WO2023241492A1 - Vhh antibody specifically binding to human cd318 or antigen-binding fragment thereof, preparation method therefor, and use thereof - Google Patents

Vhh antibody specifically binding to human cd318 or antigen-binding fragment thereof, preparation method therefor, and use thereof Download PDF

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WO2023241492A1
WO2023241492A1 PCT/CN2023/099572 CN2023099572W WO2023241492A1 WO 2023241492 A1 WO2023241492 A1 WO 2023241492A1 CN 2023099572 W CN2023099572 W CN 2023099572W WO 2023241492 A1 WO2023241492 A1 WO 2023241492A1
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seq
sequence
cells
antibody
binding molecule
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PCT/CN2023/099572
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Chinese (zh)
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王海鹰
李靖
刘真莹
胡红明
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上海恒润达生生物科技股份有限公司
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Publication of WO2023241492A1 publication Critical patent/WO2023241492A1/en

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    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2896—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against molecules with a "CD"-designation, not provided for elsewhere
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    • A61K39/001129—Molecules with a "CD" designation not provided for elsewhere
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    • 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
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    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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Definitions

  • the present invention relates to the technical field of biological immunotherapy. Specifically, it relates to an antibody that specifically binds to CD318 or an antigen-binding fragment thereof and its preparation method and application.
  • CD318, also known as CDCP1 (CUB domain-containing protein 1), encodes a highly glycosylated, single-channel type I transmembrane protein that is expressed in mesenchymal stem cells, neural stem cells, fibroblasts, and hematopoietic progenitors. expressed in cells.
  • CDCP1 consists of a large extracellular domain (ECD) containing three CUB (respectively: complement C1r/C1s, Uegf and Bmp1) domains and a short intracellular part. The protein undergoes tyrosine phosphorylation. play a role in dependence regulation.
  • CD318 is abnormally elevated in a variety of malignant tumors, including colon cancer, breast cancer, pancreatic cancer, lung cancer, kidney cancer, liver cancer, etc.
  • CD318 has been identified as a stem cell marker for benign and malignant progenitor cells.
  • Significant CD318 expression was observed in a subset of CD34CD133 leukemia cells enriched in leukemia stem cells in AML patients. Therefore, CD318 is a very promising target for tumor immunotherapy.
  • Chimeric Antigen Receptor T cell (CAR T) therapy is a new immunotherapy method that targets specific antigens on the surface of tumor cells.
  • VHH antibodies also known as nanobodies, are a type of naturally missing light chain antibody found in the peripheral blood of alpacas. The antibody only contains one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions. Compared with traditional artificially modified scFv, it has the advantages of small molecular weight, easy expression, strong specificity, high affinity, weak immunogenicity to humans, and short development cycle. Combining the advantages of immunotherapy and VHH antibodies, highly efficient CAR-T therapy can be developed.
  • the invention provides a CD318-binding molecule, comprising an anti-CD318 Nanobody or an antigen-binding fragment thereof.
  • the complementarity determining region CDR of the anti-CD318 Nanobody includes CDR1, CDR2 and CDR3, wherein CDR1 includes SEQ ID NO: 1, 4, and 7.
  • CDR1 includes SEQ ID NO: 1, 4, and 7.
  • CDR2 includes the sequence shown in any one of SEQ ID NO: 2, 5, 8, 11, 14 and 17, and
  • CDR3 includes SEQ ID NO: 3, 6, Sequences shown in any of 9, 12, 15 and 18.
  • the CDRs are selected from any of the following:
  • CDR1 whose sequence is shown in SEQ ID NO:1, CDR2 whose sequence is shown in SEQ ID NO:2, and CDR3 whose sequence is shown in SEQ ID NO:3,
  • the heavy chain variable region sequence of the anti-CD318 Nanobody is set forth in any one of SEQ ID NOs: 19-24.
  • the FR1 of the anti-CD318 Nanobody can be selected from the FR1 of the VHH shown in any one of SEQ ID NO: 19-24, and the FR2 can be selected from any of SEQ ID NO: 19-24.
  • FR2 and FR3 of the VHH shown in SEQ ID NO:19-24 can be selected from the FR3 of the VHH shown in any one of SEQ ID NO:19-24, and FR4 can be selected from the FR4 of the VHH shown in any one of SEQ ID NO:19-24.
  • the CD318 binding molecule is a monovalent or multivalent Nanobody or single domain antibody, or a multispecific Nanobody comprising one, two or more anti-CD318 Nanobodies or antigen-binding fragments thereof or single domain antibodies.
  • the multivalent binding molecule or multispecific binding molecule is linked to multiple anti-CD318 Nanobodies or antigen-binding fragments thereof via a linker.
  • the linker consists of 1-15 amino acids selected from G and S.
  • the Nanobody is a camel heavy chain antibody or a cartilaginous fish heavy chain antibody.
  • the Nanobody further comprises a heavy chain constant region.
  • the heavy chain constant region is that of a camel heavy chain antibody, comprising CH2 and CH3.
  • the CH2 and CH3 are those of a human IgG Fc, such as those of an IgG1.
  • the heavy chain constant region is that of a cartilaginous fish heavy chain antibody, comprising CH1, CH2, CH3, CH4, and CH5.
  • the CD318-binding molecule according to any embodiment of the present invention is a chimeric antibody or a fully human antibody; preferably, it is a fully human antibody.
  • Another aspect of the invention provides a chimeric antigen receptor comprising an optional signal peptide sequence, a CD318 binding molecule according to any embodiment herein, a hinge region, a transmembrane region and an intracellular region.
  • the intracellular domain includes an intracellular costimulatory domain and/or an intracellular signaling domain.
  • the chimeric antigen receptor sequentially contains a signal peptide, the CD318-binding molecule described in any embodiment herein, a hinge region, a transmembrane region, and intracellular costimulation. domain and intracellular signaling domain.
  • the invention also provides nucleic acid molecules having a sequence selected from any of the following:
  • the fragment is a primer.
  • the invention also provides a nucleic acid construct comprising a nucleic acid molecule described herein.
  • the nucleic acid construct is a cloning vector, an expression vector, or an integration vector.
  • the invention also provides a host cell selected from:
  • the host cells are immune effector cells, preferably T cells.
  • the invention also provides a method for producing a CD318-binding molecule according to any embodiment herein, comprising: producing a CD318-binding molecule (e.g., a Nanobody or an antigen-binding fragment thereof, a monovalent or multivalent Nanobody or a single domain antibody, or a multispecific
  • a CD318-binding molecule e.g., a Nanobody or an antigen-binding fragment thereof, a monovalent or multivalent Nanobody or a single domain antibody, or a multispecific
  • the host cells described herein are cultured under the conditions of specific Nanobodies or single domain antibodies), and the CD318 binding molecules are optionally purified from the culture.
  • the present invention also provides a pharmaceutical composition, comprising the CD318 binding molecule, nucleic acid molecule, nucleic acid construct or host cell described in any embodiment herein, and pharmaceutically acceptable excipients.
  • the pharmaceutical composition is used to treat a disease or condition associated with CD318 expression.
  • the present invention also provides the use of the CD318 binding molecule, chimeric antigen receptor, nucleic acid molecule, nucleic acid construct or host cell of any embodiment herein for producing activated immune cells (eg, T cells).
  • activated immune cells eg, T cells
  • the present invention also provides the use of the CD318 binding molecule, chimeric antigen receptor, nucleic acid molecule, nucleic acid construct or host cell according to any embodiment herein in the preparation of a medicament for preventing or treating diseases or conditions related to CD318 expression.
  • the disease or condition is selected from one or more of the following: breast cancer, lung cancer, liver cancer, pancreatic cancer, ovarian cancer, kidney cancer, and colorectal cancer.
  • the present invention also provides a method for treating or preventing diseases or conditions related to CD318 expression, which method includes administering to a patient in need a therapeutically effective amount of the CD318-binding molecule or host cell according to any embodiment of the present invention, or the present invention.
  • the present invention also provides a kit for detecting CD318, which is used, for example, to evaluate drug treatment effects or diagnose cancer.
  • the kit includes the CD318-binding molecule, nucleic acid molecule, nucleic acid construct or host cell described in any embodiment of this document.
  • the kit further includes a reagent for detecting binding of CD318 to the CD318 binding molecule.
  • the bound reagent is detected, for example, by enzyme-linked immunoassay.
  • the reagent that detects binding is a detectable label, such as biotin, that can be linked to a CD318 binding molecule.
  • the detectable label is connected to the CD318 binding molecule or is present separately in the kit.
  • the present invention also provides a non-diagnostic method for detecting the presence of CD318 in a sample.
  • the method includes: incubating the sample with the CD318-binding molecule described in any embodiment of this document, and detecting the binding of CD318 to the CD318-binding molecule, This determines the presence of CD318 in the sample.
  • the detection is an enzyme-linked immunoreaction assay.
  • the present invention also provides the use of the CD318-binding molecule described in any embodiment herein in the preparation of a kit for detecting CD318 in a sample, evaluating the effect of drug treatment, or diagnosing cancer.
  • the present invention provides a new Nanobody that specifically recognizes CD318 and CAR-modified cells containing the antibody.
  • the antibody and cells have good therapeutic effects targeting CD318 and provide new treatments for diseases related to CD318 expression. or ways to improve.
  • Figure 1 shows the SDS electrophoresis pattern of recombinant human CD318-avi-his antigen protein.
  • Figure 2 is a schematic diagram of different cloned CD318 CARs.
  • Figure 3 shows the CAR expression positive rate of CD318 CAR-T cells of different clones.
  • Figure 4 shows the expression of CD107a in CD318 CAR-T cells of different clones.
  • Figure 5 shows the INF ⁇ secretion of CD318 CAR-T cells of different clones when the effect-to-target ratio is 10:1.
  • Figure 6 shows the INF ⁇ secretion of CD318 CAR-T cells of different clones when the effect-to-target ratio is 2:1.
  • Figure 7 shows the IL-2 secretion of CD318 CAR-T cells of different clones when the effect-to-target ratio is 10:1.
  • Figure 8 shows the IL-2 secretion of CD318 CAR-T cells of different clones when the effect-to-target ratio is 2:1.
  • Figure 9 shows the results of the killing experiment of different clones of CD318 CAR-T cells on target cells.
  • Figure 10 shows the results of the killing experiment of different clones of CD318 CAR-T cells on target cells.
  • the present invention uses CD318 protein to immunize alpacas to obtain a high-quality single domain antibody gene library. Phage display technology was then used to screen the antibody gene library, thereby obtaining a CD318-specific single domain antibody gene. This gene is then transferred into mammalian cells, thereby obtaining an antibody strain with high specificity that can be expressed efficiently in mammalian cells.
  • the antibody or its antigen-binding fragment has good safety and targeting properties, and can specifically bind to the extracellular domain of human CD318.
  • the present invention also provides chimeric antigen receptors (CARs) containing the Nanobodies.
  • CARs chimeric antigen receptors
  • a vector containing the coding sequence of the CAR to infect immune cells, immune effector cells with significant killing ability against tumor cells overexpressing CD318 can be obtained.
  • the immune effector cells can be used to treat or improve diseases related to CD318 expression, thereby This lays the foundation for the treatment of CD318-positive tumors.
  • CD318-binding molecules are proteins that specifically bind to CD318, including but not limited to antibodies, heavy chain antibodies, Nanobodies or their antigen-binding fragments.
  • antibody includes monoclonal antibodies (including full-length antibodies having an immunoglobulin Fc region), antibody compositions with multiple epitope specificities, multispecific antibodies (e.g., bispecific antibodies), diabodies and single-chain molecules, as well as antibody fragments, especially antigen-binding fragments, such as Fab, F(ab')2, Fd and Fv.
  • antibody and “immunoglobulin” are used interchangeably.
  • Antibodies contain the basic 4-chain antibody unit, which is a heterotetrameric glycoprotein composed of two identical light chains (L) and two identical heavy chains (H). Each heavy chain has a variable domain (VH) at the N-terminus, followed by three (for each alpha and gamma chain, CH1, CH2 and CH3) and four (for the ⁇ and epsilon isoforms, CH1, CH2, CH3 and CH4) constant domains (CH) and a hinge region (Hinge) located between the CH1 and CH2 domains. Each light chain has a variable domain (VL) at the N-terminus, followed by a constant domain (CL) at its other end. Pairs of VH and VL together form an antigen-binding site.
  • VH variable domain
  • CL constant domain
  • Light chains from any vertebrate species can be assigned to one of two distinct types called kappa and lambda, based on their constant domain amino acid sequence.
  • the gamma and alpha classes can be further divided into subclasses based on relatively small differences in CH sequence and function, for example humans express the following subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1 and IgA2.
  • Heavy chain antibodies as used herein are antibodies derived from camelids or cartilaginous fishes. Compared with the above 4-chain antibodies, the heavy chain antibody lacks the light chain and heavy chain constant region 1 (CH1), and only contains 2 heavy chains composed of variable regions (VHH) and other constant regions. The variable region has a hinge-like structure. connected to the constant region. Each heavy chain of camelid heavy chain antibodies contains 1 variable region (VHH) and 2 constant regions (CH2 and CH3), and each heavy chain of chondrichthyan heavy chain antibodies contains 1 variable region and 5 constant regions. Constant region (CH1-CH5).
  • Antigen-binding fragments of heavy chain antibodies include VHH and single-chain heavy chain antibodies.
  • the heavy chain antibody can have CH2 and CH3 of human IgG Fc by fusion to the constant region of human IgG Fc.
  • single domain antibody As used herein, the terms “single domain antibody”, “anti-CD318 single domain antibody”, “heavy chain variable region domain of a heavy chain antibody” and “VHH” are used interchangeably and all refer to specific recognition and binding to CD318 of single domain antibodies.
  • Single domain antibodies are the variable regions of heavy chain antibodies. Typically, single domain antibodies contain three CDRs and four FRs.
  • the single domain antibody of the present invention has CDR1 shown in SEQ ID NO:1, CDR2 shown in SEQ ID NO:2, and CDR3 shown in SEQ ID NO:3; or has CDR3 shown in SEQ ID NO:4 CDR1, CDR2 shown in SEQ ID NO:5, and CDR3 shown in SEQ ID NO:6; or having CDR1 shown in SEQ ID NO:7, CDR2 shown in SEQ ID NO:8, and SEQ ID NO : CDR3 shown in SEQ ID NO: 9; or having CDR1 shown in SEQ ID NO: 10, CDR2 shown in SEQ ID NO: 11, and CDR3 shown in SEQ ID NO: 12; or having CDR3 shown in SEQ ID NO: 13 CDR1, CDR2 represented by SEQ ID NO:14, and CDR3 represented by SEQ ID NO:15; or having CDR1 represented by SEQ ID NO:16, CDR2 represented by SEQ ID NO:17, and SEQ ID NO: CDR3 shown in 18.
  • Single domain antibodies are the smallest functional antigen-binding fragments. Usually, after obtaining an antibody that naturally lacks the light chain and heavy chain constant region 1 (CH1), the variable region of the antibody heavy chain is cloned to construct a single-domain antibody consisting of only one heavy chain variable region.
  • CH1 light chain and heavy chain constant region 1
  • “Nanobody” refers to an antibody containing a VHH as described herein. It can be a heavy chain antibody as described above, or a multivalent or multispecific antibody containing multiple VHHs, or it can be obtained by recombining VHH and antibody Fc (such as CH2 and CH3 or CH2, CH3 and CH4) Recombinant antibodies. Binding molecules containing two or more single domain antibodies are multivalent single domain antibodies; binding molecules containing two or more single domain antibodies with different specificities are multispecific single domain antibodies. Multivalent single domain antibodies or multispecific single domain antibodies connect multiple single domain antibodies via a linker. The linker usually consists of 1-15 amino acids selected from G and S.
  • heavy chain antibodies and antibodies are intended to distinguish different combinations of antibodies. Due to the structural similarity between the two, the following structural descriptions of antibodies are applicable to heavy chain antibodies in addition to the light chain.
  • variable region refers to the amino-terminal domain of the heavy or light chain of the antibody.
  • variable domains of the heavy and light chains may be referred to as "VH” and “VL” respectively. These domains are typically the most variable parts of the antibody (relative to other antibodies of the same type) and contain the antigen-binding site.
  • variable refers to the situation where certain segments of the variable domain vary widely among antibody sequences. Variable domains mediate antigen binding and define the specificity of a particular antibody for its particular antigen. However, variability is not evenly distributed across all amino acids spanned by the variable domain. Instead, it is concentrated in three segments called hypervariable regions (HVRs) (found in both light and heavy chain variable domains), namely HCDR1, HCDR2, and HCDR3 (heavy chain variable domains), respectively.
  • HVRs hypervariable regions
  • chain antibodies can be abbreviated as CDR1, CDR2, CDR3
  • the more highly conserved part of the variable domain is called the framework region (FR).
  • variable domains of the native heavy and light chains each contain four FR regions (FR1, FR2, FR3, and FR4), which mostly adopt a ⁇ -sheet conformation by forming loop connections and in some cases forming a ⁇ -sheet structure Part of the three HVR connections.
  • the HVRs in each chain are held in close proximity by the FR region and together with the HVRs of the other chain contribute to the formation of the antibody's antigen-binding site.
  • the structure of the light chain variable region is FR1-LCDR1-FR2-LCDR2-FR3-LCDR3-FR4
  • the structure of the heavy chain variable region is FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4.
  • the constant domain is not directly involved in binding of the antibody to the antigen, but exhibits a variety of effector functions, such as the involvement of the antibody in antibody-dependent cell-mediated cytotoxicity.
  • effector functions such as the involvement of the antibody in antibody-dependent cell-mediated cytotoxicity.
  • Fc region (crystallizable fragment region) or “Fc domain” or “Fc” refers to the C-terminal region of an antibody heavy chain that mediates binding of immunoglobulins to host tissues or factors, including those located in the immune system Binding to Fc receptors on various cells (e.g., effector cells) or to the first component (C1q) of the classical complement system.
  • the Fc region is composed of two identical protein fragments from the CH2 and CH3 domains of the two heavy chains of the antibody; the Fc regions of IgM and IgE are present in each polypeptide chain. Contains three heavy chain constant domains (CH domains 2-4).
  • an Fc region of an immunoglobulin heavy chain is generally defined as the sequence segment from the amino acid residue at position C226 or P230 of the heavy chain to the carboxyl terminus, where this numbering is according to the EU index, as in Same in Kabat.
  • an Fc region may be a native sequence Fc or a variant Fc.
  • Antibody fragment includes a portion of an intact antibody, preferably the antigen-binding region and/or variable region of an intact antibody.
  • the antibody fragment is preferably an antigen-binding fragment of an antibody.
  • antibody fragments include Fab, Fab', F(ab'), F(ab')2, Fd, and Fv fragments, disulfide-linked Fv; diabodies; linear antibodies; single chain antibody molecules; scFv-Fc fragments; multispecific antibodies formed from antibody fragments; and any fragment that should be capable of increasing half-life, either by chemical modification or by incorporation into liposomes.
  • Antigen-binding fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies, and expression of host cells containing the antigen-binding fragments.
  • Fv is the smallest antibody fragment containing intact antigen recognition and binding sites. This fragment consists of a dimer of a heavy chain variable domain and a light chain variable domain in tight, non-covalent association. Six hypervariable loops (3 loops each in the heavy and light chains) protrude from the fold of these two domains, contributing amino acid residues for antigen binding and conferring antigen-binding specificity to the antibody. However, even a single variable domain (or half an Fv containing only three HVRs specific for the antigen) has the ability to recognize and bind antigen, albeit with lower affinity than the full binding site.
  • Single-chain Fv also abbreviated as “sFv” or “scFv”
  • sFv is an antibody fragment containing the VH and VL domains of an antibody linked into a polypeptide chain.
  • the sFv polypeptide also contains a polypeptide linker between the VH and VL domains so that the sFv forms the desired antigen-binding structure.
  • the scFv is the VHH.
  • the term "monoclonal antibody” refers to an antibody obtained from a population of antibodies that are essentially homogeneous, i.e., except for possible naturally occurring mutations and/or post-translational modifications that may be present in small amounts (e.g. isomerization, amidation ), the individual antibodies that make up the population are identical. Monoclonal antibodies are highly specific and target a single antigenic site. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.
  • monoclonal antibodies are synthesized by hybridoma culture and are not contaminated by other immunoglobulins.
  • the modifier "monoclonal" indicates that the antibody is derived from a substantially homogeneous population of antibodies and should not be construed as requiring that the antibody be produced by any particular method.
  • monoclonal antibodies to be used in accordance with the present invention can be produced by a variety of techniques, including, for example, hybridoma methods, phage display methods, recombinant DNA methods, and methods for producing antibodies containing part or all of a human immunoglobulin locus or encoding a human immunoglobulin locus. Techniques and single-cell sequencing methods for producing human or human-like antibodies from animals using immunoglobulin sequence genes.
  • Monoclonal antibodies are also included herein as "chimeric" antibodies in which a portion of the heavy chain and/or light chain is derived from a specific species or belongs to a specific The corresponding sequence in an antibody of an antibody class or subclass is identical or homologous, and the remainder of the chain is identical or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, and such Fragments of antibodies as long as they exhibit the desired biological activity.
  • “Humanized” forms of non-human (eg, murine) antibodies refer to chimeric antibodies that contain minimally sequence derived from a non-human immunoglobulin.
  • a “humanized antibody” generally refers to a non-human antibody in which the variable domain framework regions are exchanged with sequences found in human antibodies.
  • the entire antibody except for the CDRs
  • CDRs some or all of which are encoded by nucleic acids derived from non-human organisms, are grafted into the beta-sheet backbone of human antibody variable regions to produce antibodies whose specificity is determined by the grafted CDRs.
  • Methods for producing such antibodies are well known in the art, for example using mice with genetically engineered immune systems.
  • antibodies, single domain antibodies, heavy chain antibodies, etc. all include humanized variants of each of the antibodies.
  • Human antibody refers to an antibody that has an amino acid sequence corresponding to that of an antibody produced by a human and/or is produced using any of the techniques disclosed herein for producing human antibodies. This definition of human antibodies specifically excludes humanized antibodies containing non-human antigen-binding residues. Human antibodies can be generated using a variety of techniques known in the art, including phage display libraries.
  • the invention also provides Nanobodies, heavy chain antibodies, antibodies or antigen-binding fragments thereof that bind the same epitope on human CD318 as the antigen-joining region of any anti-CD318 Nanobody of the invention (e.g., single domain antibody VHH ), that is, a Nanobody, heavy chain antibody, antibody or antigen-binding fragment thereof that can cross-compete with the antigen-binding region of any Nanobody of the present invention for binding to CD318.
  • any anti-CD318 Nanobody of the invention e.g., single domain antibody VHH
  • the anti-CD318 single domain antibody has CDR1 shown in SEQ ID NO:1, CDR2 shown in SEQ ID NO:2, and CDR3 shown in SEQ ID NO:3; or has CDR3 shown in SEQ ID NO:4 CDR1, CDR2 shown in SEQ ID NO:5, and CDR3 shown in SEQ ID NO:6; or having CDR1 shown in SEQ ID NO:7, CDR2 shown in SEQ ID NO:8, and SEQ ID NO : CDR3 shown in SEQ ID NO: 9; or having CDR1 shown in SEQ ID NO: 10, CDR2 shown in SEQ ID NO: 11, and CDR3 shown in SEQ ID NO: 12; or having CDR3 shown in SEQ ID NO: 13 CDR1, CDR2 represented by SEQ ID NO:14, and CDR3 represented by SEQ ID NO:15; or having CDR1 represented by SEQ ID NO:16, CDR2 represented by SEQ ID NO:17, and SEQ ID NO: CDR3 shown in 18.
  • FR1, FR2, FR3 and FR4 of the anti-CD318 single domain antibody described herein can be independently selected from FR1, FR2, FR3 and FR4 of the single domain antibody shown in any one of SEQ ID NO: 19-24.
  • the amino acid sequence of the anti-CD318 single domain antibody is as shown in any one of SEQ ID NO: 19-24.
  • a Nanobody is a heavy chain antibody comprising a single domain antibody as described herein.
  • the heavy chain constant region may be that of a camel heavy chain antibody, including CH2 and CH3.
  • the antibody constant region is derived from: the constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD, more preferably derived from any one of IgG1, IgG2, IgG3, IgG4 or's constant region.
  • the heavy chain constant region is CH2 and CH3 of a human IgG Fc, e.g., CH2 and CH3 of IgG1.
  • the CD318 binding molecules described herein can be monovalent or multivalent Nanobodies or single domain antibodies, or multispecific Nanobodies or single domain antibodies, including one, two or more anti-CD318 Nanobodies or single domain antibodies described herein.
  • Multispecificity can be against CD318 and another antigen, or it can be against two different epitopes of CD318.
  • the invention also includes such antibody derivatives and analogs.
  • “Derivatives” and “analogues” refer to polypeptides that substantially retain the same biological function or activity of the antibodies of the invention.
  • Derivatives or analogs of the present invention may be (i) a polypeptide having substituent groups in one or more amino acid residues, or (ii) a mature polypeptide combined with another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol).
  • polypeptide formed by fusion of an additional amino acid sequence to this polypeptide sequence such as a leader sequence or secretion sequence or a sequence used to purify this polypeptide or a protein sequence, or with a 6His tag fusion protein formed.
  • those skilled in the art can change one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more) amino acids to obtain variants of the antibody or functional fragment sequence thereof.
  • These variants include (but are not limited to): deletion of one or more (usually 1-50, preferably 1-30, more preferably 1-20, optimally 1-10) amino acids , insertion and/or substitution, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and/or N-terminus.
  • conservative substitutions with amino acids with similar or similar properties generally do not change the function of the protein. For example, amino acids with similar properties are substituted in the FR and/or Fc region.
  • amino acid residues that are subject to conservative substitutions are well known in the art. Such substituted amino acid residues may or may not be encoded by the genetic code. As another example, adding one or more amino acids to the C-terminus and/or N-terminus usually does not change the function of the protein. They are all considered to be included in the scope of protection of the present invention.
  • Variant forms of the antibodies described herein include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, hybridization to the DNA encoding the antibody of the invention under high or low stringency conditions Proteins encoded by DNA, and polypeptides or proteins obtained using antiserum against the antibodies of the present invention.
  • the sequence of a variant described herein may be at least 95%, 96%, 97%, 98% or 99% identical to the sequence from which it is derived.
  • the sequence identity described in the present invention can be measured using sequence analysis software.
  • the computer program BLAST uses default parameters, especially BLASTP or TBLASTN.
  • the present invention also includes those molecules having antibody heavy chain variable regions with CDRs as long as the CDRs are more than 90% (preferably more than 95%, optimally more than 98%) homologous to the CDRs identified herein .
  • the antibodies of the present invention can be prepared using conventional methods in the art, such as hybridoma technology.
  • Nanobodies of the present invention can be prepared using conventional methods in the art, such as phage display technology, which is well known in the art.
  • the antibodies or Nanobodies of the invention can be expressed in other cell lines.
  • Suitable mammalian host cells can be transformed with sequences encoding the antibodies of the invention, and the host cells can then be cultured and the antibodies purified. Transformation can be performed using any known method, including, for example, packaging the polynucleotide in a virus (or viral vector) and transducing the host cell with the virus (or vector). The transformation procedure used depends on the host being transformed.
  • Methods for introducing heterologous polynucleotides into mammalian cells include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation , encapsulating polynucleotides in liposomes and microinjecting DNA directly into the nucleus, etc.
