧光值。根据计算公式：(靶细胞荧光均值-样品荧光均值)/靶细胞荧光均值×100％，计算得到杀伤效率，结果如图10所示。
可以看出，在各种效靶比下，BH329V0-CAR T、BH329V1-CAR T、BH329V2-CAR T、BH329V3-CAR T、BH329V4-CAR T、BH329V5-CAR T、BH329V6-CAR T细胞显示出对靶细胞(Hela细胞、NUGC4细胞、Huh7细胞)的强烈杀伤作用，而对非靶细胞Huh7细胞的杀伤则较弱，表明各CAR T细胞仅对表达B7-H3的细胞呈现特异性杀伤。
6.3检测CAR T细胞的脱颗粒作用
以1×105个细胞/孔的浓度将靶细胞(Hela细胞、NUGC4细胞、Huh7细胞)和非靶细胞(K562细胞)分别铺于96孔板中，按1:1的比例加入各CAR T细胞和NT细胞(阴性对照)，然后向各孔加入10μL PE Mouse anti-human CD107a antibody(BD,货号555801)，并于37℃、5％CO2条件下避光孵育。1h后，向各孔加入20μL Golgi Stop(BD,货号51-2092K2)，并于37℃、5％CO2条件下避光孵育2.5h。然后向各孔加入10μL APC anti-human CD8(BD,货号555369)，并于37℃、5％CO2条件下避光孵育0.5h。通过流式细胞术检测各孔细胞样品，并分析CD107a阳性群、CD107a CD8双阳性细胞占T细胞的比例，结果图11所示。
可以看出，与NT细胞相比，BH329V0-CAR T、BH329V1-CAR T、BH329V2-CAR T、BH329V3-CAR T、BH329V4-CAR T、BH329V5-CAR T、BH329V6-CAR T细胞对靶细胞(Hela细胞、NUGC4细胞、Huh7细胞)均显示出显著升高的特异性的脱颗粒作用，而对非靶细胞(K562细胞)则没有观察到显著升高的脱颗粒作用。
6.4检测CAR T细胞的细胞因子释放水平
以1×105个细胞/孔的浓度将靶细胞(Hela细胞、NUGC4细胞、Huh7细胞)和非靶细胞(K562细胞)铺于96孔板中，按1:1的比例分别加入各CAR T细胞和NT细胞(阴性对照)，共培养18-24小时后收集细胞共培养上清液。
按照制造商的建议，分别使用Human IL-2 DuoSet ELISA Kit(R&D systems,货号DY202)、Human IFN-gamma DuoSet ELISA Kit(R&D systems,货号DY285)检测共培养上清液中IL2和IFN-γ的含量，结果如图12所示。
可以看出，与NT细胞相比，BH329V0-CAR T、BH329V1-CAR T、BH329V2-CAR T、BH329V3-CAR T、BH329V4-CAR T、BH329V5-CAR T、BH329V6-CAR T细胞与靶细胞共培养后，细胞因子IL2和IFN-γ的释放水平均显著升高，并且这种细胞因子释放是特异性的。
实施例7.验证人源化CAR T细胞的肿瘤抑制效果
将45只约7周龄的健康雌性NPI小鼠分成9组：G1NT组(阴性对照)、G2 BH329组(鼠源CAR T对照)、G3 BH329V0组、G4 BH329V1组、G5 BH329V2组、G6 BH329V3组、G7 BH329V4组、G8 BH329V5组、 G9 BH329V6组。
在第0天(D0)，向每只小鼠皮下注射4×106个NUGC4细胞。21天后(D21)，根据分组情况向每只小鼠尾静脉注射10×106个NT细胞或相应CAR T细胞。62天后(D62)，因笼位紧张，对BH329V2、BH329V3、BH329V4三组小鼠执行安乐死，94天后(D94)，其余各组小鼠统一安乐死。
每周评估小鼠肿瘤负荷的变化，结果如图13所示。可以看出，与NT组相比，各人源化CAR T细胞均表现出明显的肿瘤抑制效果，且其效果不劣于BH329组(鼠源CAR T对照)。
各组小鼠的体重变化如图14所示。可以看出，施用CAR T细胞后，各组小鼠体重无显著降低，提示本发明制备的CAR T无明显毒性。
各组小鼠的存活曲线如图15所示。与NT组和BH329组(鼠源CAR T对照)相比，各人源化CAR T组小鼠的存活率均显著提高，尤其是BH329V0、BH329V2、BH329V5、BH329V6组，相比于BH329V1组，小鼠存活率更高。
以上结果表明，本发明的抗B7-H3鼠源抗体、人源化抗体均能够特异性结合B7-H3蛋白，利用上述抗B7-H3鼠源抗体、人源化抗体制备得到的CAR T细胞能够在体内外有效地对肿瘤靶细胞产生特异性杀伤。
