A VHH antibody that specifically binds to human CD318 or its antigen-binding fragment, its preparation method, and its application.

Abstract

This invention relates to a VHH antibody that specifically binds to human CD318, or an antigen-binding fragment thereof, as well as its preparation method and applications. This invention provides a CD318-binding molecule comprising an anti-CD318 nanobody or an antigen-binding fragment thereof, wherein the complementarity-determining region (CDR) of the anti-CD318 nanobody comprises CDR1, CDR2, and CDR3. This invention also provides a chimeric antigen receptor containing the CD318-binding molecule and its expressing cells. The antibody and cells described herein exhibit good therapeutic effects targeting CD318.

Description

Technical Field

[0001] This invention relates to the field of biological immunotherapy technology, and more specifically, to an antibody that specifically binds to CD318 or its antigen-binding fragment, as well as its preparation method and application. Background Technology

[0002] CD318, also known as CDCP1 (protein 1 containing CUB domains), is a gene that encodes a highly glycosylated, single-channel type I transmembrane protein expressed in mesenchymal stem cells, neural stem cells, fibroblasts, and hematopoietic progenitor cells. CDCP1 consists of a large extracellular domain (ECD) containing three CUB domains (complement C1r / C1s, Uegf, and Bmp1) and a short intracellular portion. This protein plays a role in tyrosine phosphorylation-dependent regulation.

[0003] CD318 is abnormally elevated in various malignant tumors, including colon cancer, breast cancer, pancreatic cancer, lung cancer, kidney cancer, and liver cancer. In hematopoietic cells, CD318 has been identified as a stem cell marker for both benign and malignant progenitor cells. Significant CD318 expression has been observed in the CD34CD133 leukemia cell subset, which involves leukemia stem cell enrichment, in AML patients. Therefore, CD318 is a very promising target for tumor immunotherapy.

[0004] Chimeric antigen receptor T cell (CAR T) therapy is a novel immunotherapy method targeting specific antigens on the surface of tumor cells. VHH antibodies, also known as nanobodies, are naturally occurring antibodies found in alpaca peripheral blood that lack a light chain. These antibodies contain only one heavy chain variable region (VHH) and two conventional CH2 and CH3 regions. Compared to traditional artificially modified scFvs, they offer advantages such as smaller molecular weight, easier expression, higher specificity, higher affinity, weaker immunogenicity in humans, and shorter development cycles. Combining the advantages of immunotherapy and VHH antibodies can lead to the development of highly effective CAR-T therapies.

[0005] There are currently no reports of anti-CD318VHH antibodies as described in this application, or immune effector cells containing such anti-CD318VHH antibodies. Summary of the Invention

[0006] This invention provides a CD318 binding molecule comprising an anti-CD318 nanobody or an antigen-binding fragment thereof, wherein the complementarity-determining region (CDR) of the anti-CD318 nanobody comprises CDR1, CDR2, and CDR3, wherein CDR1 comprises any of the sequences shown in SEQ ID NO: 1, 4, 7, 10, 13, and 16, CDR2 comprises any of the sequences shown in SEQ ID NO: 2, 5, 8, 11, 14, and 17, and CDR3 comprises any of the sequences shown in SEQ ID NO: 3, 6, 9, 12, 15, and 18.

[0007] In one or more embodiments, the CDR is selected from any of the following:

[0008] (1) CDR1 with sequence as shown in SEQ ID NO:1, CDR2 with sequence as shown in SEQ ID NO:2, and CDR3 with sequence as shown in SEQ ID NO:3.

[0009] (2) CDR1 with sequence as shown in SEQ ID NO:4, CDR2 with sequence as shown in SEQ ID NO:5, and CDR3 with sequence as shown in SEQ ID NO:6.

[0010] (3) CDR1 with sequence as shown in SEQ ID NO:7, CDR2 with sequence as shown in SEQ ID NO:8, and CDR3 with sequence as shown in SEQ ID NO:9.

[0011] (4) CDR1 with sequence as shown in SEQ ID NO:10, CDR2 with sequence as shown in SEQ ID NO:11, and CDR3 with sequence as shown in SEQ ID NO:12.

[0012] (5) CDR1 with sequence as shown in SEQ ID NO:13, CDR2 with sequence as shown in SEQ ID NO:14, and CDR3 with sequence as shown in SEQ ID NO:15.

[0013] (6) CDR1 with sequence as shown in SEQ ID NO:16, CDR2 with sequence as shown in SEQ ID NO:17, and CDR3 with sequence as shown in SEQ ID NO:18.

[0014] In one or more embodiments, the heavy chain variable region sequence of the anti-CD318 nanobody is as shown in any of SEQ ID NO:19-24.

[0015] In one or more embodiments, the FR1 of the anti-CD318 nanobody may be selected from the FR1 of any of the VHHs shown in SEQ ID NO:19-24, the FR2 may be selected from the FR2 of any of the VHHs shown in SEQ ID NO:19-24, the FR3 may be selected from the FR3 of any of the VHHs shown in SEQ ID NO:19-24, and the FR4 may be selected from the FR4 of any of the VHHs shown in SEQ ID NO:19-24.

[0016] In one or more embodiments, the CD318 binding molecule is a monovalent or multivalent nanobody or single-domain antibody, or a multispecific nanobody or single-domain antibody, comprising one, two or more anti-CD318 nanobodies or their antigen-binding fragments.

[0017] In one or more embodiments, the multivalent or multispecific binding molecule is linked to multiple anti-CD318 nanobodies or their antigen-binding fragments via a linker. The linker consists of 1-15 amino acids selected from G and S.

[0018] In one or more embodiments, the nanobody is a camel heavy chain antibody or a cartilaginous fish heavy chain antibody.

[0019] In one or more embodiments, the nanobody further comprises a heavy chain constant region.

[0020] In one or more embodiments, the heavy chain constant region is the constant region of a camel heavy chain antibody, comprising CH2 and CH3.

[0021] In one or more embodiments, CH2 and CH3 are CH2 and CH3 of human IgG Fc, such as CH2 and CH3 of IgG1.

[0022] In one or more embodiments, the heavy chain constant region is a constant region of a cartilaginous fish heavy chain antibody, comprising CH1, CH2, CH3, CH4 and CH5.

[0023] In one or more embodiments, the CD318 binding molecule described in any embodiment of the present invention is a chimeric antibody or a fully human antibody; preferably a fully human antibody.

[0024] Another aspect of the present invention provides a chimeric antigen receptor comprising an optional signal peptide sequence, a CD318 binding molecule as described in any embodiment herein, a hinge region, a transmembrane region, and an intracellular region.

[0025] In one or more embodiments, the intracellular region includes an intracellular co-stimulatory domain and / or an intracellular signaling domain.

[0026] In one or more embodiments, from the N-terminus to the C-terminus, the chimeric antigen receptor sequentially comprises a signal peptide, a CD318 binding molecule as described in any of the embodiments herein, a hinge region, a transmembrane region, an intracellular co-stimulatory domain, and an intracellular signaling domain.

[0027] The present invention also provides a nucleic acid molecule having a sequence selected from any of the following:

[0028] (1) The coding sequence of the CD318 binding molecule or chimeric antigen receptor described in any of the embodiments herein;

[0029] (2) and (1) are complementary sequences;

[0030] A 5-50bp fragment of any sequence from (3), (1), or (2).

[0031] In one or more embodiments, the fragment is a primer.

[0032] The present invention also provides a nucleic acid construct comprising the nucleic acid molecules described herein.

[0033] In one or more embodiments, the nucleic acid construct is a cloning vector, an expression vector, or an integration vector.

[0034] The present invention also provides a host cell selected from:

[0035] (1) Expressing and / or secreting the CD318 binding molecule or chimeric antigen receptor as described in any of the embodiments herein;

[0036] (2) Contains the nucleic acid molecules described herein; and / or

[0037] (3) Includes the nucleic acid constructs described in this article.

[0038] In one or more embodiments, the host cell is an immune effector cell, preferably a T cell.

[0039] The present invention also provides a method for generating a CD318 binding molecule according to any embodiment herein, comprising: culturing the host cells described herein under conditions suitable for generating a CD318 binding molecule (e.g., nanobodies or antigen-binding fragments thereof, monovalent or multivalent nanobodies or single-domain antibodies, or multispecific nanobodies or single-domain antibodies), and optionally purifying the CD318 binding molecule from the culture.

[0040] The present invention also provides a pharmaceutical composition comprising a CD318 binding molecule, a nucleic acid molecule, a nucleic acid construct or a host cell as described in any embodiment herein, and pharmaceutically acceptable excipients.

[0041] In one or more embodiments, the pharmaceutical composition is used to treat diseases or conditions related to CD318 expression.

[0042] The present invention also provides the use of the CD318 binding molecule, chimeric antigen receptor, nucleic acid molecule, nucleic acid construct or host cell described in any embodiment herein in the preparation of activated immune cells (e.g. T cells).

[0043] The present invention also provides the use of the CD318 binding molecule, chimeric antigen receptor, nucleic acid molecule, nucleic acid construct or host cell described in any embodiment herein in the preparation of a medicament for the prevention or treatment of diseases or conditions associated with CD318 expression.

[0044] 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.

[0045] The present invention also provides a method for treating or preventing diseases or conditions related to CD318 expression, the method comprising administering to a patient in need a therapeutically effective amount of the CD318 binding molecule or host cell as described in any embodiment of the present invention, or a pharmaceutical composition as described in any embodiment of the present invention.

[0046] The present invention also provides a kit for detecting CD318, for example, to evaluate the efficacy of drug treatment or to diagnose cancer, said kit comprising a CD318 binding molecule, nucleic acid molecule, nucleic acid construct or host cell as described in any embodiment herein.

[0047] In one or more embodiments, the kit further includes reagents for detecting the binding of CD318 to the CD318 binding molecule. For example, reagents for detecting the binding by an enzyme-linked immunosorbent assay (ELISA).

[0048] In one or more embodiments, the detection binding reagent is a detectable marker, such as biotin, that can be linked to the CD318 binding molecule. The detectable marker is either linked to the CD318 binding molecule or is present separately in the kit.

[0049] This invention also provides a non-diagnostic method for detecting the presence of CD318 in a sample, the method comprising: incubating the sample with the CD318 binding molecule as described in any embodiment of this invention, and detecting the binding of CD318 to the CD318 binding molecule, thereby determining the presence of CD318 in the sample. The detection is performed using an enzyme-linked immunosorbent assay (ELISA).

