WO2022037323A1 - 人源化抗体、嵌合抗原受体、核酸、载体、细胞及应用 - Google Patents

人源化抗体、嵌合抗原受体、核酸、载体、细胞及应用 Download PDF

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WO2022037323A1
WO2022037323A1 PCT/CN2021/106097 CN2021106097W WO2022037323A1 WO 2022037323 A1 WO2022037323 A1 WO 2022037323A1 CN 2021106097 W CN2021106097 W CN 2021106097W WO 2022037323 A1 WO2022037323 A1 WO 2022037323A1
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antibody
chimeric antigen
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French (fr)
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沈青山
邢凯旋
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上海易慕峰生物科技有限公司
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Priority to US18/041,723 priority Critical patent/US20230357427A1/en
Priority to EP21857418.4A priority patent/EP4190808A4/en
Priority to JP2023509649A priority patent/JP7570653B2/ja
Publication of WO2022037323A1 publication Critical patent/WO2022037323A1/zh

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    • C—CHEMISTRY; METALLURGY
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    • 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
    • C07K16/2821—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 ICAM molecules, e.g. CD50, CD54, CD102
    • A—HUMAN NECESSITIES
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    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
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    • A61K39/461—Cellular immunotherapy characterised by the cell type used
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    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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    • C07K14/70503—Immunoglobulin superfamily
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    • G01—MEASURING; TESTING
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K2239/00—Indexing codes associated with cellular immunotherapy of group A61K39/46
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    • A61K2239/11—Antigen recognition domain
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    • A61K2239/21—Transmembrane domain
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    • A61K2239/22—Intracellular domain
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    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
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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]
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    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
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    • C07K2319/00—Fusion polypeptide
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    • 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
    • G01N33/57492—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 involving compounds localized on the membrane of tumor or cancer cells

Definitions

  • the present invention relates to the technical field of antibodies, in particular to humanized antibodies, chimeric antigen receptors, nucleic acids, vectors, cells and applications.
  • Epithelial Cell Adhesion Molecule (EpCAM) is closely related to human esophageal cancer, gastric cancer, colorectal cancer, prostate cancer,
  • EpCAM Epithelial Cell Adhesion Molecule
  • the major surface antigens associated with epithelial tumors such as lung, breast, ovarian, renal, hepatocellular and pancreatic cancer can be highly expressed in various human epithelial tumor cells and their progenitor cells and stem cells, so they have the potential to be anti-tumor Important target for immunotherapy.
  • mouse-derived antibodies are still an important source of immunotherapy drugs, but mouse-derived antibodies are immunogenic to humans and can be easily eliminated as heterologous proteins in the human body, thereby weakening their therapeutic effectiveness.
  • Mouse-derived antibodies are humanized to evade recognition by the human immune system and avoid inducing human anti-mouse antibody (HAMA) responses.
  • HAMA human anti-mouse antibody
  • the object of the present invention is to provide a humanized antibody, a chimeric antigen receptor comprising the humanized antibody, a nucleic acid encoding the humanized antibody and the chimeric antigen receptor, and a vector comprising the nucleic acid and cells.
  • the antibody, chimeric antigen receptor, nucleic acid, vector and cell can be used to prepare medicines for treating human EpCAM-positive tumors and reagents for detecting human EpCAM-positive tumors.
  • the humanized antibody of the present invention includes a heavy chain variable region and a light chain variable region
  • the heavy chain variable region sequence includes the sequence shown in SEQ ID.1, the sequence shown in SEQ ID.2.
  • the sequence shown and the sequence shown in SEQ ID.3 are respectively used as the sequence of the first heavy chain complementarity determining region, the second heavy chain complementarity determining region sequence and the third heavy chain complementarity determining region sequence; the light chain variable region
  • the sequence includes the sequence shown in SEQ ID.4, the sequence shown in SEQ ID.5 and the sequence shown in SEQ ID.6, respectively as the sequence of the first light chain complementarity determining region, the second light chain complementarity determining region sequence and The sequence of the third light chain complementarity determining region.
  • the heavy chain variable region sequence is at least 90% identical to any one of SEQ ID.7, SEQ ID.8, SEQ ID.9, SEQ ID.10 and SEQ ID.11; the The sequence of the light chain variable region is at least 90% identical to any one of SEQ ID. 12, SEQ ID. 13, SEQ ID. 14 and SEQ ID. 15.
  • the antibody that specifically recognizes EpCAM can be: single chain antibody (scFV), monoclonal antibody, domain antibody, Fab fragment, Fd fragment, Fv fragment, F(ab')2 fragment and its derivatives, or other forms of antibodies.
  • scFV single chain antibody
  • monoclonal antibody domain antibody
  • Fab fragment fragment
  • Fd fragment fragment
  • Fv fragment fragment
  • F(ab')2 fragment fragment and its derivatives, or other forms of antibodies.
  • the antibody that specifically recognizes EpCAM can be a humanized antibody.
  • the humanized antibody is a full-length antibody, and the subtype is any one of hIgG1, hIgG2, hIgG3 and hIgG4.
  • first heavy chain complementarity determining region and the second heavy chain complementarity determining region between the first heavy chain complementarity determining region and the second heavy chain complementarity determining region, between the second heavy chain complementarity determining region and the third heavy chain complementarity determining region, and the The third heavy chain complementarity determining region is respectively connected with the first heavy chain framework region, the second heavy chain framework region and the third heavy chain framework region.
  • the first light chain complementarity determining region and the second light chain complementarity determining region between the second light chain complementarity determining region and the third light chain complementarity determining region, and the The third light chain complementarity determining region is respectively connected with the first light chain framework region, the second light chain framework region and the third light chain framework region.
  • the nucleic acid of the present invention comprises an antibody sequence or sequence fragment encoding the humanized antibody, and a nucleotide sequence or sequence fragment of the chimeric antigen receptor.
  • the vector of the present invention contains the nucleic acid.
  • the cells of the present invention comprise the nucleic acid or the vector.
  • the present invention provides the application of any one of the humanized antibodies, chimeric antigen receptors, nucleic acids, vectors and cells in the preparation of reagents for detecting human tumors.
  • the present invention provides the application of any one of the humanized antibodies, chimeric antigen receptors, nucleic acids, vectors and cells in the preparation of drugs for treating human tumors.
  • drugs for treating human tumors.
  • the preparation of anti-human EpCAM monoclonal antibody, bispecific antibody, multispecific antibody, antibody conjugated drug (ADC) and other drugs for example, in the preparation of anti-human EpCAM monoclonal antibody, bispecific antibody, multispecific antibody, antibody conjugated drug (ADC) and other drugs.
  • ADC antibody conjugated drug
  • the extracellular antigen binding region of the chimeric antigen receptor of the present invention comprises the humanized antibody or antibody fragment.
  • the chimeric antigen receptor further comprises a signal peptide, and the sequence of the signal peptide is derived from at least one of GM-CSF, CD8 ⁇ and CD28.
  • the chimeric antigen receptor further comprises a hinge region, the sequence of which is derived from at least one of CD8 ⁇ , CD28, 4-1BB, ICOS, OX40, CD40, CD80 and IgG.
  • the chimeric antigen receptor further comprises a transmembrane region, the sequence of which is derived from at least one of CD8 ⁇ , CD28, 4-1BB, ICOS, OX40, CD40, CD80 and CD3.
  • the chimeric antigen receptor further comprises an intracellular signal region, the sequence of which is derived from Toll-like receptor, CD2, CD27, LFA-1, CD8a, CD28, 4-1BB, ICOS , at least one of OX40, CD40, CD80, DAP10, DAP12, CD3 ⁇ and CD3 ⁇ , and the like.
  • the sequence of the intracellular signal region is derived from CD11a.
  • the sequence of the intracellular signal region is derived from CD18.
