WO2024002252A1 - Nanocorps anti-trop2 et son utilisation - Google Patents

Nanocorps anti-trop2 et son utilisation Download PDF

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WO2024002252A1
WO2024002252A1 PCT/CN2023/103907 CN2023103907W WO2024002252A1 WO 2024002252 A1 WO2024002252 A1 WO 2024002252A1 CN 2023103907 W CN2023103907 W CN 2023103907W WO 2024002252 A1 WO2024002252 A1 WO 2024002252A1
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trop2
cells
antibody
sequence
binding molecule
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Chinese (zh)
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王海鹰
熊青卉
王彦
胡红明
江鹏斐
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上海恒润达生生物科技股份有限公司
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    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
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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 to anti-Trop2 nanobodies and their uses.
  • Trop2 is a cell surface glycoprotein encoded and expressed by the TACSTD2 gene, whose full name is human trophoblast cell surface glycoprotein antigen 2 (Trophoblast Cell Surface Antigens 2).
  • Trop2 consists of a hydrophobic leader peptide, an extracellular domain, a transmembrane domain and a cytoplasmic tail. It is a single-pass transmembrane glycoprotein with a size of 35.7KD. It is a type of calcium ion channel signal converter.
  • the N-terminus of Trop2 protein is the extracellular domain (Trop2EC), which is connected to the intracellular short tail (Trop2IC) through a unidirectional transmembrane helix (TM), thereby fixing it to the cell membrane.
  • cytoplasmic tail has a highly conserved phosphatidylinositol 4,5-bisphosphate (PIP2) binding sequence, indicating that PIP2 plays an important role in Trop2 signal transduction. In addition to the PIP2 binding motif, it contains conserved tyrosine and serine phosphorylation sites.
  • PIP2 phosphatidylinositol 4,5-bisphosphate
  • Trop2 is not expressed or has low expression in normal tissues. It is overexpressed in various malignant tumors such as breast cancer, gastric cancer, colorectal cancer, pancreatic cancer, prostate cancer, cervical cancer, head and neck cancer, and ovarian cancer, and can promote tumor occurrence and invasion. , metastasis and spread, etc., play a key role in the process of tumor growth, so Trop2 is considered a candidate target for tumor immunotherapy.
  • Chimeric Antigen Receptor-T cells refer to T cells that have been genetically modified to recognize specific target antigens in an MHC-unrestricted manner and to continuously activate and expand.
  • the 2012 International Cell Therapy Association Annual Meeting pointed out that biological immune cell therapy has become the fourth method of treating tumors in addition to surgery, radiotherapy, and chemotherapy. It is a new immunotherapy method that targets specific antigens on the surface of tumor cells.
  • a large number of studies have shown that CAR-T cells can effectively recognize tumor antigens, induce specific anti-tumor immune responses, and significantly improve the survival status of patients.
  • Chimeric Antigen Receptor is the core component of CAR-T, which gives T cells the ability to recognize tumor antigens in an HLA-independent manner. This allows CAR-modified T cells to be more capable than the natural T cell surface receptor TCR. Identify broader goals.
  • CAR consists of three main domains: extracellular antigen-binding domain, transmembrane domain and intracellular signaling domain.
  • antibodies have a decisive impact on CAR-T's specific and efficient killing effect and reduction of toxicity. Therefore, antibodies that can bind target antigens with high specificity and have low immunogenicity are the focus of developing CAR-T products.
  • VHH heavy chain variable region
  • CH2 and CH3 regions The cloned and expressed VHH structure has the same structural stability and antigen-binding activity as the original heavy chain antibody, and is the smallest unit currently known that can bind the target antigen.
  • the VHH crystal is 2.5nm, 4nm long, and has a molecular weight of only 15KDa, so it is also called Nanobody (Nb).
  • VHH is extremely soluble, easy to manufacture and modify, has a short circulation half-life, has high tissue penetration, is not easy to aggregate, and can withstand denaturing conditions such as high temperature, strong acid, and strong alkali. It is suitable for prokaryotic expression and various eukaryotic expression systems and is widely used.
  • diagnostic reagents In the fields of developing therapeutic antibody drugs, diagnostic reagents, affinity purification matrices and scientific research. Nanobodies have unique properties such as small molecular weight, good water solubility, strong stability, strong antigen recognition ability, easy production, etc. In recent years, they have become increasingly popular as diagnostic tools and are also considered to be a promising candidate for CAR-T and targeted drug delivery. important basis.