需要说明的是，以上仅为本发明的优选实施例而已，并不用于限制本发明，对于本领域的技术人员来说，本发明可以有各种更改和变化。本领域技术人员理解的是，凡在本发明的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (26)

  1. 一种靶向B7-H3的抗体，其包含：
    (1)如SEQ ID NO：1所示的CDR-L1、如SEQ ID NO：2所示的CDR-L2、如SEQ ID NO：3所示的CDR-L3、如SEQ ID NO：4所示的CDR-H1、如SEQ ID NO：5所示的CDR-H2和如SEQ ID NO：6所示的CDR-H3；
    (2)如SEQ ID NO：10所示的CDR-L1、如SEQ ID NO：11所示的CDR-L2、如SEQ ID NO：12所示的CDR-L3、如SEQ ID NO：13所示的CDR-H1、如SEQ ID NO：14所示的CDR-H2和如SEQ ID NO：15所示的CDR-H3；
    (3)如SEQ ID NO：19所示的CDR-L1、如SEQ ID NO：20所示的CDR-L2、如SEQ ID NO：21所示的CDR-L3、如SEQ ID NO：22所示的CDR-H1、如SEQ ID NO：23所示的CDR-H2和如SEQ ID NO：24所示的CDR-H3；
    (4)如SEQ ID NO：28所示的CDR-L1、如SEQ ID NO：29所示的CDR-L2、如SEQ ID NO：30所示的CDR-L3、如SEQ ID NO：31所示的CDR-H1、如SEQ ID NO：32所示的CDR-H2和如SEQ ID NO：33所示的CDR-H3；或
    (5)如SEQ ID NO：37所示的CDR-L1、如SEQ ID NO：38所示的CDR-L2、如SEQ ID NO：39所示的CDR-L3、如SEQ ID NO：40所示的CDR-H1、如SEQ ID NO：41所示的CDR-H2和如SEQ ID NO：42所示的CDR-H3。
  2. 根据权利要求1所述的抗体，所述抗体包含重链可变区和轻链可变区，所述重链可变区与选自SEQ ID NO：8、17、26、35、44、47、50、53、56、59、62和65的氨基酸序列具有至少90％同一性，或与选自SEQ ID NO：8、17、26、35、44、47、50、53、56、59、62和65的氨基酸序列相比具有一个或几个氨基酸的保守性修饰；所述轻链可变区与选自SEQ ID NO：7、16、25、34、43、46、49、52、55、58、61和64的氨基酸序列具有至少90％同一性，或与选自SEQ ID NO：7、16、25、34、43、46、49、52、55、58、61和64的氨基酸序列相比具有一个或几个氨基酸的保守性修饰。
  3. 根据权利要求1所述的抗体，其中所述抗体的氨基酸序列与选自SEQ ID NO：9、18、27、36、45、48、51、54、57、60、63和66的氨基酸序列具有至少90％同一性，或与选自SEQ ID NO：9、18、27、36、45、48、51、54、57、60、63和66的氨基酸序列相比具有一个或几个氨基酸的保守性修饰。
  4. 根据权利要求1-3任一项所述的抗体，其中所述抗体是鼠源抗体、嵌合抗体、人源化抗体或人抗体。
  5. 根据权利要求1-4任一项所述的抗体，其中所述抗体选自IgG、Fab、Fab'、F(ab')2、Fd、Fd′、Fv、scFv、sdFv、线性抗体和双体。
  6. 一种多特异性抗体，其包含权利要求1-5任一项所述的抗体和一个或多个与其他抗原特异性结合的第二抗体或其抗原结合片段。
  7. 根据权利要求6所述的多特异性抗体，其中所述第二抗体或其抗原结合片段选自全长抗体、Fab、Fab'、(Fab')2、Fv、scFv、scFv-scFv、微抗体、双抗体或sdAb。
  8. 一种核酸分子，其编码权利要求1-5任一项所述的抗体或权利要求6或7所述的多特异性抗体。
  9. 一种载体，其包含编码权利要求1-5任一项所述的抗体或权利要求6或7所述的多特异性抗体的核酸分子。
  10. 一种宿主细胞，其表达权利要求1-5任一项所述的抗体或权利要求6或7所述的多特异性抗体。
  11. 一种嵌合多肽，其包含权利要求1-5任一项所述的抗体或权利要求6或7所述的多特异性抗体，所述嵌合多肽选自CAR、TCR、TRuC、TAC或ImmTAC。
  12. 根据权利要求11所述的嵌合多肽，其进一步包含跨膜结构域和胞内信号传导结构域；所述胞内信号传导结构域包括初级信号传导结构域和/或共刺激结构域。