  • Figure 1 shows the binding performance curves of mHmL, H1L1, H2L1, H3L1, H3L2, H4L1, H5L1, H2L2, H2L3, H2L4, H3L3 and SW480;
  • Figure 2 is the binding performance curve of H1L4 and H1L3 and SW480;
  • Figure 3 is the binding performance curve of mHmL, H1L1, H2L1, H3L1, H3L2, H4L1, H5L1, H2L2, H2L3, H2L4, H3L3 and HCT116;
  • Fig. 4 is the binding performance curve of H1L4 and H1L3 and HCT116;
  • Fig. 5 is the structural representation of the chimeric antigen receptor targeting EpCAM
  • Figure 6 is a comparison chart of the flow analysis results of the reference sample and CAR-T;
  • Figure 7 is a comparison chart of the EpCAM expression levels of HCT116-Luc, SW480, KATO-III, A549 and MIAPaCA-2;
  • Figure 8 is a comparison chart of the killing efficiency of CAR-T and reference T cell unT on different tumor cells under different effector-target ratios
  • Figure 9 is a comparison chart of the amount of IFN- ⁇ released after the action of CAR-T and reference T cells unT with different tumor cells;
  • Figure 10 is a comparison chart of the average tumor volume changes of each group of mice within the experimental time
  • Figure 11 is a comparison chart of the average body weight changes of each group of mice during the experimental period
  • Figure 12 shows the results of histological anatomy of mice in G1, G2 and G3 groups.
  • Antibody in the present invention refers to an antigen-binding protein of the immune system, including an intact full-length antibody having an antigen-binding region and any fragment thereof in which the "antigen-binding portion” or “antigen-binding region” remains, or a single chain thereof such as Single-chain variable fragments (scFv).
  • a native antibody refers to a glycoprotein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, or antigen-binding fragments thereof.
  • “Antibody” also includes all recombinant forms of antibodies, particularly those described herein, such as antibodies expressed in prokaryotic cells, unglycosylated antibodies, and antibody fragments and derivatives that bind to an antigen.
  • Each heavy chain consists of a heavy chain variable region (abbreviated as VH) and a heavy chain constant region (abbreviated as CH).
  • Each light chain consists of a light chain variable region (abbreviated as VL) and a light chain constant region (abbreviated as CL).
  • VH and VL can be further subdivided into hypervariable regions called complementarity determining regions (CDRs) interspersed in more conserved regions called framework regions (FRs).
  • CDRs complementarity determining regions
  • FRs framework regions
  • Each VH and VL consists of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.
  • variable regions of the heavy and light chains contain binding domains that interact with the antigen.
  • the constant region of the antibody mediates the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (eg, effector cells) and the first component (Clq) of the classical complement system.
  • Antibody fragments include, but are not limited to:
  • Fc or "Fc region” in the present invention includes a polypeptide comprising an antibody constant region other than the first constant region immunoglobulin domain.
  • Fc refers to the last two constant region immunoglobulin domains of IgA, IgD and IgG, and the last three constant region immunoglobulin domains of IgE and IgM, and the flexible hinge at the N-terminus of these domains.
  • the Fc can include the J chain.
  • Fc includes the immunoglobulin domains C ⁇ 2 and C ⁇ 3 and a hinge between C ⁇ 1 and C ⁇ 2.
  • the Fc region of a human IgG heavy chain is generally defined as comprising residues C226 or P230 at its carboxy terminus, where the numbering is according to the Kabat EU index.
  • Fc is defined herein as comprising residue P232 to its carboxy terminus, where the numbering is according to the EU index in Kabat.
  • Fc can refer to this region in isolation, or in the context of an Fc polypeptide, eg, an antibody.
  • the aforementioned "hinge” includes a flexible polypeptide comprising amino acids between the first and second constant domains of an antibody. Structurally, the IgG CH1 domain ends at EU220 and the IgG CH2 domain begins at residue EU237.
  • mutated sequence obtained by substitution in the present invention means replacing an amino acid at a specific position in the parent polypeptide sequence with another amino acid.
  • deletion in the “mutated sequence obtained by substitution or deletion” of the present invention means the removal of an amino acid at a specific position in the parent polypeptide sequence.
  • chimeric antigen receptor refers to an extracellular antigen binding region, a hinge region, a transmembrane region and an intracellular signal region capable of binding an antigen
  • the intracellular signal region refers to a signal transduction through a defined Pathways proteins that transmit information into cells to regulate cellular activity by generating secondary messengers, or proteins that act as effectors by responding to such messengers, comprise primary signaling domains, and may also include stimuli derived from stimuli as defined below The functional signaling domain of the molecule (ie, the costimulatory signaling domain).
  • Intracellular signaling domains generate signals that can promote the immune effector function of cells of the CAR (e.g., CAR-T cells), examples of immune effector functions, such as in CAR-T cells, include cytolytic activity and helper activity, including cellular factor secretion.
  • CAR-T cells include cytolytic activity and helper activity, including cellular factor secretion.
  • costimulatory signaling domain refers to a "costimulatory molecule", which is a cognate binding partner on a T cell that specifically binds a costimulatory ligand, thereby mediating a costimulatory response of the T cell, such as, but not limited to ,proliferation.
  • Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for an effective immune response.
  • Costimulatory molecules include, but are not limited to, Toll-like receptors, and CD2, CD27, LFA-1 (CD11a/CD18), CD8 ⁇ , CD28, 4-1BB, ICOS, OX40, CD40, CD80, DAP10, DAP12, CD3 ⁇ , and CD3 ⁇ Wait.
  • CD3 ⁇ is defined as the protein provided by GenBan Accession No. BAG36664.1, or the equivalent residues from non-human species such as mouse, rodent, monkey, ape, and the like.
  • a "CD3 zeta domain” is defined as the amino acid residues from the cytoplasmic domain of the zeta chain sufficient to functionally transmit the initial signals required for T cell activation.
  • the cytoplasmic domain of zeta comprises residues 52 to 163 of GenBank Accession No. BAG36664.1, functionally directed to homologues - equivalents from non-human species such as mouse, rodent, monkey, ape, etc. Residues.
  • CD28 is defined as the protein provided by GenBan Accession No. NP_006130.1, or the equivalent residue from a non-human species such as mouse, rodent, monkey, ape, and the like.
  • the "CD28 signaling region” is defined as the amino acid residues from the cytoplasmic domain of CD28 that are capable of transmitting costimulatory signals required for T cell activation; its sequence comprises residues 180 to 220 of GenBank Accession No. NP_006130.1, its function Sexually directed homologues - equivalent residues from non-human species such as mouse, rodent, monkey, ape, etc.
  • the "CD28 hinge region” comprises residues 114 to 152 of GenBank Accession No.
  • NP_006130.1 which are functionally directed to homologues - equivalent residues from non-human species such as mouse, rodent, monkey, ape, etc.
  • the "CD28 hinge transmembrane region” comprises residues 153 to 179 of GenBank Accession No. NP_006130.1, which are functionally directed to homologues - equivalent residues from non-human species such as mouse, rodent, monkey, ape, etc. .
  • 4-1BB refers to a member of the TNFR superfamily having the amino acid sequence of GenBank Accession No. NP_001552.2, or equivalent residues from non-human species such as mouse, rodent, monkey, ape, and the like.
  • the "4-1BB signaling region” is defined as the amino acid residues from the cytoplasmic domain of 4-1BB capable of transmitting costimulatory signals required for T cell activation; its sequence comprises residues 214 to 1 of GenBank Accession No. NP_006130.1 255. Its functionality is directed to homologues - equivalent residues from non-human species such as mouse, rodent, monkey, ape, etc.
  • the EpCAM described in the embodiments of the present invention is an epithelial cell adhesion molecule.
  • the humanized antibody specifically binds to EpCAM expressed in the relevant tumor.
  • the related tumors include epithelial tumors, specifically esophageal cancer, gastric cancer, colorectal cancer, prostate cancer, lung cancer, ovarian cancer, breast cancer, kidney cancer, hepatocellular carcinoma and pancreatic cancer.
  • the humanized antibody is a humanized full-length antibody, and the subtype is hIgG1.
  • the humanized antibody is a single-chain antibody, and the subtype is any one of hIgG1, hIgG2, hIgG3 and hIgG4.
  • the heavy chain variable region sequence includes the sequence shown in SEQ ID.1, the sequence shown in SEQ ID.2 and the sequence shown in SEQ ID.3, respectively denoted as the sequence of CDR-H1,
  • the sequence of H2 and the sequence of CDR-H3, CDR-H1, CDR-H2 and CDR-H3 are the first heavy chain complementary absolute region, the second heavy chain complementary absolute region and the third heavy chain complementarity determining region, respectively.
  • the sequence of the first heavy chain framework region between CDR-H1 and CDR-H2 is QVQLVQSGAEVKKPGX6SVKVSCKASGX7TFX8, wherein X6 is any one of A and S, and X7 is any one of Y and G , X8 is either T or S.