[0050] 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 efficacy of drug treatment, or diagnosing cancer.

[0051] The present invention has the following beneficial effects:

[0052] This invention provides a novel nanobody that specifically recognizes CD318 and CAR-modified cells containing the antibody. The antibody and cells have good therapeutic effects targeting CD318, providing a new treatment or improvement approach for diseases related to CD318 expression. Attached Figure Description

[0053] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings 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 should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

[0054] Figure 1 This is an SDS-PAGE image of the recombinant human CD318-avi-his antigen protein.

[0055] Figure 2 A schematic diagram of different cloned CD318 CARs.

[0056] Figure 3 The CAR expression positivity rate of CD318 CAR-T cells from different clones.

[0057] Figure 4 CD107a expression in CD318 CAR-T cells of different clones.

[0058] Figure 5 INFγ secretion in CD318 CAR-T cells of different clones at an effector-target ratio of 10:1.

[0059] Figure 6 INFγ secretion in CD318 CAR-T cells of different clones at an effector-target ratio of 2:1.

[0060] Figure 7 IL-2 secretion in CD318 CAR-T cells of different clones at an effector-target ratio of 10:1.

[0061] Figure 8 IL-2 secretion in CD318 CAR-T cells of different clones at an effector-target ratio of 2:1.

[0062] Figure 9 The results of experiments on the killing of target cells by different clones of CD318 CAR-T cells.

[0063] Figure 10 The results of experiments on the killing of target cells by different clones of CD318 CAR-T cells. Detailed Implementation

[0064] Through extensive and in-depth research and 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.

[0065] Specifically, this invention utilizes CD318 protein to immunize alpacas, obtaining a high-quality single-domain antibody gene library. Then, phage display technology is used to screen the antibody gene library, thereby obtaining CD318-specific single-domain antibody genes. These genes are then transferred into mammalian cells, resulting in antibody strains that can be efficiently expressed in mammalian cells and exhibit high specificity. The antibodies or their antigen-binding fragments possess good safety and targeting properties, specifically binding to the extracellular domain of human CD318.

[0066] The present invention also provides a chimeric antigen receptor (CAR) containing the nanobody. Using a vector containing the coding sequence of the CAR to infect immune cells can yield immune effector cells with significant killing ability against tumor cells overexpressing CD318. These immune effector cells can be used to treat or improve CD318-related diseases, thus laying the foundation for the treatment of CD318-positive tumors.

[0067] Antibody

[0068] In this article, "CD318 binding molecule" refers to a protein that specifically binds to CD318, including but not limited to antibodies, heavy chain antibodies, nanobodies, or their antigen-binding fragments.

[0069] In this document, the term "antibody" includes monoclonal antibodies (including full-length antibodies having the immunoglobulin Fc region), antibody compositions with multi-epitope specificity, multispecific antibodies (e.g., bispecific antibodies), biantibodies and single-chain molecules, and antibody fragments, especially antigen-binding fragments, such as Fab, F(ab')2, Fd, and Fv. In this document, "antibody" and "immunoglobulin" are used interchangeably.

[0070] Traditional "antibodies" contain a basic four-chain antibody unit, 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 its N-terminus, followed by three (CH1, CH2, and CH3 for each α and γ chain) and four (CH1, CH2, CH3, and CH4 for μ and ε isoforms) constant domains (CH), and a hinge region located between the CH1 and CH2 domains. Each light chain has a variable domain (VL) at its N-terminus, followed by a constant domain (CL) at its other end. Pairs of VH and VL together form an antigen-binding site. For information on the structure and properties of different classes of antibodies, see Basic and Clinical Immunology, 8th Edition, edited by Daniel P. Sties, Abba I. Terr, and Tristram G. Parsolw, Appleton & Lange, Norwalk, CT, 1994, p. 71 and Chapter 6. Light chains from any vertebrate species can be classified into one of two distinct types, called κ and λ, based on their constant domain amino acid sequences. Based on relatively minor differences in CH sequence and function, the γ and α types can be further subdivided into subclasses, such as those expressed in humans: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1, and IgA2.

[0071] The "heavy chain antibody" described in this article refers to antibodies derived from camelid or cartilaginous fish. Compared to the aforementioned four-chain antibodies, heavy chain antibodies lack the light chain and heavy chain constant region 1 (CH1), containing only two heavy chains composed of a variable region (VHH) and other constant regions. The variable region is linked to the constant region via a hinge-like structure. Each heavy chain of camelid heavy chain antibodies contains one variable region (VHH) and two constant regions (CH2 and CH3), while each heavy chain of cartilaginous fish heavy chain antibodies contains one variable region and five constant regions (CH1-CH5). The antigen-binding fragment of heavy chain antibodies includes VHH and single-chain heavy chain antibodies. By fusing with the constant region of human IgG Fc, heavy chain antibodies can possess the CH2 and CH3 regions of human IgG Fc.

[0072] 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 single-domain antibodies that specifically recognize and bind to CD318. A single-domain antibody is the variable region of a heavy chain antibody. Typically, a single-domain antibody contains 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 CDR1 shown in SEQ ID NO:4, CDR2 shown in SEQ ID NO:5, and CDR3 shown in SEQ ID NO:6; or has CDR1 shown in SEQ ID NO:7, CDR2 shown in SEQ ID NO:8, and CDR3 shown in SEQ ID NO:9; or has CDR1 shown in SEQ ID NO:10, CDR2 shown in SEQ ID NO:11, and CDR3 shown in SEQ ID NO:12; or has CDR1 shown in SEQ ID NO:13, CDR2 shown in SEQ ID NO:14, and CDR3 shown in SEQ ID NO:15; or has CDR1 shown in SEQ ID NO:16, CDR2 shown in SEQ ID NO:17, and CDR3 shown in SEQ ID NO:18. A single-domain antibody is the smallest functional antigen-binding fragment. Typically, an antibody that naturally lacks both the light chain and the heavy chain constant region 1 (CH1) is first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single-domain antibody consisting of only one heavy chain variable region.

[0073] In this document, "nanobody" refers to an antibody containing the VHH described herein. It can be a heavy chain antibody as described above, a multivalent or multispecific antibody containing multiple VHHs, or a recombinant antibody obtained by recombination of VHHs and antibody Fc (e.g., CH2 and CH3 or CH2, CH3, and CH4). A binding molecule containing two or more single-domain antibodies is a multivalent single-domain antibody; a binding molecule containing two or more single-domain antibodies with different specificities is a multispecific single-domain antibody. Multivalent or multispecific single-domain antibodies are linked together by a linker. The linker typically consists of 1-15 amino acids selected from G and S.

[0074] In this article, heavy chain antibodies and antibodies (traditional four-chain antibodies) are used to distinguish different combinations of antibodies. Due to their structural similarities, the structural descriptions of antibodies below, except for those involving light chains, also apply to heavy chain antibodies.

[0075] The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of either the heavy or light chain. The variable domains of the heavy and light chains are 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 antigen-binding sites.

[0076] The term "variable" refers to the wide variation in certain segments within a variable domain within the antibody sequence. Variable domains mediate antigen binding and define the specificity of a particular antibody for its specific antigen. However, variability is not uniformly distributed across all amino acids spanned by the variable domain. Instead, it is concentrated in three segments called hypervariable regions (HVRs) (present in both light and heavy chain variable domains): HCDR1, HCDR2, and HCDR3 in the heavy chain variable domain (simply referred to as CDR1, CDR2, and CDR3 in heavy chain antibodies) and LCDR1, LCDR2, and LCDR3 in the light chain variable domain. The more highly conserved portions of the variable domain are called backbone regions (FRs). The variable domains of both the natural heavy and light chains each contain four FR regions (FR1, FR2, FR3, and FR4), which mostly adopt a β-sheet conformation and are linked by three HVRs that form a ring connection and, in some cases, part of a β-sheet structure. The HVRs in each chain are held together very closely by the FR regions and, together with the HVRs of the other chain, contribute to the formation of the antibody's antigen-binding site. Typically, the structure of the variable region in the light chain is FR1-LCDR1-FR2-LCDR2-FR3-LCDR3-FR4, and the structure of the variable region in the heavy chain is FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4. Constant domains do not directly participate in antibody-antigen binding but exhibit various effector functions, such as antibody involvement in antibody-dependent cell-mediated cytotoxicity. There are several labeling schemes for antibody variable regions, including Chothia, Kabat, IMGT, and Contact. This article uses the IMGT labeling scheme as an example.

[0077] The “Fc region” (crystallizable fragment region), “Fc domain”, or simply “Fc” refers to the C-terminal region of an antibody heavy chain that mediates the binding of immunoglobulins to host tissues or factors, including binding to Fc receptors on various cells of the immune system (e.g., effector cells) or to the first component (C1q) of the classical complement system. In IgG, IgA, and IgD antibody isotypes, the Fc region consists of two identical protein fragments from the CH2 and CH3 domains of the two antibody heavy chains; the Fc regions of IgM and IgE contain three heavy chain constant domains (CH domains 2–4) in each polypeptide chain. Although the boundaries of the Fc region of the 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 the numbering is based on the EU index, as in Kabat. As used herein, the Fc region can be a native sequence Fc or a variant Fc.

[0078] An "antibody fragment" comprises a portion of a complete antibody, preferably the antigen-binding region and / or variable region of the complete antibody. The antibody fragment is preferably an antigen-binding fragment of the antibody. Examples of antibody fragments include Fab, Fab', F(ab'), F(ab')2, Fd, and Fv fragments; disulfide-linked Fv fragments; biantibodies; linear antibodies; single-chain antibody molecules; scFv-Fc fragments; multispecific antibodies formed from antibody fragments; and any fragment whose half-life should be increased by chemical modification or by incorporation into liposomes. Antigen-binding fragments can be prepared using a variety of techniques, including but not limited to hydrolyzing and digesting complete antibody proteins, and by expression in host cells containing the antigen-binding fragment.