  • Mammalian cell lines that can be used as hosts for expression are well known in the art and include, but are not limited to, a variety of immortalized cell lines available from the American Type Culture Collection (ATCC), including, but not limited to, Chinese Hamster Ovary (CHO) ) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (eg, HepG2), etc.
  • ATCC American Type Culture Collection
  • the present invention also provides a chimeric antigen receptor (CAR) targeting CD318.
  • the CAR contains an optional signal peptide sequence, an antigen recognition region, namely the anti-CD318 binding molecule described herein, a hinge region, a transmembrane region and an intracellular region.
  • the intracellular region includes one or more intracellular costimulatory domains and/or one or more intracellular signaling domains.
  • the "hinge region”, “transmembrane region” and “intracellular region” in this article can all be selected from the sequences of the hinge region, transmembrane region and intracellular region in known CAR-T technology.
  • a signal peptide is a peptide sequence that targets a polypeptide to a desired location in a cell.
  • the signal peptide targets the polypeptide to the secretory pathway of the cell and will allow the polypeptide to integrate and anchor into the lipid bilayer; the signal peptide may also be a membrane-localized signal peptide.
  • Exemplary signal peptides such as CD8 signal peptide, CD28 signal peptide, CD4 signal peptide or light chain signal peptide, the sequences of which are within the knowledge of those skilled in the art.
  • the CD8 signal peptide suitable for the present invention can be various human CD8 signal peptide sequences commonly used for CAR in the art.
  • the amino acid sequence of the CD8 signal peptide includes the sequence shown in SEQ ID NO: 25.
  • the hinge region of the chimeric antigen receptor is located between the extracellular antigen-binding region and the transmembrane region.
  • the hinge region is a segment of amino acids that usually exists between two domains of a protein and can allow for the flexibility of the protein and the separation of the two domains. move relative to each other.
  • the hinge region may be of a naturally occurring protein Hinge area or part thereof.
  • Hinge regions of antibodies such as IgG, IgA, IgM, IgE or IgD antibodies may also be used in the chimeric antigen receptors described herein.
  • Non-naturally occurring peptides may also be used as hinge regions of the chimeric antigen receptors described herein.
  • the hinge region of the CAR is selected from the group consisting of CD8 ⁇ hinge region, IgD hinge region, IgG1 Fc CH2CH3 hinge region or IgG4 Fc CH2CH3 hinge region, the sequences of which are within the knowledge of those skilled in the art.
  • the CD8 ⁇ hinge region suitable for the present invention can be various human CD8 ⁇ hinge region sequences commonly used for CAR in the art.
  • the human CD8 alpha hinge region comprises the sequence set forth in SEQ ID NO:26.
  • the transmembrane region of a chimeric antigen receptor can form an alpha helix, a complex of more than one alpha helix, a beta barrel, or any other stable structure capable of spanning the cellular phospholipid bilayer.
  • the transmembrane region may be of natural or synthetic origin.
  • the transmembrane region may be selected from those of the following proteins: CD3 ⁇ , CD4, CD5, CD8 ⁇ , CD9, CD16, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, T cell receptor ⁇ , ⁇ or ⁇ chain of the body.
  • the human CD8 ⁇ transmembrane region suitable for the present invention can be various human CD8 ⁇ transmembrane region sequences commonly used for CAR in the art.
  • the amino acid sequence of the human CD8 ⁇ transmembrane region includes the sequence shown in SEQ ID NO: 27.
  • the intracellular signaling domain (or intracellular signaling domain) is responsible for the activation of at least one normal effector function of the immune effector cell expressing the chimeric antigen receptor.
  • the effector function of a T cell may be cytolytic activity or auxiliary activity, including secretion of cytokines.
  • cytolytic activity or auxiliary activity, including secretion of cytokines.
  • auxiliary activity including secretion of cytokines.
  • an intracellular signaling domain includes any truncated form of an intracellular signaling domain that is sufficient to transduce an effector function signal.
  • the intracellular signaling domain of the CAR can be selected as needed, including but not limited to those derived from at least one of CD3 ⁇ , FcR ⁇ (FCER1G), FcR ⁇ (Fc ⁇ Rib), CD3 ⁇ , CD3 ⁇ , CD3 ⁇ , CD5, CD22, CD79a, CD79b and CD66d Intracellular signaling domain.
  • the intracellular signal region is derived from the human CD3 ⁇ intracellular signal region.
  • the human CD3 ⁇ intracellular signal region has the amino acid sequence shown in SEQ ID NO: 29.
  • costimulatory domain may be the cytoplasmic portion of a costimulatory molecule.
  • costimulatory molecule refers to an associated binding partner on an immune cell, such as a T cell, that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the immune cell, such as, but not limited to, proliferation and survival. .
  • Suitable intracellular costimulatory domains can be selected as needed, including intracellular domains with costimulatory signaling molecules, such as those derived from 4-1BB, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54, CD83, OX40 , CD137, CD134, CD150, CD152, CD223, CD270, PD-L2, PD-L1, CD278, DAP10, LAT, NKD2C, SLP76, TRIM, Fc ⁇ RI ⁇ , MyD88, and at least one of the intracellular domains of 41BBL.
  • the amino acid sequence of the 4-1BB costimulatory domain comprises the sequence set forth in SEQ ID NO: 28.
  • the above-mentioned parts that form the chimeric antigen receptor of the present invention interact with each other.
  • the linkage can be direct or can be linked via a linker sequence.
  • the linker sequence may be one well known in the art and suitable for use with antibodies, such as a G and S-containing linker sequence.
  • linkers typically contain one or more repeating motifs.
  • the motif may be GGGS, GGGGS, SSSSG, GSGSA, and GGSGG.
  • the motifs are contiguous in the linker sequence, with no intervening amino acid residues between repeats.
  • Linker sequences can contain 1, 2, 3, 4 or 5 repeating motifs.
  • the length of the linker can be 3 to 25 amino acid residues, such as 3 to 15, 5 to 15, or 10 to 20 amino acid residues.
  • the linker sequence is a polyglycine linker sequence.
  • the number of glycines in the linker sequence is not particularly limited, but is usually 2 to 20, such as 2 to 15, 2 to 10, or 2 to 8.
  • the linker can also contain other known amino acid residues, such as alanine (A), leucine (L), threonine (T), glutamic acid (E), phenylalanine Acid (F), arginine (R), glutamine (Q), etc.
  • the linker sequence is a (GGGGS)n linkage, where n is an integer from 1 to 5.
  • the CAR contains CD8 signal peptide, anti-CD318 Nanobody or antigen-binding fragment thereof described herein, CD8 ⁇ hinge region, CD8 ⁇ transmembrane region, 4-1BB costimulatory domain, in sequence from N-terminus to C-terminus. CD3 ⁇ intracellular signaling domain.
  • an exemplary CAR having the above structure is as shown in any of SEQ ID NOs: 30-35.
  • the amino terminus or carboxyl terminus of the CAR of the present invention may also contain one or more polypeptide fragments as protein tags.
  • Any suitable tag may be used for this article.
  • the tags may be FLAG, HA, HA1, c-Myc, Poly-His, Poly-Arg, Strep-TagII, AU1, EE, T7, 4A6, ⁇ , B, gE and Ty1. These tags can be used to purify proteins.
  • the antigen recognition region in the CAR of the present invention can be a variant of the aforementioned anti-CD318 Nanobody or its functional fragment sequence.
  • other parts of the CAR can also undergo sequence changes, and the resulting mutant has at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 97% sequence identity with the CAR and retains the sequence identity.
  • Biological activity of CAR (such as activated T cells). Sequence identity between two aligned sequences can be calculated using, for example, NCBI's BLASTp.
  • Mutants also include amino acid sequences that have one or several mutations (insertions, deletions, or substitutions) in the amino acid sequence of the CAR described in any embodiment while still retaining the biological activity of the CAR.
  • the number of mutations usually refers to within 1-10, such as 1-8, 1-5 or 1-3.
  • Substitutions are preferably conservative substitutions.
  • conservative substitutions with amino acids with similar or similar properties generally do not change the function of the protein or polypeptide.
  • amino acids with similar or similar properties include, for example, families of amino acid residues with similar side chains.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • amino acids with acidic side chains chain amino acids (e.g., aspartic acid, glutamic acid)
  • amino acids with uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine amino acids
  • amino acids with non-polar side chains such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • Amino acids with ⁇ -branched side chains eg threonine, valine, isoleucine
  • amino acids with aromatic side chains eg tyrosine, phenylalanine, tryptophan, histidine
  • the invention also provides polynucleotides encoding the above-mentioned antibodies or CARs.
  • the polynucleotides of the invention may be in DNA form or RNA form. Forms of DNA include cDNA, genomic DNA, or synthetic DNA. DNA can be single-stranded or double-stranded. DNA can be a coding strand or a non-coding strand.
  • the present invention also encompasses degenerate variants of the polynucleotide sequence encoding the fusion protein, ie, nucleotide sequences encoding the same amino acid sequence but with different nucleotide sequences.
  • the present invention also relates to polynucleotides that hybridize to the above-mentioned polynucleotide sequences and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences.
  • the invention particularly relates to polynucleotides that hybridize under stringent conditions to the polynucleotides of the invention.
  • stringent conditions refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 ⁇ SSC, 0.1% SDS, 60°C; or (2) adding water during hybridization There are denaturants, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only the identity between the two sequences is at least 90%, more It is best when hybridization occurs only when the ratio is above 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.
  • the full-length nucleotide sequence of the antibody of the present invention or its fragment can usually be obtained by PCR amplification, recombinant or artificial synthesis.
  • one A feasible method is to use artificial synthesis to synthesize the relevant sequences, especially when the fragment length is short. Often, fragments with long sequences are obtained by first synthesizing multiple small fragments and then ligating them.
  • the coding sequence of the heavy chain and the expression tag (such as 6His) can also be fused together to form a fusion protein.
  • the sequence of the CAR can also be obtained as above.
  • the sequences of each part of the CAR (signal peptide, antigen recognition region, hinge region, transmembrane region or intracellular region) can be obtained as above and then connected to obtain the full length of the CAR.
  • Biomolecules (nucleic acids, proteins, etc.) involved in the present invention include biomolecules in isolated form.
  • the DNA sequence encoding the protein of the present invention (or its fragments, or its derivatives) can be obtained entirely through chemical synthesis.
  • the DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors) and cells known in the art.
  • mutations can also be introduced into the protein sequence of the invention through chemical synthesis.
  • Each part of the CAR can be cloned sequentially into the vector or can be integrated into the full-length CAR and then cloned.
  • the present invention also relates to nucleic acid constructs containing the polynucleotide sequences described herein, and one or more regulatory sequences operably linked to these sequences.
  • the polynucleotide sequences of the invention can be manipulated in various ways to ensure the expression of the antibody or CAR. Before inserting the nucleic acid construct into the vector, the nucleic acid construct can be manipulated according to the differences or requirements of the expression vector. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.
  • the control sequence may be a suitable promoter sequence.
  • the promoter sequence is usually operably linked to the coding sequence of the protein to be expressed.
  • the promoter can be any nucleotide sequence that exhibits transcriptional activity in the host cell of choice, including mutant, truncated, and hybrid promoters, and can be derived from genes encoding extracellular genes that are homologous or heterologous to the host cell. or genetic acquisition of intracellular polypeptides.
  • An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence.
  • CMV immediate early cytomegalovirus
  • the promoter sequence is a strong constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operably linked thereto.
  • EF-1 ⁇ elongation growth factor-1 ⁇
  • SV40 simian virus 40
  • MMTV mouse mammary tumor virus
  • HSV human immunodeficiency virus
  • LTR long terminal repeat
  • MoMuLV avian leukemia virus promoter
  • Epstein-Barr virus immediate early promoter Epstein-Barr virus immediate early promoter
  • Ruth's sarcoma virus promoter and human gene promoters such as but not limited to actin promoter, myosin promoter, heme promoter and creatine kinase promoter.
  • inducible promoters may also be considered.
  • an inducible promoter provides a molecular switch capable of turning on expression of a polynucleotide sequence operably linked to the inducible promoter when expression is required and turning off expression when expression is undesirable.
  • inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.
  • the control sequence may also be a suitable transcription terminator sequence, a sequence recognized by the host cell to terminate transcription.
  • the terminator sequence is operably linked to the 3' end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the host cell of choice can be used in the present invention.
  • the regulatory sequence may also be a suitable leader sequence, an untranslated region of the mRNA important for translation by the host cell. The leader sequence is operably linked to the 5' end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the host cell of choice can be used in the present invention.
  • the nucleic acid construct is a vector, such as a cloning vector, an expression vector, and an integration vector.
  • Expression of a polynucleotide sequence of the invention is generally accomplished by operably linking the polynucleotide sequence of the invention to an expression vector.
  • Typical cloning vectors contain transcriptional and translational terminators, initiation sequences, and promoters that can be used to regulate expression of the desired nucleic acid sequence.
  • Integration vectors contain components that integrate target sequences into the cellular genome. These vectors can be used to transform appropriate host cells to enable expression of the protein.
  • Vectors typically contain sequences for plasmid maintenance and for cloning and expression of exogenous nucleotide sequences.
  • the sequences (collectively referred to in certain embodiments as “flanking sequences”) generally include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a donor-containing sequence and acceptor splice site, a sequence encoding a leader for secretion of the polypeptide, a ribosome binding site, a polyadenylation sequence, and a polylinker for insertion of nucleic acid encoding the antibody to be expressed. area and optional marker elements.
  • the type of vector is not limited, for example, plasmids, phagemids, phage derivatives, animal viruses, and cosmids, and may vary depending on the host cell to be introduced.
  • Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals.
  • Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses.
  • the vector introduced into the cell may also contain either or both a selectable marker gene or a reporter gene to facilitate identification of the population of cells sought to be transfected or infected by the viral vector. Identification and selection of expressing cells.
  • Host cells suitable for introducing the nucleic acid constructs described herein can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells, especially immune cells, Immune effector cells are preferred.
  • prokaryotic cells such as bacterial cells
  • lower eukaryotic cells such as yeast cells
  • higher eukaryotic cells such as mammalian cells, especially immune cells
  • Immune effector cells are preferred.
  • Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells of CHO, COS7, 293 cells, etc.
  • Immuno effector cells are immune cells that perform immune effector functions.
  • the immune effector cells express at least Fc ⁇ RIII and perform ADCC effector functions.
  • Examples of immune effector cells that mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells, neutrophils, and eosinophils.
  • PBMC peripheral blood mononuclear cells
  • NK natural killer
  • monocytes cytotoxic T cells
  • neutrophils neutrophils
  • eosinophils eosinophils.
  • the immune effector cells are selected from: at least one of immune cells cultured and differentiated from pluripotent stem cells or embryonic stem cells, T lymphocytes, NK cells, peripheral blood mononuclear cells (PBMC) and hematopoietic stem cells. More preferably, the immune effector cells are T lymphocytes (the same as T cells).
  • T cells can be CD4+/CD8-, CD4-/CD8+, CD4+/CD8+, CD4-/CD8-, or combinations thereof.
  • T cells produce IL-2, IFN, and/or TNF when expressing chimeric antigen receptors and binding to target cells.
  • CD8+ T cells lyse antigen-specific target cells when expressing chimeric antigen receptors and binding to target cells.
  • T cells suitable for use in the present invention can be various types of T cells from various sources.
  • T cells can be derived from PBMCs of patients with malignant solid tumors, such as pancreatic cancer.
  • T cells after T cells are obtained, they can be first stimulated and activated with an appropriate amount (for example, 30-80ng/ml, such as 50ng/ml) of CD3 antibodies, and then containing an appropriate amount (for example, 30-80IU/ml, such as 50ng/ml) of CD3 antibody for activation. 50IU/ml) IL2 medium for culture and use.
  • nucleic acids or vectors into mammalian cells
  • the vectors may be introduced into the cells by physical, chemical, or biological means.
  • the host is a prokaryotic organism such as E. coli
  • competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl2 method.
  • the steps used are well known in the art.
  • the host is a eukaryotic organism
  • the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.
  • transduced or transfected immune effector cells are propagated ex vivo following introduction of nucleic acid or vector.
  • the obtained transformants can be cultured using conventional methods to express the antibody or CAR encoded by the gene of the present invention.
  • the medium used in culture can be selected from various conventional media. Cultivate under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced using an appropriate method (such as temperature shift or chemical induction), and the cells are cultured for a further period of time.
  • the polypeptide in the above method can be expressed within the cell, on the cell membrane, or secreted outside the cell.
  • the recombinant protein can be isolated and purified by various separation methods utilizing its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitating agents (salting out method), centrifugation, osmotic sterilization, ultratreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid phases Chromatographic techniques and combinations of these methods.
  • Nanobodies that could bind CD318.
  • the inventors constructed CAR and CAR-T cells. After experimental verification at the cell level, the CAR-T has strong immune function, better CD107a expression, IFN- ⁇ and IL-2 secretion, and resistance to The specific killing function of target cells has significant efficacy in vivo.
  • All aspects of the antibodies, CARs, coding sequences, nucleic acid constructs, and cells described herein can be used to prepare medicaments for the prevention or treatment of various conditions and diseases described herein that are associated with CD318 expression.
  • Diseases or conditions refer to diseases caused directly or indirectly by abnormal expression of CD318, usually referring to diseases caused by overexpression of CD318, such as cancer, including but not limited to: breast cancer, lung cancer, liver cancer, pancreatic cancer, ovarian cancer , kidney cancer and colorectal cancer.
  • the invention also encompasses a type of cell therapy that involves expressing a CAR as described herein in immune cells (eg, T cells) and administering to a recipient in need thereof a therapeutically effective amount of the cells capable of killing tumor cells in the recipient.
  • immune cells eg, T cells
  • CAR-T cells are able to replicate in vivo, producing long-term persistence that can lead to sustained tumor control.
  • the anti-tumor immune response caused by CAR-T cells can be an active or passive immune response.
  • a CAR-mediated immune response can be part of an adoptive immunotherapy step, in which CAR-T cells induce an immune response specific for the antigen-binding portion of the CAR.
  • the antibodies, nucleic acids or CAR-modified cells of the invention can be administered alone or as pharmaceutical compositions in combination with diluents and/or with other components such as relevant cytokines or cell populations.
  • the pharmaceutical composition may be prepared in the form of a lyophilized preparation or aqueous solution by mixing the active agent with the desired purity and optionally a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers are nontoxic to the recipient at the dosage and concentration used and may include buffers (e.g., neutral buffered saline, sulfate buffered saline), antioxidants, preservatives, isotonic agents, stabilizing agents at least one of an agent, a chelating agent (such as EDTA or glutathione), an adjuvant (such as aluminum hydroxide), and a surfactant.
  • buffers e.g., neutral buffered saline, sulfate buffered saline
  • antioxidants e.g., sulfate buffered saline
  • preservatives e.g., isotonic agents
  • stabilizing agents at least one of an agent e.g., a chelating agent (such as EDTA or glutathione), an adjuvant (such as aluminum hydroxide), and a surfactant.
  • an adjuvant such as aluminum hydroxide
  • surfactant such as aluminum
  • compositions may contain at least one additive from the group consisting of cytotoxic agents, chemotherapeutic agents, cytokines, immunosuppressants, growth inhibitors, and active agents required for the particular indication to be treated.
  • the specific amount of additives can be adjusted according to actual needs.
  • the pharmaceutical composition of the present invention may be administered in an "immunologically effective amount", “anti-tumor effective amount”, “tumor-inhibitory effective amount” or "therapeutic amount”.
  • Treatment refers to a subject taking a treatment regimen described herein to achieve at least one positive therapeutic effect (for example, reduction in the number of cancer cells, reduction in tumor volume, reduction in the rate of cancer cell infiltration into surrounding organs, or reduction in tumor metastasis or tumor growth) rate decreases).
  • an "immunologically effective amount”, “anti-tumor effective amount”, “tumor-suppressive effective amount” or “therapeutic amount” is indicated, the precise amount of the composition of the invention to be administered can be determined by the physician, who takes into account the patient (subject) ) age, weight, tumor size, degree of infection or metastasis, and individual differences in disease.
  • compositions including T cells described herein may be administered at a dose of 10 4 to 10 9 cells/kg body weight, preferably 10 5 to 10 6 cells/kg body weight. T cell compositions can also be administered multiple times at these dosages. Cells can be administered using infusion techniques well known in immunotherapy (see, eg, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dosage and treatment regimen for a particular patient can be readily determined by one skilled in the medical field by monitoring the patient for signs of disease and adjusting treatment accordingly.
  • compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intraspinally, intramuscularly, by intravenous injection, or intraperitoneally.
  • the T cell composition of the invention is administered to the patient by intradermal or subcutaneous injection.
  • the T cell composition of the invention is preferably administered by intravenous injection.
  • the composition of T cells can be injected directly into the tumor, lymph node or site of infection.
  • the CAR-T cells of the invention or compositions thereof may be combined with other therapies known in the art.
  • described Therapies include, but are not limited to, chemotherapy, radiation therapy, and immunosuppressants.
  • treatment may be combined with radiotherapy or chemotherapy agents known in the art to treat CD318-mediated diseases.
  • anti-tumor effect refers to a biological effect, which can be expressed by a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in the number of metastases, an increase in life expectancy, or an improvement in various physiological symptoms related to cancer.
  • Patient “Patient,” “subject,” “individual” and the like are used interchangeably herein to refer to a living organism, such as a mammal, that can elicit an immune response. Examples include, but are not limited to, humans, dogs, cats, mice, rats, and transgenic species thereof.
  • the binding molecules of the invention can be used in assays due to their high affinity for CD318, such as binding assays to detect and/or quantify CD318 expressed in tissues or cells. Binding molecules such as single domain antibodies can be used in studies to further investigate the role of CD318 in disease.
  • the method for detecting CD318 is roughly as follows: obtain cell and/or tissue samples; detect the level of CD318 in the samples.
  • the CD318-binding molecules of the invention may be used for diagnostic purposes to detect, diagnose or monitor CD318-related diseases and/or conditions.
  • the present invention provides for the detection of the presence of CD318 in a sample using classical immunohistological methods known to those skilled in the art. Detection of CD318 can be performed in vivo or in vitro. Examples of methods suitable for detecting the presence of CD318 include ELISA, FACS, RIA, etc.
  • binding molecules such as single domain antibodies are often labeled with detectable labeling groups.
  • Suitable labeling groups include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 3H, 14C, 15N, 35S, 90Y, 99Tc, 111In, 125I, 131I), fluorescent groups (e.g., FITC, Rodan fluorophores, lanthanide phosphors), enzymatic groups (e.g., horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), chemiluminescent groups, biotinyl groups or a predetermined polypeptide epitope recognized by a secondary reporter (e.g., leucine zipper pair sequence, binding site for secondary antibody, metal binding domain, epitope tag), MRI (magnetic resonance imaging) or CT (Computed X-ray tomography) contrast agent.
  • a secondary reporter e.
  • Another aspect of the invention provides a method of detecting the presence of a test molecule that competes with an antibody of the invention for binding to CD318.
  • An example of such an assay would involve detecting the amount of free antibody in a solution containing an amount of CD318 in the presence or absence of a test molecule.
  • An increased amount of free antibody i.e., antibody that does not bind CD318, will indicate that the test molecule is able to compete with the antibody for binding to CD318.
  • the antibody is labeled with a labeling group.
  • the test molecule is labeled and the amount of free test molecule is monitored in the presence or absence of antibody.
  • the invention also provides a detection kit for detecting CD318 levels.
  • the kit includes an antibody that recognizes CD318 protein, a lysis medium for dissolving the sample, and general reagents and buffers required for detection, such as various buffers, detection Labeling, detection substrate, etc.
  • the detection kit may be an in vitro diagnostic device.
  • Example 1 Construction and eukaryotic expression of recombinant human CD318 protein expression vector
  • a one-step method was used to construct a nanobody phage display library, that is, the alpaca nanobody VHH gene was connected to the phage display vector.
  • Alpaca immunization services are provided by Chengdu Apak Biotechnology Co., Ltd.
  • the specific operation process is as follows:
  • Alpaca selection Choose alpacas that are healthy, strong, in good spirits, and of moderate size. The selected alpacas have bright wool and no symptoms of injury or discomfort. Select good animals and raise them for about a week first to eliminate some unqualified animals so that later experiments can proceed smoothly.
  • Immunization plan B Select the alpaca and ensure that the animal is suitable. Record the ear number and start the immunity experiment. A total of 4 immunizations were performed.
  • the immunization plan is as follows: D0, take 10 mL of blood before immunization, and use it as a negative serum control. Mix 0.5 mg of antigen and 1 ml of CFA and then inject it subcutaneously; D21, mix 0.25 mg of antigen and 1 ml of CFA and inject it subcutaneously; D28, take 10 ml of blood.
  • D42 mix 0.25 mg of antigen and 1 ml of CFA and then inject subcutaneously
  • D49 collect 50 mL of peripheral blood to isolate lymphocytes
  • D63 mix 0.25 mg of antigen and 1 ml of CFA and inject subcutaneously
  • D70 collect 50 mL of peripheral blood to isolate lymphocytes.
  • Extract PBMC total RNA Dissolve peripheral blood lymphocytes preserved in Trizol on ice and transfer to a 1.5 mL centrifuge tube. Add 1/5 volume of chloroform and shake to mix, let stand at room temperature for 5 minutes, then centrifuge at 4°C at 12000g for 15 minutes; Transfer the centrifuged supernatant to a new centrifuge tube, add an equal volume of isopropanol to the new centrifuge tube, let it stand at room temperature for 10 minutes, then centrifuge at 12000g for 10 minutes at 4°C, wash the precipitate with 75% ethanol, 7500g at 4°C After centrifugation for 5 minutes, discard the supernatant, dry the pellet at room temperature and dissolve it in an appropriate amount of RNase-free water. Analyze RNA extraction purity from A260/280 and prepare for RNA transcription.
  • cDNA synthesis Use the SuperScript TM IV First-Strand Synthesis System kit to reverse-transcribe cDNA and store it at -80°C.
  • VHH-F forward primer
  • CH2-R reverse primer
  • the PCR reaction conditions are as follows: pre-denature at 98°C for 45 seconds and then enter the temperature cycle. Denaturation at 98°C for 15 seconds, annealing at 58°C for 20 seconds, extension at 72°C 45 seconds, 30 cycles, final extension at 72°C for 7 minutes. After the PCR product was subjected to 1.5% agarose gel electrophoresis, a gel recovery kit (Promega) was used to recover the 750 bp target band.
  • VHH-CH2-F forward primer
  • VHJ-R reverse primer
  • the PCR reaction conditions are as follows: pre-denature at 98°C for 45 seconds and then enter the temperature cycle, 98 Denaturation at °C for 15 seconds, annealing at 60°C for 20 seconds, extension at 72°C for 45 seconds, 30 cycles, and final extension at 72°C for 7 minutes. After the PCR product was electrophoresed on a 1.5% agarose gel, a 400 bp target band was recovered using a gel recovery kit (Promega).
  • the phagemid vector pcomb3X and VHH genes were digested with SfiI DNA endonuclease for 16 hours at 50°C.
  • the digested pcomb3X vector was subjected to 1% agarose gel electrophoresis and recovered using a gel recovery kit (Promega). 4000bp vector fragment.
  • the digested VHH gene was directly purified through the column using a gel recovery kit (400 bp).
  • the VHH gene was ligated into the phagemid vector using T4 DNA ligase kit (Invitrogen), and the ligation was carried out overnight at 16°C. A small amount of the ligation product was taken for agarose gel electrophoresis to detect the ligation efficiency.