  • the invention provides a Trop2-binding molecule, comprising an anti-Trop2 nanobody or an antigen-binding fragment thereof.
  • the complementarity determining region CDR of the anti-Trop2 nanobody includes CDR1, CDR2 and CDR3, wherein CDR1 includes the sequence shown in SEQ ID NO: 1 , CDR2 includes the sequence shown in SEQ ID NO:2, and CDR3 includes the sequence shown in SEQ ID NO:3.
  • the heavy chain variable region sequence of the anti-Trop2 Nanobody is set forth in any one of SEQ ID NOs: 4-5.
  • the FR1 of the anti-Trop2 Nanobody can be selected from the FR1 of the VHH shown in any one of SEQ ID NO:4-5, and the FR2 can be selected from any of SEQ ID NO:4-5.
  • the FR2 and FR3 of the VHH shown in SEQ ID NO:4-5 can be selected from the FR3 of the VHH shown in any one of SEQ ID NO:4-5, and the FR4 can be selected from the FR4 of the VHH shown in any one of SEQ ID NO:4-5.
  • the Trop2 binding molecule is a monovalent or multivalent Nanobody or single domain antibody, or a multispecific Nanobody comprising one, two or more anti-Trop2 Nanobodies or antigen-binding fragments thereof or single domain antibodies.
  • the multivalent binding molecule or multispecific binding molecule is linked via a linker to multiple anti-Trop2 Nanobodies or antigen-binding fragments thereof.
  • 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 Trop2-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 Trop2 binding molecule as described in 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 Trop2 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 Trop2-binding molecule according to any embodiment herein, comprising: producing a Trop2-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 Nanobodies or single domain antibodies)
  • a Trop2-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 Nanobodies or single domain antibodies
  • the host cells described herein are cultured under the conditions described herein, and the Trop2 binding molecules are optionally purified from the culture.
  • the present invention also provides a pharmaceutical composition, comprising the Trop2 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 Trop2 expression.
  • the present invention also provides the use of Trop2 binding molecules, chimeric antigen receptors, nucleic acid molecules, nucleic acid constructs or host cells according to 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 Trop2 binding molecules, chimeric antigen receptors, nucleic acid molecules, nucleic acid constructs or host cells according to any embodiment herein in the preparation of medicaments for preventing or treating diseases or conditions related to Trop2 expression.
  • the disease or condition is selected from one or more of the following: breast cancer, gastric cancer, colorectal cancer, pancreatic cancer, prostate cancer, cervical cancer, head and neck cancer, lung cancer, esophageal cancer, Kidney, bladder, uterine and ovarian cancer.
  • the present invention also provides a method for treating or preventing diseases or conditions related to Trop2 expression, which method includes administering to a patient in need a therapeutically effective amount of the Trop2-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 Trop2, which is used, for example, to evaluate drug treatment effects or diagnose cancer.
  • the kit includes a Trop2-binding molecule, a nucleic acid molecule, a nucleic acid construct or a host cell according to any embodiment of this document.
  • the kit further includes reagents for detecting binding of Trop2 to the Trop2 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 Trop2 binding molecule.
  • the detectable label is linked to the Trop2 binding molecule or is present separately in the kit.
  • the present invention also provides a non-diagnostic method for detecting the presence of Trop2 in a sample.
  • the method includes: incubating the sample with the Trop2-binding molecule described in any embodiment of this document, and detecting the binding of Trop2 to the Trop2-binding molecule, This determines the presence of Trop2 in the sample.
  • the detection is an enzyme-linked immunoreaction assay.
  • the present invention also provides the use of the Trop2-binding molecule described in any embodiment herein in the preparation of a kit for detecting Trop2 in a sample, evaluating the effect of drug treatment, or diagnosing cancer.
  • the present invention provides a new Nanobody that specifically recognizes Trop2 and a CAR-modified cell containing the antibody.
  • the antibody and cells have good therapeutic effects targeting Trop2 and provide new treatments for diseases related to Trop2 expression. or ways to improve.
  • Figure 1 shows the SDS electrophoresis pattern of recombinant human Trop2-avi-his antigen protein.
  • FIG. 2 is a schematic diagram of different cloned Trop2CARs.
  • Figure 3 shows the CAR expression positive rate of Trop2CAR-T cells of different clones.