[0079] "Fv" is the smallest antibody fragment containing a complete antigen recognition and binding site. This fragment consists of a dimer of a tightly bound, non-covalently linked heavy chain variable domain and a light chain variable domain. Six hypervariable rings (three from the heavy chain and three from the light chain) protrude from the folds of these two domains, contributing the 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 the three antigen-specific HVRs) can recognize and bind antigens, although with lower affinity than a complete binding site. A "single-chain Fv," also abbreviated as "sFv" or "scFv," is an antibody fragment containing antibody VH and VL domains linked together into a single polypeptide chain. Preferably, the sFv polypeptide also contains a polypeptide linker between the VH and VL domains, allowing the sFv to form the desired antigen-binding structure. For heavy chain antibodies or nanobodies, scFv is VHH.

[0080] In this document, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous group of antibodies, meaning that the individual antibodies constituting the group are identical except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in small amounts. Monoclonal antibodies are highly specific, targeting a single antigenic site. Compared to polyclonal antibody formulations (which typically consist of different antibodies targeting different determinants (epitopes), each monoclonal antibody targets a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage that they are synthesized through hybridoma culture, free from contamination by other immunoglobulins. The modifier "monoclonal" indicates the characteristic that the antibody is obtained from a substantially homogeneous group of antibodies and should not be interpreted as requiring the production of the antibody by any particular method. For example, the monoclonal antibodies to be used according to the invention can be generated by a variety of techniques, including, for example, hybridoma methods, phage display methods, recombinant DNA methods, and techniques for generating human or human-like antibodies from animals having partial or whole human immunoglobulin loci or genes encoding human immunoglobulin sequences, single-cell sequencing methods.

[0081] Monoclonal antibodies also include “chimeric” antibodies in this article, wherein a portion of the heavy chain and / or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remaining portion of the chain 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, provided they exhibit the desired biological activity.

[0082] The “humanized” form of a non-human (e.g., mouse) antibody refers to a chimeric antibody that minimally contains sequences derived from non-human immunoglobulins. Therefore, a “humanized antibody” generally refers to a non-human antibody with a variable domain framework region that exchanges sequences found in human antibodies. Typically, in a humanized antibody, the entire antibody (except for the CDR) is encoded by a human-derived polynucleotide or is identical to that antibody (except for the CDR). The CDR (some or all of which are encoded by nucleic acids derived from non-human organisms) is transplanted into the β-sheet backbone of the variable region of the human antibody to produce an antibody whose specificity is determined by the transplanted CDR. Methods for producing such antibodies are well known in the art, for example, using mice with genetically engineered immune systems. In this invention, antibodies, single-domain antibodies, heavy-chain antibodies, etc., all include humanized variants of the aforementioned antibodies.

[0083] "Human antibody" refers to an antibody having an amino acid sequence corresponding to that of antibodies generated by humans and / or produced using any of the techniques disclosed herein for generating human antibodies. This definition of human antibody explicitly 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.

[0084] In some embodiments, the present invention also provides nanobodies, heavy chain antibodies, antibodies or antigen-binding fragments thereof (e.g., single-domain antibody VHH) that bind to the same epitope on human CD318 as the antigen-binding region of any anti-CD318 nanobody of the present invention, i.e., nanobodies, heavy chain antibodies, antibodies or antigen-binding fragments thereof that can cross-compete with the antigen-binding region of any nanobody of the present invention for binding to CD318.

[0085] In this 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 CDR1 shown in SEQ ID NO:4, CDR2 shown in SEQ ID NO:5, and CDR3 shown in SEQ ID NO:6; or has CDR1 shown in SEQ ID NO:7, CDR2 shown in SEQ ID NO:8, and CDR3 shown in SEQ ID NO:9; or has CDR1 shown in SEQ ID NO:10, CDR2 shown in SEQ ID NO:11, and CDR3 shown in SEQ ID NO:12; or has CDR1 shown in SEQ ID NO:13, CDR2 shown in SEQ ID NO:14, and CDR3 shown in SEQ ID NO:15; or has CDR1 shown in SEQ ID NO:16, CDR2 shown in SEQ ID NO:17, and CDR3 shown in SEQ ID NO:18.

[0086] The FR1, FR2, FR3, and FR4 of the anti-CD318 single-domain antibody described herein can be independently selected from the FR1, FR2, FR3, and FR4 of any of the single-domain antibodies shown in SEQ ID NO:19-24. Preferably, the amino acid sequence of the anti-CD318 single-domain antibody is as shown in any of SEQ ID NO:19-24.

[0087] When a single-domain antibody is attached to a heavy chain constant region, the nanobody is a heavy chain antibody comprising the single-domain antibody described herein. The heavy chain constant region may be a constant region of a camel heavy chain antibody, comprising CH2 and CH3. Preferably, the antibody constant region is derived from the constant regions of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD; more preferably, it is derived from the constant regions of any one of IgG1, IgG2, IgG3, and IgG4. In one or more embodiments, the heavy chain constant region is CH2 and CH3 of human IgG Fc, such as CH2 and CH3 of IgG1.

[0088] The CD318 binding molecules described herein can be monovalent or multivalent nanobodies or single-domain antibodies, or multispecific nanobodies or single-domain antibodies, comprising one, two, or more of the anti-CD318 nanobodies or single-domain antibodies described herein. Multispecificity can be against CD318 and another antigen, or against two different epitopes of CD318.

[0089] This invention also includes the antibody derivatives and analogs described herein. “Derivatives” and “analytes” refer to polypeptides that substantially retain the same biological function or activity as the antibodies of this invention. The derivatives or analogs of this invention may be (i) polypeptides having substituents in one or more amino acid residues, or (ii) polypeptides formed by fusing a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iii) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (e.g., a leader sequence, a secretory sequence, a sequence used to purify this polypeptide, or a proteogenic sequence, or a fusion protein formed with a 6His tag). Based on the teachings herein, these derivatives and analogs are within the scope well known to those skilled in the art.

[0090] Without substantially affecting antibody activity, those skilled in the art can modify the antibody sequence of the present invention by one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) to obtain variants of the antibody or its functional fragment sequence. These variants include (but are not limited to): deletions, insertions, and / or substitutions of one or more amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10), and the addition of one or more amino acids (typically up to 20, preferably up to 10, more preferably up to 5) at the C-terminus and / or N-terminus. In the art, conservative substitutions with amino acids of similar or comparable properties generally do not alter protein function. For example, substitutions of amino acids with similar properties in the FR and / or Fc regions. Amino acid residues that can be conservatively substituted are well known in the art. Such substituted amino acid residues may or may not be encoded by the genetic code. For example, adding one or more amino acids to the C-terminus and / or N-terminus generally does not change the function of the protein. These are all considered to be included within the scope of protection of this invention.

[0091] The variants of the antibodies described herein include: homologous sequences, conserved variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of the antibodies of the present invention under high or low stringency conditions, and polypeptides or proteins obtained using antiserum against the antibodies of the present invention. In some embodiments, the sequences of the variants described herein may have at least 95%, 96%, 97%, 98%, or 99% homology with their source sequences. The sequence homology described herein can be measured using sequence analysis software, such as the computer program BLAST with default parameters, especially BLASTP or TBLASTN. The present invention also includes molecules having antibody heavy chain variable regions with CDRs, provided that their CDRs have at least 90% (preferably at least 95%, most preferably at least 98%) homology with the CDRs identified herein.

[0092] The antibodies of the present invention can be prepared using methods conventional in the art, such as hybridoma techniques. The nanobodies of the present invention can be prepared using methods conventional in the art, such as phage display techniques well known in the art. Alternatively, the antibodies or nanobodies of the present invention can be expressed in other cell lines. Suitable mammalian host cells can be transformed with sequences encoding the antibodies of the present invention, followed by culturing the host cells and purifying the antibodies. Transformation can be performed using any known method, including, for example, packaging polynucleotides in a virus (or viral vector) and transducing host cells with the virus (or vector). The transformation procedure used depends on the host to be transformed. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art, including dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of polynucleotides in liposomes, and direct microinjection of DNA into the nucleus. Mammalian cell lines that can be used as hosts for expression are well known in the art, including but 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, young hamster kidney (BHK) cells, monkey kidney cells (COS) cells, human hepatocellular carcinoma cells (e.g., HepG2).

[0093] CAR

[0094] This invention also provides a chimeric antigen receptor (CAR) targeting CD318. The CAR contains an optional signal peptide sequence, an antigen recognition region (i.e., 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 co-stimulatory domains and / or one or more intracellular signaling domains. The terms "hinge region," "transmembrane region," and "intracellular region" as used herein can all be selected from sequences of the hinge region, transmembrane region, and intracellular region in known CAR-T technologies.

[0095] The signal peptide, optionally selected for the CAR, can be chosen as needed. Generally, a signal peptide is a peptide sequence that directs the polypeptide to a desired site within the cell. The signal peptide directs the polypeptide to the cell's secretory pathway and allows the polypeptide to integrate and anchor to the lipid bilayer; the signal peptide can also be a membrane-localizing signal peptide. Exemplary signal peptides include 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 use in this invention can be any of the various human CD8 signal peptide sequences commonly used in CARs in the art. In some embodiments, the amino acid sequence of the CD8 signal peptide comprises the sequence shown in SEQ ID NO:25.

[0096] The hinge region of a chimeric antigen receptor is located between the extracellular antigen-binding region and the transmembrane region. The hinge region is an amino acid segment that typically exists between two domains of a protein and allows for protein flexibility and relative movement between the two domains. The hinge region can be a hinge region of a naturally occurring protein or a portion thereof. The hinge region of an antibody (such as IgG, IgA, IgM, IgE, or IgD antibodies) can also be used in the chimeric antigen receptor described herein. Non-naturally occurring peptides can also be used as the hinge region of the chimeric antigen receptor described herein. Exemplarily, the hinge region of a CAR is selected from the CD8α hinge region, the IgD hinge region, the IgG1FcCH2CH3 hinge region, or the IgG4FcCH2CH3 hinge region, the sequences of which are within the knowledge of those skilled in the art. The CD8α hinge region suitable for use in this invention can be any of the various human CD8α hinge region sequences commonly used in CARs in the art. In some embodiments, the human CD8α hinge region comprises the sequence shown in SEQ ID NO:26.