  • sequence of the second heavy chain framework region between CDR-H2 and CDR-H3 is any one of WVRQAPGQGLEWMG and WVRQAPGQGLEWIG.
  • the sequence of the third heavy chain framework region connecting CDR-H3 is X9VTX10TX11DX12STSTX13YMELSSLRSEDTAVYYCAR, X9 is any one of R and K, X10 is any one of M, L and I, X11 is R, T, Any one of A and V, X12 is any one of T and E, and X13 is any one of V and A.
  • the light chain variable region sequence includes the sequence shown in SEQ ID.4, the sequence shown in SEQ ID.5 and the sequence shown in SEQ ID.6, respectively denoted as the sequence of CDR-L1, the sequence of CDR -
  • the sequence of L2 and the sequence of CDR-L3, CDR-L1, CDR-L2 and CDR-L3 are the first light chain complementarity determining region, the second light chain complementarity determining region and the third light chain complementarity determining region.
  • the embodiment of the present invention also provides a chimeric antigen receptor that specifically targets EpCAM, and the antigen binding region of the chimeric antigen receptor comprises the humanized antibody, or a fragment of the humanized antibody.
  • the antibody fragment targeting EpCAM is a single-chain variable region (scFv); the single-chain variable region structure is VH-Linker-VL or VL-Linker-VH; the The amino acid sequence of Linker is shown in SEQ ID NO.30, which is GGGGSGGGGSGGGGS;
  • the chimeric antigen receptor further comprises a hinge region, the sequence of which is derived from at least one of CD8 ⁇ , CD28, 4-1BB, ICOS, OX40, CD40, CD80 and IgG.
  • the chimeric antigen receptor further comprises a transmembrane region, the sequence of which is derived from at least one of CD8 ⁇ , CD28, 4-1BB, ICOS, OX40, CD40, CD80 and CD3.
  • the chimeric antigen receptor further comprises an intracellular signal region, the sequence of which is derived from CD8 ⁇ , CD28, 4-1BB, ICOS, OX40, CD40, CD80, DAP10, DAP12, CD3 ⁇ and at least one of CD3 ⁇ .
  • the CAR molecular sequence is shown in the following table:
  • the embodiments of the present invention also provide the application of the chimeric antigen receptor, including specifically targeting the EpCAM antigen that is highly expressed in related tumors.
  • Embodiments of the present invention also provide a nucleic acid comprising a nucleotide sequence encoding the chimeric antigen receptor.
  • Embodiments of the present invention also provide a vector comprising the nucleic acid.
  • Embodiments of the present invention also provide cells comprising the vector.
  • the present embodiment provides a method for designing the humanized antibody, including the following steps:
  • S3 Computer modeling, simulating the sites in the antibody structure that may affect the binding to the antigen, backmutating key sites and combinations, and then screening out the optimal structural solution.
  • IGHV1 with the highest homology was selected as the humanized design template for the design of VH
  • IGKV1, IGKV3 and IGKV4 were preferentially selected as the humanized design template for the design of VL.
  • IGHV1-KV1 and IGHV1-KV3 are common pairing methods.
  • Discovery Studio and Antibody Modeling adopts homology modeling method to select 5-10 optimal structural solutions
  • the Loop region is generally modeled using a homology modeling method. If the CDR amino acid sequence alignment result shows that the homology is less than 50%, a de novo modeling method is used to build a CDR3 structural model. Use PDB BLAST to retrieve the 10 antibody crystal structure models with a structural resolution higher than 2.5 angstroms that are closest to the sequence, compare the automatic modeling models, and select the optimal structural model.
  • Humanized Antibody Name Humanized % Number of back mutation sites H1L1 98.5% 10 H2L1 98.8% 8 H3L1 99.1% 6 H3L2 99.3% 5 H4L1 99.15 6 H5L1 99.3% 5 H2L2 99.0% 7 H2L3 99.0% 7 H2L4 99.1% 6 H3L3 99.3% 5 H1L4 98.8% 8 H1L3 98.7% 9
  • Embodiment 2 Expression and purification of humanized antibody and detection of EpCAM specific binding ability
  • the application of the humanized antibody includes using pCDNA3.4 vector and ExpiCHO-s expression system to express the humanized antibody.
  • each sequence of SEQ ID NO.7-11 was cloned into the pCDNA3.4 vector to obtain several heavy chain gene expression vectors
  • each sequence of SEQ ID NO.12-15 was cloned into the pCDNA3.4 vector to obtain Several light chain gene expression vectors are then paired with the several heavy chain gene expression vectors and the several light chain gene expression vectors are respectively transfected through the ExpiCHO-s expression system to obtain several recombinant antibodies, which are respectively denoted as H1L1, H2L1, H3L1, H3L2, H4L1, H5L1, H2L2, H2L3, H2L4, H3L3, H1L4 and H1L3.
  • mHmL is the sequence of the EpCAM antibody. The specific cloning and transfection processes are conventional technical means of those skilled in the art, and will not be repeated here.
  • the present invention determines the binding and dissociation curves of empty probe, mHmL, H1L1, H2L1, H3L1, H3L2, H4L1, H5L1, H2L2, H2L3, H2L4, H3L3, H1L4 and H1L3 and Human-EpCAM-Protetin-His respectively .
  • the specific assay method is as follows: using PBST buffer with a concentration of 0.025% formed by dissolving Tween 20 in PBS buffer, diluting each recombinant antibody to a concentration of 50 nM, and diluting Human-EpCAM-Protetin-His to a concentration of 0.025%.
  • the binding activity of the recombinant antibody to human colon cancer cells SW480, human colorectal cancer cells HCT116 and colon cancer RKO cells was also investigated by flow cytometry fluorescence sorting. The results showed that each recombinant antibody had poor binding to RKO cells, but had good binding activity to human colon cancer cell SW460 and human colorectal cancer cell HCT116.
  • the number of cells in each flow tube was 1 ⁇ 10 5 , and the cells were centrifuged and washed with FACS buffer; after resuspension, 100 ⁇ l of FACS diluent was added to each flow tube, and the cells were incubated at 4 degrees Celsius.
  • the embodiment of the present invention provides a chimeric antigen receptor targeting EpCAM (abbreviated as EpCAM-CAR). Internal costimulatory signaling domain.
  • EpCAM-CAR chimeric antigen receptor targeting EpCAM
  • EpCAM-CAR is shown in SEQ ID NO.19, see SEQ ID NO.19:
  • the sequence of the GM-CSF signal peptide is MLLLVTSLLLCELPHPAFLLIP;
  • the VH sequence of the humanized antibody is:
  • the sequence of the G4S x3 linker region is: GGGGSGGGGSGGGGS;
  • the VL sequence of the humanized antibody is:
  • the sequence of the CD28 hinge region is:
  • CD28 transmembrane region is: FWVLVVVGGVLACYSLLVTVAFIIFW;
  • the sequence of the CD28 costimulatory signal region is:
  • the sequence of the CD3 ⁇ costimulatory signal region is:
  • the lentiviral vector plasmid encoding EpCAM-CAR is packaged into lentivirus, and the CAR plasmid composed of EpCAM-CAR and the backbone part of the third-generation lentiviral vector comes from Heyuan Biotechnology Co., Ltd.,
  • the specific packaging method is as follows:
  • the number of control cells in the culture flask is 5 ⁇ 10 6 , and the culture volume is 20 ml;
  • Tube A and Tube B After mixing Tube A and Tube B, incubate at room temperature for 15 minutes, then add the sample to be transfected and incubate for 48 hours;
  • the supernatant was collected and centrifuged at 500g for 10min.
  • the supernatant was filtered and sealed in a centrifuge tube, and centrifuged at 10,000g overnight at 4°C to obtain a white virus precipitate; the white virus precipitate was extracted and 200 ⁇ l of AIM-V medium was used. After dissolving, take 2ul to measure the titer according to the subsequent steps, and store the rest at -80°C.
  • the above lentivirus is used to infect human peripheral blood mononuclear cells (PBMC) to construct CAR-T cells (abbreviated as CAR-T).