  • Figure 4 shows the expression of CD107a in Trop2CAR-T cells of different clones.
  • Figure 5 shows the INF ⁇ secretion of Trop2CAR-T cells of different clones when the effect-to-target ratio is 10:1.
  • Figure 6 shows the INF ⁇ secretion of Trop2CAR-T cells of different clones when the effect-to-target ratio is 2:1.
  • Figure 7 shows the IL-2 secretion of Trop2CAR-T cells of different clones when the effect-to-target ratio is 10:1.
  • Figure 8 shows the IL-2 secretion of Trop2CAR-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 Trop2CAR-T cells on the target cell BxPC3-LUC-GFP.
  • Figure 10 shows the results of the killing experiment of different clones of Trop2CAR-T cells against the target cell U251-LUC-GFP.
  • Figure 11 shows the CAR expression positive rates of Trop2CAR-T cells of different clones in Example 6.
  • Figure 12 shows the short-term killing results of Trop2CAR-T cells against MDA-MB-231, MDA-MB-468 and HCC1395.
  • Figure 13 shows the long-term killing results of Trop2CAR-T cells against MDA-MB-231 and MDA-MB-468.
  • the present invention uses Trop2 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 Trop2-specific single domain antibody genes. 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 Trop2.
  • 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 Trop2 can be obtained.
  • the immune effector cells can be used to treat or improve diseases related to Trop2 expression, thereby It lays the foundation for the treatment of Trop2-positive tumors.
  • Trop2-binding molecules are proteins that specifically bind Trop2, 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 mu and epsilon isoforms, CH1, CH2, and CH3). CH2, CH3 and CH4) constant domains (CH) and the hinge region (Hinge) located between the CH1 domain and the CH2 domain. Each light chain has a variable domain (VL) at the N-terminus, followed by a constant domain (CL) at its other end.
  • VH variable domain
  • CL constant domain
  • Pairs of VH and VL together form an antigen-binding site On the structure and properties of different classes of antibodies, see e.g. 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.
  • Heavy chain antibodies as used herein are antibodies derived from camelids or cartilaginous fishes. Compared with the above 4-chain antibodies, heavy chain antibodies are missing
  • the light chain and heavy chain constant region 1 (CH1) only contain two heavy chains consisting of a variable region (VHH) and other constant regions. The variable region is connected to the constant region through a hinge-like structure.
  • 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-Trop2 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 Trop2 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.
  • 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) and LCDR1, LCDR2 and LCDR3 of the light chain variable region.
  • 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 antibody of antigen-binding fragments.
  • 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 also include herein "chimeric" antibodies in which a portion of the heavy chain and/or light chain is identical or homologous to the corresponding sequence in an antibody derived from a specific species or belonging to a specific antibody class or subclass, and the chain The remainder is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so 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 Trop2 as the antigen-joining region of any anti-Trop2 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 Trop2.
  • any anti-Trop2 Nanobody of the invention e.g., single domain antibody VHH
  • the anti-Trop2 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.
  • FR1, FR2, FR3 and FR4 of the anti-Trop2 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: 4-5.
  • the amino acid sequence of the anti-Trop2 single domain antibody is as shown in any one of SEQ ID NO: 4-5.
  • a Nanobody is a heavy chain antibody comprising a single domain antibody as described herein.
  • the heavy chain constant region can be The constant region of the camel heavy chain antibody contains 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, such as CH2 and CH3 of IgGl.
  • Trop2 binding molecules described herein may be monovalent or multivalent Nanobodies or single domain antibodies, or multispecific Nanobodies or single domain antibodies comprising one, two or more anti-Trop2 Nanobodies or single domain antibodies described herein .
  • Multispecificity can be against Trop2 and another antigen, or it can be against two different epitopes of Trop2.
  • 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 Trop2.
  • the CAR contains an optional signal peptide sequence, an antigen recognition region, which is the anti-Trop2 binding molecule described herein, a hinge region, a transmembrane region and an intracellular region. wherein the intracellular region contains one or more intracellular costimulatory domains and/or a 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: 6.
  • 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 that of a naturally occurring protein or a portion 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 a CD8 ⁇ hinge region, an IgD hinge region, an IgG1FcCH2CH3 hinge region, or an IgG4FcCH2CH3 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:7.
  • 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: 8.
  • 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: 10.
  • 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 shown in SEQ ID NO:9.