  • the ligation product is desalted using MECK MILLIPOREF microporous filter membrane.
  • the above-mentioned ligation product was added to the self-made TG1 electroconversion competent cell, and then electroporation was performed using an electroporation instrument. Take out 50 ⁇ L of bacterial solution and perform gradient dilution 10 2 -10 5 times with PBS. Streamline 10 ⁇ L of each gradient dilution on an Amp/2YT plate and incubate at 37°C overnight to count and calculate the size of the phage antibody library. Add 2YT to the remaining electrotransformed bacteria to 500 ml, add ampicillin containing 100 ⁇ g/mL, and culture at 30°C and 220 rpm overnight. Finally, a VHH immune library exceeding 9E9 was obtained. The antibody library transformed by electroporation was amplified overnight, and the library cells were collected by centrifugation. The final concentration of 20% glycerol was added and stored at -80 degrees.
  • the avi-tag of the recombinant human CD318 protein was biotin-modified to obtain biotinylated CD318 protein.
  • Example 3 Preparation of retrovirus stock solution containing anti-human CD318 chimeric antigen receptor element
  • the chimeric antigen receptor sequence containing the single-domain antibody VHH, hinge region, transmembrane region and intracellular signal segment against human CD318 antigen is genetically synthesized or cloned. Its structure is shown in Figure 2. According to the different loaded VHHs, the chimeric antigen receptors are named 4A4-BBz, 4A8-BBz, 4A10-BBz, 4B2-BBz, 4B7-BBz, 4B12-BBz, 4F5-BBz, 4G2-BBz, and 4A4-BBz.
  • amino acid sequences of , 4A8-BBz, 4A10-BBz, 4B2-BBz, 4B7-BBz, and 4G2-BBz are shown in SEQ ID NO.30-35 respectively, and the nucleotide sequences are shown in SEQ ID NO.36 to SEQ respectively. Shown as ID NO.41.
  • chimeric antigen receptors expressing 4A4-BBz, 4A8-BBz, 4A10-BBz, 4B2-BBz, 4B7-BBz, 4B12-BBz, 4F5-BBz, and 4G2-BBz clones were constructed. of retroviral plasmids. Select the correctly sequenced clone, inoculate the bacterial solution into 200ml 2YT medium, shake the culture overnight, and follow the instructions of the NucleoBondXtra Maxi EF kit to complete the plasmid purification.
  • PEI cationic polymer
  • the process is as follows: dilute 36 ⁇ L PEI and retrovirus packaging plasmid (viral main plasmid 6 ⁇ g, Gag-pol 3.8 ⁇ g, vsvg 1.5 ⁇ g) with 600 ⁇ L serum-free DMEM respectively; then PEI/DMEM Add the plasmid/DMEM mixture, vortex to mix, and let stand at room temperature for 15 minutes; add the plasmid-PEI complex to the pre-plated 293T cells.
  • PBMC Receive a PBMC, verify that the patient's individual identification number is correct, and then perform resuscitation.
  • PBMC that had been recovered overnight were pipetted gently, filtered with a 70 ⁇ m cell mesh, transferred to a 50 ml centrifuge tube, centrifuged at room temperature, 1500 rpm, for 5 min, and the supernatant was discarded.
  • Remove the magnetic stand from the flow tube take an equal volume of DPBS or X-VIVO15 and resuspend the cell suspension, add the magnetic beads and cells in the cell suspension, mix in a 15ml centrifuge tube, and incubate on a rotating mixer. Incubate at room temperature for 30 minutes. After the incubation is completed, gently transfer the cells to a sterile flow tube, rinse the 15ml centrifuge tube with 1ml DPBS, and merge the rinse solution into the same flow tube. Move the sterile flow tube to the magnetic stand, let it stand for 1 minute, and then aspirate the unadsorbed liquid.
  • CAR-T culture medium to adjust the cell density to 1 ⁇ 10 6 ml, add IL-2 to a final concentration of 200IU/ml, and culture in a 37°C, 5% CO 2 incubator for two days.
  • Adjust the activated T cells to 5 ⁇ 10 5 /mL add 1 ml of T cells and 1 ml of virus stock solution to a 24-well plate, add 2 ⁇ L polybrene to each well, and centrifuge at 32°C, 2500 rpm for 1.5 h. Discard the supernatant and add 1 ml of T cell culture medium (containing IL-2 300IU/ml) to each well. Place the culture plate in a 37°C, 5% CO2 incubator. 24 hours after infection, transfer to a 6-well plate, observe the cell density every day, and add T cell culture medium containing IL-2 300IU/ml in a timely manner to maintain the density of T cells at about 1 ⁇ 10 6 /ml and allow the cells to expand. increase.
  • the positive rate of CAR was detected in retrovirus-infected T lymphocytes 72 hours after virus infection.
  • the chimeric antigen receptor group containing 4A4, 4A8, 4A10, 4B2, 4B7, 4B12, 4F5, and 4G2 clones and the negative uninfected control group NT take 1 ⁇ 10 6 cells respectively, centrifuge to remove the culture medium, and use 500 ⁇ L PBS Wash the cells once and resuspend them in 100 ⁇ L in a flow cytometry tube (BD). Biotin-labeled CD318 antigen (1:200) was added and incubated four times for 30 minutes.
  • Example 5 Functional analysis of CAR-T cells based on anti-human CD318
  • CAR-T cells containing different antibody clones were used with target cells (CD318-positive pancreatic cancer cell line BxPC3) and control target cells (CD318-negative human glioma cell U251) at a ratio of 1:1. After a total of 4 hours of incubation in a 37°C, 5% CO 2 incubator, the proportion of cells expressing CD107a in each group of samples that were positive for CAR-T was measured by flow cytometry. Cell number ratio. Evaluate the degranulation response of CAR-T cells after stimulation by target cells. The results of flow cytometry analysis of CD107a expression are shown in Figure 4.
  • CAR-T cells containing different antibody clones were compared with target cells (CD318-positive cell line BxPC3) and control target cells (CD318-negative cell line U251) at an effect-to-target ratio of 10:1 and 2:1 respectively.
  • target cells CD318-positive cell line BxPC3
  • control target cells CD318-negative cell line U251
  • Target cells are 3 ⁇ 10 4
  • the supernatant was collected, and the secretion of IFN- ⁇ and IL-2 was detected using ELISA (enzyme-linked immunoassay) method.
  • IFN- ⁇ and IL-2 were detected using the Human IFN-gamma ELISA Kit, Human IL-2ELISA kit, and the experimental steps were performed according to the product instructions.
  • the detection results of IFN- ⁇ secretion are shown in Figure 5 and Figure 6.
  • the detection results of IL-2 secretion are shown in Figure 7 and Figure 8.
  • the CAR-T killing toxicity experiment evaluates the in vitro function of CAR-T cells by detecting the killing effect of CAR-T cells on target cells in vitro.
  • the T cells were treated with CD318-positive target cells stably expressing firefly luciferase BxPC3-LUC- at different effective-to-target ratios (based on 3 ⁇ 10 4 target cells, the effective-to-target ratios were 10:1 and 2:1 respectively).
  • GFP, and CD318 negative control target cells U251-LUC-GFP were co-cultured, and a positive control with only target cells was set up.
  • Killing efficiency (fluorescence value of positive control well - fluorescence value of experimental well)/fluorescence of positive control well value) ⁇ 100%.

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Abstract

The present invention relates to a VHH antibody that specifically binds to human CD318 or an antigen-binding fragment thereof, a preparation method therefor, and a use thereof. Provided are a CD318 binding molecule comprising an anti-CD318 nano-antibody or an antigen-binding fragment thereof. Further provided are a chimeric antigen receptor comprising the CD318 binding molecule and an expression cell thereof. The antibody and cell described in the present application have good therapeutic effects in targeting CD318.

Description

一种特异性结合人CD318的VHH抗体或其抗原结合片段及其制备方法和应用A VHH antibody that specifically binds to human CD318 or an antigen-binding fragment thereof and its preparation method and application 技术领域Technical field
本发明涉及生物免疫治疗技术领域，具体而言，涉及一种特异性结合CD318的抗体或其抗原结合片段及其制备方法和应用。The present invention relates to the technical field of biological immunotherapy. Specifically, it relates to an antibody that specifically binds to CD318 or an antigen-binding fragment thereof and its preparation method and application.
背景技术Background technique
CD318，也称为CDCP1(含CUB结构域的蛋白1)，该基因编码一种高度糖基化的，单通道I型跨膜蛋白，在间充质干细胞、神经干细胞、成纤维细胞和造血祖细胞中表达。CDCP1由一个大的细胞外结构域(ECD)组成，其中包含三个CUB(分别为：补体C1r/C1s，Uegf和Bmp1)结构域和一个短的细胞内部分，该蛋白在酪氨酸磷酸化依赖性调节中起作用。CD318, also known as CDCP1 (CUB domain-containing protein 1), encodes a highly glycosylated, single-channel type I transmembrane protein that is expressed in mesenchymal stem cells, neural stem cells, fibroblasts, and hematopoietic progenitors. expressed in cells. CDCP1 consists of a large extracellular domain (ECD) containing three CUB (respectively: complement C1r/C1s, Uegf and Bmp1) domains and a short intracellular part. The protein undergoes tyrosine phosphorylation. play a role in dependence regulation.
CD318在多种恶性肿瘤中表达均异常升高，包括结肠癌、乳腺癌、胰腺癌、肺癌、肾癌、肝癌等。在造血细胞中，CD318已被鉴定为良性和恶性祖细胞的干细胞标志物。在AML患者涉及白血病干细胞富集的CD34CD133白血病细胞亚群中可观察到明显的CD318表达。因此，CD318是一个非常有前景的肿瘤免疫治疗靶标。The expression of CD318 is abnormally elevated in a variety of malignant tumors, including colon cancer, breast cancer, pancreatic cancer, lung cancer, kidney cancer, liver cancer, etc. In hematopoietic cells, CD318 has been identified as a stem cell marker for benign and malignant progenitor cells. Significant CD318 expression was observed in a subset of CD34CD133 leukemia cells enriched in leukemia stem cells in AML patients. Therefore, CD318 is a very promising target for tumor immunotherapy.
嵌合抗原受体T细胞(Chimeric Antigen Receptor T cell，CAR T)疗法是一种针对肿瘤细胞表面特异性抗原的新型免疫治疗方法。VHH抗体又称纳米抗体，是羊驼外周血液中存在的一种天然缺失轻链的抗体，该抗体只包含一个重链可变区(VHH)和两个常规的CH2与CH3区。与传统的人工改造的scFv相比具有分子量小，易于表达，特异性强，亲和力高，对人的免疫原性弱，开发周期短等优势。结合免疫疗法和VHH抗体的优势，能够开发出高效的CAR-T疗法。Chimeric Antigen Receptor T cell (CAR T) therapy is a new immunotherapy method that targets specific antigens on the surface of tumor cells. VHH antibodies, also known as nanobodies, are a type of naturally missing light chain antibody found in the peripheral blood of alpacas. The antibody only contains one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions. Compared with traditional artificially modified scFv, it has the advantages of small molecular weight, easy expression, strong specificity, high affinity, weak immunogenicity to humans, and short development cycle. Combining the advantages of immunotherapy and VHH antibodies, highly efficient CAR-T therapy can be developed.
目前尚未见如本申请所记载的抗CD318 VHH抗体以及含该抗CD318 VHH抗体的免疫效应细胞的报道。There have been no reports of anti-CD318 VHH antibodies and immune effector cells containing the anti-CD318 VHH antibodies as described in this application.
发明内容Contents of the invention
本发明提供一种CD318结合分子，包含抗CD318纳米抗体或其抗原结合片段，所述抗CD318纳米抗体的互补决定区CDR包含CDR1、CDR2和CDR3，其中CDR1包括SEQ ID NO:1、4、7、10、13和16中任一所示的序列、CDR2包括SEQ ID NO:2、5、8、11、14和17中任一所示的序列、和CDR3包括SEQ ID NO:3、6、9、12、15和18中任一所示的序列。The invention provides a CD318-binding molecule, comprising an anti-CD318 Nanobody or an antigen-binding fragment thereof. The complementarity determining region CDR of the anti-CD318 Nanobody includes CDR1, CDR2 and CDR3, wherein CDR1 includes SEQ ID NO: 1, 4, and 7. , the sequence shown in any one of 10, 13 and 16, CDR2 includes the sequence shown in any one of SEQ ID NO: 2, 5, 8, 11, 14 and 17, and CDR3 includes SEQ ID NO: 3, 6, Sequences shown in any of 9, 12, 15 and 18.
在一个或多个实施方案中，所述CDR选自以下任一项：In one or more embodiments, the CDRs are selected from any of the following:
(1)序列如SEQ ID NO:1所示的CDR1、序列如SEQ ID NO:2所示的CDR2、序列如SEQ ID NO:3所示的CDR3，(1) CDR1 whose sequence is shown in SEQ ID NO:1, CDR2 whose sequence is shown in SEQ ID NO:2, and CDR3 whose sequence is shown in SEQ ID NO:3,
(2)序列如SEQ ID NO:4所示的CDR1、序列如SEQ ID NO:5所示的CDR2、序列如SEQ ID NO:6所示的CDR3，(2) CDR1 whose sequence is shown in SEQ ID NO:4, CDR2 whose sequence is shown in SEQ ID NO:5, and CDR3 whose sequence is shown in SEQ ID NO:6,
(3)序列如SEQ ID NO:7所示的CDR1、序列如SEQ ID NO:8所示的CDR2、序列如SEQ ID NO:9所示的CDR3，(3) CDR1 whose sequence is shown in SEQ ID NO:7, CDR2 whose sequence is shown in SEQ ID NO:8, and CDR3 whose sequence is shown in SEQ ID NO:9,
(4)序列如SEQ ID NO:10所示的CDR1、序列如SEQ ID NO:11所示的CDR2、序列如SEQ ID NO:12所示的CDR3， (4) CDR1 whose sequence is shown in SEQ ID NO:10, CDR2 whose sequence is shown in SEQ ID NO:11, and CDR3 whose sequence is shown in SEQ ID NO:12,
(5)序列如SEQ ID NO:13所示的CDR1、序列如SEQ ID NO:14所示的CDR2、序列如SEQ ID NO:15所示的CDR3，(5) CDR1 whose sequence is shown in SEQ ID NO:13, CDR2 whose sequence is shown in SEQ ID NO:14, and CDR3 whose sequence is shown in SEQ ID NO:15,
(6)序列如SEQ ID NO:16所示的CDR1、序列如SEQ ID NO:17所示的CDR2、序列如SEQ ID NO:18所示的CDR3。(6) CDR1 whose sequence is shown in SEQ ID NO:16, CDR2 whose sequence is shown in SEQ ID NO:17, and CDR3 whose sequence is shown in SEQ ID NO:18.
在一个或多个实施方案中，所述抗CD318纳米抗体的重链可变区序列如SEQ ID NO:19-24中任一所示。In one or more embodiments, the heavy chain variable region sequence of the anti-CD318 Nanobody is set forth in any one of SEQ ID NOs: 19-24.
在一个或多个实施方案中，所述抗CD318纳米抗体的FR1可选自SEQ ID NO:19-24中任一所示的VHH的FR1，FR2可选自SEQ ID NO:19-24中任一所示的VHH的FR2，FR3可选自SEQ ID NO:19-24中任一所示的VHH的FR3，FR4可选自SEQ ID NO:19-24中任一所示的VHH的FR4。In one or more embodiments, the FR1 of the anti-CD318 Nanobody can be selected from the FR1 of the VHH shown in any one of SEQ ID NO: 19-24, and the FR2 can be selected from any of SEQ ID NO: 19-24. FR2 and FR3 of the VHH shown in SEQ ID NO:19-24 can be selected from the FR3 of the VHH shown in any one of SEQ ID NO:19-24, and FR4 can be selected from the FR4 of the VHH shown in any one of SEQ ID NO:19-24.
在一个或多个实施方案中，所述CD318结合分子是包含一条、两条或多条抗CD318纳米抗体或其抗原结合片段的单价或多价纳米抗体或单域抗体、或多特异性纳米抗体或单域抗体。In one or more embodiments, the CD318 binding molecule is a monovalent or multivalent Nanobody or single domain antibody, or a multispecific Nanobody comprising one, two or more anti-CD318 Nanobodies or antigen-binding fragments thereof or single domain antibodies.
在一个或多个实施方案中，所述多价结合分子或多特异性结合分子通过连接子连接多个抗CD318纳米抗体或其抗原结合片段。所述连接子由选自G和S的1-15个氨基酸组成。In one or more embodiments, the multivalent binding molecule or multispecific binding molecule is linked to multiple anti-CD318 Nanobodies or antigen-binding fragments thereof via a linker. The linker consists of 1-15 amino acids selected from G and S.
在一个或多个实施方案中，所述纳米抗体是骆驼重链抗体或软骨鱼重链抗体。In one or more embodiments, the Nanobody is a camel heavy chain antibody or a cartilaginous fish heavy chain antibody.
在一个或多个实施方案中，所述纳米抗体还包含重链恒定区。In one or more embodiments, the Nanobody further comprises a heavy chain constant region.
在一个或多个实施方案中，所述重链恒定区是骆驼重链抗体的恒定区，包含CH2和CH3。In one or more embodiments, the heavy chain constant region is that of a camel heavy chain antibody, comprising CH2 and CH3.
在一个或多个实施方案中，所述CH2和CH3是人IgG Fc的CH2和CH3，例如IgG1的CH2和CH3。In one or more embodiments, the CH2 and CH3 are those of a human IgG Fc, such as those of an IgG1.
在一个或多个实施方案中，所述重链恒定区是软骨鱼重链抗体的恒定区，包含CH1、CH2、CH3、CH4和CH5。In one or more embodiments, the heavy chain constant region is that of a cartilaginous fish heavy chain antibody, comprising CH1, CH2, CH3, CH4, and CH5.
在一个或多个实施方案中，本发明任一实施方案所述的CD318结合分子为嵌合抗体或完全人抗体；优选为完全人抗体。In one or more embodiments, the CD318-binding molecule according to any embodiment of the present invention is a chimeric antibody or a fully human antibody; preferably, it is a fully human antibody.
本发明另一方面提供一种嵌合抗原受体，包含任选的信号肽序列、本文任一实施方案所述的CD318结合分子、铰链区、跨膜区和胞内区。Another aspect of the invention provides a chimeric antigen receptor comprising an optional signal peptide sequence, a CD318 binding molecule according to any embodiment herein, a hinge region, a transmembrane region and an intracellular region.
在一个或多个实施方案中，胞内区包括胞内共刺激域和/或胞内信号域。In one or more embodiments, the intracellular domain includes an intracellular costimulatory domain and/or an intracellular signaling domain.
在一个或多个实施方案中，从N端到C端，该嵌合抗原受体依次含有信号肽、本文任一实施方案所述的CD318结合分子、铰链区、跨膜区、胞内共刺激域和胞内信号域。In one or more embodiments, from the N-terminus to the C-terminus, the chimeric antigen receptor sequentially contains a signal peptide, the CD318-binding molecule described in any embodiment herein, a hinge region, a transmembrane region, and intracellular costimulation. domain and intracellular signaling domain.
本发明还提供核酸分子，其具有选自以下任一项的序列：The invention also provides nucleic acid molecules having a sequence selected from any of the following:
(1)本文任一实施方案所述CD318结合分子或嵌合抗原受体的编码序列；(1) The coding sequence of the CD318 binding molecule or chimeric antigen receptor described in any embodiment of this article;
(2)(1)的互补序列；(2) The complementary sequence of (1);
(3)(1)或(2)中任一序列的5-50bp的片段。(3) A 5-50 bp fragment of any sequence in (1) or (2).
在一个或多个实施方案中，所述片段是引物。In one or more embodiments, the fragment is a primer.
本发明还提供一种核酸构建物，包含本文所述的核酸分子。The invention also provides a nucleic acid construct comprising a nucleic acid molecule described herein.
在一个或多个实施方案中，所述核酸构建物是克隆载体、表达载体或整合载体。In one or more embodiments, the nucleic acid construct is a cloning vector, an expression vector, or an integration vector.
本发明还提供一种宿主细胞，选自： The invention also provides a host cell selected from:
(1)表达和/或分泌本文任一实施方案所述CD318结合分子或嵌合抗原受体；(1) Express and/or secrete the CD318 binding molecule or chimeric antigen receptor described in any embodiment herein;
(2)包含本文所述的核酸分子；和/或(2) Contains a nucleic acid molecule described herein; and/or
(3)包含本文所述的核酸构建物。(3) Comprising a nucleic acid construct described herein.
在一个或多个实施方案中，所述宿主细胞是免疫效应细胞，优选T细胞。In one or more embodiments, the host cells are immune effector cells, preferably T cells.
本发明还提供一种产生本文任一实施方案CD318结合分子的方法，包括：在适合产生CD318结合分子(例如纳米抗体或其抗原结合片段，单价或多价纳米抗体或单域抗体、或多特异性纳米抗体或单域抗体)的条件下培养本文所述的宿主细胞，和任选的从培养物中纯化所述CD318结合分子。The invention also provides a method for producing a CD318-binding molecule according to any embodiment herein, comprising: producing a CD318-binding molecule (e.g., a Nanobody or an antigen-binding fragment thereof, a monovalent or multivalent Nanobody or a single domain antibody, or a multispecific The host cells described herein are cultured under the conditions of specific Nanobodies or single domain antibodies), and the CD318 binding molecules are optionally purified from the culture.
本发明还提供一种药物组合物，包含本文任一实施方案所述CD318结合分子、核酸分子、核酸构建物或宿主细胞，和药学上可接受的辅料。The present invention also provides a pharmaceutical composition, comprising the CD318 binding molecule, nucleic acid molecule, nucleic acid construct or host cell described in any embodiment herein, and pharmaceutically acceptable excipients.
在一个或多个实施方案中，所述药物组合物用于治疗CD318表达相关的疾病或病况。In one or more embodiments, the pharmaceutical composition is used to treat a disease or condition associated with CD318 expression.
本发明还提供本文任一实施方案所述CD318结合分子、嵌合抗原受体、核酸分子、核酸构建物或宿主细胞在制备活化的免疫细胞(例如T细胞)中的用途。The present invention also provides the use of the CD318 binding molecule, chimeric antigen receptor, nucleic acid molecule, nucleic acid construct or host cell of any embodiment herein for producing activated immune cells (eg, T cells).
本发明还提供本文任一实施方案所述CD318结合分子、嵌合抗原受体、核酸分子、核酸构建物或宿主细胞在制备用于预防或治疗CD318表达相关的疾病或病况的药物中的用途。The present invention also provides the use of the CD318 binding molecule, chimeric antigen receptor, nucleic acid molecule, nucleic acid construct or host cell according to any embodiment herein in the preparation of a medicament for preventing or treating diseases or conditions related to CD318 expression.
在一个或多个实施方案中，所述疾病或病况选自以下的一种或多种：乳腺癌、肺癌、肝癌、胰腺癌、卵巢癌、肾癌和结直肠癌。In one or more embodiments, the disease or condition is selected from one or more of the following: breast cancer, lung cancer, liver cancer, pancreatic cancer, ovarian cancer, kidney cancer, and colorectal cancer.
本发明还提供一种治疗或预防CD318表达相关的疾病或病况的方法，所述方法包括给予需要的患者治疗有效量的本发明任一实施方案所述的CD318结合分子或宿主细胞，或本发明任一实施方案所述的药物组合物。The present invention also provides a method for treating or preventing diseases or conditions related to CD318 expression, which method includes administering to a patient in need a therapeutically effective amount of the CD318-binding molecule or host cell according to any embodiment of the present invention, or the present invention. The pharmaceutical composition of any embodiment.
本发明还提供一种检测CD318的试剂盒，用于例如评估药物治疗效果或诊断癌症，所述的试剂盒包含本文任一实施方案所述CD318结合分子、核酸分子、核酸构建物或宿主细胞。The present invention also provides a kit for detecting CD318, which is used, for example, to evaluate drug treatment effects or diagnose cancer. The kit includes the CD318-binding molecule, nucleic acid molecule, nucleic acid construct or host cell described in any embodiment of this document.
在一个或多个实施方案中，所述试剂盒还包括用于检测CD318与所述CD318结合分子的结合的试剂。例如通过酶联免疫反应法检测所述结合的试剂。In one or more embodiments, the kit further includes a reagent for detecting binding of CD318 to the CD318 binding molecule. The bound reagent is detected, for example, by enzyme-linked immunoassay.
在一个或多个实施方案中，所述检测结合的试剂是能与CD318结合分子连接的可检测标记物，例如生物素。所述的可检测标记物被连接于所述CD318结合分子或分离地存在于试剂盒中。In one or more embodiments, the reagent that detects binding is a detectable label, such as biotin, that can be linked to a CD318 binding molecule. The detectable label is connected to the CD318 binding molecule or is present separately in the kit.
本发明还提供一种检测样品中CD318存在情况的非诊断性方法，所述方法包括：以本文任一实施方案所述CD318结合分子与样品孵育，和检测CD318与所述CD318结合分子的结合，从而确定样品中CD318存在情况。所述检测是酶联免疫反应法检测。The present invention also provides a non-diagnostic method for detecting the presence of CD318 in a sample. The method includes: incubating the sample with the CD318-binding molecule described in any embodiment of this document, and detecting the binding of CD318 to the CD318-binding molecule, This determines the presence of CD318 in the sample. The detection is an enzyme-linked immunoreaction assay.
本发明还提供本文任一实施方案所述CD318结合分子在制备用于检测样品中CD318、评估药物治疗效果或诊断癌症的试剂盒中的用途。The present invention also provides the use of the CD318-binding molecule described in any embodiment herein in the preparation of a kit for detecting CD318 in a sample, evaluating the effect of drug treatment, or diagnosing cancer.
本发明具有以下有益效果： The invention has the following beneficial effects:
本发明提供了一种新的特异性识别CD318的纳米抗体以及含有该抗体的CAR修饰细胞，该抗体和细胞具有良好的靶向CD318的治疗效果，为与CD318表达相关的疾病提供了新的治疗或改善途径。The present invention provides a new Nanobody that specifically recognizes CD318 and CAR-modified cells containing the antibody. The antibody and cells have good therapeutic effects targeting CD318 and provide new treatments for diseases related to CD318 expression. or ways to improve.
附图说明Description of the drawings
为了更清楚地说明本发明实施例的技术方案，下面将对实施例中所需要使用的附图作简单地介绍，应当理解，以下附图仅示出了本发明的某些实施例，因此不应被看作是对范围的限定，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他相关的附图。In order to explain the technical solutions of the embodiments of the present invention more clearly, the drawings required to be used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and therefore do not It should be regarded as a limitation of the scope. For those of ordinary skill in the art, other relevant drawings can be obtained based on these drawings without exerting creative efforts.
图1为重组人CD318-avi-his抗原蛋白的SDS电泳图。Figure 1 shows the SDS electrophoresis pattern of recombinant human CD318-avi-his antigen protein.
图2为不同克隆CD318 CAR的示意图。Figure 2 is a schematic diagram of different cloned CD318 CARs.
图3为不同克隆CD318 CAR-T细胞的CAR表达阳性率。Figure 3 shows the CAR expression positive rate of CD318 CAR-T cells of different clones.
图4为不同克隆CD318 CAR-T细胞的CD107a表达。Figure 4 shows the expression of CD107a in CD318 CAR-T cells of different clones.