  • PBMC peripheral blood mononuclear cells
  • CAR-T CAR-T cells
  • PBMC Human peripheral blood mononuclear cells
  • AIM-V medium 5% fetal bovine serum, 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 300 IU/mL IL-2
  • the CD2/CD3/CD28 T cell activation and expansion kit from Miltenyi Company activates T cells, that is, the coated magnetic beads are mixed with cells in a ratio of 1:2, and the final cell density is 5 ⁇ 10 6 cells/mL/cm 2 , mixed and placed in a 37°C, 5% CO2 incubator for stimulation for 48h; RetroNectin from Takara was diluted to 20 ⁇ g/ml and then coated on a culture plate (non-tissue culture treated), the coating solution was 4 ⁇ g/cm2, and the Refrigerate at 4°C overnight.
  • FIG. 7 is a comparison chart of the EpCAM expression levels of HCT116-Luc, SW480, KATO-III, A549 and MIAPaCA-2.
  • the cell lines that positively express EpCAM are HCT116-Luc, SW480, KATO-III and A549; the cell line negatively expressing EpCAM was MIAPaCA-2.
  • the examples of the present invention examine the in vitro killing of HCT116-Luc, SW480, KATO-III, A549 and MIAPaCA-2 by CAR-T.
  • the prepared CAR-T and reference T cells unT were mixed and cultured with 1 ⁇ 10 4 tumor cells according to different effect-target ratios.
  • FIG 8 is a comparison chart of the killing efficiency of CAR-T and reference T cell unT on different tumor cells under different effector-target ratios. Referring to Figure 8, it can be seen that CAR-T cells specifically kill EpCAM-positive tumor cells HCT116-Luc, SW480, KATO-III, and A549, but do not kill EpCAM-negative tumor cells MIAPaCA-2.
  • Figure 9 is a comparison chart of the amount of IFN- ⁇ released by CAR-T and reference T cells unT and different tumor cells. Consistent with the results of the killing experiment, referring to Figure 9, the IFN- ⁇ release of EpCAM CAR-T cells was specifically activated by EpCAM-positive tumor cells HCT116-Luc, SW480, KATO-III, and A549, but co-existed with EpCAM-negative tumor cells MIAPaCA-2. There was no significant activation of IFN- ⁇ release upon incubation.
  • the examples of the present invention examine the therapeutic effect and safety of CAR-T on HCT116 subcutaneous tumor formation in M-NSG mice.
  • M-NSG mice were subcutaneously inoculated with HCT116 to establish the HCT116 tumor model. On day 10 after inoculation, the average tumor volume in mice was approximately 170 mm 3 .
  • the mice were divided into 5 groups by random block method: G1-G5. Each group was given PBS by tail vein administration of G1, G2 was unT, G3, G4, G5 groups were given CAR-T with 20E6, 2E6, 0.2E6 cells to each mouse.
  • mice in each group were collected blood from the intraocular canthus on the 1st, 8th, 15th, 22nd, 28th, 36th and 42nd days after injection, respectively, and the persistence of human CD45+ and human CD3+ in the peripheral blood of mice was detected by flow cytometry (FACS). and the proportion of CAR+ cells.
  • the tumor diameter was measured twice a week, the body weight of the animals was weighed, the living conditions of the animals were observed, and the abnormal conditions were recorded.
  • the mice were euthanized according to the experimental requirements, and the mice were dissected and collected for slice analysis. The number of mice in each group was 6 for statistical analysis.
  • Figure 10 is a comparison chart of the average tumor volume changes in each group of mice within the experimental time.
  • the average tumor volume of the G1 group was 1586.69 ⁇ 439.96mm 3
  • the average tumor volumes of the other groups were 1770.23 ⁇ 460.15mm 3 , 272.00 ⁇ 26.42mm 3 , 272.00 ⁇ 26.42mm 3 , and 498.22 ⁇ 47.85mm in turn. 3 and 3607.44 ⁇ 510.54mm 3
  • the corresponding tumor inhibition rates were -13.11%, 92.97%, 76.99%, and -142.95%, respectively.
  • the average tumor volume of the G3 and G4 groups was significantly lower than that of the G1 group, indicating that CAR-T at the experimental concentrations of 20E6 and 2E6 showed a certain effect of inhibiting tumor growth.