  • 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 motif is in the linker
  • the sequences are contiguous, with no intervening amino acid residues between the 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-Trop2 Nanobody or antigen-binding fragment thereof as 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: 11-12.
  • 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-Trop2 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 two sequences Hybridization occurs when the identity between columns is at least 90%, preferably more than 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.
  • 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 various other liquid chromatography techniques and combinations of these methods.
  • Nanobodies that could bind Trop2.
  • the inventors constructed CAR and CAR-T cells were obtained. It was verified by cell-level experiments that the CAR-T has strong immune function, better CD107a expression, IFN- ⁇ and IL-2 secretion, and specific killing function of target cells. The medicinal effect is remarkable in the body.
  • 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 Trop2 expression.
  • Diseases or conditions refer to diseases caused directly or indirectly by abnormal expression of Trop2, usually referring to diseases caused by overexpression of Trop2, such as cancer, including but not limited to: breast cancer, gastric cancer, colorectal cancer, pancreatic cancer, Prostate, cervical, head and neck, lung and ovarian 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.
  • Such therapies include, but are not limited to, chemotherapy, radiotherapy, and immunosuppressants.
  • treatment may be combined with radiotherapy or chemotherapy agents known in the art to treat Trop2-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 refers to a living organism, such as a mammal, that can elicit an immune response.
  • a living organism such as a mammal
  • 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 Trop2, for example binding assays to detect and/or quantify Trop2 expressed in tissues or cells. Binding molecules such as single domain antibodies can be used in studies to further investigate the role of Trop2 in disease.
  • the method for detecting Trop2 is roughly as follows: obtain cell and/or tissue samples; detect the level of Trop2 in the samples.
  • Trop2-binding molecules of the invention may be used for diagnostic purposes to detect, diagnose or monitor Trop2-related diseases and/or conditions.
  • the present invention provides for the detection of the presence of Trop2 in a sample using classical immunohistological methods known to those skilled in the art. Detection of Trop2 can be performed in vivo or in vitro. Examples of methods suitable for detecting the presence of Trop2 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 Trop2.
  • An example of such an assay would involve detecting the amount of free antibody in a solution containing an amount of Trop2 in the presence or absence of a test molecule. An increase in the amount of free antibody (i.e., antibody that does not bind Trop2) will indicate that the test molecule is able to compete with the antibody for binding to Trop2.
  • 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 Trop2 levels.
  • the kit includes an antibody that recognizes Trop2 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 Trop2 protein expression vector
  • the fusion gene sequence encodes the amino acid sequence shown in SEQ ID NO:15.
  • the nucleotide sequence of the fusion gene is as SEQ ID NO: 16 shown.
  • 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 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 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 and 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, 98°C Denaturation for 15 seconds, annealing at 58°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 subjected to 1.5% agarose gel electrophoresis, a gel recovery kit (Promega) was used to recover the 750 bp target band.
  • a gel recovery kit Promega
  • 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 pcomb3xTT and VHH genes were digested with SfiI DNA endonuclease for 16 hours at 50°C.
  • the digested pcomb3xTT 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 the Amp/2YT plate, incubate at 37°C overnight, and count accordingly. And count 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 Trop2 protein was biotin-modified to obtain biotinylated Trop2 protein.
  • Example 3 Preparation of retrovirus stock solution containing anti-human Trop2 chimeric antigen receptor element
  • a chimeric antigen receptor sequence containing a single domain antibody VHH against human Trop2 antigen, hinge region, transmembrane region and intracellular signal segment. Its structure is shown in Figure 2. According to the difference in loaded VHH, the chimeric antigen receptors were named 1A4-BBz and 1F2-BBz respectively. Their amino acid sequences are as shown in SEQ ID NO:11 and SEQ ID NO:12 respectively, and their nucleotide sequences are as shown in SEQ ID NO:13 and SEQ ID NO:14 are shown.
  • retroviral plasmids expressing chimeric antigen receptors cloned 1A4 and 1F2 were constructed. Select the correctly sequenced clone, inoculate the bacterial solution into 200ml 2YT medium, shake the culture overnight, and complete the plasmid purification according to the instructions of the NucleoBond Xtra Maxi EF kit.
  • 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 it to the cell suspension, mix the magnetic beads and cells 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 and rinse 15ml with 1ml DPBS. Centrifuge the tube 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 respectively to a 24-well plate, add 2 ⁇ l of 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.