[0097] The transmembrane region of a chimeric antigen receptor can form an α-helix, a complex of more than one α-helix, a β-barrel, or any other stable structure capable of translocating the cellular phospholipid bilayer. The transmembrane region can be of natural or synthetic origin. It can be selected from the transmembrane regions of the following proteins: CD3ε, CD4, CD5, CD8α, CD9, CD16, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, or the α, β, or ζ chains of T-cell receptors. The human CD8α transmembrane region suitable for this invention can be any of the various human CD8α transmembrane region sequences commonly used in the art for CARs. In some embodiments, the amino acid sequence of the human CD8α transmembrane region comprises the sequence shown in SEQ ID NO:27.

[0098] Intracellular signaling regions (or intracellular signal transduction regions) are responsible for activating at least one normal effector function of immune effector cells expressing chimeric antigen receptors. For example, the effector function of T cells can be lytic activity or helper activity, including cytokine secretion. While the entire intracellular signal transduction region can generally be used, in many cases, using the whole chain is unnecessary. Regarding the use of truncated portions of intracellular signal transduction regions, such truncated portions can be used instead of the whole chain as long as they transduce effector function signals. Therefore, intracellular signal transduction regions include any truncated form of intracellular signal transduction regions sufficient to transduce effector function signals. The intracellular signaling domain of a CAR can be selected as needed, including but not limited to intracellular signaling domains derived from at least one of CD3ζ, FcRγ (FCER1G), FcRβ (FcεRib), CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d. Preferably, the intracellular signaling region is derived from the human CD3ζ intracellular signaling region. Furthermore, the human CD3ζ intracellular signaling region has the amino acid sequence shown in SEQ ID NO:29.

[0099] In addition to stimulation by antigen-specific signals, many immune effector cells require co-stimulation to promote cell proliferation, differentiation, and survival, as well as to activate effector functions. The "co-stimulatory domain" can be the cytoplasmic portion of a co-stimulatory molecule. The term "co-stimulatory molecule" refers to an associated binding chaperone on immune cells (such as T cells) that specifically binds to a co-stimulatory ligand, thereby enabling the immune cell to mediate a co-stimulatory response, such as, but not limited to, proliferation and survival. Suitable intracellular co-stimulatory domains can be selected as needed, including intracellular domains containing co-stimulatory signaling molecules, such as at least one of the intracellular domains 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 41BBL. In some embodiments, the amino acid sequence of the 4-1BB co-stimulatory domain comprises the sequence shown in SEQ ID NO:28.

[0100] The aforementioned portions forming the chimeric antigen receptor of the present invention, such as the CD8 signal peptide, the anti-CD318 nanobody, the CD8α hinge region, the CD8α transmembrane region, the CD3ζ intracellular signal domain, and the 4-1BB co-stimulatory domain, can be directly linked to each other or linked via adapter sequences. The adapter sequence can be a known antibody-compatible adapter sequence, such as a G and S-containing adapter sequence. Typically, the adapter contains one or more repeating motifs. For example, the motif can be GGGS, GGGGS, SSSSG, GSGSA, and GGSGG. Preferably, the motifs are adjacent in the adapter sequence, with no inserted amino acid residues between the repeats. The adapter sequence can consist of 1, 2, 3, 4, or 5 repeating motifs. The length of the adapter can be 3 to 25 amino acid residues, for example, 3 to 15, 5 to 15, or 10 to 20 amino acid residues. In some embodiments, the adapter sequence is a polyglycine adapter sequence. The number of glycine residues in the linker sequence is not particularly limited, typically ranging from 2 to 20, for example, 2 to 15, 2 to 10, or 2 to 8. Besides glycine and serine, the linker may also contain other known amino acid residues, such as alanine (A), leucine (L), threonine (T), glutamic acid (E), phenylalanine (F), arginine (R), and glutamine (Q). In some embodiments, the linker sequence is (GGGGS)n-linked, where n is an integer from 1 to 5.

[0101] In an exemplary embodiment, the CAR contains, from the N-terminus to the C-terminus, a CD8 signal peptide, the anti-CD318 nanobody described herein or its antigen-binding fragment, a CD8α hinge region, a CD8α transmembrane region, a 4-1BB co-stimulatory domain, and a CD3ζ intracellular signaling domain. In a specific embodiment, an exemplary CAR having the above structure is shown in any of SEQ ID NO:30-35.

[0102] It should be understood that in gene cloning, it is often necessary to design suitable restriction enzyme sites, which inevitably introduces one or more irrelevant residues at the end of the expressed amino acid sequence, without affecting the activity of the target sequence. To construct fusion proteins, promote the expression of recombinant proteins, obtain recombinant proteins that are automatically secreted outside the host cell, or facilitate the purification of recombinant proteins, it is often necessary to add some amino acids to the N-terminus, C-terminus, or other suitable regions within the recombinant protein, such as, but not limited to, suitable adaptor peptides, signal peptides, leader peptides, and terminal extensions. Therefore, the amino or carboxyl terminus of the CAR of the present invention may also contain one or more polypeptide fragments as protein tags. Any suitable tag can be used herein. 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 for protein purification.

[0103] The antigen recognition region in the CAR of the present invention can be the aforementioned anti-CD318 nanobody or a variant of its functional fragment sequence. Furthermore, other parts of the CAR can also undergo sequence changes, resulting in a mutant with 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 retaining the biological activity of the CAR (e.g., activation of T cells). Sequence identity between two aligned sequences can be calculated using, for example, NCBI's BLASTp.

[0104] The mutant also includes an amino acid sequence having one or more 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 typically refers to 1-10, for example 1-8, 1-5, or 1-3. Substitution is preferably conserved. For example, in the art, conserved substitution with amino acids of similar or comparable properties generally does not alter the function of the protein or peptide. "Amino acids with similar or comparable properties" includes, for example, families of amino acid residues having similar side chains. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, replacing one or more sites in the polypeptide of the present invention with another amino acid residue from the same side chain class will not substantially affect its activity.

[0105] Nucleic acid

[0106] This invention also provides polynucleotides encoding the aforementioned antibodies or CARs. The polynucleotides of this invention can be in DNA or RNA form. DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand. This invention also includes degenerate variants of polynucleotide sequences encoding fusion proteins, i.e., nucleotide sequences encoding the same amino acid sequence but with different nucleotide sequences.

[0107] Therefore, the present invention also relates to polynucleotides that hybridize with the above-mentioned polynucleotide sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that are hybridizable with the polynucleotides described herein under stringent conditions. In the present invention, “stringent conditions” means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with a denaturing agent, such as 50% (v / v) formamide, 0.1% fetal bovine serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization only occurs when the identity between the two sequences is at least 90%, preferably at least 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0108] The full-length nucleotide sequence or fragments of the antibody of the present invention can generally be obtained by PCR amplification, recombinant methods, or artificial synthesis. One feasible method is to synthesize the relevant sequence artificially, especially when the fragment length is short. Typically, a long fragment can be obtained by first synthesizing multiple small fragments and then ligating them. Furthermore, the coding sequence of the heavy chain and an expression tag (such as 6His) can be fused together to form a fusion protein. The CAR sequence can also be obtained as described above. Alternatively, the sequences of the various parts of the CAR (signal peptide, antigen recognition region, hinge region, transmembrane region, or intracellular region) can be obtained as described above and then ligated to obtain the full-length CAR.

[0109] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transforming it into cells, and then isolating the sequence from the proliferated host cells using conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in this invention include biomolecules existing in isolated forms. Currently, DNA sequences encoding the proteins of this invention (or fragments thereof, or derivatives thereof) can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors, etc.) and cells known in the art. Furthermore, mutations can be introduced into the protein sequences of this invention through chemical synthesis. The CAR portions can be sequentially cloned into a vector or integrated into a full-length CAR before cloning.

[0110] This invention also relates to nucleic acid constructs containing the polynucleotide sequences described herein, and one or more regulatory sequences operatively linked to these sequences. The polynucleotide sequences described herein can be manipulated in various ways to ensure the expression of the antibody or CAR. The nucleic acid constructs can be manipulated depending on the expression vector or requirements before insertion into a vector. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.

[0111] The regulatory sequence can be a suitable promoter sequence. Promoter sequences are typically operatively linked to the coding sequence of the protein to be expressed. A promoter can be any nucleotide sequence that exhibits transcriptional activity in the chosen host cell, including mutant, truncated, and heterozygous promoters, and can be obtained from a gene encoding an extracellular or intracellular polypeptide that is homologous or heterologous to that host cell. An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strongly constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operatively linked to it. 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 the early promoter of simian virus 40 (SV40), mouse mammary cancer virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Russ'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 inducible promoters provides a molecular switch that can turn on the expression of the polynucleotide sequence operatively linked to the inducible promoter during time-limited expression and turn 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.

[0112] The regulatory sequence can also be a suitable transcription terminator sequence, a sequence recognized by the host cell to terminate transcription. The terminator sequence is operatively linked to the 3' end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in a selected host cell can be used in this invention. The regulatory sequence can also be a suitable leader sequence, the untranslated region of mRNA important for translation by the host cell. The leader sequence is operatively linked to the 5' end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in a selected host cell can be used in this invention.

[0113] In some embodiments, the nucleic acid construct is a vector, such as a cloning vector, an expression vector, and an integration vector. Expression of the polynucleotide sequences of the present invention is typically achieved by operably linking the polynucleotide sequences of the present invention to an expression vector. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters that can be used to regulate the expression of the desired nucleic acid sequence. Integration vectors contain components for integrating the target sequence into the cellular genome. These vectors can be used to transform appropriate host cells to enable them to express proteins. Vectors typically contain sequences for plasmid maintenance and for cloning and expressing exogenous nucleotide sequences. These sequences (collectively referred to as “flanking sequences” in some embodiments) typically 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 complete intron sequence containing donor and acceptor splicing sites, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylated sequence, a multi-connector region for inserting a nucleic acid encoding an antibody to be expressed, and optional marker elements.

[0114] Furthermore, the type of vector is not limited; for example, plasmids, phage particles, phage derivatives, animal viruses, and entrapments can be modified depending on the host cell to be introduced. Viral vector technology is well known in the art and has been 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.

[0115] To assess the expression of CAR peptides or portions thereof, the vector introduced into cells may also contain one or both of an optional marker gene or reporter gene to facilitate the identification and selection of expressing cells from a population of cells seeking transfection or infection via a viral vector.