  • the tumor tissue of all mice in the G4 group was completely removed, achieving complete remission.
  • Figure 11 is a comparison chart of the average body weight changes of each group of mice during the experimental period. Referring to FIG. 11 , on the 21st day, the weight growth rates of mice in each group were -4.42%, -5.21%, 6.89%, 1.45% and 7.28%. The mice in the G3, G4 and G5 groups could maintain their body weight well during the experiment, indicating that CAR-T did not show obvious toxicity at the three experimental concentrations of 20E6, 2E6, and 0.2E6, while the G1, G2 Groups of mice were not able to maintain body weight well during the experiment.
  • mice were taken for histological anatomical detection, and the representative samples of the formed tissue sections are shown in Figure 12.
  • mice in group G1 showed moderate tumor cell infiltration or metastasis; mice in group G2 infiltrated perivascular inflammatory cells in the lungs, slightly increased neutrophils in the spleen, and slightly infiltrated inflammatory cells in the portal area of the liver;
  • the inflammatory cells around the pulmonary blood vessels of the mice in the G3 group showed mild infiltration, slight infiltration of inflammatory cells around the pulmonary blood vessels, and mild infiltration of inflammatory cells around the pulmonary blood vessels, but they did not show tumor cell infiltration or metastasis.
  • the histopathological changes in G3 tissue sections may be related to the pharmacological effects of tumor-bearing mice and the test substance, namely CAR-T, and there is no CAR-T-related toxicity.

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Abstract

一种特异性结合EpCAM的人源化抗体、所述人源化抗体相关的嵌合抗原受体、核酸、载体、细胞以及应用。所述人源化抗体包括重链可变区和轻链可变区，所述重链可变区序列包括如SEQ ID.1所示序列，SEQ ID.2所示序列以及SEQ ID.3所示序列，所述轻链可变区序列包括如SEQ ID.4所示序列，SEQ ID.5所示的序列以及SEQ ID.6所示序列。所述人源化抗体构建为嵌合抗原受体T细胞后，能够特异性识别和杀伤EpCAM阳性肿瘤细胞，且能够在动物体内清除肿瘤。

Description

人源化抗体、嵌合抗原受体、核酸、载体、细胞及应用 技术领域
本发明涉及抗体技术领域，尤其涉及人源化抗体、嵌合抗原受体、核酸、载体、细胞及应用。
背景技术
抗肿瘤免疫治疗的关键在于所选用的抗体能够选择性识别肿瘤部位高表达的抗原，上皮细胞粘附分子(Epithelial Cell Adhesion Molecule，EpCAM)是与人类食管癌、胃癌、结直肠癌、前列腺癌、肺癌、乳腺癌、卵巢癌、肾癌、肝细胞癌和胰腺癌等上皮性肿瘤相关的主要表面抗原，可在各种人类上皮肿瘤细胞及其祖细胞和干细胞中高表达，因而有潜力成为抗肿瘤免疫治疗的重要靶点。
目前为止，鼠源抗体仍是免疫治疗药物的重要来源，但鼠源抗体对人具有免疫原性，在人体内作为异源蛋白容易被清除掉，从而削弱其治疗的有效性，因此，需要对鼠源抗体进行人源化改造，从而逃避人免疫系统的识别，避免诱导人抗鼠抗体(human anti-mouse antibody，HAMA)反应。
因此，有必要设计用于特异性结合EpCAM的人源化抗体及其应用以避免现有技术中存在的上述问题。
发明内容
本发明的目的在于提供一种人源化抗体、包含所述人源化抗体的嵌合抗原受体、编码所述人源化抗体和嵌合抗原受体的核酸，和包含所述核酸的载体和细胞。该抗体、嵌合抗原受体、核酸、载体和细胞能够应用于制备治疗人类EpCAM阳性肿瘤的药物，及检测人类EpCAM阳性肿瘤的试剂。
为实现上述目的，本发明的所述人源化抗体包括重链可变区和轻链可变区，所述重链可变区序列包括如SEQ ID.1所示序列、SEQ ID.2所示序列以及SEQ ID.3所示序列，分别作为第一重链互补决定区的序列、第二重链互补决定区的序列和第三重链互补决定区的序列；所述轻链可变区序列包括如SEQ ID.4所示序列,SEQ ID.5所示的序列以及SEQ ID.6所示序列，分别作为第一轻链互补决定区的序列、第二轻链互补决定区的序列和第三轻链互补决定区的序列。
一些优选例中，所述重链可变区序列与SEQ ID.7、SEQ ID.8、SEQ ID.9、SEQ ID.10和SEQ ID.11中任一种的至少90％相同；所述轻链可变区的序列与SEQ ID.12、SEQ ID.13、SEQ ID.14和SEQ ID.15中任一种的至少90％相同。
一些优选例中，所述的特异性识别EpCAM的抗体可以是：单链抗体(scFV)，单克隆抗体，结构域抗体，Fab片段，Fd片段，Fv片段，F(ab’)2片段和其衍生物，或其它形式的抗体。
一些优选例中，所述的特异性识别EpCAM的抗体可以是人源化抗体。
一些优选例中，所述人源化抗体为全长抗体，亚型为hIgG1、hIgG2、hIgG3和hIgG4的任意一种。
一些实施例中，所述第一重链互补决定区和第二重链互补决定区之间、所述第二重链互补决定区和所述第三重链互补决定区之间，以及所述第三重链互补决定区分别连接有第一重链骨架区、第二重链骨架区和第三重链骨架区。
一些实施例中，所述第一轻链互补决定区和第二轻链互补决定区之间、所述第二轻链互补决定区和所述第三轻链互补决定区之间，以及所述第三轻链互补决定区分别连接有第一轻链骨架区、第二轻链骨架区和第三轻链骨架区。
本发明的核酸包含编码所述人源化抗体的抗体序列或序列片段，和所述嵌合抗原受体的核苷酸序列或序列片段。
本发明的载体包含所述核酸。
本发明的细胞包含所述核酸或所述载体。
本发明提供了所述人源化抗体、嵌合抗原受体、核酸、载体和细胞的任意一种应用在制备检测人类肿瘤试剂中的应用。
本发明提供了所述人源化抗体、嵌合抗原受体、核酸、载体和细胞的任意一种在制备治疗人类肿瘤的药物中的应用。例如，在制备抗人EpCAM单抗、双特异性抗体、多特异性抗体、抗体偶联药物(ADC)等药物中的应用。
本发明的所述嵌合抗原受体的胞外抗原结合区包含所述人源化抗体或抗体片段。
优选的，所述嵌合抗原受体还包含信号肽，所述信号肽的序列来源于GM-CSF、CD8α和CD28中至少一种。
优选的，所述嵌合抗原受体还包含铰链区，所述铰链区的序列来源于CD8α、CD28、4-1BB、ICOS、OX40、CD40、CD80和IgG的至少一种。
优选的，所述嵌合抗原受体还包含跨膜区，所述跨膜区的序列来源于CD8α、CD28、4-1BB、ICOS、OX40、CD40、CD80和CD3的至少一种。
优选的，所述嵌合抗原受体还包含胞内信号区，所述胞内信号区的序列来源于Toll样受体、以及CD2、CD27、LFA-1、CD8a、CD28、4-1BB、ICOS、OX40、CD40、CD80、DAP10、DAP12、CD3ζ和CD3ε等的至少一种。
一些实施例中，所述胞内信号区的序列来源于CD11a。
一些实施例中，所述胞内信号区的序列来源于CD18。
附图说明
图1为mHmL、H1L1、H2L1、H3L1、H3L2、H4L1、H5L1、H2L2、H2L3、H2L4、H3L3与SW480的结合性能曲线；
图2为H1L4和H1L3与SW480的结合性能曲线；
图3为mHmL、H1L1、H2L1、H3L1、H3L2、H4L1、H5L1、H2L2、H2L3、H2L4、H3L3与HCT116的结合性能曲线；