图5为效靶比10:1时不同克隆CD318 CAR-T细胞的INFγ分泌。Figure 5 shows the INFγ secretion of CD318 CAR-T cells of different clones when the effect-to-target ratio is 10:1.
图6为效靶比2:1时不同克隆CD318 CAR-T细胞的INFγ分泌。Figure 6 shows the INFγ secretion of CD318 CAR-T cells of different clones when the effect-to-target ratio is 2:1.
图7为效靶比10:1时不同克隆CD318 CAR-T细胞的IL-2分泌。Figure 7 shows the IL-2 secretion of CD318 CAR-T cells of different clones when the effect-to-target ratio is 10:1.
图8为效靶比2:1时不同克隆CD318 CAR-T细胞的IL-2分泌。Figure 8 shows the IL-2 secretion of CD318 CAR-T cells of different clones when the effect-to-target ratio is 2:1.
图9为不同克隆CD318 CAR-T细胞对靶细胞的杀伤实验结果。Figure 9 shows the results of the killing experiment of different clones of CD318 CAR-T cells on target cells.
图10为不同克隆CD318 CAR-T细胞对靶细胞的杀伤实验结果。Figure 10 shows the results of the killing experiment of different clones of CD318 CAR-T cells on target cells.
具体实施方式Detailed ways
本发明人经过广泛而深入地研究，经过大量的筛选，发现一类抗CD318纳米抗体及其抗原结合片段，其能够特异性识别CD318，以高亲和力与CD318结合，具有良好的功能活性。After extensive and in-depth research and extensive screening, the inventors discovered a class of anti-CD318 nanobodies and their antigen-binding fragments, which can specifically recognize CD318, bind to CD318 with high affinity, and have good functional activity.
具体地，本发明利用CD318蛋白免疫羊驼，获得高质量的单域抗体基因文库。然后利用噬菌体展示技术筛选抗体基因库，从而获得了CD318特异性的单域抗体基因。再将此基因转至哺乳动物细胞中，从而获得了能在哺乳动物细胞中高效表达的、且特异性高的抗体株。所述抗体或其抗原结合片段具有良好的安全性和靶向性，能够特异性结合人CD318的胞外域。Specifically, the present invention uses CD318 protein to immunize alpacas to obtain a high-quality single domain antibody gene library. Phage display technology was then used to screen the antibody gene library, thereby obtaining a CD318-specific single domain antibody gene. This gene is then transferred into mammalian cells, thereby obtaining an antibody strain with high specificity that can be expressed efficiently in mammalian cells. The antibody or its antigen-binding fragment has good safety and targeting properties, and can specifically bind to the extracellular domain of human CD318.
本发明还提供含有所述纳米抗体的嵌合抗原受体(CAR)。将包含该CAR的编码序列的载体用于感染免疫细胞，能够获得对过表达CD318的肿瘤细胞具有显著杀伤能力的免疫效应细胞，该免疫效应细胞能够应用于治疗或改善CD318表达相关的疾病，从而为CD318阳性肿瘤的治疗奠定了基础。The present invention also provides chimeric antigen receptors (CARs) containing the Nanobodies. By using a vector containing the coding sequence of the CAR to infect immune cells, immune effector cells with significant killing ability against tumor cells overexpressing CD318 can be obtained. The immune effector cells can be used to treat or improve diseases related to CD318 expression, thereby This lays the foundation for the treatment of CD318-positive tumors.
抗体Antibody
本文中，“CD318结合分子”是特异性结合CD318的蛋白质，包括但不仅限于，抗体、重链抗体、纳米抗体或它们的抗原结合片段。As used herein, "CD318-binding molecules" are proteins that specifically bind to CD318, including but not limited to antibodies, heavy chain antibodies, Nanobodies or their antigen-binding fragments.
本文中，术语“抗体”包括单克隆抗体(包括全长抗体，其具有免疫球蛋白Fc区)，具有多表位特异性的抗体组合物，多特异性抗体(例如，双特异性抗体)，双抗体和单链分子，以及抗体片段，尤其是抗原结合片段，例如，Fab，F(ab’)2，Fd和Fv。本文中，“抗体”与“免疫球蛋白”可互换使用。As used herein, the term "antibody" includes monoclonal antibodies (including full-length antibodies having an immunoglobulin Fc region), antibody compositions with multiple epitope specificities, multispecific antibodies (e.g., bispecific antibodies), diabodies and single-chain molecules, as well as antibody fragments, especially antigen-binding fragments, such as Fab, F(ab')2, Fd and Fv. Herein, "antibody" and "immunoglobulin" are used interchangeably.
传统的“抗体”含有基本的4链抗体单元，是由两条相同的轻链(L)和两条相同的重链(H)构成的异四聚体糖蛋白。每条重链在N-末端具有可变结构域(VH)，接着是三个(对于每种α和γ链，CH1、CH2 和CH3)和四个(对于μ和ε同种型，CH1、CH2、CH3和CH4)恒定结构域(CH)以及位于CH1结构域与CH2结构域之间的绞链区(Hinge)。每条轻链在N-末端具有可变结构域(VL)，接着是其另一端的恒定结构域(CL)。成对的VH和VL一起形成一个抗原结合位点。关于不同类别抗体的结构和性质，参见如Basic and Clinical Immunology，第八版，Daniel P.Sties，Abba I.Terr和Tristram G.Parsolw编辑，Appleton&Lange，Norwalk，CT，1994，第71页和第6章。来自任何脊椎动物物种的轻链，根据其恒定结构域氨基酸序列，可归入两种称作κ和λ的截然不同型中的一种。根据CH序列和功能的相对较小差异，γ和α类可进一步分为亚类，例如人表达下列亚类：IgG1、IgG2A、IgG2B、IgG3、IgG4、IgA1和IgA2。Traditional "antibodies" contain the basic 4-chain antibody unit, which is a heterotetrameric glycoprotein composed of two identical light chains (L) and two identical heavy chains (H). Each heavy chain has a variable domain (VH) at the N-terminus, followed by three (for each alpha and gamma chain, CH1, CH2 and CH3) and four (for the μ and epsilon isoforms, CH1, CH2, CH3 and CH4) constant domains (CH) and a hinge region (Hinge) located between the CH1 and CH2 domains. Each light chain has a variable domain (VL) at the N-terminus, followed by a constant domain (CL) at its other end. Pairs of VH and VL together form an antigen-binding site. On the structure and properties of different classes of antibodies, see, for example, Basic and Clinical Immunology, 8th edition, edited by Daniel P. Sties, Abba I. Terr, and Tristram G. Parsolw, Appleton & Lange, Norwalk, CT, 1994, pp. 71 and 6 chapter. Light chains from any vertebrate species can be assigned to one of two distinct types called kappa and lambda, based on their constant domain amino acid sequence. The gamma and alpha classes can be further divided into subclasses based on relatively small differences in CH sequence and function, for example humans express the following subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1 and IgA2.
本文所述“重链抗体”是源自骆驼科生物或软骨鱼科生物的抗体。相比上述4链抗体，重链抗体缺失轻链和重链恒定区1(CH1)，仅包含2条由可变区(VHH)和其他恒定区组成重链，可变区通过类似铰链区结构与恒定区相连。骆驼科重链抗体的每条重链包含1个可变区(VHH)和2个恒定区(CH2和CH3)，软骨鱼科重链抗体的每条重链含有1个可变区和5个恒定区(CH1-CH5)。重链抗体的抗原结合片段包括VHH和单链重链抗体。通过与人IgG Fc的恒定区融合，重链抗体可以具有人IgG Fc的CH2和CH3。"Heavy chain antibodies" as used herein are antibodies derived from camelids or cartilaginous fishes. Compared with the above 4-chain antibodies, the heavy chain antibody lacks the light chain and heavy chain constant region 1 (CH1), and only contains 2 heavy chains composed of variable regions (VHH) and other constant regions. The variable region has a hinge-like structure. connected to the constant region. Each heavy chain of camelid heavy chain antibodies contains 1 variable region (VHH) and 2 constant regions (CH2 and CH3), and each heavy chain of chondrichthyan heavy chain antibodies contains 1 variable region and 5 constant regions. Constant region (CH1-CH5). Antigen-binding fragments of heavy chain antibodies include VHH and single-chain heavy chain antibodies. The heavy chain antibody can have CH2 and CH3 of human IgG Fc by fusion to the constant region of human IgG Fc.
如本文所用，术语“单域抗体”、“抗CD318单域抗体”、“重链抗体的重链可变区结构域”、“VHH”可互换使用，均指特异性识别和结合于CD318的单域抗体。单域抗体是重链抗体的可变区。通常，单域抗体含有三个CDR和四个FR。优选地，本发明的单域抗体具有SEQ ID NO:1所示的CDR1、SEQ ID NO:2所示的CDR2、和SEQ ID NO:3所示的CDR3；或具有SEQ ID NO:4所示的CDR1、SEQ ID NO:5所示的CDR2、和SEQ ID NO:6所示的CDR3；或具有SEQ ID NO:7所示的CDR1、SEQ ID NO:8所示的CDR2、和SEQ ID NO:9所示的CDR3；或具有SEQ ID NO:10所示的CDR1、SEQ ID NO:11所示的CDR2、和SEQ ID NO:12所示的CDR3；或具有SEQ ID NO:13所示的CDR1、SEQ ID NO:14所示的CDR2、和SEQ ID NO:15所示的CDR3；或具有SEQ ID NO:16所示的CDR1、SEQ ID NO:17所示的CDR2、和SEQ ID NO:18所示的CDR3。单域抗体是最小的功能性抗原结合片段。通常先获得天然缺失轻链和重链恒定区1(CH1)的抗体后，再克隆抗体重链的可变区，构建仅由一个重链可变区组成的单域抗体。As used herein, the terms "single domain antibody", "anti-CD318 single domain antibody", "heavy chain variable region domain of a heavy chain antibody" and "VHH" are used interchangeably and all refer to specific recognition and binding to CD318 of single domain antibodies. Single domain antibodies are the variable regions of heavy chain antibodies. Typically, single domain antibodies contain three CDRs and four FRs. Preferably, the single domain antibody of the present invention has CDR1 shown in SEQ ID NO:1, CDR2 shown in SEQ ID NO:2, and CDR3 shown in SEQ ID NO:3; or has CDR3 shown in SEQ ID NO:4 CDR1, CDR2 shown in SEQ ID NO:5, and CDR3 shown in SEQ ID NO:6; or having CDR1 shown in SEQ ID NO:7, CDR2 shown in SEQ ID NO:8, and SEQ ID NO : CDR3 shown in SEQ ID NO: 9; or having CDR1 shown in SEQ ID NO: 10, CDR2 shown in SEQ ID NO: 11, and CDR3 shown in SEQ ID NO: 12; or having CDR3 shown in SEQ ID NO: 13 CDR1, CDR2 represented by SEQ ID NO:14, and CDR3 represented by SEQ ID NO:15; or having CDR1 represented by SEQ ID NO:16, CDR2 represented by SEQ ID NO:17, and SEQ ID NO: CDR3 shown in 18. Single domain antibodies are the smallest functional antigen-binding fragments. Usually, after obtaining an antibody that naturally lacks the light chain and heavy chain constant region 1 (CH1), the variable region of the antibody heavy chain is cloned to construct a single-domain antibody consisting of only one heavy chain variable region.
本文中，“纳米抗体”是指含有本文所述VHH的抗体。其可以是如上所述的重链抗体，还可以是含有多条VHH的多价或多特异性抗体，也可以是将VHH和抗体Fc(例如CH2和CH3或CH2、CH3和CH4)重组获得的重组抗体。包含两条或多条单域抗体的结合分子是多价单域抗体；包含两条或多条不同特异性单域抗体的结合分子是多特异性单域抗体。多价单域抗体或多特异性单域抗体通过连接子连接多个单域抗体。所述连接子通常由选自G和S的1-15个氨基酸组成。As used herein, "Nanobody" refers to an antibody containing a VHH as described herein. It can be a heavy chain antibody as described above, or a multivalent or multispecific antibody containing multiple VHHs, or it can be obtained by recombining VHH and antibody Fc (such as CH2 and CH3 or CH2, CH3 and CH4) Recombinant antibodies. Binding molecules containing two or more single domain antibodies are multivalent single domain antibodies; binding molecules containing two or more single domain antibodies with different specificities are multispecific single domain antibodies. Multivalent single domain antibodies or multispecific single domain antibodies connect multiple single domain antibodies via a linker. The linker usually consists of 1-15 amino acids selected from G and S.
本文中，重链抗体和抗体(传统四链抗体)旨在区分抗体的不同组合方式。由于二者的结构具有相似性，下述针对抗体的结构描述除涉及轻链外也均适用于重链抗体。In this article, heavy chain antibodies and antibodies (traditional four-chain antibodies) are intended to distinguish different combinations of antibodies. Due to the structural similarity between the two, the following structural descriptions of antibodies are applicable to heavy chain antibodies in addition to the light chain.
抗体的“可变区”或“可变结构域”是指抗体的重链或轻链的氨基末端结构域。重链和轻链的可变结构域可分别称为“VH”和“VL”。这些结构域通常是抗体的最可变的部分(相对于相同类型的其它抗体)并含有抗原结合位点。The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. The variable domains of the heavy and light chains may be referred to as "VH" and "VL" respectively. These domains are typically the most variable parts of the antibody (relative to other antibodies of the same type) and contain the antigen-binding site.
术语“可变的”指可变结构域中的某些区段在抗体序列中差异广泛的情况。可变结构域介导抗原结合并限定特定抗体对其特定抗原的特异性。然而，变异性并非均匀分布于可变结构域跨越的全部氨基酸。相反，其集中在三个称为高变区(HVR)的区段(在轻链和重链可变结构域中均有)，即分别为重链可变区的HCDR1、HCDR2、HCDR3(重链抗体中可简称为CDR1、CDR2、CDR3)以及轻链可变区的LCDR1、 LCDR2和LCDR3。可变结构域中更为高度保守的部分称为骨架区(FR)。天然重链和轻链的可变结构域各自包含四个FR区(FR1、FR2、FR3和FR4)，它们大多采取β-折叠构象，通过形成环状连接且在有些情况中形成β-折叠结构一部分的三个HVR连接。每条链中的HVR通过FR区非常接近的保持在一起，并与另一条链的HVR一起促成抗体的抗原结合位点的形成。通常，轻链可变区的结构为FR1-LCDR1-FR2-LCDR2-FR3-LCDR3-FR4，重链可变区的结构为FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4。恒定结构域不直接参与抗体与抗原的结合，但展现出多种效应子功能，如在抗体依赖性细胞介导的细胞毒性中抗体的参与。抗体的可变区有多种标注方案，包括：Chothia、Kabat、IMGT和Contact。本文示例性使用IMGT标注方案。The term "variable" refers to the situation where certain segments of the variable domain vary widely among antibody sequences. Variable domains mediate antigen binding and define the specificity of a particular antibody for its particular antigen. However, variability is not evenly distributed across all amino acids spanned by the variable domain. Instead, it is concentrated in three segments called hypervariable regions (HVRs) (found in both light and heavy chain variable domains), namely HCDR1, HCDR2, and HCDR3 (heavy chain variable domains), respectively. chain antibodies (can be abbreviated as CDR1, CDR2, CDR3) and LCDR1, LCDR2 and LCDR3. The more highly conserved part of the variable domain is called the framework region (FR). The variable domains of the native heavy and light chains each contain four FR regions (FR1, FR2, FR3, and FR4), which mostly adopt a β-sheet conformation by forming loop connections and in some cases forming a β-sheet structure Part of the three HVR connections. The HVRs in each chain are held in close proximity by the FR region and together with the HVRs of the other chain contribute to the formation of the antibody's antigen-binding site. Generally, the structure of the light chain variable region is FR1-LCDR1-FR2-LCDR2-FR3-LCDR3-FR4, and the structure of the heavy chain variable region is FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4. The constant domain is not directly involved in binding of the antibody to the antigen, but exhibits a variety of effector functions, such as the involvement of the antibody in antibody-dependent cell-mediated cytotoxicity. There are various annotation schemes for the variable regions of antibodies, including: Chothia, Kabat, IMGT, and Contact. This article uses the IMGT annotation scheme as an example.
“Fc区”(可结晶片段区域)或“Fc结构域”或“Fc”是指抗体重链的C-末端区域，其介导免疫球蛋白与宿主组织或因子的结合，包括与位于免疫***的各种细胞(例如，效应细胞)上的Fc受体的结合，或者与经典补体***的第一组分(C1q)的结合。在IgG，IgA和IgD抗体同种型中，Fc区由来自抗体两条重链的CH2结构域和CH3结构域的两个相同的蛋白片段构成；IgM和IgE的Fc区在每个多肽链中包含三个重链恒定结构域(CH结构域2-4)。虽然免疫球蛋白重链的Fc区的边界可以变化，但是人IgG重链Fc区通常定义为从重链位置C226或P230的氨基酸残基到羧基端的序列段，其中该编号是根据EU索引，如在Kabat中一样。如本文所使用的，Fc区可以是天然序列Fc或变体Fc。"Fc region" (crystallizable fragment region) or "Fc domain" or "Fc" refers to the C-terminal region of an antibody heavy chain that mediates binding of immunoglobulins to host tissues or factors, including those located in the immune system Binding to Fc receptors on various cells (e.g., effector cells) or to the first component (C1q) of the classical complement system. In the IgG, IgA, and IgD antibody isotypes, the Fc region is composed of two identical protein fragments from the CH2 and CH3 domains of the two heavy chains of the antibody; the Fc regions of IgM and IgE are present in each polypeptide chain. Contains three heavy chain constant domains (CH domains 2-4). Although the boundaries of the Fc region of an immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is generally defined as the sequence segment from the amino acid residue at position C226 or P230 of the heavy chain to the carboxyl terminus, where this numbering is according to the EU index, as in Same in Kabat. As used herein, an Fc region may be a native sequence Fc or a variant Fc.
“抗体片段”包含完整抗体的一部分，优选完整抗体的抗原结合区和/或可变区。抗体片段优选为抗体的抗原结合片段。抗体片段的例子包括Fab、Fab’、F(ab’)、F(ab’)2、Fd、和Fv片段、二硫键连接的Fv；双抗体；线性抗体；单链抗体分子；scFv-Fc片段；由抗体片段形成的多特异性抗体；以及通过化学修饰或通过掺入脂质体中应能够增加半衰期的任何片段。抗原结合片段可以通过多种技术制备，包括但不限于将完整的抗体蛋白水解消化，以及由包含抗原结合片段的宿主细胞表达产生。"Antibody fragment" includes a portion of an intact antibody, preferably the antigen-binding region and/or variable region of an intact antibody. The antibody fragment is preferably an antigen-binding fragment of an antibody. Examples of antibody fragments include Fab, Fab', F(ab'), F(ab')2, Fd, and Fv fragments, disulfide-linked Fv; diabodies; linear antibodies; single chain antibody molecules; scFv-Fc fragments; multispecific antibodies formed from antibody fragments; and any fragment that should be capable of increasing half-life, either by chemical modification or by incorporation into liposomes. Antigen-binding fragments can be produced by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies, and expression of host cells containing the antigen-binding fragments.
“Fv”是含有完整抗原识别和结合位点的最小抗体片段。该片段由紧密、非共价结合的一个重链可变结构域和一个轻链可变结构域的二聚体组成。从这两个结构域的折叠中突出了六个高变环(重链和轻链各3个环)，贡献出抗原结合的氨基酸残基并赋予抗体以抗原结合特异性。然而，即使是单个可变结构域(或只包含对抗原特异的三个HVR的半个Fv)也具有识别和结合抗原的能力，尽管亲合力低于完整结合位点。“单链Fv”也可缩写为“sFv”或“scFv”，是包含抗体VH和VL结构域的连接成一条多肽链的抗体片段。优选的是，sFv多肽在VH和VL结构域之间还包含多肽接头，使得sFv形成期望的抗原结合结构。对于重链抗体或纳米抗体而言，scFv即为VHH。"Fv" is the smallest antibody fragment containing intact antigen recognition and binding sites. This fragment consists of a dimer of a heavy chain variable domain and a light chain variable domain in tight, non-covalent association. Six hypervariable loops (3 loops each in the heavy and light chains) protrude from the fold of these two domains, contributing amino acid residues for antigen binding and conferring antigen-binding specificity to the antibody. However, even a single variable domain (or half an Fv containing only three HVRs specific for the antigen) has the ability to recognize and bind antigen, albeit with lower affinity than the full binding site. "Single-chain Fv", also abbreviated as "sFv" or "scFv", is an antibody fragment containing the VH and VL domains of an antibody linked into a polypeptide chain. Preferably, the sFv polypeptide also contains a polypeptide linker between the VH and VL domains so that the sFv forms the desired antigen-binding structure. For heavy chain antibodies or Nanobodies, the scFv is the VHH.
本文中，术语“单克隆抗体”指从一群基本上同质的抗体中获得的抗体，即除了可能以少量存在的可能的天然出现的突变和/或翻译后修饰(例如异构化、酰胺化)之外，构成群体的各个抗体是相同的。单克隆抗体是高度特异性的，针对单个抗原位点。与多克隆抗体制剂(其典型地包括针对不同决定簇(表位)的不同抗体)相比，每个单克隆抗体针对抗原上的单个决定簇。除它们的特异性外，单克隆抗体的优势在于它们通过杂交瘤培养合成，未受到其它免疫球蛋白的污染。修饰语“单克隆”表明抗体从基本上同质的抗体群获得的特征，不应解释为要求通过任何特定方法来生产抗体。例如，将根据本发明使用的单克隆抗体可通过多种技术来生成，包括例如杂交瘤法、噬菌体展示法、重组DNA法、及用于从具有部分或整个人免疫球蛋白基因座或编码人免疫球蛋白序列的基因的动物生成人或人样抗体的技术、单细胞测序法。As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of antibodies that are essentially homogeneous, i.e., except for possible naturally occurring mutations and/or post-translational modifications that may be present in small amounts (e.g. isomerization, amidation ), the individual antibodies that make up the population are identical. Monoclonal antibodies are highly specific and target a single antigenic site. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, the advantage of monoclonal antibodies is that they are synthesized by hybridoma culture and are not contaminated by other immunoglobulins. The modifier "monoclonal" indicates that the antibody is derived from a substantially homogeneous population of antibodies and should not be construed as requiring that the antibody be produced by any particular method. For example, monoclonal antibodies to be used in accordance with the present invention can be produced by a variety of techniques, including, for example, hybridoma methods, phage display methods, recombinant DNA methods, and methods for producing antibodies containing part or all of a human immunoglobulin locus or encoding a human immunoglobulin locus. Techniques and single-cell sequencing methods for producing human or human-like antibodies from animals using immunoglobulin sequence genes.
单克隆抗体在本文中也包括“嵌合”抗体，其中重链和/或轻链的一部分与衍生自特定物种或属于特定 抗体类别或亚类的抗体中的相应序列相同或同源，而链的剩余部分与衍生自另一物种或属于另一抗体类别或亚类的抗体中的相应序列相同或同源，以及此类抗体的片段，只要它们展现出期望的生物学活性。Monoclonal antibodies are also included herein as "chimeric" antibodies in which a portion of the heavy chain and/or light chain is derived from a specific species or belongs to a specific The corresponding sequence in an antibody of an antibody class or subclass is identical or homologous, and the remainder of the chain is identical or homologous to a corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, and such Fragments of antibodies as long as they exhibit the desired biological activity.
非人(例如鼠)抗体的“人源化”形式指最低限度包含衍生自非人免疫球蛋白的序列的嵌合抗体。因此，“人源化抗体”通常指可变结构域构架区与在人抗体中发现的序列交换的非人抗体。通常在人源化抗体中，整个抗体(除CDR以外)由人来源的多核苷酸编码或与这种抗体相同(除CDR以外)。CDR(其中一些或全部由源自非人生物体的核酸编码)被移植到人抗体可变区的β-折叠骨架中以产生抗体，其特异性由被移植的CDR来决定。这类抗体的产生方法本领域周知，例如使用具有基因工程免疫***的小鼠而产生。本发明中，抗体、单域抗体、重链抗体等均包括各所述抗体的经人源化的变体。"Humanized" forms of non-human (eg, murine) antibodies refer to chimeric antibodies that contain minimally sequence derived from a non-human immunoglobulin. Thus, a "humanized antibody" generally refers to a non-human antibody in which the variable domain framework regions are exchanged with sequences found in human antibodies. Typically in a humanized antibody, the entire antibody (except for the CDRs) is encoded by a polynucleotide of human origin or is identical to the antibody (except for the CDRs). CDRs, some or all of which are encoded by nucleic acids derived from non-human organisms, are grafted into the beta-sheet backbone of human antibody variable regions to produce antibodies whose specificity is determined by the grafted CDRs. Methods for producing such antibodies are well known in the art, for example using mice with genetically engineered immune systems. In the present invention, antibodies, single domain antibodies, heavy chain antibodies, etc. all include humanized variants of each of the antibodies.
“人抗体”指这样的抗体，其具有与由人生成的抗体的氨基酸序列对应的氨基酸序列和/或使用本文所公开的用于生成人抗体的任何技术产生。人抗体的这种定义明确排除包含非人抗原结合残基的人源化抗体。人抗体可使用本领域已知的多种技术来生成，包括噬菌体展示文库。"Human antibody" refers to an antibody that has an amino acid sequence corresponding to that of an antibody produced by a human and/or is produced using any of the techniques disclosed herein for producing human antibodies. This definition of human antibodies specifically excludes humanized antibodies containing non-human antigen-binding residues. Human antibodies can be generated using a variety of techniques known in the art, including phage display libraries.
在一些实施方案中，本发明还提供与本发明的任何抗CD318纳米抗体的抗原接合区结合人CD318上相同表位的纳米抗体、重链抗体、抗体或其抗原结合片段(例如单域抗体VHH)，即能够与本发明的任何纳米抗体的抗原结合区交叉竞争与CD318的结合的纳米抗体、重链抗体、抗体或其抗原结合片段。In some embodiments, the invention also provides Nanobodies, heavy chain antibodies, antibodies or antigen-binding fragments thereof that bind the same epitope on human CD318 as the antigen-joining region of any anti-CD318 Nanobody of the invention (e.g., single domain antibody VHH ), that is, a Nanobody, heavy chain antibody, antibody or antigen-binding fragment thereof that can cross-compete with the antigen-binding region of any Nanobody of the present invention for binding to CD318.
本发明中，抗CD318单域抗体具有SEQ ID NO:1所示的CDR1、SEQ ID NO:2所示的CDR2、和SEQ ID NO:3所示的CDR3；或具有SEQ ID NO:4所示的CDR1、SEQ ID NO:5所示的CDR2、和SEQ ID NO:6所示的CDR3；或具有SEQ ID NO:7所示的CDR1、SEQ ID NO:8所示的CDR2、和SEQ ID NO:9所示的CDR3；或具有SEQ ID NO:10所示的CDR1、SEQ ID NO:11所示的CDR2、和SEQ ID NO:12所示的CDR3；或具有SEQ ID NO:13所示的CDR1、SEQ ID NO:14所示的CDR2、和SEQ ID NO:15所示的CDR3；或具有SEQ ID NO:16所示的CDR1、SEQ ID NO:17所示的CDR2、和SEQ ID NO:18所示的CDR3。In the present invention, the anti-CD318 single domain antibody has CDR1 shown in SEQ ID NO:1, CDR2 shown in SEQ ID NO:2, and CDR3 shown in SEQ ID NO:3; or has CDR3 shown in SEQ ID NO:4 CDR1, CDR2 shown in SEQ ID NO:5, and CDR3 shown in SEQ ID NO:6; or having CDR1 shown in SEQ ID NO:7, CDR2 shown in SEQ ID NO:8, and SEQ ID NO : CDR3 shown in SEQ ID NO: 9; or having CDR1 shown in SEQ ID NO: 10, CDR2 shown in SEQ ID NO: 11, and CDR3 shown in SEQ ID NO: 12; or having CDR3 shown in SEQ ID NO: 13 CDR1, CDR2 represented by SEQ ID NO:14, and CDR3 represented by SEQ ID NO:15; or having CDR1 represented by SEQ ID NO:16, CDR2 represented by SEQ ID NO:17, and SEQ ID NO: CDR3 shown in 18.