[0116] cell

[0117] The host cells suitable for introducing the nucleic acid constructs described herein can be prokaryotic cells, such as bacterial cells; lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells, especially immune cells, preferably immune effector cells. Representative examples include: Escherichia coli, Streptomyces; Salmonella typhimurium bacterial cells; fungal cells such as yeast; Drosophila S2 or Sf9 insect cells; and animal cells such as CHO, COS7, and 293 cells.

[0118] "Immune effector cells" are immune cells capable of performing immune effector functions. In some embodiments, immune effector cells express at least FcγRIII and perform ADCC effector functions. Examples of immune effector cells mediating ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, neutrophils, and eosinophils. Preferably, 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 (PBMCs), and hematopoietic stem cells. More preferably, the immune effector cells are T lymphocytes (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 a chimeric antigen receptor and binding to target cells. In some implementations, CD8+ T cells lyse antigen-specific target cells when expressing chimeric antigen receptors and binding to target cells.

[0119] The T cells suitable for use in this invention can be of various types and origins. For example, T cells can be derived from PBMCs of patients with malignant solid tumors (e.g., pancreatic cancer). In some embodiments, after obtaining T cells, they can be activated by stimulation with an appropriate amount (e.g., 30–80 ng / ml, such as 50 ng / ml) of CD3 antibody, and then cultured in IL2 medium containing an appropriate amount (e.g., 30–80 IU / ml, such as 50 IU / ml) for later use.

[0120] Methods for introducing nucleic acids or vectors into mammalian cells are known in the art, and the vectors can be transferred into cells by physical, chemical, or biological methods. When the host is a prokaryote such as *Escherichia coli*, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with CaCl2, the steps of which are well known in the art. When the host is a eukaryote, DNA transfection methods such as calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, and liposome packaging can be used. In some embodiments, transduced or transfected immune effector cells proliferate in vitro after the introduction of nucleic acids or vectors.

[0121] The obtained transformants can be cultured using conventional methods to express the antibody or CAR encoded by the gene of this invention. Depending on the host cells used, the culture medium can be selected from various conventional media. Culture is carried out under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature adjustment or chemical induction), and the cells are cultured for a further period.

[0122] The peptides used in the methods described above can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be separated and purified using various separation methods based on their 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 precipitants (salting out), centrifugation, permeation, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0123] Uses and methods

[0124] By constructing a nanobody library, the inventors screened nanobodies that could bind to CD318. Using these nanobodies, the inventors constructed CAR and CAR-T cells. Cell-level experiments verified that the CAR-T cells have strong immune function, superior CD107a expression, IFN-γ and IL-2 secretion, and specific killing function against target cells, demonstrating significant in vivo efficacy.

[0125] All aspects of the antibodies, CARs, coding sequences, nucleic acid constructs, and cells described herein can be used to prepare drugs for the prevention or treatment of the various conditions and diseases described herein, which are diseases or conditions related to CD318 expression, referring to diseases directly or indirectly caused by abnormal CD318 expression, usually referring to diseases caused by CD318 overexpression, such as cancer, including but not limited to: breast cancer, lung cancer, liver cancer, pancreatic cancer, ovarian cancer, kidney cancer, and colorectal cancer.

[0126] This invention also includes a class of cell therapies comprising expressing the CAR described herein in immune cells (e.g., T cells) and administering a therapeutically effective amount of the cells to a recipient who requires them, the cells being capable of killing the recipient's tumor cells. Compared to antibody therapies, CAR-T cells can replicate in vivo, producing long-lasting durability that can lead to sustained tumor control. The anti-tumor immune response induced by CAR-T cells can be an active or passive immune response. Additionally, CAR-mediated immune responses can be part of an adoptive immunotherapy step, wherein CAR-T cells induce an immune response specific to the antigen-binding portion of the CAR.

[0127] The antibodies, nucleic acids, or CAR-modified cells of the present invention can be administered alone or as a pharmaceutical composition in combination with a diluent and / or other components such as associated cytokines or cell populations. In this regard, the pharmaceutical compositions can be prepared by mixing an active pharmaceutical agent of desired purity with an optional pharmaceutically acceptable carrier in the form of a lyophilized formulation or an aqueous solution. Pharmaceutically acceptable carriers are non-toxic to the recipient at the dose and concentration used and may include at least one of buffers (e.g., neutral buffered saline, sulfate buffered saline), antioxidants, preservatives, isotonic agents, stabilizers, chelating agents (e.g., EDTA or glutathione), adjuvants (e.g., aluminum hydroxide), and surfactants. Furthermore, in order for the pharmaceutical compositions to be usable for in vivo administration, they must be sterile. The pharmaceutical compositions can be sterilized by filtration through a sterile filter membrane.

[0128] In some embodiments, the pharmaceutical composition may contain at least one additive selected from: a cytotoxic agent, a chemotherapeutic agent, a cytokine, an immunosuppressant, a growth inhibitor, and an active pharmaceutical agent required for the specific indication to be treated. The specific amount of the additive may be adjusted as needed. The pharmaceutical composition of the present invention may be administered in amounts described as “immunologically effective,” “antitumor effective,” “tumor-inhibitory effective,” or “therapeutic.” “Therapeutic” refers to a subject receiving the treatment regimen described herein to achieve at least one positive therapeutic effect (e.g., a reduction in the number of cancer cells, a reduction in tumor volume, a decrease in the rate of cancer cell invasion into surrounding organs, or a decrease in the rate of tumor metastasis or tumor growth). When “immunologically effective,” “antitumor effective,” “tumor-inhibitory effective,” or “therapeutic” is indicated, the precise amount of the composition of the present invention to be administered may be determined by a physician, taking into account individual differences in the patient’s (subject’s) age, weight, tumor size, degree of infection or metastasis, and disease. Typically, a pharmaceutical composition including T cells as described herein may be administered in an amount of 10 4 Up to 10 9 A dose of cells / kg body weight, preferably 10. 5 Up to 10 6 The dosage is cells per kg of body weight. T-cell compositions can also be administered at these dosages multiple times. Cells can be administered using infusion techniques known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dosage and treatment regimen for a specific patient can be readily determined by a physician skilled in the medical field by monitoring the patient's disease signs and thus adjusting the treatment accordingly.

[0129] The composition can be administered in any convenient manner, including by spraying, injection, swallowing, infusion, implantation, or transplantation. The composition described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intraspinally, intramuscularly, intravenously, or intraperitoneally. In one embodiment, the T-cell composition of the present invention is administered to a patient by intradermal or subcutaneous injection. In another embodiment, the T-cell composition of the present invention is preferably administered by intravenous injection. The T-cell composition can be injected directly into the tumor, lymph node, or site of infection.

[0130] In some embodiments of the present invention, the CAR-T cells or compositions thereof of the present invention can be combined with other therapies known in the art. These therapies include, but are not limited to, chemotherapy, radiotherapy, and immunosuppressants. For example, treatment may be combined with radiotherapy or chemotherapy agents known in the art for treating mesothelin-mediated diseases.

[0131] In this article, "anti-tumor effect" refers to a biological effect that can be represented 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 cancer-related physiological symptoms.

[0132] The terms "patient," "subject," and "individual" are used interchangeably in this article 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 their transgenic species.

[0133] Diagnostics, tests and kits

[0134] The binding molecules of this invention, due to their high affinity for CD318, can be used for assays, 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 further investigating the role of CD318 in disease. The method for detecting CD318 generally involves obtaining cell and / or tissue samples; detecting the level of CD318 in the samples.

[0135] The CD318 binding molecule of this invention can be used for diagnostic purposes to detect, diagnose, or monitor CD318-related diseases and / or conditions. This invention provides methods for detecting the presence of CD318 in samples using classical immunohistochemical 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.

[0136] For diagnostic applications, binding molecules such as single-domain antibodies are typically 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, rhodamine, lanthanide phosphors), enzymatic groups (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent groups, biotinylated groups, or predetermined polypeptide epitopes recognized by secondary reporter molecules (e.g., leucine zipper pairs, binding sites for secondary antibodies, metal-binding domains, epitope tags), MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents. Various methods for labeling proteins are known in the art and can be used in carrying out this invention.

[0137] Another aspect of the invention provides a method for detecting the presence of a test molecule that competes with the antibody of the invention for binding to CD318. An example of such a determination would involve detecting the amount of free antibody in a solution containing a certain amount of CD318, in the presence or absence of the test molecule. An increase in the amount of free antibody (i.e., antibody not bound to CD318) would indicate that the test molecule is able to competitively bind to CD318 with the antibody. 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 the antibody.

[0138] This invention also provides a detection kit for detecting CD318 levels, comprising an antibody that recognizes the CD318 protein, a lysis medium for dissolving samples, and universal reagents and buffers required for detection, such as various buffers, detection labels, and detection substrates. This detection kit can be used as an in vitro diagnostic device.

[0139] The present invention will be described below by way of specific embodiments. It should be understood that these embodiments are merely illustrative and are not intended to limit the scope of the invention. Unless otherwise stated, the methods and materials used in the embodiments are conventional materials and methods in the art.

[0140] Example

[0141] Example 1: Construction and eukaryotic expression of recombinant human CD318 protein expression vector

[0142] 1. Synthesis of gene sequences and construction of protein expression vectors

[0143] The human CD318 protein sequence was downloaded from Uniport (https: / / www.uniprot.org / uniprot), optimized using an online codon optimization tool (http: / / www.jcat.de / #opennewwindow), and then synthesized by Sangon Biotech (Shanghai) Co., Ltd. Simultaneously, avi-tag and 6×his-tag nucleic acid sequences were added to the 3' end of this gene sequence, and the fusion gene sequence encodes the amino acid sequence shown in SEQ ID NO:42. The fusion gene sequence is shown in SEQ ID NO:43. Molecular cloning was used to clone the splicing product into pTT5 using the TaKaRa seamless cloning kit to obtain the expression vector.

[0144] 2. Expression and purification of recombinant human CD318 protein

[0145] Five days after transfecting 293T cells (ATCC) with the obtained expression vector, the culture supernatant was collected, and recombinant human CD318 protein was purified using AKTA Explorer 100 (GE). The CD318 protein, after SDS-PAGE electrophoresis and Coomassie Brilliant Blue staining, showed a size of approximately 110 kDa. The results are as follows... Figure 1 As shown.

[0146] Example 2: Preparation of CD318VHH antibody

[0147] 1. Construction of VHH phage display library

[0148] A one-step method was used to construct a nanobody phage display library, which involved ligating the alpaca nanobody VHH gene into a phage display vector.