图4为H1L4和H1L3与HCT116的结合性能曲线；
图5为靶向EpCAM的嵌合抗原受体的结构示意图；
图6为参比样品和CAR-T的流式分析结果对比图；
图7为HCT116-Luc、SW480、KATO-III、A549和MIAPaCA-2的EpCAM表达水平的对比图；
图8为不同效靶比下CAR-T和参比T细胞unT对不同肿瘤细胞的杀伤效率对比图；
图9为CAR-T和参比T细胞unT与不同肿瘤细胞作用后的IFN-γ释放量的对比图；
图10为各组小鼠在实验时间内的平均肿瘤体积变化情况对比图；
图11为各组小鼠在实验时间内的平均体重变化情况对比图；
图12为G1、G2和G3组小鼠的组织学解剖检测结果。
具体实施方式
为使本发明实施例的目的、技术方案和优点更加清楚，下面将对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。除非另外定义，此处使用的技术术语或者科学术语应当为本发明所属领域内具有一般技能的人士所理解的通常意义。本文中使用的“包括”等类似的词语意指出现该词前面的元件或者物件涵盖出现在该词后面列举的元件或者物件及其等同，而不排除其他元件或者物件。
本发明采用的科技术语具有与本领域技术人员常规理解的相同或相似的含义。为便于理解本发明，一些术语定义如下：
本发明中的“抗体”指免疫系统的抗原结合蛋白，包括具有抗原结合区域的完整的全长抗体及其中“抗原结合部分”或“抗原结合区域”保留的其任何片段、或其单链例如单链可变片段(scFv)。天然抗体指包含通过二硫键互联的至少两条重(H)链和两条轻(L)链或其抗原结合片段的糖蛋白。“抗体”还包括抗体(特别是本文所述抗体)的所有重组形式，例如在原核细胞中表达的抗体，未糖基化的抗体以及与抗原结合的抗体片段和衍生物。每条重链由重链可变区(简记为VH)和重链恒定区(简记为CH)组成。每条轻链由轻链可变区(简记为VL)和轻链恒定区(简记为CL)组成。VH和VL可进一步细分为称为互补性决定区(CDR)的高变区，他们散布在称为骨架区(FR)的更保守区域中。每条VH和VL由三个CDR和四个FR组成，从氨基端至羧基端按以下顺序排列：FR1、CDR1、FR2、CDR2、FR3、CDR3、FR4。重链和轻链的可变区含有与抗原相互作用的结合结构域。抗体的恒定区可介导该免疫球蛋白与宿主组织或因子的结合，所述宿主组织或因子包括免疫系统的多种细胞(如效应细胞)和经典补体系统的第一成分(C1q)。
抗体片断包括但不限于：
(i)由VL、VH、CL和CH1结构域组成的Fab片段，包括Fab’和Fab’-SH，(ii)VH和CH1结构域组成的Fd片段，(iii)由单个抗体的VL和VH结构域组成的Fv片段；(iv)由单个可变区组成的dAb片段；(v)F(ab’)2片段，包含2个连接的Fab片段的二价片段；(vi)单链Fv分子抗原结合位点；(vii)双特异性单链Fv二聚体；(viii)“二体”或“三体”，通过基因融合构建的多价或多特异性片段和(ix)与相同或不同抗体遗传融合的scFv。
本发明中的术语“Fc”或“Fc区”包括包含除第一恒定区免疫球蛋白结构域以外的抗体恒定区的多肽。因而，Fc指IgA、IgD和IgG的最后两个恒定区免 疫球蛋白结构域，和IgE和IgM的最后三个恒定区免疫球蛋白结构域，和这些结构域N端的柔性铰链。对于IgA和IgM，Fc可包括J链。对于IgG，Fc包括免疫球蛋白结构域Cγ2和Cγ3和在Cγ1和Cγ2之间的铰链。虽然Fc区的边界可以改变，但人IgG重链Fc区通常定义为在其羧基端包含残基C226或P230，其中编号是根据Kabat的EU索引。对于人IgG1，Fc在本文定义为包含残基P232至其羧基端，其中编号是根据Kabat中的EU索引。Fc可以指分离的该区域，或者位于Fc多肽，例如抗体，环境中的该区域。上述“铰链”包括包含在抗体的第一和第二恒定结构域之间的氨基酸的柔性多肽。结构上，IgG CH1结构域中止于EU220位，IgG CH2结构域始于残基EU237位。
本发明所述“经替换得到的突变序列”意指用另一种氨基酸替换亲本多肽序列中特定位置上的氨基酸。本发明所述“经替换或缺失得到的突变序列”中的“缺失”意指去除亲本多肽序列中特定位置上的氨基酸。
本发明所述“嵌合抗原受体”或“CAR”指：包含能够结合抗原的胞外抗原结合区、铰链区、跨膜区和胞内信号区，胞内信号区指经由确定的信号传导途径通过产生第二信使而将信息传递到细胞内以调节细胞活性的蛋白质、或通过相应于此类信使而作为效应子发挥作用的蛋白质，包含初级信号域，还可以包括源自下文定义的刺激分子的功能信号传导结构域(即共刺激信号域)。胞内信号域产生可以促进CAR的细胞(例如CAR-T细胞)的免疫效应子功能的信号，免疫效应子功能的例子，例如在CAR-T细胞中，包括细胞裂解活性和辅助活性，包括细胞因子分泌。
术语“共刺激信号域”指“共刺激分子”，为T细胞上的关连结合性配偶体，其特异性结合共刺激配体，由此介导T细胞的共刺激反应，例如，但不限于，增殖。共刺激分子是有效免疫反应所需的、非抗原受体的细胞表面分子或其配体。共刺激分子包括但不限于，Toll样受体、以及CD2、CD27、LFA-1(CD11a/CD18)、CD8α、CD28、4-1BB、ICOS、OX40、CD40、CD80、DAP10、 DAP12、CD3ζ和CD3ε等。
本发明中：
“CD3ζ”定义为GenBan登录号BAG36664.1提供的蛋白质、或来自非人类物种例如小鼠、啮齿类动物、猴、猿等的等价残基。“CD3ζ结构域”定义为来自ζ链的胞质结构域的氨基酸残基，其足以功能性地传递T细胞活化所需的初始信号。一方面，ζ的胞质结构域包含GenBank登录号BAG36664.1的残基52至163、其功能性指向同源物—来自非人物种例如小鼠、啮齿类动物、猴、猿等的等价残基。
“CD28”定义为GenBan登录号NP_006130.1提供的蛋白质、或来自非人类物种例如小鼠、啮齿类动物、猴、猿等的等价残基。“CD28信号区”定义为来自CD28的胞质结构域的氨基酸残基，其能够传递T细胞活化所需的共刺激信号；其序列包含GenBank登录号NP_006130.1的残基180至220、其功能性指向同源物—来自非人物种例如小鼠、啮齿类动物、猴、猿等的等价残基。“CD28铰链区”包含GenBank登录号NP_006130.1的残基114至152、其功能性指向同源物—来自非人物种例如小鼠、啮齿类动物、猴、猿等的等价残基。“CD28铰跨膜区”包含GenBank登录号NP_006130.1的残基153至179、其功能性指向同源物—来自非人物种例如小鼠、啮齿类动物、猴、猿等的等价残基。
“4-1BB”指TNFR超家族的成员，其具有GenBank登录号NP_001552.2的氨基酸序列、或来自非人物种例如小鼠、啮齿类动物、猴、猿等的等价残基。“4-1BB信号区”定义为来自4-1BB的胞质结构域的氨基酸残基，其能够传递T细胞活化所需的共刺激信号；其序列包含GenBank登录号NP_006130.1的残基214至255、其功能性指向同源物—来自非人物种例如小鼠、啮齿类动物、猴、猿等的等价残基。
本发明实施例所述的EpCAM为上皮细胞细胞黏附分子。所述的人源化抗体 特异性结合表达于相关肿瘤的EpCAM。所述相关肿瘤包括上皮性肿瘤，具体包括食管癌、胃癌、结直肠癌、前列腺癌、肺癌、卵巢癌、乳腺癌、肾癌、肝细胞癌和胰腺癌。
本发明实施例中，所述人源化抗体为人源化全长抗体，亚型为hIgG1。
一些实施例中，所述人源化抗体为单链抗体，亚型为hIgG1、hIgG2、hIgG3和hIgG4的任意一种。
一些实施例中，所述重链可变区序列包括如SEQ ID.1所示序列、SEQ ID.2所示序列以及SEQ ID.3所示序列，分别记为CDR-H1的序列、CDR-H2的序列和CDR-H3的序列，CDR-H1、CDR-H2和CDR-H3分别为第一重链互补绝对区、第二重链互补绝对区和第三重链互补决定区。
一些实施例中，CDR-H1和CDR-H2之间的第一重链骨架区的序列为QVQLVQSGAEVKKPG X6SVKVSCKASGX7TFX8，其中，X6为A和S中的任意一种，X7为Y和G中的任意一种，X8为T和S中的任意一种。
一些实施例中，CDR-H2和CDR-H3之间的第二重链骨架区的序列为WVRQAPGQGLEWMG和WVRQAPGQGLEWIG的任意一种。
一些实施例中，连接CDR-H3的第三重链骨架区的序列为X9VTX10TX11DX12STSTX13YMELSSLRSEDTAVYYCAR，X9为R和K的任意一种，X10为M、L和I中的任意一种，X11为R、T、A和V中的任意一种，X12为T和E中的任意一种，X13为V和A中的任意一种。
一些实施例中，所述轻链可变区序列包括如SEQ ID.4所示序列,SEQ ID.5所示的序列以及SEQ ID.6所示序列，分别记为CDR-L1的序列、CDR-L2的序列和CDR-L3的序列，CDR-L1、CDR-L2和CDR-L3依次为第一轻链互补决定区、第二轻链互补决定区和第三轻链互补决定区。
本发明实施例还提供了特异性靶向EpCAM的嵌合抗原受体，所述嵌合抗原 受体的抗原结合区包含所述人源化抗体，或所述人源化抗体的片段。
优选的，在一些实施例中，所述靶向EpCAM的抗体片段为单链可变区(scFv)；所述单链可变区结构为VH-Linker-VL或VL-Linker-VH；所述Linker氨基酸序列如SEQ ID NO.30所示，为GGGGSGGGGSGGGGS；