本文所述的抗CD318单域抗体的FR1、FR2、FR3和FR4可分别独立选自SEQ ID NO:19-24中任一所示的单域抗体的FR1、FR2、FR3和FR4。优选地，抗CD318单域抗体的氨基酸序列如SEQ ID NO:19-24中任一所示。FR1, FR2, FR3 and FR4 of the anti-CD318 single domain antibody described herein can be independently selected from FR1, FR2, FR3 and FR4 of the single domain antibody shown in any one of SEQ ID NO: 19-24. Preferably, the amino acid sequence of the anti-CD318 single domain antibody is as shown in any one of SEQ ID NO: 19-24.
当单域抗体与重链恒定区连接时，纳米抗体是包含本文所述单域抗体的重链抗体。重链恒定区可以是骆驼重链抗体的恒定区，包含CH2和CH3。优选地，所述抗体恒定区源自：IgG1、IgG2、IgG3、IgG4、IgA、IgM、IgE和IgD中的任意一者的恒定区，更优选源自IgG1、IgG2、IgG3、IgG4中的任意一者的恒定区。在一个或多个实施方案中，所述重链恒定区是人IgG Fc的CH2和CH3，例如IgG1的CH2和CH3。When a single domain antibody is linked to a heavy chain constant region, a Nanobody is a heavy chain antibody comprising a single domain antibody as described herein. The heavy chain constant region may be that of a camel heavy chain antibody, including CH2 and CH3. Preferably, the antibody constant region is derived from: the constant region of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD, more preferably derived from any one of IgG1, IgG2, IgG3, IgG4 or's constant region. In one or more embodiments, the heavy chain constant region is CH2 and CH3 of a human IgG Fc, e.g., CH2 and CH3 of IgG1.
本文所述CD318结合分子可以是包含一条、两条或多条本文所述的抗CD318纳米抗体或单域抗体的单价或多价纳米抗体或单域抗体、或多特异性纳米抗体或单域抗体。多特异性可以是针对CD318和另一种抗原，也可以是针对CD318的两种不同表位。The CD318 binding molecules described herein can be monovalent or multivalent Nanobodies or single domain antibodies, or multispecific Nanobodies or single domain antibodies, including one, two or more anti-CD318 Nanobodies or single domain antibodies described herein. . Multispecificity can be against CD318 and another antigen, or it can be against two different epitopes of CD318.
本发明还包括所述抗体衍生物和类似物。“衍生物”和“类似物”是指基本上保持本发明抗体相同的生物学功能或活性的多肽。本发明的衍生物或类似物可以是(i)在一个或多个氨基酸残基中具有取代基团的多肽，或(ii)成熟多肽与另一个化合物(比如延长多肽半衰期的化合物，例如聚乙二醇)融合所形成的多肽，或(iii)附加的氨基酸序列融合到此多肽序列而形成的多肽(如前导序列或分泌序列或用来纯化此多肽的序列或蛋白原序列，或与6His标签形成的融合蛋白)。根据本文的教导，这些衍生物和类似物属于本领域熟练技术人员公知的范围。 The invention also includes such antibody derivatives and analogs. "Derivatives" and "analogues" refer to polypeptides that substantially retain the same biological function or activity of the antibodies of the invention. Derivatives or analogs of the present invention may be (i) a polypeptide having substituent groups in one or more amino acid residues, or (ii) a mature polypeptide combined with another compound (such as a compound that extends the half-life of the polypeptide, such as polyethylene glycol). diol), or (iii) a polypeptide formed by fusion of an additional amino acid sequence to this polypeptide sequence (such as a leader sequence or secretion sequence or a sequence used to purify this polypeptide or a protein sequence, or with a 6His tag fusion protein formed). These derivatives and analogs are within the scope of those skilled in the art in light of the teachings herein.
在不实质性影响抗体活性的前提下，本领域技术人员可以对本发明的抗体序列改变一个或更多个(例如1、2、3、4、5、6、7、8、9或10个或更多个)氨基酸，以获得所述抗体或其功能性片段序列的变体。这些变体包括(但并不限于)：一个或多个(通常为1-50个，较佳地1-30个，更佳地1-20个，最佳地1-10个)氨基酸的缺失、***和/或取代，以及在C末端和/或N末端添加一个或数个(通常为20个以内，较佳地为10个以内，更佳地为5个以内)氨基酸。在本领域中，用性能相近或相似的氨基酸进行保守性取代时，通常不会改变蛋白质的功能。如在FR和/或Fc区中将具有类似性质的氨基酸进行取代。可进行保守性取代的氨基酸残基为本领域所周知。这样的取代的氨基酸残基可以是也可以不是由遗传密码编码的。又比如，在C末端和/或N末端添加一个或数个氨基酸通常也不会改变蛋白质的功能。它们都被视为包括在本发明保护的范围内。Without substantially affecting the activity of the antibody, those skilled in the art can change one or more (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more) amino acids to obtain variants of the antibody or functional fragment sequence thereof. These variants include (but are not limited to): deletion of one or more (usually 1-50, preferably 1-30, more preferably 1-20, optimally 1-10) amino acids , insertion and/or substitution, and addition of one or several (usually within 20, preferably within 10, more preferably within 5) amino acids at the C-terminus and/or N-terminus. In the art, conservative substitutions with amino acids with similar or similar properties generally do not change the function of the protein. For example, amino acids with similar properties are substituted in the FR and/or Fc region. Amino acid residues that are subject to conservative substitutions are well known in the art. Such substituted amino acid residues may or may not be encoded by the genetic code. As another example, adding one or more amino acids to the C-terminus and/or N-terminus usually does not change the function of the protein. They are all considered to be included in the scope of protection of the present invention.
本文所述抗体的变异形式包括：同源序列、保守性变异体、等位变异体、天然突变体、诱导突变体、在高或低的严紧度条件下能与本发明抗体的编码DNA杂交的DNA所编码的蛋白、以及利用抗本发明抗体的抗血清获得的多肽或蛋白。在一些实施方案中，本发明所述变体的序列可以与其来源序列有至少有95％、96％、97％、98％或99％的一致性。本发明所述的序列一致性可以使用序列分析软件测量。例如使用缺省参数的计算机程序BLAST，尤其是BLASTP或TBLASTN。本发明还包括那些具有带CDR的抗体重链可变区的分子，只要其CDR与此处鉴定的CDR具有90％以上(较佳地95％以上，最佳地98％以上)的同源性。Variant forms of the antibodies described herein include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, hybridization to the DNA encoding the antibody of the invention under high or low stringency conditions Proteins encoded by DNA, and polypeptides or proteins obtained using antiserum against the antibodies of the present invention. In some embodiments, the sequence of a variant described herein may be at least 95%, 96%, 97%, 98% or 99% identical to the sequence from which it is derived. The sequence identity described in the present invention can be measured using sequence analysis software. For example, the computer program BLAST uses default parameters, especially BLASTP or TBLASTN. The present invention also includes those molecules having antibody heavy chain variable regions with CDRs as long as the CDRs are more than 90% (preferably more than 95%, optimally more than 98%) homologous to the CDRs identified herein .
可采用本领域常规的方法制备本发明的抗体，如杂交瘤技术。可采用本领域常规的方法制备本发明的纳米抗体，如本领域熟知的噬菌体展示技术。或者，本发明的抗体或纳米抗体可在其他细胞系中表达。可用编码本发明抗体的序列转化合适的哺乳动物宿主细胞，然后培养宿主细胞并纯化抗体。转化可采用任何已知的方法进行，例如包括将多核苷酸包装在病毒(或病毒载体中)并用病毒(或载体)转导宿主细胞。所用的转化程序取决于将转化的宿主。用于将异源多核苷酸引入哺乳动物细胞中的方法为本领域所熟知，包括葡聚糖介导的转染、磷酸钙沉淀、聚凝胺介导的转染、原生质体融合、电穿孔、将多核苷酸囊封在脂质体中和将DNA直接微注射至核中等。可用作用于表达的宿主的哺乳动物细胞系为本领域所熟知，包括但不限于可从美国典型培养物保藏中心(ATCC)获得的多种永生化细胞系，包括但不限于中国仓鼠卵巢(CHO)细胞、HeLa细胞、幼仓鼠肾(BHK)细胞、猴肾细胞(COS)、人肝细胞癌细胞(例如，HepG2)等。The antibodies of the present invention can be prepared using conventional methods in the art, such as hybridoma technology. Nanobodies of the present invention can be prepared using conventional methods in the art, such as phage display technology, which is well known in the art. Alternatively, the antibodies or Nanobodies of the invention can be expressed in other cell lines. Suitable mammalian host cells can be transformed with sequences encoding the antibodies of the invention, and the host cells can then be cultured and the antibodies purified. Transformation can be performed using any known method, including, for example, packaging the polynucleotide in a virus (or viral vector) and transducing the host cell with the virus (or vector). The transformation procedure used depends on the host being transformed. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation , encapsulating polynucleotides in liposomes and microinjecting DNA directly into the nucleus, etc. Mammalian cell lines that can be used as hosts for expression are well known in the art and include, but are not limited to, a variety of immortalized cell lines available from the American Type Culture Collection (ATCC), including, but not limited to, Chinese Hamster Ovary (CHO) ) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (eg, HepG2), etc.
CARCAR
本发明还提供一种靶向CD318的嵌合抗原受体(CAR)。该CAR含有任选的信号肽序列、抗原识别区即本文所述的抗CD318结合分子、铰链区、跨膜区和胞内区。其中胞内区包含一个或多个胞内共刺激域和/或一个或多个胞内信号域。本文中的“铰链区”、“跨膜区”和“胞内区”均可选自已知的CAR-T技术中的铰链区、跨膜区和胞内区的序列。The present invention also provides a chimeric antigen receptor (CAR) targeting CD318. The CAR contains an optional signal peptide sequence, an antigen recognition region, namely the anti-CD318 binding molecule described herein, a hinge region, a transmembrane region and an intracellular region. The intracellular region includes one or more intracellular costimulatory domains and/or one or more intracellular signaling domains. The "hinge region", "transmembrane region" and "intracellular region" in this article can all be selected from the sequences of the hinge region, transmembrane region and intracellular region in known CAR-T technology.
CAR上任选的信号肽可以根据需要选择。一般而言，信号肽是使多肽靶向细胞中的所需部位的肽序列。信号肽使多肽靶向细胞的分泌通路，并且将允许多肽整合和锚定至脂双层；信号肽还可以是膜定位信号肽。示例性的信号肽例如CD8信号肽、CD28信号肽、CD4信号肽或轻链信号肽，其序列在本领域技术人员的知识范围内。适用于本发明的CD8信号肽可以是本领域常用于CAR的各种人CD8信号肽序列。在某些实施方案中，所述CD8信号肽的氨基酸序列包含SEQ ID NO:25所示序列。Optional signal peptides on the CAR can be selected as desired. Generally speaking, a signal peptide is a peptide sequence that targets a polypeptide to a desired location in a cell. The signal peptide targets the polypeptide to the secretory pathway of the cell and will allow the polypeptide to integrate and anchor into the lipid bilayer; the signal peptide may also be a membrane-localized signal peptide. Exemplary signal peptides such as CD8 signal peptide, CD28 signal peptide, CD4 signal peptide or light chain signal peptide, the sequences of which are within the knowledge of those skilled in the art. The CD8 signal peptide suitable for the present invention can be various human CD8 signal peptide sequences commonly used for CAR in the art. In certain embodiments, the amino acid sequence of the CD8 signal peptide includes the sequence shown in SEQ ID NO: 25.
嵌合抗原受体的铰链区位于胞外抗原结合区和跨膜区之间，铰链区是通常在蛋白质的两个域之间存在的氨基酸区段，并且可以允许蛋白质的柔性和两个域的彼此相对运动。铰链区可以是天然存在的蛋白质的 铰链区或其部分。抗体(诸如IgG、IgA、IgM、IgE或IgD抗体)的铰链区也可用于本文所述的嵌合抗原受体。非天然存在的肽也可用作本文所述的嵌合抗原受体的铰链区。示例性地，CAR的铰链区选自CD8α铰链区、IgD铰链区、IgG1Fc CH2CH3铰链区或IgG4Fc CH2CH3铰链区，其序列在本领域技术人员的知识范围内。适用于本发明的CD8α铰链区可以是本领域常用于CAR的各种人CD8α铰链区序列。在某些实施方案中，所述人CD8α铰链区包含SEQ ID NO:26所示序列。The hinge region of the chimeric antigen receptor is located between the extracellular antigen-binding region and the transmembrane region. The hinge region is a segment of amino acids that usually exists between two domains of a protein and can allow for the flexibility of the protein and the separation of the two domains. move relative to each other. The hinge region may be of a naturally occurring protein Hinge area or part thereof. Hinge regions of antibodies such as IgG, IgA, IgM, IgE or IgD antibodies may also be used in the chimeric antigen receptors described herein. Non-naturally occurring peptides may also be used as hinge regions of the chimeric antigen receptors described herein. Exemplarily, the hinge region of the CAR is selected from the group consisting of CD8α hinge region, IgD hinge region, IgG1 Fc CH2CH3 hinge region or IgG4 Fc CH2CH3 hinge region, the sequences of which are within the knowledge of those skilled in the art. The CD8α hinge region suitable for the present invention can be various human CD8α hinge region sequences commonly used for CAR in the art. In certain embodiments, the human CD8 alpha hinge region comprises the sequence set forth in SEQ ID NO:26.
嵌合抗原受体的跨膜区可以形成α螺旋、多于一个α螺旋的复合物、β桶或能够跨域细胞磷脂双层的任何其它稳定结构。跨膜区可以是天然或合成来源的。跨膜区可选自以下蛋白的跨膜区：CD3ε、CD4、CD5、CD8α、CD9、CD16、CD22、CD28、CD33、CD37、CD45、CD64、CD80、CD86、CD134、CD137、CD154、T细胞受体的α、β或ζ链。适用于本发明的人CD8α跨膜区可以是本领域常用于CAR的各种人CD8α跨膜区序列。在某些实施方案中，所述人CD8α跨膜区的氨基酸序列包含SEQ ID NO:27所示序列。The transmembrane region of a chimeric antigen receptor can form an alpha helix, a complex of more than one alpha helix, a beta barrel, or any other stable structure capable of spanning the cellular phospholipid bilayer. The transmembrane region may be of natural or synthetic origin. The transmembrane region may be selected from those of the following proteins: CD3ε, CD4, CD5, CD8α, CD9, CD16, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, T cell receptor α, β or ζ chain of the body. The human CD8α transmembrane region suitable for the present invention can be various human CD8α transmembrane region sequences commonly used for CAR in the art. In certain embodiments, the amino acid sequence of the human CD8α transmembrane region includes the sequence shown in SEQ ID NO: 27.
胞内信号区(或胞内信号传导区)负责表达嵌合抗原受体的免疫效应细胞的至少一种正常效应子功能的活化。例如，T细胞的效应子功能可以是细胞裂解活性或辅助活性，包括细胞因子的分泌。虽然通常可以利用整个胞内信号传导区，但是在很多情况下，使用整个链是不必要的。就使用胞内信号传导区的截短部分而言，只要其转导效应子功能信号，就可以使用这种截短部分代替完整链。因此，胞内信号传导区包括足以转导效应子功能信号的胞内信号传导区的任何截短形式。CAR的胞内信号域可以根据需要选择，包括但不限于来源于CD3ζ、FcRγ(FCER1G)、FcRβ(FcεRib)、CD3γ、CD3δ、CD3ε、CD5、CD22、CD79a、CD79b和CD66d中的至少一种的胞内信号域。优选地，所述胞内信号区来源于人CD3ζ胞内信号区。进一步地，所述人CD3ζ胞内信号区具有SEQ ID NO:29所示氨基酸序列。The intracellular signaling domain (or intracellular signaling domain) is responsible for the activation of at least one normal effector function of the immune effector cell expressing the chimeric antigen receptor. For example, the effector function of a T cell may be cytolytic activity or auxiliary activity, including secretion of cytokines. Although it is often possible to utilize the entire intracellular signaling domain, in many cases it is not necessary to use the entire chain. To the extent that truncated portions of the intracellular signaling region are used, such truncated portions can be used in place of the intact chain as long as they transduce effector function signals. Thus, an intracellular signaling domain includes any truncated form of an intracellular signaling domain that is sufficient to transduce an effector function signal. The intracellular signaling domain of the CAR can be selected as needed, including but not limited to those derived from at least one of CD3ζ, FcRγ (FCER1G), FcRβ (FcεRib), CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b and CD66d Intracellular signaling domain. Preferably, the intracellular signal region is derived from the human CD3ζ intracellular signal region. Further, the human CD3ζ intracellular signal region has the amino acid sequence shown in SEQ ID NO: 29.
在抗原特异性信号的刺激以外，很多免疫效应细胞还需要共刺激来促进细胞增殖、分化和存活，以及活化细胞的效应子功能。“共刺激域”可以是共刺激分子的胞质部分。术语“共刺激分子”是指免疫细胞(诸如T细胞)上的关联结合伴侣，该关联结合伴侣与共刺激配体特异性结合，从而由免疫细胞介导共刺激响应，诸如但不限于增殖和存活。可以根据需要选择适合的胞内共刺激域，包括具有共刺激信号分子的胞内结构域，例如源自4-1BB，CARD11，CD2，CD7，CD27，CD28，CD30，CD40，CD54，CD83，OX40，CD137，CD134，CD150，CD152，CD223，CD270，PD-L2，PD-L1，CD278，DAP10，LAT，NKD2C，SLP76，TRIM，FcεRIγ，MyD88，和41BBL的胞内结构域的至少一种。在某些实施方案中，4-1BB共刺激域的氨基酸序列包含SEQ ID NO:28所示序列。In addition to the stimulation of antigen-specific signals, many immune effector cells also require costimulation to promote cell proliferation, differentiation, and survival, as well as to activate the effector functions of the cells. A "costimulatory domain" may be the cytoplasmic portion of a costimulatory molecule. The term "costimulatory molecule" refers to an associated binding partner on an immune cell, such as a T cell, that specifically binds to a costimulatory ligand, thereby mediating a costimulatory response by the immune cell, such as, but not limited to, proliferation and survival. . Suitable intracellular costimulatory domains can be selected as needed, including intracellular domains with costimulatory signaling molecules, such as those derived from 4-1BB, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54, CD83, OX40 , CD137, CD134, CD150, CD152, CD223, CD270, PD-L2, PD-L1, CD278, DAP10, LAT, NKD2C, SLP76, TRIM, FcεRIγ, MyD88, and at least one of the intracellular domains of 41BBL. In certain embodiments, the amino acid sequence of the 4-1BB costimulatory domain comprises the sequence set forth in SEQ ID NO: 28.
形成本发明的嵌合抗原受体的上述各部分，如CD8信号肽、抗CD318纳米抗体、CD8α铰链区、CD8α跨膜区、CD3ζ胞内信号域、4-1BB共刺激域等，相互之间可直接连接，或者可通过接头序列连接。接头序列可以是本领域周知的适用于抗体的接头序列，例如含G和S的接头序列。通常，接头含有一个或多个前后重复的基序。例如，该基序可以是GGGS、GGGGS、SSSSG、GSGSA和GGSGG。优选地，该基序在接头序列中是相邻的，在重复之间没有***氨基酸残基。接头序列可以包含1、2、3、4或5个重复基序组成。接头的长度可以是3～25个氨基酸残基，例如3～15、5～15、10～20个氨基酸残基。在某些实施方案中，接头序列是多甘氨酸接头序列。接头序列中甘氨酸的数量无特别限制，通常为2～20个，例如2～15、2～10、2～8个。除甘氨酸和丝氨酸来，接头中还可含有其它已知的氨基酸残基，例如丙氨酸(A)、亮氨酸(L)、苏氨酸(T)、谷氨酸(E)、苯丙氨酸(F)、精氨酸(R)、谷氨酰胺(Q)等。在某些实施方案中，接头序列为(GGGGS)n连接，其中n为1～5的整数。 The above-mentioned parts that form the chimeric antigen receptor of the present invention, such as CD8 signal peptide, anti-CD318 nanobody, CD8α hinge region, CD8α transmembrane region, CD3ζ intracellular signaling domain, 4-1BB costimulatory domain, etc., interact with each other. The linkage can be direct or can be linked via a linker sequence. The linker sequence may be one well known in the art and suitable for use with antibodies, such as a G and S-containing linker sequence. Typically, linkers contain one or more repeating motifs. For example, the motif may be GGGS, GGGGS, SSSSG, GSGSA, and GGSGG. Preferably, the motifs are contiguous in the linker sequence, with no intervening amino acid residues between repeats. Linker sequences can contain 1, 2, 3, 4 or 5 repeating motifs. The length of the linker can be 3 to 25 amino acid residues, such as 3 to 15, 5 to 15, or 10 to 20 amino acid residues. In certain embodiments, the linker sequence is a polyglycine linker sequence. The number of glycines in the linker sequence is not particularly limited, but is usually 2 to 20, such as 2 to 15, 2 to 10, or 2 to 8. In addition to glycine and serine, the linker can also contain other known amino acid residues, such as alanine (A), leucine (L), threonine (T), glutamic acid (E), phenylalanine Acid (F), arginine (R), glutamine (Q), etc. In certain embodiments, the linker sequence is a (GGGGS)n linkage, where n is an integer from 1 to 5.
在示例性实施方案中，CAR从N端到C端依次含有CD8信号肽、本文所述的抗CD318纳米抗体或其抗原结合片段、CD8α铰链区、CD8α跨膜区、4-1BB共刺激域、CD3ζ胞内信号域。在具体实施例中，具有上述结构的示例性CAR如SEQ ID NO:30-35中任一所示。In an exemplary embodiment, the CAR contains CD8 signal peptide, anti-CD318 Nanobody or antigen-binding fragment thereof described herein, CD8α hinge region, CD8α transmembrane region, 4-1BB costimulatory domain, in sequence from N-terminus to C-terminus. CD3ζ intracellular signaling domain. In specific embodiments, an exemplary CAR having the above structure is as shown in any of SEQ ID NOs: 30-35.
应理解，在基因克隆操作中，常常需要设计合适的酶切位点，这势必在所表达的氨基酸序列末端引入了一个或多个不相干的残基，而这并不影响目的序列的活性。为了构建融合蛋白、促进重组蛋白的表达、获得自动分泌到宿主细胞外的重组蛋白、或利于重组蛋白的纯化，常常需要将一些氨基酸添加至重组蛋白的N-末端、C-末端或该蛋白内的其它合适区域内，例如，包括但不限于，适合的接头肽、信号肽、前导肽、末端延伸等。因此，本发明的CAR的氨基端或羧基端还可含有一个或多个多肽片段，作为蛋白标签。任何合适的标签都可以用于本文。例如，所述的标签可以是FLAG，HA，HA1，c-Myc，Poly-His，Poly-Arg，Strep-TagII，AU1，EE，T7，4A6，ε，B，gE以及Ty1。这些标签可用于对蛋白进行纯化。It should be understood that in gene cloning operations, it is often necessary to design appropriate enzyme cutting sites, which will inevitably introduce one or more irrelevant residues at the end of the expressed amino acid sequence, but this does not affect the activity of the target sequence. In order to construct fusion proteins, promote the expression of recombinant proteins, obtain recombinant proteins that are automatically secreted out of host cells, or facilitate the purification of recombinant proteins, it is often necessary to add some amino acids to the N-terminus, C-terminus of the recombinant protein or within the protein. Other suitable regions include, for example, but are not limited to, suitable linker peptides, signal peptides, leader peptides, terminal extensions, etc. Therefore, the amino terminus or carboxyl terminus of the CAR of the present invention may also contain one or more polypeptide fragments as protein tags. Any suitable tag may be used for this article. For example, the tags may be FLAG, HA, HA1, c-Myc, Poly-His, Poly-Arg, Strep-TagII, AU1, EE, T7, 4A6, ε, B, gE and Ty1. These tags can be used to purify proteins.
本发明的CAR中的抗原识别区可以是前述的抗CD318纳米抗体或其功能性片段序列的变体。此外，CAR的其他部分也可以发生序列变化，得到的突变体与该CAR具有至少80％，优选至少85％，优选至少90％，优选至少95％，优选至少97％的序列相同性并保留该CAR的生物学活性(如活化T细胞)。可采用例如NCBI的BLASTp计算两条比对的序列之间的序列相同性。The antigen recognition region in the CAR of the present invention can be a variant of the aforementioned anti-CD318 Nanobody or its functional fragment sequence. In addition, other parts of the CAR can also undergo sequence changes, and the resulting mutant has at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 97% sequence identity with the CAR and retains the sequence identity. Biological activity of CAR (such as activated T cells). Sequence identity between two aligned sequences can be calculated using, for example, NCBI's BLASTp.
突变体还包括：在任一实施方案所述的CAR的氨基酸序列中具有一个或数个突变(***、缺失或取代)、同时仍保留该CAR的生物学活性的氨基酸序列。所述数个突变通常指1-10个以内，例如1-8个、1-5个或1-3个。取代优选是保守性取代。例如，在本领域中，用性能相近或相似的氨基酸进行保守性取代时，通常不会改变蛋白质或多肽的功能。“性能相近或相似的氨基酸”包括例如，具有相似侧链的氨基酸残基的家族，这些家族包括具有碱性侧链的氨基酸(例如赖氨酸、精氨酸、组氨酸)、具有酸性侧链的氨基酸(例如天冬氨酸、谷氨酸)、具有不带电荷的极性侧链的氨基酸(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)、具有非极性侧链的氨基酸(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)、具有β-分支侧链的氨基酸(例如苏氨酸、缬氨酸、异亮氨酸)和具有芳香侧链的氨基酸(例如酪氨酸、苯丙氨酸、色氨酸、组氨酸)。因此，在本发明多肽中用来自同一侧链类的另一氨基酸残基替换一个或几个位点，将不会在实质上影响其活性。Mutants also include amino acid sequences that have one or several mutations (insertions, deletions, or substitutions) in the amino acid sequence of the CAR described in any embodiment while still retaining the biological activity of the CAR. The number of mutations usually refers to within 1-10, such as 1-8, 1-5 or 1-3. Substitutions are preferably conservative substitutions. For example, in the art, conservative substitutions with amino acids with similar or similar properties generally do not change the function of the protein or polypeptide. "Amino acids with similar or similar properties" include, for example, families of amino acid residues with similar side chains. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains, chain amino acids (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine amino acids), amino acids with non-polar side chains (such as alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), with Amino acids with β-branched side chains (eg threonine, valine, isoleucine) and amino acids with aromatic side chains (eg tyrosine, phenylalanine, tryptophan, histidine). Therefore, substitution of one or more positions in a polypeptide of the invention with another amino acid residue from the same side chain class will not materially affect its activity.