[0149] 1) Alpaca Immunization

[0150] Alpaca immunization services are provided by Chengdu Apak Biotechnology Co., Ltd., and the specific operating procedures are as follows:

[0151] Alpaca Selection: Choose healthy, strong alpacas with good spirits and moderate size. Select alpacas with glossy coats and no signs of injury or discomfort. After selecting the animals, pre-raise them for about one week to weed out any unsuitable animals, ensuring the smooth progress of later experiments.

[0152] Immunization Protocol B: Select suitable alpacas and ensure they are fit. Record their ear tags and begin the immunization experiment. A total of four immunizations will be performed. The immunization protocol is as follows: D0, collect 10 mL of blood before immunization as a negative serum control. Mix 0.5 mg of antigen with 1 mL of CFA and inject subcutaneously; D21, mix 0.25 mg of antigen with 1 mL of CFA and inject subcutaneously; D28, collect 10 mL of blood; D42, mix 0.25 mg of antigen with 1 mL of CFA and inject subcutaneously; D49, collect 50 mL of peripheral blood and separate lymphocytes; D63, mix 0.25 mg of antigen with 1 mL of CFA and inject subcutaneously; D70, collect 50 mL of peripheral blood and separate lymphocytes.

[0153] C serum test:

[0154] a) Dilute the antigen to 2 μg / mL with 0.05 M carbonate buffer (pH 9.6), and coat it overnight at 4°C at a rate of 100 μL / well;

[0155] 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 h;

[0156] c) Wash 3 times with PBST, add 100 μL / well of serum diluent (start with serial dilution from 1:2000), incubate at 37°C for 45 min;

[0157] d) Wash 5 times with PBST, add 100 μL of horseradish peroxidase-labeled goat anti-Alpaca secondary antibody (diluted with PBS at a ratio of 1:1W) to each well, and incubate at 37°C for 45 min.

[0158] e) Wash the plate 5 times with PBST. Add TMB chromogenic solution (100 μL / well) for color development, incubate at 37°C for 5 min, then add stop solution (50 μL / well) to terminate the reaction. Measure the optical density at 450 nm.

[0159] 2) cDNA synthesis

[0160] Total RNA extraction from PBMCs: Peripheral blood lymphocytes preserved in Trizol were thawed on ice and transferred to 1.5 mL centrifuge tubes. 1 / 5 volume of chloroform was added, and the mixture was vortexed and incubated at room temperature for 5 minutes. The mixture was then centrifuged at 12000 g for 15 minutes at 4°C. The supernatant was transferred to a new centrifuge tube, and an equal volume of isopropanol was added. The mixture was incubated at room temperature for 10 minutes and then centrifuged at 12000 g for 10 minutes at 4°C. The precipitate was washed with 75% ethanol, centrifuged at 7500 g for 5 minutes at 4°C, and the supernatant was discarded. The precipitate was air-dried at room temperature and dissolved in an appropriate amount of RNase-free water. RNA purity was analyzed using the A260 / 280 ratio to prepare for RNA transcription.

[0161] cDNA Synthesis: Using SuperScript TM IV First-Strand Synthesis System kit, reverse transcribed to obtain cDNA and stored at -80℃.

[0162] 3) VHH gene amplification

[0163] Using alpaca PBMC cDNA as a template, the VHH-CH2 gene was amplified by PCR under the following conditions: pre-denaturation at 98℃ for 45 seconds, followed by temperature cycling: denaturation at 98℃ for 15 seconds, annealing at 58℃ for 20 seconds, extension at 72℃ for 45 seconds, for 30 cycles, and a final extension at 72℃ for 7 minutes. The PCR product was then subjected to 1.5% agarose gel electrophoresis, and the 750 bp target band was recovered using a gel extraction kit (Promega). Using VHH-CH2 as a template, the VHH gene was amplified by PCR under the following conditions: pre-denaturation at 98℃ for 45 seconds, followed by temperature cycling: denaturation at 98℃ for 15 seconds, annealing at 60℃ for 20 seconds, extension at 72℃ for 45 seconds, for 30 cycles, and a final extension at 72℃ for 7 minutes. After electrophoresis of the PCR products on a 1.5% agarose gel, the 400bp target band was recovered using a gel recovery kit (Promega).

[0164] 4) Construction of the VHH Library

[0165] The phage vector pcomb3X and the VHH gene were digested with SfiI DNA endonuclease at 50°C for 16 h. The digested pcomb3X vector was then subjected to 1% agarose gel electrophoresis, and the 4000 bp fragment was recovered using a Promega gel extraction kit. The digested VHH gene (400 bp) was directly recovered by column chromatography using a gel extraction kit. The VHH gene was ligated into the phage vector using an Invitrogen T4 DNA ligase kit and incubated overnight at 16°C. A small amount of the ligation product was taken for agarose gel electrophoresis to assess ligation efficiency. The ligation product was desalted using a MECK MILLIPOREF microporous membrane.

[0166] The ligation product was added to self-made TG1 electroporation competent cells, and then electroporation was performed using an electroporator. 50 μL of the bacterial culture was then serially diluted 10⁻⁶ times with PBS. 2 -10 510 μL of each serial dilution was flow-lined onto an Amp / 2YT plate and incubated overnight at 37°C. The size of the phage antibody library was then counted and determined. The remaining electroporated bacteria were replenished with 2YT to a final volume of 500 mL, and then incubated overnight at 30°C and 220 rpm with 100 μg / mL ampicillin. The final VHH immunotherapy library exceeded 9E9 cells. The electroporated antibody library was amplified overnight, and the bacterial cells were collected by centrifugation and stored at -80°C with 20% glycerol to a final concentration.

[0167] A portion of the frozen alpaca natural antibody phage display library was inoculated into a 2YT culture set at an inoculation density of 0.1 OD. The bacterial culture was incubated at 37°C and 220 rpm for approximately 1.5 hours, until the bacterial density reached 0.6 OD. At this point, 20 times the number of M13KO7 phage cells were added and allowed to stand for 30 minutes for infection. The culture was then incubated overnight at 30°C and 220 rpm. The next day, the bacterial culture was centrifuged at 10,000 g, and the supernatant was collected. 1 / 4 volume of PEG / NaCl solution (20% PEG8000, 2.5 M NaCl) was added to the supernatant, mixed well, and incubated on ice for 1 hour. After the ice incubation, the culture was centrifuged at 8,000 g for 10 minutes, and the precipitate was collected. The precipitate was dissolved in 10% Glycerol / PBST to obtain the VHH phage display library. The OD268 was measured, and the library was aliquoted into 1.5 mL centrifuge tubes (6 OD / tube) and stored at -80°C.

[0168] 2. Select CD318VHH antibody

[0169] 1) Biotinylation of recombinant human CD318 protein

[0170] Using a biotinylation kit (EasyBio) following the kit instructions, the avi-tag of recombinant human CD318 protein was biotinylated to obtain biotinylated CD318 protein. 10 μg of the biotinylated recombinant protein was added to 100 μL of streptavidin magnetic beads (DynaBeads 280) that had been washed three times with PBS. The mixture was placed on a rotary shaker at 20 rpm and coupled at room temperature for 1 hour, followed by washing three times with PBS.

[0171] 2) Blocking phage libraries and negative magnetic beads

[0172] Take one vial of VHH lib, thaw it at room temperature, add 200 μL of 5% BSA / PBST, place it on a rotary shaker at 20 rpm, and incubate at room temperature for 1 hour. These phages are designated Input1. Simultaneously, take 100 μL of unconjugated DynaBeads280, wash it three times with PBS, add 1 mL of 1% BSA / PBS, and incubate under the same conditions for 1 hour.

[0173] 3) Blocking positive magnetic beads

[0174] Add 1 mL of 1% BSA / PBS to the magnetic beads coupled with CD318 and rotate at 20 rpm for 1 hour at room temperature.

[0175] 4) Negative screening

[0176] To remove antibodies that interact with the magnetic beads, negative panning is necessary. A BSA-blocked phage library and unconjugated magnetic beads are mixed and incubated under the conditions described above for 1 hour. After incubation, the phage-magnetic bead mixture is placed on a magnetic rack. Once the beads have adhered to the walls, the supernatant is transferred to a new EP tube.

[0177] 5) Positive screening

[0178] The blocked magnetic beads conjugated with CD318 protein were added to the negatively screened phage supernatant for positive screening. The mixture was rotated at 20 rpm for 1 hour at room temperature. After incubation, the magnetic beads were washed with 1 mL of PBST (0.1% Tween-20 in PBS), repeating the wash 10 times. After washing, 1 mL of 100 mM glycine (pH 2.0) was added, and the mixture was placed on a rotary shaker at 20 rpm for elution for 10 minutes. After elution, the EP tube was placed on a magnetic rack, and once the magnetic beads adhered to the walls, the eluent was transferred to a new EP tube. 0.2 mL of 1 M Tris-HCl solution (pH 8.0) was added to the eluent for neutralization. The neutralized eluent was added to 30 mL of TG1 bacterial culture with an OD600 of approximately 0.6 and allowed to stand for 30 minutes. Then, 20 times the number of M13KO7 phages were added and allowed to stand for another 30 minutes. Finally, 100 mL of 2YT medium and ampicillin and kanamycin at a final concentration of 100 μg / mL were added, and the mixture was incubated overnight at 30°C and 220 rpm. The next day, phages were harvested using the same method described above for harvesting phage libraries. The resulting phage was Input2.

[0179] 6) Repeated positive screening

[0180] The above selection method was repeated twice, that is, Input2 was subjected to a second round of negative and positive selection to obtain Input3. The difference was that after the eluent obtained from Input3 was infected with TG1, M13KO7 was not added. Instead, 10 μL of bacterial culture was serially diluted with PBS, and 10 μL of the solution was used. 3 10 4 10 5 Spread 100 μL of bacterial culture from each of the three dilution gradients onto 2YT / Amp plates and incubate overnight at 30°C. Incubate the remaining bacterial culture overnight at 30°C and 220 rpm.