  • Example 5 Functional analysis based on anti-human Trop2 CAR-T cells
  • CAR-T cells containing different antibody clones were compared with target cells (Trop2-positive human pancreatic cancer cell line BxPC3) and control target cells (human glioma cells U251) at an effect-to-target ratio of 1:1 (effector).
  • target cells Trop2-positive human pancreatic cancer cell line BxPC3
  • control target cells human glioma cells U251
  • effect-to-target ratio 1:1 (effector)
  • cells and target cells both 3 ⁇ 10 5
  • flow cytometry was used to detect the proportion of CD107a-expressing cells in each group of samples to the number of CD3+ cells. 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.
  • the CAR-T cells containing different antibody clones were compared with target cells (Trop2-positive cell line BxPC3) and control target cells (Trop2-negative cell line U251) at an effect-to-target ratio of 10:1 and 2:1 respectively ( After a total of 24 hours of incubation (3 ⁇ 10 4 target cells), the supernatant was collected, and the secretion of IFN- ⁇ and IL-2 was detected using ELISA (enzyme-linked immunosorbent assay). 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 Trop2-positive target cells BxPC3-LUC- that stably express firefly luciferase 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 Trop2 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%.
  • a chimeric antigen receptor sequence containing a single domain antibody VHH against human Trop2 antigen, hinge region, transmembrane region and intracellular signal segment. Its structure is shown in Figure 2. According to the different loaded VHHs, the chimeric antigen receptors were named 1A4-BBz and 1F2-BBz respectively. Their amino acid sequences are as shown in SEQ ID NO:11 and SEQ ID NO:12 respectively, and their nucleotide sequences are as shown in SEQ ID NO:13 and SEQ ID NO:14 shown.
  • retroviral plasmids expressing chimeric antigen receptors cloned 1A4 and 1F2 were constructed. Select the correctly sequenced clone, inoculate the bacterial solution into 200ml 2YT medium, shake the culture overnight, and complete the plasmid purification according to the instructions of the NucleoBond Xtra Maxi EF kit.
  • PEI cationic polymer
  • the process is as follows: dilute 18 ⁇ l PEI and 3 ⁇ g of retrovirus packaging master plasmid with 300 ⁇ l of serum-free Opti-MEM TM (gibco) respectively; then add PEI/Opti-MEM TM to the plasmid/Opti- MEM TM mixture, vortex to mix, and let stand at room temperature for 10 minutes; add the plasmid-PEI complex to the pre-plated 293V cells (4E6 cells). Change the medium 16 hours after transfection. Collect the first viral supernatant after 48 hours and store it at 4°C.
  • a PBMC Receives a PBMC, verify that the patient's individual identification number is correct, and then perform resuscitation.
  • 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, resuspend the magnetic beads with cells containing DPBS, and add them to the cell suspension; mix the magnetic beads and cells in a 5mL sterile flow tube, incubate on a rotating mixer, and incubate at room temperature for 30 minutes. , after the incubation is completed, place the cells on the magnetic stand for 2 minutes.
  • CAR-T culture medium to adjust the cell density to 1 ⁇ 10 6 /ml, add IL-2 to a final concentration of 300IU/ml, and culture in a 37°C, 5% CO 2 incubator for two days.
  • the CAR positivity rate was detected after the seventh day of CAR-T cell preparation.
  • Targeting the target cells MDA-MB-231, MDA-MB-468 and HCC1395 (MDA-MB-468 highly expresses Trop2, MDA-MB-231 expresses low Trop2, and HCC1395 barely expresses Trop2, see https://www.proteinatlas.
  • target cells MDA-MB-231 and MDA-MB-468
  • Trop2-positive target cells MDA-MB-231-Luc-GFP and MDA-MB-468-Luc-GFP were co-incubated at 37°C, and a positive control with only target cells was set at the same time.

Abstract

L'invention concerne un nanocorps anti-Trop2 et son utilisation. L'invention concerne également une molécule de liaison à Trop2, comprenant un nanocorps anti-Trop2 ou un fragment de liaison à l'antigène de celui-ci. Des régions déterminant la complémentarité CDR du nanocorps anti-Trop2 comprennent CDR1, CDR2 et CDR3. L'invention concerne en outre un récepteur antigénique chimérique comprenant la molécule de liaison à Trop2 et une cellule l'expression.
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