核酸nucleic acid
本发明还提供了编码上述抗体或CAR的多核苷酸。本发明的多核苷酸可以是DNA形式或RNA形式。DNA形式包括cDNA、基因组DNA或人工合成的DNA。DNA可以是单链的或是双链的。DNA可以是编码链或非编码链。本发明也包括编码融合蛋白的多核苷酸序列的简并变异体，即编码相同的氨基酸序列但核苷酸序列有所不同的核苷酸序列。The invention also provides polynucleotides encoding the above-mentioned antibodies or CARs. The polynucleotides of the invention may be in DNA form or RNA form. Forms of DNA include cDNA, genomic DNA, or synthetic DNA. DNA can be single-stranded or double-stranded. DNA can be a coding strand or a non-coding strand. The present invention also encompasses degenerate variants of the polynucleotide sequence encoding the fusion protein, ie, nucleotide sequences encoding the same amino acid sequence but with different nucleotide sequences.
所以，本发明还涉及与上述多核苷酸序列杂交且两个序列之间具有至少50％，较佳地至少70％，更佳地至少80％相同性的多核苷酸。本发明特别涉及在严谨条件下与本发明所述多核苷酸可杂交的多核苷酸。在本发明中，“严谨条件”是指：(1)在较低离子强度和较高温度下的杂交和洗脱，如0.2×SSC，0.1％SDS，60℃；或(2)杂交时加有变性剂，如50％(v/v)甲酰胺，0.1％小牛血清/0.1％Ficoll，42℃等；或(3)仅在两条序列之间的相同性至少在90％以上，更好是95％以上时才发生杂交。并且，可杂交的多核苷酸编码的多肽与成熟多肽有相同的生物学功能和活性。Therefore, the present invention also relates to polynucleotides that hybridize to the above-mentioned polynucleotide sequences and have at least 50%, preferably at least 70%, more preferably at least 80% identity between the two sequences. The invention particularly relates to polynucleotides that hybridize under stringent conditions to the polynucleotides of the invention. In the present invention, "stringent conditions" refer to: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) adding water during hybridization There are denaturants, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42°C, etc.; or (3) only the identity between the two sequences is at least 90%, more It is best when hybridization occurs only when the ratio is above 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.
本发明的抗体的核苷酸全长序列或其片段通常可以用PCR扩增法、重组法或人工合成的方法获得。一 种可行的方法是用人工合成的方法来合成有关序列，尤其是片段长度较短时。通常，通过先合成多个小片段，然后再进行连接可获得序列很长的片段。此外，还可将重链的编码序列和表达标签(如6His)融合在一起，形成融合蛋白。CAR的序列也可以如上获得。或者，可以如上得到CAR的各部分(信号肽、抗原识别区、铰链区、跨膜区或胞内区)的序列后再连接得到CAR的全长。The full-length nucleotide sequence of the antibody of the present invention or its fragment can usually be obtained by PCR amplification, recombinant or artificial synthesis. one A feasible method is to use artificial synthesis to synthesize the relevant sequences, especially when the fragment length is short. Often, fragments with long sequences are obtained by first synthesizing multiple small fragments and then ligating them. In addition, the coding sequence of the heavy chain and the expression tag (such as 6His) can also be fused together to form a fusion protein. The sequence of the CAR can also be obtained as above. Alternatively, the sequences of each part of the CAR (signal peptide, antigen recognition region, hinge region, transmembrane region or intracellular region) can be obtained as above and then connected to obtain the full length of the CAR.
一旦获得了有关的序列，就可以用重组法来大批量地获得有关序列。这通常是将其克隆入载体，再转入细胞，然后通过常规方法从增殖后的宿主细胞中分离得到有关序列。本发明所涉及的生物分子(核酸、蛋白等)包括以分离的形式存在的生物分子。目前，已经可以完全通过化学合成来得到编码本发明蛋白(或其片段，或其衍生物)的DNA序列。然后可将该DNA序列引入本领域中已知的各种现有的DNA分子(或如载体)和细胞中。此外，还可通过化学合成将突变引入本发明蛋白序列中。CAR各部分可以顺序克隆到载体中或者可以整合为全长CAR后再克隆。Once the relevant sequence is obtained, recombination can be used to obtain the relevant sequence in large quantities. This is usually done by cloning it into a vector, transforming it into cells, and then isolating the relevant sequence from the propagated host cells by conventional methods. Biomolecules (nucleic acids, proteins, etc.) involved in the present invention include biomolecules in isolated form. At present, the DNA sequence encoding the protein of the present invention (or its fragments, or its derivatives) can be obtained entirely through chemical synthesis. The DNA sequence can then be introduced into a variety of existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into the protein sequence of the invention through chemical synthesis. Each part of the CAR can be cloned sequentially into the vector or can be integrated into the full-length CAR and then cloned.
本发明也涉及核酸构建物，该核酸构建物含有本文所述的多核苷酸序列，以及与这些序列操作性连接的一个或多个调控序列。本发明所述的多核苷酸序列可以多种方式***作以保证所述抗体或CAR的表达。在将核酸构建物***载体之前可根据表达载体的不同或要求而对核酸构建物进行操作。利用重组DNA方法来改变多核苷酸序列的技术是本领域已知的。The present invention also relates to nucleic acid constructs containing the polynucleotide sequences described herein, and one or more regulatory sequences operably linked to these sequences. The polynucleotide sequences of the invention can be manipulated in various ways to ensure the expression of the antibody or CAR. Before inserting the nucleic acid construct into the vector, the nucleic acid construct can be manipulated according to the differences or requirements of the expression vector. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.
调控序列可以是合适的启动子序列。启动子序列通常与待表达蛋白的编码序列操作性连接。启动子可以是在所选择的宿主细胞中显示转录活性的任何核苷酸序列，包括突变的、截短的和杂合启动子，并且可以从编码与该宿主细胞同源或异源的胞外或胞内多肽的基因获得。合适的启动子的一个例子为即时早期巨细胞病毒(CMV)启动子序列。该启动子序列是能够驱动可操作地连接至其上的任何多核苷酸序列高水平表达的强组成型启动子序列。合适的启动子的另一个例子为延伸生长因子-1α(EF-1α)。然而，也可使用其他组成型启动子序列，包括但不限于类人猿病毒40(SV40)早期启动子、小鼠乳癌病毒(MMTV)、人免疫缺陷病毒(HIV)长末端重复(LTR)启动子、MoMuLV启动子、鸟类白血病病毒启动子、EB病毒即时早期启动子、鲁斯氏肉瘤病毒启动子、以及人基因启动子，诸如但不限于肌动蛋白启动子、肌球蛋白启动子、血红素启动子和肌酸激酶启动子。进一步地，也可考虑使用诱导型启动子。诱导型启动子的使用提供了分子开关，其能够在期限表达时打开可操作地连接诱导型启动子的多核苷酸序列的表达，而在当表达是不期望的时关闭表达。诱导型启动子的例子包括但不限于金属硫蛋白启动子、糖皮质激素启动子、孕酮启动子和四环素启动子。The control sequence may be a suitable promoter sequence. The promoter sequence is usually operably linked to the coding sequence of the protein to be expressed. The promoter can be any nucleotide sequence that exhibits transcriptional activity in the host cell of choice, including mutant, truncated, and hybrid promoters, and can be derived from genes encoding extracellular genes that are homologous or heterologous to the host cell. or genetic acquisition of intracellular polypeptides. An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. The promoter sequence is a strong constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including, but not limited to, simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Ruth's sarcoma virus promoter, and human gene promoters such as but not limited to actin promoter, myosin promoter, heme promoter and creatine kinase promoter. Furthermore, the use of inducible promoters may also be considered. The use of an inducible promoter provides a molecular switch capable of turning on expression of a polynucleotide sequence operably linked to the inducible promoter when expression is required and turning off expression when expression is undesirable. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.
调控序列也可以是合适的转录终止子序列，由宿主细胞识别以终止转录的序列。终止子序列与编码该多肽的核苷酸序列的3’末端操作性连接。在选择的宿主细胞中有功能的任何终止子都可用于本发明。调控序列也可以是合适的前导序列，对宿主细胞翻译重要的mRNA的非翻译区。前导序列与编码该多肽的核苷酸序列的5’末端可操作连接。在选择的宿主细胞中有功能的任何终止子都可用于本发明。The control sequence may also be a suitable transcription terminator sequence, a sequence recognized by the host cell to terminate transcription. The terminator sequence is operably linked to the 3' end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the host cell of choice can be used in the present invention. The regulatory sequence may also be a suitable leader sequence, an untranslated region of the mRNA important for translation by the host cell. The leader sequence is operably linked to the 5' end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in the host cell of choice can be used in the present invention.
在某些实施方案中，所述核酸构建物是载体，例如克隆载体、表达载体和整合载体。通常通过可操作地连接本发明的多核苷酸序列至表达载体，实现本发明多核苷酸序列的表达。典型的克隆载体包含可用于调节期望核酸序列表达的转录和翻译终止子、起始序列和启动子。整合载体含有将靶序列整合到细胞基因组上的组件。这些载体可以用于转化适当的宿主细胞，以使其能够表达蛋白质。载体通常含有用于质粒维系和用于克隆与表达外源性核苷酸序列的序列。所述序列(在某些实施方案中总称为“侧翼序列”)通常包括一个或多个以下核苷酸序列：启动子、一个或多个增强子序列、复制起点、转录终止序列、含有供体 和受体剪接位点的完全内含子序列、编码用于多肽分泌的前导序列的序列、核糖体结合位点、聚腺苷酸化序列、用于***编码将要表达的抗体的核酸的多连接子区和可选标记元件。In certain embodiments, the nucleic acid construct is a vector, such as a cloning vector, an expression vector, and an integration vector. Expression of a polynucleotide sequence of the invention is generally accomplished by operably linking the polynucleotide sequence of the invention to an expression vector. Typical cloning vectors contain transcriptional and translational terminators, initiation sequences, and promoters that can be used to regulate expression of the desired nucleic acid sequence. Integration vectors contain components that integrate target sequences into the cellular genome. These vectors can be used to transform appropriate host cells to enable expression of the protein. Vectors typically contain sequences for plasmid maintenance and for cloning and expression of exogenous nucleotide sequences. The sequences (collectively referred to in certain embodiments as "flanking sequences") generally include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a donor-containing sequence and acceptor splice site, a sequence encoding a leader for secretion of the polypeptide, a ribosome binding site, a polyadenylation sequence, and a polylinker for insertion of nucleic acid encoding the antibody to be expressed. area and optional marker elements.
此外，载体的类型不受限制，例如，质粒、噬菌粒、噬菌体衍生物、动物病毒和粘粒，可根据待导入的宿主细胞而改变。病毒载体技术在本领域中是公知的并在例如Sambrook等(2001，Molecular Cloning:A Laboratory Manual，Cold Spring Harbor Laboratory，New York)和其他病毒学和分子生物学手册中进行了描述。可用作载体的病毒包括但不限于逆转录病毒、腺病毒、腺伴随病毒、疱疹病毒和慢病毒。Furthermore, the type of vector is not limited, for example, plasmids, phagemids, phage derivatives, animal viruses, and cosmids, and may vary depending on the host cell to be introduced. Viral vector technology is well known in the art and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses.
为了评估CAR多肽或其部分的表达，被引入细胞的载体也可包含可选择的标记基因或报道基因中的任一个或两者，以便于从通过病毒载体寻求被转染或感染的细胞群中鉴定和选择表达细胞。To assess expression of a CAR polypeptide or portion thereof, the vector introduced into the cell may also contain either or both a selectable marker gene or a reporter gene to facilitate identification of the population of cells sought to be transfected or infected by the viral vector. Identification and selection of expressing cells.
细胞cell
适用于导入本文所述核酸构建物的宿主细胞可以是原核细胞，如细菌细胞；或是低等真核细胞，如酵母细胞；或是高等真核细胞，如哺乳动物细胞，特别是免疫细胞，优选免疫效应细胞。代表性例子有：大肠杆菌，链霉菌属；鼠伤寒沙门氏菌的细菌细胞；真菌细胞如酵母；果蝇S2或Sf9的昆虫细胞；CHO、COS7、293细胞的动物细胞等。Host cells suitable for introducing the nucleic acid constructs described herein can be prokaryotic cells, such as bacterial cells; or lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells, especially immune cells, Immune effector cells are preferred. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; animal cells of CHO, COS7, 293 cells, etc.
“免疫效应细胞”是可执行免疫效应功能的免疫细胞。在一些实施方案中，免疫效应细胞表达至少FcγRIII并执行ADCC效应子功能。介导ADCC的免疫效应细胞的实例包括外周血单个核细胞(PBMC)、天然杀伤(NK)细胞、单核细胞、细胞毒性T细胞、中性粒细胞和嗜酸性粒细胞。优选地，免疫效应细胞选自：由多能干细胞或胚胎干细胞培养分化的免疫细胞、T淋巴细胞、NK细胞、外周血单个核细胞(PBMC)和造血干细胞中的至少一种。更优选地，所述免疫效应细胞为T淋巴细胞(同T细胞)。在一些实施方案中，T细胞可以为CD4+/CD8-、CD4-/CD8+、CD4+/CD8+、CD4-/CD8-或它们的组合。在一些实施方案中，T细胞在表达嵌合抗原受体并结合至靶细胞时产生IL-2、IFN和/或TNF。在一些实施方案中，CD8+T细胞在表达嵌合抗原受体并结合至靶细胞时裂解抗原特异性靶细胞。"Immune effector cells" are immune cells that perform immune effector functions. In some embodiments, the immune effector cells express at least FcγRIII and perform ADCC effector functions. Examples of immune effector cells that mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells, neutrophils, and eosinophils. Preferably, the immune effector cells are selected from: at least one of immune cells cultured and differentiated from pluripotent stem cells or embryonic stem cells, T lymphocytes, NK cells, peripheral blood mononuclear cells (PBMC) and hematopoietic stem cells. More preferably, the immune effector cells are T lymphocytes (the same as T cells). In some embodiments, T cells can be CD4+/CD8-, CD4-/CD8+, CD4+/CD8+, CD4-/CD8-, or combinations thereof. In some embodiments, T cells produce IL-2, IFN, and/or TNF when expressing chimeric antigen receptors and binding to target cells. In some embodiments, CD8+ T cells lyse antigen-specific target cells when expressing chimeric antigen receptors and binding to target cells.
适用于本发明的T细胞可以是各种来源的各种类型的T细胞。例如，T细胞可来源于恶性实体瘤(例如胰腺癌)患者的PBMC。在某些实施方案中，获得T细胞后，可先用适量的(例如30～80ng/ml，如50ng/ml)的CD3抗体刺激活化，然后在含有适量的(例如30～80IU/ml，如50IU/ml)的IL2培养基进行培养备用。T cells suitable for use in the present invention can be various types of T cells from various sources. For example, T cells can be derived from PBMCs of patients with malignant solid tumors, such as pancreatic cancer. In some embodiments, after T cells are obtained, they can be first stimulated and activated with an appropriate amount (for example, 30-80ng/ml, such as 50ng/ml) of CD3 antibodies, and then containing an appropriate amount (for example, 30-80IU/ml, such as 50ng/ml) of CD3 antibody for activation. 50IU/ml) IL2 medium for culture and use.
将核酸或载体引入哺乳动物细胞的方法是本领域已知的，所述载体可以通过物理、化学或生物方法转入细胞。当宿主为原核生物如大肠杆菌时，能吸收DNA的感受态细胞可在指数生长期后收获，用CaCl2法处理，所用的步骤在本领域众所周知。当宿主是真核生物，可选用如下的DNA转染方法：磷酸钙共沉淀法，常规机械方法如显微注射、电穿孔，脂质体包装等。在一些实施方案中，转导的或转染的免疫效应细胞在引入核酸或载体之后离体繁殖。Methods for introducing nucleic acids or vectors into mammalian cells are known in the art, and the vectors may be introduced into the cells by physical, chemical, or biological means. When the host is a prokaryotic organism such as E. coli, competent cells that can absorb DNA can be harvested after the exponential growth phase and treated with the CaCl2 method. The steps used are well known in the art. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate co-precipitation method, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc. In some embodiments, transduced or transfected immune effector cells are propagated ex vivo following introduction of nucleic acid or vector.
获得的转化子可以用常规方法培养，表达本发明的基因所编码的抗体或CAR。根据所用的宿主细胞，培养中所用的培养基可选自各种常规培养基。在适于宿主细胞生长的条件下进行培养。当宿主细胞生长到适当的细胞密度后，用合适的方法(如温度转换或化学诱导)诱导选择的启动子，将细胞再培养一段时间。The obtained transformants can be cultured using conventional methods to express the antibody or CAR encoded by the gene of the present invention. Depending on the host cells used, the medium used in culture can be selected from various conventional media. Cultivate under conditions suitable for host cell growth. After the host cells have grown to an appropriate cell density, the selected promoter is induced using an appropriate method (such as temperature shift or chemical induction), and the cells are cultured for a further period of time.
在上面的方法中的多肽可在细胞内、或在细胞膜上表达、或分泌到细胞外。如果需要，可利用其物理的、化学的和其它特性通过各种分离方法分离和纯化重组的蛋白。这些方法是本领域技术人员所熟知的。这些方法的例子包括但并不限于：常规的复性处理、用蛋白沉淀剂处理(盐析方法)、离心、渗透破菌、超处理、超离心、分子筛层析(凝胶过滤)、吸附层析、离子交换层析、高效液相层析(HPLC)和其它各种液相 层析技术及这些方法的结合。The polypeptide in the above method can be expressed within the cell, on the cell membrane, or secreted outside the cell. If desired, the recombinant protein can be isolated and purified by various separation methods utilizing its physical, chemical and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitating agents (salting out method), centrifugation, osmotic sterilization, ultratreatment, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption layer analysis, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid phases Chromatographic techniques and combinations of these methods.
用途和方法Uses and methods
通过构建纳米抗体文库，发明人筛选了可以结合CD318的纳米抗体。利用这些纳米抗体，发明人构建了CAR以及CAR-T细胞，经细胞水平实验验证，所述CAR-T有很强的免疫功能，更优的CD107a表达、IFN-γ和IL-2分泌以及对靶细胞的特异性杀伤功能，体内药效显著。By constructing a Nanobody library, the inventors screened Nanobodies that could bind CD318. Using these nanobodies, the inventors constructed CAR and CAR-T cells. After experimental verification at the cell level, the CAR-T has strong immune function, better CD107a expression, IFN-γ and IL-2 secretion, and resistance to The specific killing function of target cells has significant efficacy in vivo.
本文所述的抗体、CAR、编码序列、核酸构建物、和细胞的所有方面都可用于制备用以预防或治疗本文所述各种病况和疾病的药物，所述病况和疾病是与CD318表达相关的疾病或病况，指由CD318表达异常所直接或间接导致的疾病，通常是指由CD318过表达所导致的疾病，例如癌症，包括但不限于：乳腺癌、肺癌、肝癌、胰腺癌、卵巢癌、肾癌和结直肠癌。All aspects of the antibodies, CARs, coding sequences, nucleic acid constructs, and cells described herein can be used to prepare medicaments for the prevention or treatment of various conditions and diseases described herein that are associated with CD318 expression. Diseases or conditions refer to diseases caused directly or indirectly by abnormal expression of CD318, usually referring to diseases caused by overexpression of CD318, such as cancer, including but not limited to: breast cancer, lung cancer, liver cancer, pancreatic cancer, ovarian cancer , kidney cancer and colorectal cancer.
本发明还包括一类细胞疗法，包括在免疫细胞(例如T细胞)中表达本文所述的CAR，和对需要其的接受者施用治疗有效量的细胞，细胞能够杀死接受者的肿瘤细胞。相比抗体疗法，CAR-T细胞能够体内复制，产生可导致持续肿瘤控制的长期持久性。由CAR-T细胞引起的抗肿瘤免疫应答可为主动或被动免疫应答。另外，CAR介导的免疫应答可为过继免疫疗法步骤的一部分，其中CAR-T细胞诱导对CAR中的抗原结合部分特异性的免疫应答。The invention also encompasses a type of cell therapy that involves expressing a CAR as described herein in immune cells (eg, T cells) and administering to a recipient in need thereof a therapeutically effective amount of the cells capable of killing tumor cells in the recipient. Compared to antibody therapies, CAR-T cells are able to replicate in vivo, producing long-term persistence that can lead to sustained tumor control. The anti-tumor immune response caused by CAR-T cells can be an active or passive immune response. Additionally, a CAR-mediated immune response can be part of an adoptive immunotherapy step, in which CAR-T cells induce an immune response specific for the antigen-binding portion of the CAR.
本发明的抗体、核酸或CAR-修饰的细胞可被单独施用或作为药物组合物与稀释剂和/或与其他组分诸如相关的细胞因子或细胞群结合施用。在此方面，药物组合物可以通过使具有所需纯度的活性药剂与任选的药学上可接受的载剂混合以冻干制剂或水溶液的形式制备。药学上可接受的载剂在所用的剂量和浓度下对接受者是无毒的，可包括缓冲剂(例如中性缓冲盐水、硫酸盐缓冲盐水)、抗氧化剂、防腐剂、等渗剂、稳定剂、螯合剂(例如EDTA或谷胱甘肽)、佐剂(例如氢氧化铝)和表面活性剂中的至少一种。此外，为了使药物组合物可用于体内施用，它们必须是无菌的。可以通过无菌过滤膜过滤使药物组合物无菌。The antibodies, nucleic acids or CAR-modified cells of the invention can be administered alone or as pharmaceutical compositions in combination with diluents and/or with other components such as relevant cytokines or cell populations. In this regard, the pharmaceutical composition may be prepared in the form of a lyophilized preparation or aqueous solution by mixing the active agent with the desired purity and optionally a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are nontoxic to the recipient at the dosage and concentration used and may include buffers (e.g., neutral buffered saline, sulfate buffered saline), antioxidants, preservatives, isotonic agents, stabilizing agents at least one of an agent, a chelating agent (such as EDTA or glutathione), an adjuvant (such as aluminum hydroxide), and a surfactant. Furthermore, in order for pharmaceutical compositions to be useful for in vivo administration, they must be sterile. Pharmaceutical compositions can be rendered sterile by filtration through a sterile filter membrane.
在一些实施方案中，药物组合物可以含有：细胞毒性剂、化学治疗剂、细胞因子、免疫抑制剂、生长抑制剂以及待治疗的具体适应症所需的活性药剂中的至少一种添加剂。添加剂的具体添加量可根据实际需要进行调整。本发明的药物组合物可以以“免疫学上有效量”、“抗肿瘤有效量”、“肿瘤-抑制有效量”或“治疗量”的量施用。“治疗”指向受试者采用本文所述治疗方案以达到至少一种阳性治疗效果(比如，癌症细胞数目减少、肿瘤体积减小、癌细胞浸润至周边器官的速率降低或肿瘤转移或肿瘤生长的速率降低)。当指出“免疫学上有效量”、“抗肿瘤有效量”、“肿瘤-抑制有效量”或“治疗量”时，待施用的本发明组合物的精确量可由医师确定，其考虑患者(对象)的年龄、重量、肿瘤大小、感染或转移程度和病症的个体差异。通常，包括本文描述的T细胞的药物组合物可以以104至109个细胞/kg体重的剂量，优选105至106个细胞/kg体重的剂量。T细胞组合物也可以以这些剂量多次施用。细胞可通过使用免疫疗法中公知的注入技术(见例如Rosenberg等，New Eng.J.of Med.319:1676，1988)施用。对于具体患者的最佳剂量和治疗方案可通过监测患者的疾病迹象并因此调节治疗由医学领域技术人员容易地确定。In some embodiments, pharmaceutical compositions may contain at least one additive from the group consisting of cytotoxic agents, chemotherapeutic agents, cytokines, immunosuppressants, growth inhibitors, and active agents required for the particular indication to be treated. The specific amount of additives can be adjusted according to actual needs. The pharmaceutical composition of the present invention may be administered in an "immunologically effective amount", "anti-tumor effective amount", "tumor-inhibitory effective amount" or "therapeutic amount". "Treatment" refers to a subject taking a treatment regimen described herein to achieve at least one positive therapeutic effect (for example, reduction in the number of cancer cells, reduction in tumor volume, reduction in the rate of cancer cell infiltration into surrounding organs, or reduction in tumor metastasis or tumor growth) rate decreases). When an "immunologically effective amount", "anti-tumor effective amount", "tumor-suppressive effective amount" or "therapeutic amount" is indicated, the precise amount of the composition of the invention to be administered can be determined by the physician, who takes into account the patient (subject) ) age, weight, tumor size, degree of infection or metastasis, and individual differences in disease. Typically, pharmaceutical compositions including T cells described herein may be administered at a dose of 10 4 to 10 9 cells/kg body weight, preferably 10 5 to 10 6 cells/kg body weight. T cell compositions can also be administered multiple times at these dosages. Cells can be administered using infusion techniques well known in immunotherapy (see, eg, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dosage and treatment regimen for a particular patient can be readily determined by one skilled in the medical field by monitoring the patient for signs of disease and adjusting treatment accordingly.
组合物的施用可以以任何方便的方式进行，包括通过喷雾法、注射、吞咽、输液、植入或移植。本文描述的组合物可被皮下、皮内、瘤内、结内、脊髓内、肌肉内、通过静脉内注射或腹膜内施用给患者。在一个实施方案中，本发明的T细胞组合物通过皮内或皮下注射被施用给患者。在另一个实施方案中，本发明的T细胞组合物优选通过静脉注射施用。T细胞的组合物可被直接注入肿瘤、***或感染位置。Administration of the composition may be by any convenient means, including by spraying, injection, swallowing, infusion, implantation or transplantation. The compositions described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intraspinally, intramuscularly, by intravenous injection, or intraperitoneally. In one embodiment, the T cell composition of the invention is administered to the patient by intradermal or subcutaneous injection. In another embodiment, the T cell composition of the invention is preferably administered by intravenous injection. The composition of T cells can be injected directly into the tumor, lymph node or site of infection.
在本发明的一些实施方案中，本发明的CAR-T细胞或其组合物可与本领域已知的其它疗法结合。所述 疗法包括但不限于化疗、放疗和免疫抑制剂。例如，可结合本领域周知的治疗CD318介导的疾病的放疗或化疗制剂进行治疗。In some embodiments of the invention, the CAR-T cells of the invention or compositions thereof may be combined with other therapies known in the art. described Therapies include, but are not limited to, chemotherapy, radiation therapy, and immunosuppressants. For example, treatment may be combined with radiotherapy or chemotherapy agents known in the art to treat CD318-mediated diseases.
本文中，“抗肿瘤效应”指一种生物学效应，其可由肿瘤体积的减少、肿瘤细胞数的减少、转移数的减少、预期寿命的增加或与癌相关的各种生理症状的改善表示。As used herein, "anti-tumor effect" refers to a biological effect, which can be expressed by a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in the number of metastases, an increase in life expectancy, or an improvement in various physiological symptoms related to cancer.
“患者”、“对象”、“个体”等等在本文中可交换使用，指可引起免疫应答的活有机体，如哺乳动物。例子包括但不限于人、狗、猫、小鼠、大鼠和其转基因物种。"Patient," "subject," "individual" and the like are used interchangeably herein to refer to a living organism, such as a mammal, that can elicit an immune response. Examples include, but are not limited to, humans, dogs, cats, mice, rats, and transgenic species thereof.