[0181] 7) ELISA screening for positive antibodies

[0182] Using a pipette tip, randomly pick TG1 monoclonal antibodies from the above plates and transfer them to 800 μL of a deep-well plate containing 10× self-induced 2 YT / Amp. Cover the deep-well plate with a breathable membrane and incubate at 37°C and 220 rpm for 3 hours, then incubate overnight at 30°C and 220 rpm. Coat each well of the ELISA plate with 100 ng of recombinant human CD318 protein. The next day, first take 50 μL of the culture medium from the deep-well plate to preserve the bacteria, and centrifuge the remaining medium at 4000 rpm for 10 minutes to remove the culture medium, retaining the bacterial pellet. Add 100 μL of TES solution (20% sucrose, 0.1 mM EDTA, 50 mM Tris-HCl, pH 8.0) to each well, shake to resuspend the bacteria, incubate on ice for 30 minutes, then add 200 μL of ultrapure water and shake to mix for 30 minutes. After shaking, centrifuge at 4000 rpm for 10 minutes. The supernatant in the deep-well plate at this point is the periplasmic extract containing the antibody. Wash the ELISA plate three times with a plate washer, then add 200 μL of 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-mentioned periplasmic extract, incubate at 37°C for 1 hour, wash three times with a plate washer, add 100 μL of Chicken anti-HA HRP (1% BSA / PBS), incubate at 37°C for 1 hour, wash three times with a plate washer, add 100 μL of TMB chromogenic solution, develop at 37°C for 10 minutes, and add 100 μL of stop solution. Read the OD450 value using a microplate reader. Perform Sanger sequencing on clones with OD450 values ​​three times higher than the background value to obtain the antibody gene sequence.

[0183] 8) Verify positive clones

[0184] Based on the sequencing results, clones with significant amino acid sequence differences in antibody CDR3 were selected, re-inoculated, and induced overnight. The ELISA method described above was then used to verify again whether the selected clones could bind to CD318. 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.

[0185] Example 3: Preparation of retroviral stock solution containing anti-human CD318 chimeric antigen receptor element

[0186] 1. Preparation of CAR targeting human CD318 antigen

[0187] Genetic synthesis or cloning of chimeric antigen receptor sequences containing single-domain antibodies (VHH), hinge regions, transmembrane regions, and intracellular signaling segments against human CD318 antigen, with structures as follows: Figure 2As shown. Based on the different VHH loading, the chimeric antigen receptors are named 4A4-BBz, 4A8-BBz, 4A10-BBz, 4B2-BBz, 4B7-BBz, 4B12-BBz, 4F5-BBz, and 4G2-BBz, respectively. The amino acid sequences of 4A4-BBz, 4A8-BBz, 4A10-BBz, 4B2-BBz, 4B7-BBz, and 4G2-BBz are shown in SEQ ID NO. 30-35, and the nucleotide sequences are shown in SEQ ID NO. 36 to SEQ ID NO. 41, respectively.

[0188] Using the reverse transcription vector MSGV as the backbone, chimeric antigen receptor retroviral plasmids expressing clones 4A4-BBz, 4A8-BBz, 4A10-BBz, 4B2-BBz, 4B7-BBz, 4B12-BBz, 4F5-BBz, and 4G2-BBz were constructed. Clones with correct sequencing were selected, inoculated into 200 ml of 2YT medium, cultured overnight, and plasmids were extracted using the NucleoBondXtra MaxiEF kit according to the manufacturer's instructions.

[0189] 2. Retroviral packaging

[0190] The retrovirus was packaged using the cationic polymer PEI as follows: 36 μL of PEI and the retrovirus packaging plasmid (6 μg of main viral plasmid, 3.8 μg of Gag-pol, and 1.5 μg of vsvg) were diluted separately with 600 μL of serum-free DMEM; then, PEI / DMEM was added to the plasmid / DMEM mixture, vortexed to mix, and incubated at room temperature for 15 minutes; the plasmid-PEI complex was added to pre-coated 293T cells. The medium was changed 16 h after transfection, and the first viral supernatant was collected after 48 h, and the second viral supernatant was collected after 72 h. The supernatant was filtered through a 0.45 μm filter, aliquoted into 1.5 mL centrifuge tubes (1 mL / tube), and stored at -80 °C for later use.

[0191] Example 4: Preparation of CD318 CAR-T cells and determination of CAR positivity rate

[0192] 1. PBMC Isolation and Activation

[0193] After receiving a PBMC and verifying the patient's individual identification code, resuscitation was initiated. The cell density was adjusted to 1×10⁶ cells / mL using X-VIVO complete culture medium. 6 / mL. Gently pipette the PBMCs that have recovered overnight, filter through a 70μm cell sieve, and transfer to a 50ml centrifuge tube. Centrifuge at 1500rpm for 5min at room temperature, discarding the supernatant. Resuspend the cells in an appropriate amount of DPBS, mix 20μl with trypan blue at a 1:1 ratio, count the cells, calculate the viability and CD3+ cell count, then take the required volume of cells, centrifuge at 1500rpm for 5min at room temperature, discarding the supernatant for sorting. Calculate the amount of magnetic beads used according to a 1:1 ratio of CD3 / CD28 magnetic beads to CD3+ cells: Magnetic bead amount = [CD3+ cell count / 4 × 10⁻⁶] 5 **μl.** Cleaning the magnetic beads: Take a sterile flow cytometry tube, add 2ml of DPBS and magnetic beads, and let it stand on a magnetic rack for 1 min. Discard the supernatant. Remove the flow cytometry tube from the magnetic rack, resuspend the cells in an equal volume of DPBS or X-VIVO15, add the magnetic beads and cells to the cell suspension, mix, and incubate on a rotary mixer. Incubate at room temperature for 30 min. After incubation, gently transfer the cells to a sterile flow cytometry tube, and rinse the 15ml centrifuge tube with 1ml of DPBS, adding the rinse solution to the same flow cytometry tube. Transfer the sterile flow cytometry tube to a magnetic rack, let it stand for 1 min, and discard any unabsorbed liquid. Remove the sterile flow cytometry tube from the magnetic rack, resuspend the cells in 1ml of CAR-T medium, and rinse the tube wall twice with CAR-T medium. Collect all the CAR-T medium and transfer it to the same centrifuge tube. Adjust the cell density to 1×10⁻⁶ cells / mL with CAR-T medium. 6 Add IL-2 to a final concentration of 200 IU / ml and incubate at 37°C in a 5% CO2 incubator for two days.

[0194] 2. Virus stock solution infection and culture

[0195] The activated T cells were adjusted to 5 × 10 5 Add 1 ml of T cells and 1 ml of viral stock solution to each well of a 24-well plate, along with 2 μL of polybrene. Centrifuge at 32°C, 2500 rpm for 1.5 h. Discard the supernatant and add 1 ml of T cell culture medium (containing 300 IU / ml IL-2) to each well. Incubate the plate at 37°C in a 5% CO2 incubator. 24 h post-infection, transfer to 6-well plates. Observe cell density daily and supplement with T cell culture medium containing 300 IU / ml IL-2 as needed to maintain T cell density at 1 × 10⁻⁶ cells / well. 6 Approximately 1 / ml, to promote cell proliferation.

[0196] 3. CAR positivity rate detection

[0197] The CAR positivity rate of retrovirally infected T lymphocytes was detected 72 hours after viral infection. NT samples (1×10⁻⁶) were collected from the chimeric antigen receptor group containing clones 4A4, 4A8, 4A10, 4B2, 4B7, 4B12, 4F5, and 4G2, and from the negative, uninfected control group. 6 Cells were centrifuged to remove the culture medium, washed once with 500 μL PBS, and resuspended in 100 μL of PBS in a flow cytometry tube (BD). Biotin-labeled CD318 antigen (1:200) was added and incubated at °C for 30 minutes. After washing once with PBS, secondary antibody PE-SA streptavidin (BioLegend) was added at a 1:100 ratio and incubated at °C in the dark for 30 minutes. After washing with 500 μL PBS, cells were resuspended in 200 μL PBS and analyzed by flow cytometry. The CAR-T positivity rate results are shown below. Figure 3 As shown. According to Figure 3 As a result, after excluding the two sequences 4B12 and 4F5 that might have false positives, the six CD318-VHH antibody sequences with better performance were finally obtained: 4A4, 4A8, 4A10, 4B2, 4B7, and 4G2.

[0198] Example 5: Functional analysis based on anti-human CD318 CAR-T cells

[0199] 1. Analysis of CD107a expression in anti-human CD318 CAR-T cells

[0200] CAR-T cells containing different antibody clones were mixed with target cells (CD318-positive pancreatic cancer cell line BxPC3) and control target cells (CD318-negative human glioma cells U251) at a 1:1 effector-target ratio (both effector cells and target cells were 3 × 10⁻⁶). 5 (The samples were) mixed and incubated at 37°C in a 5% CO2 incubator for 4 hours. Flow cytometry was then used to detect the proportion of CD107a-expressing cells among CAR-T positive cells in each group. The degranulation response of CAR-T cells after stimulation by target cells was evaluated. The flow cytometry results of CD107a expression are shown below. Figure 4 As shown.

[0201] 2. Detection of cytokine secretion capacity of anti-human CD318 CAR-T cells

[0202] CAR-T cells containing different antibody clones were mixed with target cells (CD318-positive cell line BxPC3) and control target cells (CD318-negative cell line U251) at effector-target ratios of 10:1 and 2:1, respectively (target cells were 3 × 10⁻⁶ cells per cell line). 4After incubation for 24 hours, the supernatant was collected, and the secretion of IFN-γ and IL-2 was detected using ELISA (enzyme-linked immunosorbent assay). IFN-γ and IL-2 were detected using the Erbitsin Human IFN-gamma ELISA Kit and Human IL-2 ELISA Kit, and the experimental procedures were performed according to the product instructions. The results of IFN-γ secretion detection are as follows: Figure 5 , Figure 6 As shown in the figure. The results of IL-2 secretion detection are as follows. Figure 7 , Figure 8 As shown.

[0203] 3. Anti-human CD318 CAR-T cell toxicity experiment

[0204] The CAR-T cell cytotoxicity assay evaluates the in vitro function of CAR-T cells by detecting their cytotoxic effect on target cells. Different effector-to-target ratios (3 × 10⁻⁶) were used. 4 Using a target cell ratio of 10:1 and 2:1 (with target cell ratios of 10:1 and 2:1 respectively), T cells were co-cultured with CD318-positive target cells BxPC3-LUC-GFP stably expressing firefly luciferase and CD318-negative control target cells U251-LUC-GFP, respectively. A positive control consisting only of target cells was also included. After overnight incubation at 37°C, 100 μL of luciferase reaction substrate was added to the culture system, and fluorescence values ​​were detected. The killing efficiency was calculated using the following formula: Killing efficiency = (Fluorescence value of positive control well - Fluorescence value of experimental well) / (Fluorescence value of positive control well) × 100%. Experimental grouping and analysis results are as follows. Figure 9 , Figure 10 As shown.