  13. 根据权利要求12所述的嵌合多肽，所述跨膜结构域选自以下蛋白质的跨膜结构域：TCRα链、TCRβ链、TCRγ链、TCRδ链、CD3ζ亚基、CD3ε亚基、CD3γ亚基、CD3δ亚基、CD45、CD4、CD5、CD8α、CD9、CD16、CD22、CD33、CD28、CD37、CD64、CD80、CD86、CD134、CD137和CD154。
  14. 根据权利要求12所述的嵌合多肽，所述初级信号传导结构域选自以下蛋白的胞内区：FcRγ、FcRβ、CD3γ、CD3δ、CD3ε、CD3ζ、CD22、CD79a、CD79b和CD66d。
  15. 根据权利要求12所述的嵌合多肽，所述共刺激结构域选自以下蛋白质的共刺激信号传导结构域：TLR1、TLR2、TLR3、TLR4、TLR5、TLR6、TLR7、TLR8、TLR9、TLR10、CARD11、CD2、B7-H3、CD8、CD18(LFA-1)、CD27、CD28、CD30、CD40、CD54(ICAM)、CD83、CD134(OX40)、CD137(4-1BB)、CD270(HVEM)、CD272(BTLA)、CD276(B7-H3)、CD278(ICOS)、CD357(GITR)、DAP10、LAT、NKG2C、SLP76、PD-1、LIGHT、TRIM以及ZAP70。
  16. 一种工程化免疫细胞，其包含权利要求11-15任一项所述的嵌合多肽。
  17. 根据权利要求16所述的工程化免疫细胞，其选自T细胞、NK细胞、NKT细胞、巨噬细胞以及树突细胞。
  18. 根据权利要求16所述的工程化免疫细胞，其选自CD4+CD8+T细胞、CD4+T细胞、CD8+T细胞、CD4-CD8-T细胞、肿瘤浸润细胞、记忆T细胞、幼稚T细胞、γδ-T细胞以及αβ-T细胞。
  19. 根据权利要求16或18所述的工程化免疫细胞，其还包含靶向其他一种或多种肿瘤抗原的第二嵌合抗原受体。
  20. 根据权利要求16-19任一项所述的工程化免疫细胞，还包含至少一种选自以下的基因的表达被抑制或沉默：TRAC、TRBC、HLA-A、HLA-B、HLA-C、B2M、RFX5、RFXAP、RFXANK、CIITA、PD1、LAG3、TIM3和CTLA4。
  21. 一种抗体偶联物，其包含权利要求1-5任一项所述的抗体或权利要求6或7所述的多特异性抗体，和第 二功能性结构，其中所述第二功能性结构选自Fc、放射性同位素、延长半衰期的结构部分、可检测标记物和药物。
  22. 根据权利要求21所述的抗体偶联物，其中所述延长半衰期的结构部分选自：白蛋白的结合结构、转铁蛋白的结合结构、聚乙二醇分子、重组聚乙二醇分子、人血清白蛋白、人血清白蛋白的片段和结合人血清白蛋白的白多肽；所述可检测标记物选自荧光团、化学发光化合物、生物发光化合物、酶、抗生素抗性基因和造影剂；所述药物选自细胞毒素和免疫调节剂。
  23. 一种检测试剂盒，其包含权利要求1-5任一项所述的抗体、权利要求6或7所述的多特异性抗体、权利要求11-15任一项所述的嵌合多肽、权利要求16-20任一项所述的工程化免疫细胞、或权利要求21或22所述的抗体偶联物。
  24. 一种药物组合物，包含权利要求1-5任一项所述的抗体、权利要求6或7所述的多特异性抗体、权利要求11-15任一项所述的嵌合多肽、权利要求16-20任一项所述的工程化免疫细胞、或权利要求21或22所述的抗体偶联物，和一种或多种药学上可接受的赋形剂。
  25. 权利要求1-5任一项所述的抗体、权利要求6或7所述的多特异性抗体、权利要求11-15任一项所述的嵌合多肽、权利要求16-20任一项所述的工程化免疫细胞、或权利要求21或22所述的抗体偶联物、或权利要求23所述的检测试剂盒、或权利要求24所述的药物组合物在制备用于治疗和/或预防和/或诊断与B7-H3表达相关的疾病的药物中的用途。
  26. 根据权利要求25所述的用途，其中所述与B7-H3表达相关的疾病选自膀胱癌、乳腺癌、宫颈癌、结直肠癌、食管癌、肾癌、肝癌、肺癌、卵巢癌、胰腺癌、前列腺癌、胆道癌、口腔鳞状细胞癌、子宫内膜癌、鳞状细胞癌、胃癌、骨肉癌、胶质瘤、黑色素瘤、肾上腺恶性肿瘤、急性髓细胞样白血病、急性淋巴细胞性白血病、淋巴母细胞淋巴瘤、霍奇金淋巴瘤、非霍奇金淋巴瘤以及多发性骨髓瘤。
PCT/CN2023/090397 2022-06-30 2023-04-24 靶向b7-h3的抗体及其用途 WO2024001470A1 (zh)

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