诊断、检测和试剂盒Diagnostics, tests and kits
本发明的结合分子因其与CD318的高亲合力可用于测定，例如结合测定来检测和/或定量在组织或细胞中表达的CD318。结合分子例如单域抗体可用在进一步研究CD318在疾病中的作用的研究中。检测CD318的方法大致如下：获得细胞和/或组织样本；检测样本中CD318的水平。The binding molecules of the invention can be used in assays due to their high affinity for CD318, such as binding assays to detect and/or quantify CD318 expressed in tissues or cells. Binding molecules such as single domain antibodies can be used in studies to further investigate the role of CD318 in disease. The method for detecting CD318 is roughly as follows: obtain cell and/or tissue samples; detect the level of CD318 in the samples.
本发明的CD318结合分子可用于诊断目的，用来检测、诊断或监控与CD318相关的疾病和/或病况。本发明提供使用本领域技术人员已知的经典免疫组织学方法检测样本中CD318的存在。可以体内或体外进行CD318的检测。适用于检测CD318的存在的方法实例包括ELISA、FACS、RIA等。The CD318-binding molecules of the invention may be used for diagnostic purposes to detect, diagnose or monitor CD318-related diseases and/or conditions. The present invention provides for the detection of the presence of CD318 in a sample using classical immunohistological methods known to those skilled in the art. Detection of CD318 can be performed in vivo or in vitro. Examples of methods suitable for detecting the presence of CD318 include ELISA, FACS, RIA, etc.
对于诊断应用来说，通常用可检测的标记基团来标记结合分子例如单域抗体。合适的标记基团包括(但不限于)以下：放射性同位素或放射性核素(例如，3H、14C、15N、35S、90Y、99Tc、111In、125I、131I)、荧光基团(例如，FITC、罗丹明、镧系元素磷光体)、酶促基团(例如，辣根过氧化物酶、β根半乳糖苷酶、荧光素酶、碱性磷酸酶)、化学发光基团、生物素基基团或由二级报导体识别的预定多肽表位(例如，亮氨酸拉链对序列、用于二级抗体的结合位点、金属结合结构域、表位标签)、MRI(磁共振成像)或CT(电子计算机X射线断层扫描技术)造影剂。用于标记蛋白质的各种方法在本领域中已知且可用来进行本发明。For diagnostic applications, binding molecules such as single domain antibodies are often labeled with detectable labeling groups. Suitable labeling groups include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 3H, 14C, 15N, 35S, 90Y, 99Tc, 111In, 125I, 131I), fluorescent groups (e.g., FITC, Rodan fluorophores, lanthanide phosphors), enzymatic groups (e.g., horseradish peroxidase, beta-galactosidase, luciferase, alkaline phosphatase), chemiluminescent groups, biotinyl groups or a predetermined polypeptide epitope recognized by a secondary reporter (e.g., leucine zipper pair sequence, binding site for secondary antibody, metal binding domain, epitope tag), MRI (magnetic resonance imaging) or CT (Computed X-ray tomography) contrast agent. Various methods for labeling proteins are known in the art and can be used to carry out the present invention.
本发明的另一方面提供检测与本发明的抗体竞争结合CD318的测试分子的存在的方法。一种所述测定的实例将涉及在存在或不存在测试分子的情形下检测含有一定量CD318的溶液中的游离抗体的量。游离抗体(即，未结合CD318的抗体)的量增加将表示测试分子能与该抗体竞争结合CD318。在一个实施方案中，用标记基团标记抗体。或者，标记测试分子并在存在或不存在抗体的情形下监控游离测试分子的量。Another aspect of the invention provides a method of detecting the presence of a test molecule that competes with an antibody of the invention for binding to CD318. An example of such an assay would involve detecting the amount of free antibody in a solution containing an amount of CD318 in the presence or absence of a test molecule. An increased amount of free antibody (i.e., antibody that does not bind CD318) will indicate that the test molecule is able to compete with the antibody for binding to CD318. In one embodiment, the antibody is labeled with a labeling group. Alternatively, the test molecule is labeled and the amount of free test molecule is monitored in the presence or absence of antibody.
本发明还提供了用于检测CD318水平的检测试剂盒，该试剂盒包括识别CD318蛋白的抗体，用于溶解样本的裂解介质，检测所需的通用试剂和缓冲液，如各种缓冲液、检测标记、检测底物等。该检测试剂盒可以是体外诊断装置。The invention also provides a detection kit for detecting CD318 levels. The kit includes an antibody that recognizes CD318 protein, a lysis medium for dissolving the sample, and general reagents and buffers required for detection, such as various buffers, detection Labeling, detection substrate, etc. The detection kit may be an in vitro diagnostic device.
下文将以具体实施例的方式阐述本发明。应理解，这些实施例仅仅是阐述性的，并非意图限制本发明的范围。实施例中所用到的方法和材料，除非另有说明，否则均为本领域常规的材料和方法。The present invention will be explained below by way of specific examples. It should be understood that these examples are illustrative only and are not intended to limit the scope of the invention. Unless otherwise stated, the methods and materials used in the examples are all conventional materials and methods in the art.
实施例Example
实施例1：重组人CD318蛋白表达载体构建与真核表达Example 1: Construction and eukaryotic expression of recombinant human CD318 protein expression vector
1、基因序列的合成及蛋白的表达载体的构建1. Synthesis of gene sequences and construction of protein expression vectors
从Uniport下载人CD318蛋白序列(https://www.uniprot.org/uniprot)，经密码子在线优化工具(http://www.jcat.de/#opennewwindow)优化后，交由生工生物工程(上海)股份有限公司合成CD318胞外段基因序列。同时在该基因序列的3’端添加avi-tag和6×his-tag的核酸序列，融合基因序列编码SEQ ID  NO:42所示的氨基酸序列，融合基因序列如SEQ ID NO:43所示。通过分子克隆，拼接产物用TaKaRa无缝克隆试剂盒克隆到pTT5中，获得表达载体。Download the human CD318 protein sequence from Uniport (https://www.uniprot.org/uniprot), optimize it with the codon online optimization tool (http://www.jcat.de/#opennewwindow), and submit it to Sangon Bioengineering (Shanghai) Co., Ltd. synthesized the CD318 extracellular segment gene sequence. At the same time, the nucleic acid sequences of avi-tag and 6×his-tag are added to the 3' end of the gene sequence, and the fused gene sequence encodes SEQ ID The amino acid sequence shown in NO:42 and the fusion gene sequence are shown in SEQ ID NO:43. Through molecular cloning, the spliced product was cloned into pTT5 using TaKaRa seamless cloning kit to obtain an expression vector.
2、重组人CD318蛋白的表达、纯化2. Expression and purification of recombinant human CD318 protein
以获得的表达载体转染293T细胞(ATCC)5天后，收集培养上清，用AKTA Explorer 100(GE)纯化重组人CD318蛋白。CD318蛋白经SDS-PAGE电泳后通过考马斯亮蓝染色显示其大小约110kDa左右，结果如图1所示。Five days after the obtained expression vector was transfected into 293T cells (ATCC), the culture supernatant was collected and the recombinant human CD318 protein was purified using AKTA Explorer 100 (GE). The CD318 protein was electrophoresed by SDS-PAGE and stained with Coomassie brilliant blue, showing that its size was approximately 110 kDa. The results are shown in Figure 1.
实施例2：CD318 VHH抗体的制备Example 2: Preparation of CD318 VHH antibodies
1、VHH噬菌体展示文库构建1. VHH phage display library construction
采用一步法构建纳米抗体噬菌体展示文库，即将羊驼纳米抗体VHH基因连接到噬菌体展示载体中。A one-step method was used to construct a nanobody phage display library, that is, the alpaca nanobody VHH gene was connected to the phage display vector.
1)羊驼免疫1)Alpaca immunity
羊驼免疫服务由成都阿帕克生物科技有限公司提供服务，具体操作流程如下：Alpaca immunization services are provided by Chengdu Apak Biotechnology Co., Ltd. The specific operation process is as follows:
A羊驼选择：选择健康强壮、精神状态良好、体型适中的羊驼，挑选的羊驼毛色光亮，无受伤不适症状。挑选好动物，先预养1周左右以淘汰有些不合格的动物，使后期的实验能顺利进行。A. Alpaca selection: Choose alpacas that are healthy, strong, in good spirits, and of moderate size. The selected alpacas have bright wool and no symptoms of injury or discomfort. Select good animals and raise them for about a week first to eliminate some unqualified animals so that later experiments can proceed smoothly.
B免疫方案：挑选好羊驼并确保动物适合，记录耳号后开始免疫实验。一共进行4次免疫。免疫方案如下：D0，免疫前取血10mL，作为阴性血清对照，将抗原0.5mg与CFA 1ml混匀后皮下注射；D21，将抗原0.25mg与CFA 1ml混匀后皮下注射；D28，取血10ml；D42，将抗原0.25mg与CFA 1ml混匀后皮下注射；D49，采50mL外周血分离淋巴细胞；D63，将抗原0.25mg与CFA 1ml混匀后皮下注射；D70，采50mL外周血分离淋巴细胞。Immunization plan B: Select the alpaca and ensure that the animal is suitable. Record the ear number and start the immunity experiment. A total of 4 immunizations were performed. The immunization plan is as follows: D0, take 10 mL of blood before immunization, and use it as a negative serum control. Mix 0.5 mg of antigen and 1 ml of CFA and then inject it subcutaneously; D21, mix 0.25 mg of antigen and 1 ml of CFA and inject it subcutaneously; D28, take 10 ml of blood. ; D42, mix 0.25 mg of antigen and 1 ml of CFA and then inject subcutaneously; D49, collect 50 mL of peripheral blood to isolate lymphocytes; D63, mix 0.25 mg of antigen and 1 ml of CFA and inject subcutaneously; D70, collect 50 mL of peripheral blood to isolate lymphocytes. .
C血清检测：C serum test:
a).将抗原用0.05M碳酸盐buffer(pH9.6)稀释至2μg/mL，按100μL/well，4℃包被过夜；a). Dilute the antigen to 2μg/mL with 0.05M carbonate buffer (pH9.6), and coat at 100μL/well overnight at 4°C;
b).弃包被液，PBST洗涤3次，每孔加入300μL 5％脱脂牛奶，37℃封闭1h；b). Discard the coating solution, wash 3 times with PBST, add 300 μL of 5% skim milk to each well, and block at 37°C for 1 hour;
c).PBST洗涤3次，加入100μL/孔的血清稀释液(从1:2000开始倍比稀释)，37℃，孵育45min；c). Wash 3 times with PBST, add 100 μL/well of serum dilution (dilution starting from 1:2000), incubate at 37°C for 45 minutes;
d).PBST洗涤5次，分别加入辣根过氧化物酶标记的羊抗Alpaca二抗(用PBS按1:1W稀释)，100μL/孔，37℃孵育45min；d). Wash 5 times with PBST, add horseradish peroxidase-labeled goat anti-Alpaca secondary antibody (diluted 1:1W with PBS), 100 μL/well, and incubate at 37°C for 45 minutes;
e).PBST洗板5次。加入TMB显色液显色，100μL/孔，37℃，5min，加入终止液终止反应，50μL/孔，于450nm下测光密度。e).Wash the plate 5 times with PBST. Add TMB chromogenic solution to develop color, 100 μL/well, 37°C, 5 min. Add stop solution, 50 μL/well, to terminate the reaction, and measure the optical density at 450 nm.
2)cDNA合成2) cDNA synthesis
提取PBMC总RNA：用Trizol保存的外周血淋巴细胞冰上溶解后转移至1.5mL的离心管，加入1/5体积的氯仿震荡混匀，室温静置5分钟后4℃，12000g离心15分钟；将离心后的上清液转移到新的离心管，往新离心管中加入等体积的异丙醇，室温静置10分钟后4℃12000g离心10分钟，用75％乙醇清洗沉淀，4℃7500g离心5分钟后弃去上清，沉淀室温晾干后溶解于适量的无RNA酶的水中。从A260/280来分析RNA提取纯度，准备RNA转录。Extract PBMC total RNA: Dissolve peripheral blood lymphocytes preserved in Trizol on ice and transfer to a 1.5 mL centrifuge tube. Add 1/5 volume of chloroform and shake to mix, let stand at room temperature for 5 minutes, then centrifuge at 4°C at 12000g for 15 minutes; Transfer the centrifuged supernatant to a new centrifuge tube, add an equal volume of isopropanol to the new centrifuge tube, let it stand at room temperature for 10 minutes, then centrifuge at 12000g for 10 minutes at 4°C, wash the precipitate with 75% ethanol, 7500g at 4°C After centrifugation for 5 minutes, discard the supernatant, dry the pellet at room temperature and dissolve it in an appropriate amount of RNase-free water. Analyze RNA extraction purity from A260/280 and prepare for RNA transcription.
cDNA合成：使用SuperScriptTM IV First-Strand Synthesis System试剂盒，反转录得cDNA并-80℃保存。cDNA synthesis: Use the SuperScript TM IV First-Strand Synthesis System kit to reverse-transcribe cDNA and store it at -80°C.
3)VHH基因扩增3)VHH gene amplification
使用正向引物(VHH-F)和反向引物(CH2-R)以羊驼PBMC cDNA为模板，PCR扩增VHH-CH2基因，PCR反应条件如下：98℃预变性45秒后进入温度循环，98℃变性15秒，58℃退火20秒，72℃延伸 45秒，循环30次，72℃最终延伸7分钟。PCR产物经1.5％琼脂糖凝胶电泳后，用胶回收试剂盒(Promega)回收750bp的目的条带。使用正向引物(VHH-CH2-F)和反向引物(VHJ-R)以VHH-CH2为模板，PCR扩增VHH基因，PCR反应条件如下：98℃预变性45秒后进入温度循环，98℃变性15秒，60℃退火20秒，72℃延伸45秒，循环30次，72℃最终延伸7分钟。PCR产物经1.5％琼脂糖凝胶电泳后，用胶回收试剂盒(Promega)回收400bp的目的条带。Use forward primer (VHH-F) and reverse primer (CH2-R) to use alpaca PBMC cDNA as a template to PCR amplify the VHH-CH2 gene. The PCR reaction conditions are as follows: pre-denature at 98°C for 45 seconds and then enter the temperature cycle. Denaturation at 98℃ for 15 seconds, annealing at 58℃ for 20 seconds, extension at 72℃ 45 seconds, 30 cycles, final extension at 72°C for 7 minutes. After the PCR product was subjected to 1.5% agarose gel electrophoresis, a gel recovery kit (Promega) was used to recover the 750 bp target band. Use the forward primer (VHH-CH2-F) and the reverse primer (VHJ-R) to use VHH-CH2 as the template to PCR amplify the VHH gene. The PCR reaction conditions are as follows: pre-denature at 98°C for 45 seconds and then enter the temperature cycle, 98 Denaturation at ℃ for 15 seconds, annealing at 60℃ for 20 seconds, extension at 72℃ for 45 seconds, 30 cycles, and final extension at 72℃ for 7 minutes. After the PCR product was electrophoresed on a 1.5% agarose gel, a 400 bp target band was recovered using a gel recovery kit (Promega).
4)VHH文库构建4) VHH library construction
噬菌粒载体pcomb3X及VHH基因用SfiI DNA内切酶进行单酶切，50℃酶切16h，酶切后的pcomb3X载体经1％琼脂糖凝胶电泳后，用胶回收试剂盒(Promega)回收4000bp载体片段。酶切后的VHH基因直接用胶回收试剂盒进行过柱回收(400bp)。将VHH基因用T4DNA连接酶试剂盒(Invitrogen)连接到噬菌粒载体中，16℃连接过夜，取少量连接产物琼脂糖凝胶电泳检测连接效率。连接产物用MECK MILLIPOREF微孔滤膜进行脱盐。The phagemid vector pcomb3X and VHH genes were digested with SfiI DNA endonuclease for 16 hours at 50°C. The digested pcomb3X vector was subjected to 1% agarose gel electrophoresis and recovered using a gel recovery kit (Promega). 4000bp vector fragment. The digested VHH gene was directly purified through the column using a gel recovery kit (400 bp). The VHH gene was ligated into the phagemid vector using T4 DNA ligase kit (Invitrogen), and the ligation was carried out overnight at 16°C. A small amount of the ligation product was taken for agarose gel electrophoresis to detect the ligation efficiency. The ligation product is desalted using MECK MILLIPOREF microporous filter membrane.
将上述连接产物加入自制的TG1电转化感受态中，然后使用电转仪进行电击转化。取出50μL菌液用PBS进行梯度稀释102-105倍。取10μL各梯度稀释液在Amp/2YT plate上流线，37℃孵育过夜，以此计数并统计噬菌体抗体文库的大小。剩余的电转化后的细菌补2YT至500ml，加入含100μg/mL氨苄青霉素，30℃220rpm培养过夜。最后得到超过9E9的VHH免疫文库。电击转化后的抗体文库经过夜扩增，离心收集文库菌体，加终浓度20％甘油-80度保存。The above-mentioned ligation product was added to the self-made TG1 electroconversion competent cell, and then electroporation was performed using an electroporation instrument. Take out 50 μL of bacterial solution and perform gradient dilution 10 2 -10 5 times with PBS. Streamline 10 μL of each gradient dilution on an Amp/2YT plate and incubate at 37°C overnight to count and calculate the size of the phage antibody library. Add 2YT to the remaining electrotransformed bacteria to 500 ml, add ampicillin containing 100 μg/mL, and culture at 30°C and 220 rpm overnight. Finally, a VHH immune library exceeding 9E9 was obtained. The antibody library transformed by electroporation was amplified overnight, and the library cells were collected by centrifugation. The final concentration of 20% glycerol was added and stored at -80 degrees.
取冻存的部分羊驼天然抗体噬菌体展示文库菌种接种到2YT培养集中，接种密度为0.1OD，将菌液置于37℃、220rpm条件进行培养，大约1.5小时后，菌液密度达到0.6OD，此时加入20倍于菌体数的M13KO7噬菌体静置30分钟侵染，然后30℃、220rpm培养过夜。第二天，10000g离心菌液，收集培养上清，向培养上清中加入1/4体积的PEG/NaCl溶液(20％PEG8000，2.5M NaCl)，混匀后冰浴1小时。冰浴结束后，8000g离心10分钟收集沉淀，10％Glycerol/PBST溶解沉淀即得到VHH噬菌体展示文库。测OD268，并将其分装至1.5mL离心管中，6OD/管，-80℃保存。Inoculate some of the frozen alpaca natural antibody phage display library strains into the 2YT culture set at an inoculation density of 0.1OD. Place the bacterial solution at 37°C and 220rpm for culture. After about 1.5 hours, the density of the bacterial solution reaches 0.6OD. , at this time, add 20 times the number of cells of M13KO7 phage and let it stand for 30 minutes for infection, and then culture it at 30°C and 220rpm overnight. The next day, centrifuge the bacterial liquid at 10,000g, collect the culture supernatant, add 1/4 volume of PEG/NaCl solution (20% PEG8000, 2.5M NaCl) to the culture supernatant, mix well and incubate on ice for 1 hour. After the ice bath, centrifuge at 8000g for 10 minutes to collect the precipitate. Dissolve the precipitate in 10% Glycerol/PBST to obtain the VHH phage display library. Measure OD268, and aliquot into 1.5 mL centrifuge tubes, 6 OD/tube, and store at -80°C.
2、淘选CD318 VHH抗体2. Panning for CD318 VHH antibodies
1)重组人CD318蛋白生物素化1) Biotinylation of recombinant human CD318 protein
用生物素化试剂盒(易锦生物)按试剂盒说明书，将重组人CD318蛋白的avi-tag进行生物素修饰，得到生物素化的CD318蛋白。取10μg上述生物素修饰的重组蛋白加入到经PBS洗涤3次的100μL链霉亲和素磁珠(DynaBeads 280)中，置于旋转摇床上，速度20转每分钟，室温偶联1小时后PBS洗涤3次。Using a biotinylation kit (Yijin Biotechnology) according to the kit instructions, the avi-tag of the recombinant human CD318 protein was biotin-modified to obtain biotinylated CD318 protein. Add 10 μg of the above biotin-modified recombinant protein to 100 μL streptavidin magnetic beads (DynaBeads 280) washed three times with PBS, place on a rotary shaker at a speed of 20 rpm, couple at room temperature for 1 hour and then use PBS Wash 3 times.
2)封闭噬菌体文库及阴性磁珠2) Blocked phage library and negative magnetic beads
取VHH lib一支，室温融化后，各加入200μL 5％BSA/PBST，置于旋转摇床速度20转每分钟，室温旋转封闭1小时，这些噬菌体为Input1。同时取100μL未偶联蛋白的DynaBeads 280，PBS洗涤3次后，加入1mL 1％BSA/PBS，按上述条件旋转孵育1小时。Take a tube of VHH lib, melt it at room temperature, add 200 μL of 5% BSA/PBST to each, place it on a rotating shaker at a speed of 20 rpm, and rotate and block at room temperature for 1 hour. These phages are Input1. At the same time, take 100 μL of DynaBeads 280 that is not coupled to protein, wash it three times with PBS, add 1 mL of 1% BSA/PBS, and rotate and incubate for 1 hour according to the above conditions.
3)封闭阳性磁珠3) Block positive magnetic beads
向上述偶联CD318的磁珠中加入1mL 1％BSA/PBS 20转每分钟，室温旋转封闭1小时。Add 1mL of 1% BSA/PBS to the above-mentioned CD318-coupled magnetic beads at 20 rpm, and rotate and block at room temperature for 1 hour.
4)阴性淘选4) Negative panning
为去除与磁珠相互作用的抗体，有必要进行阴性淘选。将经BSA封闭的噬菌体文库和未偶联抗原的磁珠混合，按上述条件旋转孵育1小时。孵育结束后将噬菌体磁珠混合物置于磁力架上，待磁珠都贴壁之 后，将上清转移至新的EP管中。To remove antibodies that interact with the beads, negative panning is necessary. Mix the BSA-blocked phage library and the magnetic beads without antigen coupling, and incubate for 1 hour with rotation under the above conditions. After the incubation, place the phage magnetic bead mixture on a magnetic stand and wait until all the magnetic beads adhere to the wall. Finally, transfer the supernatant to a new EP tube.
5)阳性淘选5)Positive panning
将上述封闭后的偶联有CD318蛋白的磁珠加入到阴性淘选后的噬菌体上清中进行阳性淘选，20转每分钟，室温旋转封闭1小时。孵育结束后用1mL PBST(0.1％Tween-20inPBS)洗涤磁珠，重复洗涤10次。洗涤结束后加入1mL 100mM甘氨酸(pH2.0)，置于旋转摇床上速度设定20转每分钟，旋转洗脱10分钟。洗脱结束后将EP管置于磁力架上，待磁珠都贴壁后将洗脱液转移至新的EP管中。向洗脱液中加入0.2mL 1M Tris-HCl溶液(pH 8.0)进行中和。将中和后的洗脱液加入到30mL OD600约为0.6的TG1菌液中静置侵染30分钟，然后加入20倍于菌体数的M13KO7噬菌体，静置侵染30分钟，最后加入100mL 2YT培养基及终浓度为100μg/mL的氨苄霉素和卡那霉素，30℃、220rpm培养过夜。第二天按上述收获噬菌体文库的方法收获噬菌体，此时得到的噬菌体为Input2。Add the above-mentioned blocked magnetic beads coupled with CD318 protein to the phage supernatant after negative panning to perform positive panning, and rotate and block at room temperature for 1 hour at 20 rpm. After the incubation, wash the magnetic beads with 1mL of PBST (0.1% Tween-20 in PBS) and repeat the washing 10 times. After washing, add 1mL of 100mM glycine (pH2.0), place it on a rotary shaker and set the speed to 20 rpm, and rotate for 10 minutes for elution. After the elution is completed, place the EP tube on the magnetic stand. After the magnetic beads are all attached to the wall, transfer the eluent to a new EP tube. Add 0.2mL of 1M Tris-HCl solution (pH 8.0) to the eluent for neutralization. Add the neutralized eluate to 30mL of TG1 bacterial solution with an OD600 of about 0.6 and let it stand for 30 minutes. Then add 20 times the number of M13KO7 phage and let it stand for 30 minutes. Finally, add 100mL of 2YT. Culture medium and ampicillin and kanamycin at a final concentration of 100 μg/mL were cultured overnight at 30°C and 220 rpm. The next day, harvest the phage according to the above method for harvesting the phage library. The phage obtained at this time is Input2.
6)重复阳性淘选6) Repeat positive panning
按上述淘选方法进行2次重复，即将Input2进行下一轮阴性淘选和阳性淘选得到Input3。不同之处在于Input3进行淘选得到的洗脱液侵染TG1后，不加M13KO7，而是取10μL菌液用PBS进行梯度稀释，取103、104、105三个稀释梯度各100μL菌液涂布2YT/Amp平板，30℃培养过夜，剩余的菌液30℃、220rpm培养过夜。Repeat the above panning method twice, that is, Input2 will undergo the next round of negative panning and positive panning to obtain Input3. The difference is that after the eluate obtained by panning in Input3 is infected with TG1, M13KO7 is not added. Instead, 10 μL of bacterial solution is used for gradient dilution with PBS, and 100 μL of each of the three dilution gradients of 10 3 , 10 4 , and 10 5 is taken. Spread the bacterial solution onto a 2YT/Amp plate and incubate at 30°C overnight, and incubate the remaining bacterial solution at 30°C and 220rpm overnight.
7)ELISA筛选阳性抗体7) ELISA screening for positive antibodies
用移液器枪头随机挑取上述平板中的TG1单克隆到800μL含有10×自诱导2YT/Amp的深孔板中，深孔板粘覆透气膜，37℃、220rpm培养3小时后，30℃、220rpm培养过夜。ELISA板中包被重组人CD318蛋白，每孔100ng。次日，先在深孔板中取50μL保菌，其余的4000rpm离心10分钟，去除孔内培养基保留菌体沉淀，每孔加入100μL TES溶液(20％蔗糖、0.1mM EDTA、50mM Tris-HCl，pH 8.0)，震荡使菌体重新悬浮后冰浴30分钟后再加入200μL超纯水震荡混匀30min，震荡结束后4000rpm离心10分钟，此时深孔板中的上清溶液即为含有抗体的周质腔提取物。用洗板机清洗ELISA板三次，然后加入200μL 1％BSA/PBS，37℃封闭1小时。去除ELISA板中的封闭液，加入100μL上述周质腔提取物，37℃孵育1小时，用洗板机清洗3次，加入100μL Chicken anti-HA HRP(1％BSA/PBS)，37℃孵育1小时，用洗板机清洗3次，加入100μL TMB显色液，37℃显色10分钟，加入100μL Stop solution终止液。用酶标仪读取OD450值，将读值高于背景值3倍的克隆进行sanger测序，获得抗体的基因序列。Use a pipette tip to randomly pick the TG1 monoclonal from the above plate into an 800 μL deep-well plate containing 10× auto-induction 2YT/Amp. The deep-well plate is covered with a breathable film. After culturing for 3 hours at 37°C and 220 rpm, 30 Cultivation was carried out overnight at 220 rpm. The ELISA plate was coated with recombinant human CD318 protein, 100ng per well. The next day, first take 50 μL of preserved bacteria in the deep well plate, and centrifuge the rest at 4000 rpm for 10 minutes. Remove the culture medium in the well to retain the bacterial sediment. Add 100 μL of TES solution (20% sucrose, 0.1mM EDTA, 50mM Tris-HCl, etc.) to each well. pH 8.0), shake to resuspend the bacteria, then incubate on ice for 30 minutes, then add 200 μL of ultrapure water and shake for 30 minutes. After shaking, centrifuge at 4000 rpm for 10 minutes. At this time, the supernatant solution in the deep well plate is the antibody-containing solution. Periplasmic cavity extract. Wash the ELISA plate three times with a plate washer, then add 200 μL 1% BSA/PBS and block at 37°C for 1 hour. Remove the blocking solution from the ELISA plate, add 100 μL of the above periplasmic cavity extract, incubate at 37°C for 1 hour, wash 3 times with a plate washer, add 100 μL of Chicken anti-HA HRP (1% BSA/PBS), and incubate at 37°C for 1 hour hour, wash 3 times with a plate washer, add 100 μL TMB chromogenic solution, develop color at 37°C for 10 minutes, and add 100 μL Stop solution. Use a microplate reader to read the OD450 value, and perform Sanger sequencing on the clones whose reading value is 3 times higher than the background value to obtain the gene sequence of the antibody.