一些实施例中，所述嵌合抗原受体还包含铰链区，所述铰链区的序列来源于CD8α、CD28、4-1BB、ICOS、OX40、CD40、CD80和IgG的至少一种。
一些实施例中，所述嵌合抗原受体还包含跨膜区，所述跨膜区的序列来源于CD8α、CD28、4-1BB、ICOS、OX40、CD40、CD80和CD3的至少一种。
一些实施例中，所述嵌合抗原受体还包含胞内信号区，所述胞内信号区的序列来源于CD8α、CD28、4-1BB、ICOS、OX40、CD40、CD80、DAP10、DAP12、CD3ζ和CD3ε的至少一种。
优选的，所述CAR分子序列如下表所示：
表1特异性靶向EpCAM的CAR分子序列
Figure PCTCN2021106097-appb-000001
Figure PCTCN2021106097-appb-000002
Figure PCTCN2021106097-appb-000003
Figure PCTCN2021106097-appb-000004
Figure PCTCN2021106097-appb-000005
Figure PCTCN2021106097-appb-000006
本发明实施例还提供了所述嵌合抗原受体的应用，包括特异性靶向高表达相关肿瘤的EpCAM抗原。
本发明实施例还提供了核酸，所述核酸包含编码所述嵌合抗原受体的核苷酸序列。
本发明实施例还提供了包含所述核酸的载体。
本发明实施例还提供了包含所述载体的细胞。
实施例一、人源化抗体的设计
本实施例提供了所述人源化抗体的设计方法，包括如下步骤：
S1：获取如SEQ ID NO.16所示的鼠源抗体VH序列；如SEQ ID NO.17所示的鼠源抗体VL序列
S2：将鼠源抗体序列与人源抗体数据库中进行比对，寻找同源性最高的人抗体种系；
S3：计算机建模，模拟抗体结构中可能影响和抗原结合的位点、回复突变关键位点和组合，进而筛选出最优结构解。
其中，设计VH选择同源度最高的IGHV1为人源化设计模板，设计VL优先选择IGKV1、IGKV3、IGKV4为人源化设计模板。IGHV1-KV1、IGHV1-KV3为常见配对方式。
其中，分别使用Discovery Studio和
Figure PCTCN2021106097-appb-000007
Antibody Modeling采用同源建模方法选取5-10个最优结构解；
具体的，Loop区域一般使用同源建模方法建模，如CDR氨基酸序列比对结果 显示低于同源性低于50％，则使用从头建模方法搭建CDR3结构模型。使用PDB BLAST调取序列最接近的10个结构分辨率高于2.5埃的抗体晶体结构模型，对比自动建模模型，选取最优的结构模型。
将上述若干对人源化抗体与人源Germline序列进行比对，得到的人源化程度百分比和回复突变位点数目请参见表2。
表2各人源化抗体的人源化程度评分
人源化抗体名称 人源化程度百分比 回复突变位点数目
H1L1 98.5％ 10
H2L1 98.8％ 8
H3L1 99.1％ 6
H3L2 99.3％ 5
H4L1 99.15 6
H5L1 99.3％ 5
H2L2 99.0％ 7
H2L3 99.0％ 7
H2L4 99.1％ 6
H3L3 99.3％ 5
H1L4 98.8％ 8
H1L3 98.7％ 9
实施例二、人源化抗体的表达纯化和EpCAM特异性结合能力检测
本发明实施例中，所述人源化抗体的应用包括使用pCDNA3.4载体和ExpiCHO-s表达系统对所述人源化抗体进行表达。
进一步的，将SEQ ID NO.7-11的每个序列分别克隆至pCDNA3.4载体得到若干重链基因表达载体，将SEQ ID NO.12-15的每个序列分别克隆至pCDNA3.4载体得到若干轻链基因表达载体，再将所述若干重链基因表达载体和所述若干轻链基因表达载体配对后分别通过ExpiCHO-s表达系统转染以得到若干重组抗体，分别记为H1L1、H2L1、H3L1、H3L2、H4L1、H5L1、H2L2、H2L3、H2L4、H3L3、H1L4和H1L3。mHmL为EpCAM抗体的序列。具体的克隆和转染过程为本领域技术人员的常规技术手段，在此不做赘述。
进一步的，本发明分别测定了空探针、mHmL、H1L1、H2L1、H3L1、H3L2、H4L1、H5L1、H2L2、H2L3、H2L4、H3L3、H1L4和H1L3与Human-EpCAM-Protetin-His的结合解离曲线。具体的测定方法为：使用由吐温20溶解于PBS缓冲液稀释形成的浓度为0.025％的PBST缓冲液，并将各重组抗体稀释至浓度为50nM，以及将Human-EpCAM-Protetin-His稀释成浓度梯度为500nM至31.3nM，将蛋白A偶联至生物传感器并使用上述PBST缓冲液激活10分钟；上样时间120秒，结合时间300秒，解离时间300秒。结果如表3所示的亲合力常数KD值。从表3中可以看到，重组抗体与EpCAM抗原的亲合力常数为1.0×10 -8摩尔-10 -10摩尔。
表3各抗体与EpCAM亲和力
重组抗体名称 KD(摩尔)
mHmL 1.67E-08
H1L1 1.87E-08
H2L1 1.88E-08
H3L1 2.19E-08
H3L2 6.47E-09
H4L1 8.19E-09
H5L1 1.77E-08
H2L2 4.66E-09
H2L3 1.14E-08
H2L4 1.34E-08
H3L3 1.41E-08
H1L4 1.18E-08
H1L3 9.74E-09
本发明实施例还通过流式细胞荧光分选法考察了重组抗体与人结肠癌细胞SW480、人结直肠癌细胞HCT116以及结肠癌RKO细胞的结合活性。结果显示，各重组抗体与RKO细胞的结合均不佳，但与人结肠癌细胞SW460和人结直肠癌细胞HCT116具有良好的结合活性。
具体的，每个流式管中的细胞数目为1×10 5个，使用FACS缓冲液对细胞进行离心洗涤；重悬后每个流式管加入100微升FACS稀释液，并于4摄氏度下孵育60分钟；使用FACS缓冲液进行5次离心洗涤，然后每个流式管中加入100微升的荧光素FITC与Fcγ受体片段后于4摄氏度下孵育30分钟；最后使用FACS稀释液离心洗涤后使用FACS稀释液重悬细胞，以用于测试，得到图1所示的mHmL、H1L1、H2L1、H3L1、H3L2、H4L1、H5L1、H2L2、H2L3、H2L4、H3L3与SW480的结合性能曲线以及图2所示的H1L4和H1L3与SW480的结合性能曲线，图3所示的mHmL、H1L1、H2L1、H3L1、H3L2、H4L1、H5L1、H2L2、H2L3、H2L4、H3L3与HCT116的结合性能曲线以及图4所示的H1L4和H1L3与HCT116的结合性能曲线。从图1-图4统计得到表3所示的各重组抗体分别在SW480和HCT116的EC50值。结果如表4所示，各人源化抗体结合EpCAM的EC50与原始抗体mHmL相类似，说明人源化改造并未明显影响抗体亲和力。
表4通过流式细胞术检测各抗体结合靶细胞的能力
Figure PCTCN2021106097-appb-000008
实施例三、靶向EpCAM的CAR分子设计和慢病毒包装
本发明实施例提供了一种靶向EpCAM的嵌合抗原受体(简记为EpCAM-CAR)，其结构如图5所示，包含信号肽、抗原结合区、铰链区、跨膜区和胞内共刺激信号结构域。
具体的，EpCAM-CAR的序列如SEQ ID NO.19所示，参见SEQ ID NO.19：
GM-CSF信号肽的序列为MLLLVTSLLLCELPHPAFLLIP；
人源化抗体的VH序列为：
Figure PCTCN2021106097-appb-000009
G4S x3 linker区的序列为：GGGGSGGGGSGGGGS；
人源化抗体的VL序列为：
Figure PCTCN2021106097-appb-000010
CD28铰链区的序列为：
Figure PCTCN2021106097-appb-000011
CD28跨膜区的序列为：FWVLVVVGGVLACYSLLVTVAFIIFW；
CD28共刺激信号区的序列为：
Figure PCTCN2021106097-appb-000012
CD3ζ共刺激信号区的序列为：
Figure PCTCN2021106097-appb-000013
本发明实施例将编码EpCAM-CAR的慢病毒载体质粒包装为慢病毒，EpCAM-CAR和第三代慢病毒载体骨架部分所组成的CAR质粒来源于和元生物科技有限公司，
具体包装方法如下：
在T75培养瓶中接种293T细胞并培养至汇合度在70％-80％左右，然后进行等体积换液，得到待转染样品，其中，培养瓶中控制细胞数量为5x10 6，培养体积为20毫升；
使用2毫升Opti-MEM和55微升Lipo3000配置Tube A液；使用2毫升Opti-MEM、46微升P3000、18微克辅助质粒以及6微克EpCAM-CAR主质粒配置Tube B液；
将Tube A液和Tube B液混匀后室温孵育15分钟，然后加入所述待转染样品培养48小时；
48小时转染完毕后，收集上清并在500g离心10min，过滤上清至离心管中密封，在10000g，4℃下离心过夜，得到白色病毒沉淀；提取白色病毒沉淀并用200μl AIM-V培养基溶解后，取2ul按照后续步骤测定滴度，其余置于-80℃保存。
将2ul重悬病毒上清加至198ul 1640培养基中稀释病毒，后在24孔板中按照每孔2x105数量分别加入稀释后的病毒2ul、10ul和50ul一共3个孔，并加入终浓度为5ul/ml的Polybrene辅助病毒感染48小时，得到慢病毒。病毒感染结束后，使用EpCAM-FITC标记抗原检测病毒滴度，滴度在2.5E+07-1.2E+08之间。