8)验证阳性克隆8) Verify positive clones
根据测序结果，选取抗体CDR3氨基酸序列差异较大的克隆重新接种并诱导过夜，按上述ELISA方法再次验证所选克隆能否结合CD318。最终得到4A4、4A8、4A10、4B2、4B7、4B12、4F5、4G2八条抗体序列。4A4、4A8、4A10、4B2、4B7、4G2的氨基酸序列分别为SEQ ID NO:19-24所示。Based on the sequencing results, clones with large differences in the amino acid sequences of the antibody CDR3 were selected and re-inoculated and induced overnight, and the ability of the selected clones to bind to CD318 was verified again according to the above ELISA method. Finally, eight antibody sequences were obtained, 4A4, 4A8, 4A10, 4B2, 4B7, 4B12, 4F5, and 4G2. The amino acid sequences of 4A4, 4A8, 4A10, 4B2, 4B7, and 4G2 are shown in SEQ ID NO: 19-24 respectively.
实施例3：含抗人CD318嵌合抗原受体元件的逆转录病毒原液制备Example 3: Preparation of retrovirus stock solution containing anti-human CD318 chimeric antigen receptor element
1、靶向人CD318抗原的CAR的制备1. Preparation of CAR targeting human CD318 antigen
基因合成或克隆含抗人CD318抗原的单域抗体VHH、铰链区、跨膜区和胞内信号段的嵌合抗原受体序列，其结构如图2所示。根据装载VHH的不同，将嵌合抗原受体分别命名为4A4-BBz、4A8-BBz、4A10-BBz、4B2-BBz、4B7-BBz、4B12-BBz、4F5-BBz、4G2-BBz，4A4-BBz、4A8-BBz、4A10-BBz、4B2-BBz、4B7-BBz、4G2-BBz的氨基酸序列分别如SEQ ID NO.30-35所示，核苷酸序列分别如SEQ ID NO.36至SEQ  ID NO.41所示。The chimeric antigen receptor sequence containing the single-domain antibody VHH, hinge region, transmembrane region and intracellular signal segment against human CD318 antigen is genetically synthesized or cloned. Its structure is shown in Figure 2. According to the different loaded VHHs, the chimeric antigen receptors are named 4A4-BBz, 4A8-BBz, 4A10-BBz, 4B2-BBz, 4B7-BBz, 4B12-BBz, 4F5-BBz, 4G2-BBz, and 4A4-BBz. The amino acid sequences of , 4A8-BBz, 4A10-BBz, 4B2-BBz, 4B7-BBz, and 4G2-BBz are shown in SEQ ID NO.30-35 respectively, and the nucleotide sequences are shown in SEQ ID NO.36 to SEQ respectively. Shown as ID NO.41.
以逆转录载体MSGV为骨架载体，构建了表达4A4-BBz、4A8-BBz、4A10-BBz、4B2-BBz、4B7-BBz、4B12-BBz、4F5-BBz、4G2-BBz克隆的嵌合抗原受体的逆转录病毒质粒。挑选测序正确的克隆，接种菌液至200ml 2YT培养基中，过夜摇菌，并按照NucleoBondXtra Maxi EF试剂盒说明书完成大提质粒。Using the reverse transcription vector MSGV as the backbone vector, chimeric antigen receptors expressing 4A4-BBz, 4A8-BBz, 4A10-BBz, 4B2-BBz, 4B7-BBz, 4B12-BBz, 4F5-BBz, and 4G2-BBz clones were constructed. of retroviral plasmids. Select the correctly sequenced clone, inoculate the bacterial solution into 200ml 2YT medium, shake the culture overnight, and follow the instructions of the NucleoBondXtra Maxi EF kit to complete the plasmid purification.
2、逆转录病毒包装2. Retrovirus packaging
用阳离子聚合物PEI包装逆转录病毒，流程如下：分别用无血清600μL DMEM稀释36μL PEI和逆转录病毒包装质粒(病毒主质粒6μg、Gag-pol3.8μg、vsvg1.5μg)；然后将PEI/DMEM加入质粒/DMEM混合物，涡旋震荡混匀，在室温下静置15分钟；将质粒-PEI复合物加入预先铺板的293T细胞。转染后16h换液，在48h后收集第一次病毒上清，72h后收集第二次病毒上清，并用0.45μm滤器过滤，分装至1.5mL离心管，1ml/管，-80℃保存备用。Use cationic polymer PEI to package retrovirus. The process is as follows: dilute 36 μL PEI and retrovirus packaging plasmid (viral main plasmid 6 μg, Gag-pol 3.8 μg, vsvg 1.5 μg) with 600 μL serum-free DMEM respectively; then PEI/DMEM Add the plasmid/DMEM mixture, vortex to mix, and let stand at room temperature for 15 minutes; add the plasmid-PEI complex to the pre-plated 293T cells. Change the medium 16 hours after transfection, collect the first viral supernatant after 48 hours, collect the second viral supernatant after 72 hours, filter with a 0.45 μm filter, and aliquot into 1.5 mL centrifuge tubes, 1 ml/tube, and store at -80°C spare.
实施例4：CD318 CAR-T细胞的制备和CAR阳性率测定Example 4: Preparation of CD318 CAR-T cells and determination of CAR positivity rate
1、PBMC分离与活化1. PBMC isolation and activation
领取一支PBMC，核对患者个体识别码无误后，进行复苏。用X-VIVO完全培养基调整细胞密度为1×106/mL。将复苏后恢复过夜的PBMC，轻柔吹打，用70μm细胞筛网过滤后，转入50ml离心管中，室温离心，1500rpm，离心5min，弃上清。用适量的DPBS重悬细胞，取20μl与台盼蓝1:1混匀后计数，计算活率、CD3+细胞数，然后取所需体积的细胞，室温1500rpm，离心5min，弃上清，用于分选。按照CD3/CD28磁珠与CD3+细胞1:1比例，计算磁珠用量，磁珠量＝【CD3+细胞数/4×105】μl。清洗磁珠：取无菌流式管一支，加入2ml的DPBS，同时加入磁珠，磁力架上静置1min后，弃上清。将流式管移除磁力架，取与细胞悬液等体积的DPBS或X-VIVO15重悬，加入细胞悬液中磁珠和细胞于15ml离心管混合后，在旋转混合仪上孵育。室温孵育30min。孵育完成后，轻柔将细胞转移至无菌流式管中，用1ml DPBS冲洗15ml离心管，冲洗液并入同一流式管中。将无菌流式管移至磁力架上，静置1min后，吸弃未吸附的液体。将无菌流式管移出磁力架，用1ml CAR-T培养基重悬细胞，并用CAR-T培养基冲洗管壁2次，全部收集转移至同一离心管内。用CAR-T培养基调整细胞密度为1×106ml，添加IL-2终浓度为200IU/ml，置37℃，5％CO2培养箱培养两天。Receive a PBMC, verify that the patient's individual identification number is correct, and then perform resuscitation. Use X-VIVO complete medium to adjust the cell density to 1×10 6 /mL. After resuscitation, PBMC that had been recovered overnight were pipetted gently, filtered with a 70 μm cell mesh, transferred to a 50 ml centrifuge tube, centrifuged at room temperature, 1500 rpm, for 5 min, and the supernatant was discarded. Resuspend the cells with an appropriate amount of DPBS, mix 20 μl with trypan blue 1:1 and count, calculate the viability and CD3+ cell number, then take the required volume of cells, centrifuge at 1500 rpm at room temperature for 5 min, discard the supernatant, and use sorting. Calculate the amount of magnetic beads according to the 1:1 ratio of CD3/CD28 magnetic beads to CD3+ cells. The amount of magnetic beads = [number of CD3+ cells/4×10 5 ]μl. Clean the magnetic beads: Take a sterile flow tube, add 2 ml of DPBS, and add magnetic beads at the same time. After standing on the magnetic stand for 1 minute, discard the supernatant. Remove the magnetic stand from the flow tube, take an equal volume of DPBS or X-VIVO15 and resuspend the cell suspension, add the magnetic beads and cells in the cell suspension, mix in a 15ml centrifuge tube, and incubate on a rotating mixer. Incubate at room temperature for 30 minutes. After the incubation is completed, gently transfer the cells to a sterile flow tube, rinse the 15ml centrifuge tube with 1ml DPBS, and merge the rinse solution into the same flow tube. Move the sterile flow tube to the magnetic stand, let it stand for 1 minute, and then aspirate the unadsorbed liquid. Remove the sterile flow tube from the magnetic stand, resuspend the cells in 1 ml of CAR-T culture medium, rinse the tube wall twice with CAR-T culture medium, and transfer all cells to the same centrifuge tube. Use CAR-T culture medium to adjust the cell density to 1×10 6 ml, add IL-2 to a final concentration of 200IU/ml, and culture in a 37°C, 5% CO 2 incubator for two days.
2、病毒原液感染与培养2. Infection and culture of virus stock solution
将活化后的T细胞调整为5×105/mL，在24孔板中分别加入1ml T细胞和1ml病毒原液，每孔加2μL polybrene，32℃，2500rpm，离心1.5h。弃去上清液，每孔加入1ml T细胞培养基(含IL-2 300IU/ml)。将培养板置于37℃，5％CO2培养箱中培养。感染后24h，转至6孔板，每天观察细胞的密度，适时补加含IL-2 300IU/ml的T细胞培养液，使T细胞的密度维持在1×106/ml左右，使细胞扩增。Adjust the activated T cells to 5×10 5 /mL, add 1 ml of T cells and 1 ml of virus stock solution to a 24-well plate, add 2 μL polybrene to each well, and centrifuge at 32°C, 2500 rpm for 1.5 h. Discard the supernatant and add 1 ml of T cell culture medium (containing IL-2 300IU/ml) to each well. Place the culture plate in a 37°C, 5% CO2 incubator. 24 hours after infection, transfer to a 6-well plate, observe the cell density every day, and add T cell culture medium containing IL-2 300IU/ml in a timely manner to maintain the density of T cells at about 1×10 6 /ml and allow the cells to expand. increase.
3、CAR阳性率检测3. CAR positive rate detection
逆转录病毒感染的T淋巴细胞在病毒感染72h后检测CAR阳性率。针对含4A4、4A8、4A10、4B2、4B7、4B12、4F5、4G2克隆的嵌合抗原受体组和阴性未感染对照组NT，分别取1×106个细胞，离心去除培养基，用500μL PBS洗细胞一次后用100μL重悬置于流式上样管中(BD)。加入生物素-标记的CD318抗原(1:200)四度孵育30分钟。PBS洗细胞一次后按1:100加入二抗PE-SA链霉亲合素(BioLegend)四度避光孵育30分钟。500μL PBS洗细胞后用200μL PBS重悬细胞并上机检测，CAR-T阳性率流式分析结果如图3所示。根据图3结果，排除可能存在假阳性的两条序列4B12和4F5，最终得到效果较好的6条 CD318-VHH抗体序列，分别为：4A4、4A8、4A10、4B2、4B7、4G2。The positive rate of CAR was detected in retrovirus-infected T lymphocytes 72 hours after virus infection. For the chimeric antigen receptor group containing 4A4, 4A8, 4A10, 4B2, 4B7, 4B12, 4F5, and 4G2 clones and the negative uninfected control group NT, take 1×10 6 cells respectively, centrifuge to remove the culture medium, and use 500 μL PBS Wash the cells once and resuspend them in 100 μL in a flow cytometry tube (BD). Biotin-labeled CD318 antigen (1:200) was added and incubated four times for 30 minutes. After washing the cells once with PBS, add secondary antibody PE-SA streptavidin (BioLegend) at a ratio of 1:100 and incubate at 4 degrees in the dark for 30 minutes. After washing the cells with 500 μL PBS, resuspend the cells in 200 μL PBS and run the test on the machine. The results of flow cytometry analysis of CAR-T positivity rate are shown in Figure 3. According to the results in Figure 3, the two sequences 4B12 and 4F5 that may have false positives were eliminated, and finally 6 sequences with better results were obtained. The CD318-VHH antibody sequences are: 4A4, 4A8, 4A10, 4B2, 4B7, and 4G2.
实施例5：基于抗人CD318 CAR-T细胞功能分析Example 5: Functional analysis of CAR-T cells based on anti-human CD318
1、抗人CD318 CAR-T细胞CD107a表达分析1. Analysis of CD107a expression of anti-human CD318 CAR-T cells
将含不同抗体克隆的CAR-T细胞分别与靶细胞(CD318阳性表达的胰腺癌细胞系BxPC3)与对照靶细胞(CD318阴性表达的人神经胶质细胞瘤细胞U251)按1:1的效靶比(效应细胞和靶细胞均为3×105个)混合置于37℃，5％CO2培养箱中共孵育4小时后，流式检测各组样品中表达CD107a的细胞分别占CAR-T阳性细胞数的比例。评价CAR-T细胞在受到靶细胞刺激后的脱颗粒反应。CD107a表达的流式分析结果如图4所示。CAR-T cells containing different antibody clones were used with target cells (CD318-positive pancreatic cancer cell line BxPC3) and control target cells (CD318-negative human glioma cell U251) at a ratio of 1:1. After a total of 4 hours of incubation in a 37°C, 5% CO 2 incubator, the proportion of cells expressing CD107a in each group of samples that were positive for CAR-T was measured by flow cytometry. Cell number ratio. Evaluate the degranulation response of CAR-T cells after stimulation by target cells. The results of flow cytometry analysis of CD107a expression are shown in Figure 4.
2、抗人CD318 CAR-T细胞细胞因子分泌能力检测2. Detection of cytokine secretion ability of anti-human CD318 CAR-T cells
将含不同抗体克隆的CAR-T细胞分别与靶细胞(CD318阳性表达的细胞系BxPC3)，对照靶细胞(CD318阴性表达的细胞系U251)分别均按10:1，2:1的效靶比(靶细胞为3×104个)共孵育24小时后，收集其上清，利用ELISA(酶联免疫)方法检测IFN-γ和IL-2的分泌情况。IFN-γ和IL-2检测使用爱必信试剂盒检测(Human IFN-gamma ELISA Kit，Human IL-2ELISA kit)，实验步骤依据产品说明书进行。IFN-γ分泌的检测结果如图5，图6所示。IL-2分泌的检测结果如图7，图8所示。CAR-T cells containing different antibody clones were compared with target cells (CD318-positive cell line BxPC3) and control target cells (CD318-negative cell line U251) at an effect-to-target ratio of 10:1 and 2:1 respectively. (Target cells are 3×10 4 ) After incubation for 24 hours, the supernatant was collected, and the secretion of IFN-γ and IL-2 was detected using ELISA (enzyme-linked immunoassay) method. IFN-γ and IL-2 were detected using the Human IFN-gamma ELISA Kit, Human IL-2ELISA kit, and the experimental steps were performed according to the product instructions. The detection results of IFN-γ secretion are shown in Figure 5 and Figure 6. The detection results of IL-2 secretion are shown in Figure 7 and Figure 8.
3、抗人CD318 CAR-T细胞毒性实验3. Anti-human CD318 CAR-T cell toxicity experiment
CAR-T杀伤毒性实验通过检测CAR-T细胞体外对靶细胞的杀伤效果来评估CAR-T细胞的体外功能。以不同效靶比(以3×104个靶细胞为基准，效靶比分别为10:1和2:1)将T细胞分别与稳定表达萤火虫荧光素酶的CD318阳性靶细胞BxPC3-LUC-GFP，以及CD318阴性对照靶细胞U251-LUC-GFP共同培养，同时设置只有靶细胞的阳性对照。37℃过夜孵育后在培养体系中加入100μL萤光素酶反应底物，检测荧光值，通过以下公式计算杀伤效率：杀伤效率＝(阳性对照孔荧光值－实验孔荧光值)/阳性对照孔荧光值)×100％。实验分组及分析结果如图9，图10所示。The CAR-T killing toxicity experiment evaluates the in vitro function of CAR-T cells by detecting the killing effect of CAR-T cells on target cells in vitro. The T cells were treated with CD318-positive target cells stably expressing firefly luciferase BxPC3-LUC- at different effective-to-target ratios (based on 3×10 4 target cells, the effective-to-target ratios were 10:1 and 2:1 respectively). GFP, and CD318 negative control target cells U251-LUC-GFP were co-cultured, and a positive control with only target cells was set up. After overnight incubation at 37°C, add 100 μL of luciferase reaction substrate to the culture system, detect the fluorescence value, and calculate the killing efficiency through the following formula: Killing efficiency = (fluorescence value of positive control well - fluorescence value of experimental well)/fluorescence of positive control well value)×100%. The experimental grouping and analysis results are shown in Figure 9 and Figure 10.
以上所述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。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。 The above descriptions are only preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection scope of the present invention.

Claims (10)

  1. 一种CD318结合分子，包含抗CD318纳米抗体或其抗原结合片段，所述抗CD318纳米抗体的互补决定区CDR包含CDR1、CDR2和CDR3，其中CDR1包括SEQ ID NO:1、4、7、10、13和16中任一所示的序列、CDR2包括SEQ ID NO:2、5、8、11、14和17中任一所示的序列、和CDR3包括SEQ ID NO:3、6、9、12、15和18中任一所示的序列，A CD318-binding molecule comprising an anti-CD318 Nanobody or an antigen-binding fragment thereof. The complementarity determining region CDR of the anti-CD318 Nanobody includes CDR1, CDR2 and CDR3, wherein CDR1 includes SEQ ID NO: 1, 4, 7, 10, The sequence shown in any one of 13 and 16, CDR2 includes the sequence shown in any one of SEQ ID NO: 2, 5, 8, 11, 14 and 17, and CDR3 includes SEQ ID NO: 3, 6, 9, 12 , the sequence shown in any one of 15 and 18,
    优选地，所述CDR选自以下任一项：Preferably, the CDR is selected from any of the following:
    (1)序列如SEQ ID NO:1所示的CDR1、序列如SEQ ID NO:2所示的CDR2、序列如SEQ ID NO:3所示的CDR3，(1) CDR1 whose sequence is shown in SEQ ID NO:1, CDR2 whose sequence is shown in SEQ ID NO:2, and CDR3 whose sequence is shown in SEQ ID NO:3,
    (2)序列如SEQ ID NO:4所示的CDR1、序列如SEQ ID NO:5所示的CDR2、序列如SEQ ID NO:6所示的CDR3，(2) CDR1 whose sequence is shown in SEQ ID NO:4, CDR2 whose sequence is shown in SEQ ID NO:5, and CDR3 whose sequence is shown in SEQ ID NO:6,
    (3)序列如SEQ ID NO:7所示的CDR1、序列如SEQ ID NO:8所示的CDR2、序列如SEQ ID NO:9所示的CDR3，(3) CDR1 whose sequence is shown in SEQ ID NO:7, CDR2 whose sequence is shown in SEQ ID NO:8, and CDR3 whose sequence is shown in SEQ ID NO:9,
    (4)序列如SEQ ID NO:10所示的CDR1、序列如SEQ ID NO:11所示的CDR2、序列如SEQ ID NO:12所示的CDR3，(4) CDR1 whose sequence is shown in SEQ ID NO:10, CDR2 whose sequence is shown in SEQ ID NO:11, and CDR3 whose sequence is shown in SEQ ID NO:12,
    (5)序列如SEQ ID NO:13所示的CDR1、序列如SEQ ID NO:14所示的CDR2、序列如SEQ ID NO:15所示的CDR3，(5) CDR1 whose sequence is shown in SEQ ID NO:13, CDR2 whose sequence is shown in SEQ ID NO:14, and CDR3 whose sequence is shown in SEQ ID NO:15,
    (6)序列如SEQ ID NO:16所示的CDR1、序列如SEQ ID NO:17所示的CDR2、序列如SEQ ID NO:18所示的CDR3，(6) CDR1 whose sequence is shown in SEQ ID NO:16, CDR2 whose sequence is shown in SEQ ID NO:17, and CDR3 whose sequence is shown in SEQ ID NO:18,
    优选地，所述CD318结合分子具有选自以下一项或多项的特征：Preferably, the CD318 binding molecule has characteristics selected from one or more of the following:
    所述抗CD318纳米抗体的重链可变区序列如SEQ ID NO:19-24中任一所示，The heavy chain variable region sequence of the anti-CD318 Nanobody is as shown in any one of SEQ ID NO: 19-24,
    所述CD318结合分子是包含一条、两条或多条抗CD318纳米抗体或其抗原结合片段的单价或多价纳米抗体或单域抗体、或多特异性纳米抗体或单域抗体，The CD318-binding molecule is a monovalent or multivalent Nanobody or single domain antibody, or a multispecific Nanobody or single domain antibody that contains one, two or more anti-CD318 Nanobodies or antigen-binding fragments thereof,
    所述纳米抗体是骆驼重链抗体或软骨鱼重链抗体，The Nanobody is a camel heavy chain antibody or a cartilaginous fish heavy chain antibody,
    所述纳米抗体还包含重链恒定区，The Nanobody further comprises a heavy chain constant region,
    所述CD318结合分子为嵌合抗体或完全人抗体。The CD318 binding molecule is a chimeric antibody or a fully human antibody.
  2. 一种嵌合抗原受体，包含任选的信号肽序列、权利要求1所述的CD318结合分子、铰链区、跨膜区和胞内区，A chimeric antigen receptor comprising an optional signal peptide sequence, the CD318 binding molecule of claim 1, a hinge region, a transmembrane region and an intracellular region,
    优选地，胞内区包括胞内共刺激域和/或胞内信号域，Preferably, the intracellular region includes an intracellular costimulatory domain and/or an intracellular signaling domain,
    优选地，从N端到C端，该嵌合抗原受体依次含有信号肽、权利要求1所述的CD318结合分子、铰链区、跨膜区、胞内共刺激域和胞内信号域。Preferably, from the N terminus to the C terminus, the chimeric antigen receptor contains a signal peptide, the CD318 binding molecule of claim 1, a hinge region, a transmembrane region, an intracellular costimulatory domain and an intracellular signaling domain in order.
  3. 一种核酸分子，其具有选自以下任一项的序列：A nucleic acid molecule having a sequence selected from any of the following:
    (1)权利要求1所述CD318结合分子或权利要求2所述的嵌合抗原受体的编码序列； (1) The coding sequence of the CD318 binding molecule of claim 1 or the chimeric antigen receptor of claim 2;
    (2)(1)的互补序列；(2) The complementary sequence of (1);
    (3)(1)或(2)中任一序列的5-50bp的片段，(3) A 5-50 bp fragment of any sequence in (1) or (2),
    优选地，所述片段是引物。Preferably, the fragments are primers.
  4. 一种核酸构建物，包含权利要求3所述的核酸分子，A nucleic acid construct comprising the nucleic acid molecule of claim 3,
    优选地，所述核酸构建物是克隆载体、表达载体或整合载体。Preferably, the nucleic acid construct is a cloning vector, an expression vector or an integration vector.
  5. 一种宿主细胞，选自：A host cell selected from:
    (1)表达和/或分泌权利要求1所述CD318结合分子或权利要求2所述的嵌合抗原受体；(1) Express and/or secrete the CD318 binding molecule of claim 1 or the chimeric antigen receptor of claim 2;
    (2)包含权利要求3所述的核酸分子；和/或(2) comprising the nucleic acid molecule of claim 3; and/or
    (3)包含权利要求4所述的核酸构建物，(3) comprising the nucleic acid construct of claim 4,
    优选地，所述宿主细胞是免疫效应细胞，更优选T细胞。Preferably, the host cells are immune effector cells, more preferably T cells.
  6. 一种产生权利要求1所述的CD318结合分子或权利要求2所述的嵌合抗原受体的方法，包括：在适合产生CD318结合分子的条件下培养权利要求5所述的宿主细胞，和任选的从培养物中纯化所述CD318结合分子或嵌合抗原受体的步骤。A method for producing the CD318-binding molecule of claim 1 or the chimeric antigen receptor of claim 2, comprising: culturing the host cell of claim 5 under conditions suitable for producing the CD318-binding molecule, and any Optional step of purifying the CD318 binding molecule or chimeric antigen receptor from the culture.
  7. 一种药物组合物，包含权利要求1所述的CD318结合分子、权利要求2所述的嵌合抗原受体、权利要求3所述的核酸分子、权利要求4所述的核酸构建物或权利要求5所述的宿主细胞，和药学上可接受的辅料。A pharmaceutical composition comprising the CD318 binding molecule of claim 1, the chimeric antigen receptor of claim 2, the nucleic acid molecule of claim 3, the nucleic acid construct of claim 4, or the The host cell described in 5, and pharmaceutically acceptable excipients.
  8. 权利要求1所述的CD318结合分子、权利要求2所述的嵌合抗原受体、权利要求3所述的核酸分子、权利要求4所述的核酸构建物或权利要求5所述的宿主细胞在制备活化的免疫细胞中的用途，或在制备用于预防或治疗CD318表达相关的疾病或病况的药物中的用途，The CD318 binding molecule of claim 1, the chimeric antigen receptor of claim 2, the nucleic acid molecule of claim 3, the nucleic acid construct of claim 4 or the host cell of claim 5 is used in Use in the preparation of activated immune cells, or use in the preparation of medicaments for the prevention or treatment of diseases or conditions associated with CD318 expression,
    优选地，所述疾病或病况选自以下的一种或多种：乳腺癌、肺癌、肝癌、胰腺癌、卵巢癌、肾癌和结直肠癌。Preferably, the disease or condition is selected from one or more of the following: breast cancer, lung cancer, liver cancer, pancreatic cancer, ovarian cancer, kidney cancer and colorectal cancer.
  9. 一种检测CD318的试剂盒，所述的试剂盒包含权利要求1所述的CD318结合分子、权利要求3所述的核酸分子、权利要求4所述的核酸构建物或权利要求5所述的宿主细胞，A kit for detecting CD318, said kit comprising the CD318 binding molecule of claim 1, the nucleic acid molecule of claim 3, the nucleic acid construct of claim 4 or the host of claim 5 cell,
    优选地，所述试剂盒还包括用于检测CD318与所述CD318结合分子的结合的试剂；更优选地，所述检测结合的试剂是能与CD318结合分子连接的可检测标记物。Preferably, the kit further includes a reagent for detecting the binding of CD318 to the CD318-binding molecule; more preferably, the reagent for detecting binding is a detectable label that can be linked to the CD318-binding molecule.
  10. 权利要求1所述CD318结合分子在制备用于检测样品中CD318、评估药物治疗效果或诊断癌症的试剂盒中的用途。 The use of the CD318-binding molecule of claim 1 in preparing a kit for detecting CD318 in a sample, evaluating drug treatment effects, or diagnosing cancer.
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WO2021132427A1 (en) * 2019-12-27 2021-07-01 株式会社カイオム・バイオサイエンス Anti-cdcp1 antibody

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