实施例四、靶向EpCAM的CAR-T细胞构建
本发明实施例将上述慢病毒感染人外周血单个核细胞(PBMC)以构建CAR-T细胞(简记为CAR-T)。具体构建过程如下：
人外周血单个核细胞(PBMC)采用由AIM-V培养基、5％胎牛血清、青霉素100U/mL、链霉素0.1mg/mL以及300IU/mL IL-2组成的培养基进行培养；采用来源于美天旎公司的CD2/CD3/CD28T细胞激活扩增试剂盒激活T细胞，即包被磁珠与细胞以1:2比例混合，细胞最终密度为5×10 6个/mL/cm 2，混合后置于37℃、5％CO2培养箱培养刺激48h；将来源于Takara公司的RetroNectin稀释至20μg/ml后包被培养板(non-tissue culture treated)，包被液4μg/cm2，置于4℃冰箱过夜。T细胞激活的48h后，在300g下离心5min去上清，用新鲜培养基重悬T细胞，转移至使用来源于Takara公司的RetroNectin包被好的板中，加入上述慢病毒并控制MOI＝5，置于37℃、5％CO2培养箱培养；同时制备未加入慢病毒的T细胞作为参比样品简记为unT)；加入慢病毒的24h后，300g离心5min，去上清，培养基重悬T细胞，即得CAR-T。
进一步的，加入慢病毒的48h后，取样使用流式细胞术检测转导率，其中使用EpCAM蛋白作为一抗，来源于Biolegend公司的anti-EpCAM-FITC作为二抗，得到图6所示的未转导T细胞(unT)和CAR-T的流式分析结果对比图，可知CAR 在CAR-T上得到了成功的表达。
实施例五、靶向EpCAM的CAR-T细胞体外杀伤靶细胞能力验证
本发明实施例选择不同EpCAM表达情况的肿瘤细胞系考察CAR-T的体外杀伤性能。图7为HCT116-Luc、SW480、KATO-III、A549和MIAPaCA-2的EpCAM表达水平的对比图，参照图7可知：不同EpCAM的表达水平的肿瘤细胞系分别为：阳性表达EpCAM的细胞系为HCT116-Luc、SW480、KATO-III和A549；阴性表达EpCAM的细胞系为MIAPaCA-2。
本发明实施例考察了CAR-T体外杀伤HCT116-Luc、SW480、KATO-III、A549和MIAPaCA-2的情况。
具体的，在96孔板中将制备的CAR-T和参比T细胞unT分别与1×10 4个肿瘤细胞按照不同效靶比混合培养，每组设置3个复孔，置于37℃、5％CO2培养箱共培养；24小时后，对于HCT116-luc，SW480，KATO-III，A549，MIAPaCA-2使用Sigma公司的LDH试剂盒检测靶细胞的LDH的释放，从而得出杀伤效率；同时取最高的效靶比对应的共培养孔上清，使用cisbio公司的HTRF试剂盒检测IFN-γ的量。
图8为不同效靶比下CAR-T和参比T细胞unT对不同肿瘤细胞的杀伤效率对比图。参照图8可知，CAR-T细胞特异性杀伤EpCAM阳性肿瘤细胞HCT116-Luc，SW480,KATO-III,A549，但是不杀伤EpCAM阴性肿瘤细胞MIAPaCA-2。
图9为CAR-T和参比T细胞unT与不同肿瘤细胞作用后的IFN-γ释放量的对比图。与杀伤实验结果一致，参照图9，EpCAM CAR-T细胞的IFN-γ释放特异性被EpCAM阳性肿瘤细胞HCT116-Luc，SW480,KATO-III,A549激活，但是与EpCAM阴性肿瘤细胞MIAPaCA-2共孵育的情况下无明显的IFN-γ释放激活。
实施例六、靶向EpCAM的CAR-T细胞在小鼠体内清除肿瘤细胞
本发明实施例考察了CAR-T对M-NSG小鼠HCT116皮下成瘤的治疗效果以及安全性。
具体的：在M-NSG小鼠皮下接种HCT116以建立HCT116肿瘤模型。接种后第10天，小鼠体内肿瘤平均体积约为170mm 3。采用随机区组法将小鼠分为5组：G1～G5。各组均采用尾静脉给药G1给与PBS，G2为unT,G3,G4,G5组的每只小鼠分别给予20E6,2E6,0.2E6细胞个数的CAR-T。各组小鼠分别在注射后的第1、8、15、22、28、36和42天经眼内眦采血，通过流式细胞术(FACS)检测小鼠外周血中人CD45+和人CD3+存续及CAR+细胞占比。每周测量2次瘤径、称量动物体重，观察动物生活状态，对异常情况进行记录。在整个实验过程中，按照实验要求对小鼠实施安乐死，解剖小鼠并取材做切片分析，每组用于统计分析的小鼠数目为6。
图10为各组小鼠在实验时间内的平均肿瘤体积变化情况对比图。参照图10，在day21时，G1组平均肿瘤体积为1586.69±439.96mm 3，其余各组平均肿瘤体积依次为1770.23±460.15mm 3、272.00±26.42mm 3、272.00±26.42mm 3、498.22±47.85mm 3和3607.44±510.54mm 3，对应的肿瘤抑制率依次为-13.11％、92.97％、76.99％、和-142.95％。其中G3组和G4组平均肿瘤体积明显低于G1组，表明CAR-T在20E6和2E6的实验浓度下均显示一定的抑制肿瘤生长的效果。在第42天，G4组所有小鼠的肿瘤组织均被完全清除，实现完全缓解。
图11为各组小鼠在实验时间内的平均体重变化情况对比图。参照图11，第21天时，各组小鼠的体重增长率依次为-4.42％、-5.21％、6.89％、1.45％和7.28％。其中G3组、G4组和G5组的小鼠在实验期间能较好地保持体重，表明CAR-T在20E6,2E6,0.2E6这三种实验浓度下均未显示明显毒性，而G1组、G2组的小鼠在实验期间不能较好地保持体重。
进一步的，取G1、G2和G3组小鼠做组织学解剖检测，形成的组织切片中具有代表性的样本情况如图12所示。
参照图12，G1组小鼠的肺部显示中度肿瘤细胞浸润或转移现象；G2组小鼠肺部血管周围炎细胞浸润，脾脏中性粒细胞轻微增多，肝脏汇管区炎细胞轻度浸润；G3组小鼠肺脏血管周围的炎细胞显示轻度浸润，肺部血管周围炎细胞轻 微浸润，肺部血管周围炎细胞轻度浸润，但均未显示肿瘤细胞浸润或转移现象，均显示出一定的抑制肿瘤生长的效果。G3组织切片呈现的组织病理学改变可能与荷瘤小鼠和受试物，即CAR-T的药理作用有关，不存在与CAR-T相关的毒性反应。
虽然在上文中详细说明了本发明的实施方式，但是对于本领域的技术人员来说显而易见的是，能够对这些实施方式进行各种修改和变化。但是，应理解，这种修改和变化都属于权利要求书中所述的本发明的范围和精神之内。而且，在此说明的本发明可有其它的实施方式，并且可通过多种方式实施或实现。

Claims (10)

  1. 一种人源化抗体，其特征在于，包括重链可变区和轻链可变区：
    所述重链可变区序列包括如SEQ ID.1所示序列、SEQ ID.2所示序列以及SEQ ID.3所示序列，分别作为第一重链互补决定区的序列、第二重链互补决定区的序列和第三重链互补决定区的序列；
    所述轻链可变区序列包括如SEQ ID.4所示序列,SEQ ID.5所示的序列以及SEQ ID.6所示序列，分别作为第一轻链互补决定区的序列、第二轻链互补决定区的序列和第三轻链互补决定区的序列。
  2. 根据权利要求1所述的人源化抗体，其特征在于，
    所述重链可变区序列与SEQ ID.7、SEQ ID.8、SEQ ID.9、SEQ ID.10和SEQ ID.11中任一种的至少90％相同；
    所述轻链可变区的序列与SEQ ID.12、SEQ ID.13、SEQ ID.14和SEQ ID.15中任一种的至少90％相同。
  3. 一种嵌合抗原受体，其特征在于，包含胞外抗原结合区、铰链区、跨膜区和胞内信号区，所述胞外抗原结合区包含如权利要求1-2任一项所述的人源化抗体，或所述人源化抗体的片段。
  4. 根据权利要求3所述的嵌合抗原受体，所述铰链区的序列来源于CD8α、CD28、4-1BB、ICOS、OX40、CD40、CD80和IgG的至少一种，所述跨膜区的序列来源于CD2、CD27、LFA-1、CD8α、CD28、4-1BB、ICOS、OX40、CD40、CD80、CD3ζ和CD3ε的至少一种，所述胞内信号区的序列来源于Toll样受体、以及CD2、CD27、LFA-1、CD8a、CD28、4-1BB、ICOS、OX40、CD40、CD80、DAP10、DAP12、CD3ζ和CD3ε的至少一种。
  5. 根据权利要求3所述的嵌合抗原受体，其特征在于，所述嵌合抗原受体的序 列选自SEQ ID NO.18-29所示任一序列。
  6. 一种核酸，其特征在于，包含编码权利要求1～2任一项所述的人源化抗体的抗体序列或序列片段。
  7. 一种核酸，其特征在于，包含编码权利要求3～5任一项所述的嵌合抗原受体的核苷酸序列或序列片段。
  8. 一种载体，其特征在于，包含权利要求6～7所述的核酸。
  9. 一种细胞，其特征在于，包含权利要求6～7所述的核酸或者权利要求8所述的载体。
  10. 一种生物制品的应用，其特征在于，所述生物制品应用于制备检测人类肿瘤试剂或者应用于制备治疗人类肿瘤的药物，所述生物制品为权利要求1～2任一项所述的人源化抗体，权利要求3-5任一项所述的嵌合抗原受体，权利要求6-7任一项所述的核酸，权利要求8所述的载体或权利要求9所述的细胞。
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