[0205] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention. sequence list <110> Shanghai Hengrunda Biotechnology Co., Ltd. <120> A VHH antibody that specifically binds to human CD318 or its antigen-binding fragment, its preparation method, and its application. <130> / <160> 43 <170> SIPOSequenceListing 1.0 <210> 1 <211> 8 <212> PRT <213> Artificial sequence <400> 1 Gly Ser Ile Phe Ser Gly Ser Ala 1 5 <210> 2 <211> 7 <212> PRT <213> Artificial sequence <400> 2 Glu Ser Ser Gly Gly Ser Thr 1 5 <210> 3 <211> 9 <212> PRT <213> Artificial sequence <400> 3 Arg Gly Phe Tyr Tyr Gly Met Gly Tyr 1 5 <210> 4 <211> 8 <212> PRT <213> Artificial sequence <400> 4 Gly Phe Thr Ser Asp Tyr Tyr Ala 1 5 <210> 5 <211> 8 <212> PRT <213> Artificial sequence <400> 5 Ile Asn Asn Ser Asp Gly Ser Thr 1 5 <210> 6 <211> 20 <212> PRT <213> Artificial sequence <400> 6 Ala Ala Asp Asp Asp Val Val Ala Gly Phe Ser Cys Ala Ser Tyr Arg 1 5 10 15 Tyr Leu Glu Leu 20 <210> 7 <211> 8 <212> PRT <213> Artificial sequence <400> 7 Gly Ser Asn Asp Ser Met Thr Ala 1 5 <210> 8 <211> 7 <212> PRT <213> Artificial sequence <400> 8 Ser Thr Ser Thr Gly Thr Ala 1 5 <210> 9 <211> 9 <212> PRT <213> Artificial sequence <400> 9 Arg Asn Phe Ile Arg Gly Arg Asp Tyr 1 5 <210> 10 <211> 8 <212> PRT <213> Artificial sequence <400> 10 Gly Ser Ser Asp Ser Met Asn Ala 1 5 <210> 11 <211> 7 <212> PRT <213> Artificial sequence <400> 11 Ser Thr Ser Val Gly Thr Ala 1 5 <210> 12 <211> 9 <212> PRT <213> Artificial sequence <400> 12 Arg Ile Phe Ser Arg Gly Arg Asp Tyr 1 5 <210> 13 <211> 8 <212> PRT <213> Artificial sequence <400> 13 Gly Phe Thr Leu Asp Tyr Tyr Ala 1 5 <210> 14 <211> 8 <212> PRT <213> Artificial sequence <400> 14 Ile Thr Ser Ser Asp Asp Asn Thr 1 5 <210> 15 <211> twenty four <212> PRT <213> Artificial sequence <400> 15 Ala Thr Asp Ala Leu Ser Ile Arg Arg Ser Ser Cys Gln Leu Gly Pro 1 5 10 15 Thr Ala Tyr Phe Gly Met Asp Tyr 20 <210> 16 <211> 8 <212> PRT <213> Artificial sequence <400> 16 Gly Asn Lys Tyr Asp Phe Asn Thr 1 5 <210> 17 <211> 7 <212> PRT <213> Artificial sequence <400> 17 Ile Ala Ala Gly Gly Arg Thr 1 5 <210> 18 <211> 17 <212> PRT <213> Artificial sequence <400> 18 Ser Ala Glu Arg Val Lys Asn His Tyr Ala Asp Tyr Ala Leu Asp Asp 1 5 10 15 Tyr <210> 19 <211> 115 <212> PRT <213> Artificial sequence <400> 19 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Ser Gly Ser 20 25 30 Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val 35 40 45 Val Val Glu Ser Ser Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Arg 85 90 95 Gly Phe Tyr Tyr Gly Met Gly Tyr Trp Gly Lys Gly Thr Gln Val Thr 100 105 110 Val Ser Ser 115 <210> 20 <211> 127 <212> PRT <213> Artificial sequence <400> 20 Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Ser Asp Tyr Tyr 20 25 30 Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ser Cys Ile Asn Asn Ser Asp Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ala Asp Asp Asp Val Val Ala Gly Phe Ser Cys Ala Ser Tyr Arg 100 105 110 Tyr Leu Glu Leu Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 21 <211> 115 <212> PRT <213> Artificial sequence <400> 21 Glu Val Gln Val Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Ser Asn Asp Ser Met Thr 20 25 30 Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Met Val 35 40 45 Ala Val Ser Thr Ser Thr Gly Thr Ala Ile Tyr Gly Asp Ser Met Lys 50 55 60 Gly Arg Phe Thr Ile Ala Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Asn Phe Glu Asp Thr Ala Val Tyr Tyr Cys Arg 85 90 95 Asn Phe Ile Arg Gly Arg Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr 100 105 110 Val Ser Ser 115 <210> 22 <211> 115 <212> PRT <213> Artificial sequence <400> 22 Glu Val Gln Val Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Ser Ser Asp Ser Met Asn 20 25 30 Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Thr Val 35 40 45 Ala Val Ser Thr Ser Val Gly Thr Ala Ile Tyr Gly Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Asn Phe Glu Asp Thr Ala Val Tyr Tyr Cys Arg 85 90 95 Ile Phe Ser Arg Gly Arg Asp Tyr Trp Gly Gln Gly Thr Gln Val Thr 100 105 110 Val Ser Ser 115 <210> 23 <211> 131 <212> PRT <213> Artificial Sequence <400> 23 Glu Val Gln Leu Val Glu Thr Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Tyr Tyr 20 25 30 Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val 35 40 45 Ser Cys Ile Thr Ser Ser Asp Asp Asn Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Asp Ala Leu Ser Ile Arg Arg Ser Ser Cys Gln Leu Gly Pro 100 105 110 Thr Ala Tyr Phe Gly Met Asp Tyr Trp Gly Lys Gly Thr Leu Val Thr 115 120 125 Val Ser Ser 130 <210> 24 <211> 123 <212> PRT <213> Artificial sequence <400> 24 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Phe Gly Asn Lys Tyr Asp Phe Asn 20 25 30 Thr Met Gly Trp Tyr Arg Gln Gly Pro Gly Lys Gln Arg Glu Leu Val 35 40 45 Ala Thr Ile Ala Ala Gly Gly Arg Thr Asn Tyr Ala Asp Ser Ala Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ser 85 90 95 Ala Glu Arg Val Lys Asn His Tyr Ala Asp Tyr Ala Leu Asp Asp Tyr 100 105 110 Trp Gly Gln Gly Ile Gln Val Thr Val Ser Ser 115 120 <210> 25 <211> 21 <212> PRT <213> Artificial sequence <400> 25 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro 20 <210> 26 <211> 45 <212> PRT <213> Artificial sequence <400> 26 Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 1 5 10 15 Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 20 25 30 Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp 35 40 45 <210> 27 <211> 24 <212> PRT <213> Artificial sequence <400> 27 Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu 1 5 10 15 Serum Leu Val Ile Thr Leu Tyr Cys 20 <210> 28 <211> 48 <212> PRT <213> Artificial sequence <400> 28 Arg Phe Ser Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe 1 5 10 15 Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly 20 25 30 Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg 35 40 45 <210> 29 <211> 111 <212> PRT <213> Artificial sequence <400> 29 Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln 1 5 10 15 Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp 20 25 30 Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro 35 40 45 Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp 50 55 60 Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg 65 70 75 80 Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr 85 90 95 Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 100 105 110 <210> 30 <211> 364 <212> PRT <213> Artificial sequence <400> 30 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu 20 25 30 Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser 35 40 45 Ile Phe Ser Gly Ser Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys 50 55 60 Gln Arg Glu Leu Val Val Val Glu Ser Ser Gly Gly Ser Thr Asn Tyr 65 70 75 80 Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys 85 90 95 Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala 100 105 110 Val Tyr Tyr Cys Arg Gly Phe Tyr Tyr Gly Met Gly Tyr Trp Gly Lys 115 120 125 Gly Thr Gln Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro 130 135 140 Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro 145 150 155 160 Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu 165 170 175 Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys 180 185 190 Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Arg Phe Ser 195 200 205 Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro 210 215 220 Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys 225 230 235 240 Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe 245 250 255 Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu 260 265 270 Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp 275 280 285 Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys 290 295 300 Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala 305 310 315 320 Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys 325 330 335 Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr 340 345 350 Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 355 360 <210> 31 <211> 376 <212> PRT <213> Artificial Sequence <400> 31 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu 20 25 30 Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe 35 40 45 Thr Ser Asp Tyr Tyr Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys 50 55 60 Glu Arg Glu Gly Val Ser Cys Ile Asn Asn Ser Asp Gly Ser Thr Tyr 65 70 75 80 Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala 85 90 95 Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr 100 105 110 Ala Val Tyr Tyr Cys Ala Ala Asp Asp Asp Val Val Ala Gly Phe Ser 115 120 125 Cys Ala Ser Tyr Arg Tyr Leu Glu Leu Trp Gly Gln Gly Thr Leu Val 130 135 140 Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala 145 150 155 160 Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg 165 170 175 Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys 180 185 190 Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu 195 200 205 Leu Ser Leu Val Ile Thr Leu Tyr Cys Arg Phe Ser Val Val Lys Arg 210 215 220 Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro 225 230 235 240 Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu 245 250 255 Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala 260 265 270 Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu 275 280 285 Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly 290 295 300 Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu 305 310 315 320 Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser 325 330 335 Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly 340 345 350 Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu 355 360 365 His Met Gln Ala Leu Pro Pro Arg 370 375 <210> 32 <211> 364 <212> PRT <213> Artificial sequence <400> 32 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Glu Val Gln Val Val Glu Ser Gly Gly Gly Leu 20 25 30 Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Ser 35 40 45 Asn Asp Ser Met Thr Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys 50 55 60 Gln Arg Glu Met Val Ala Val Ser Thr Ser Thr Gly Thr Ala Ile Tyr 65 70 75 80 Gly Asp Ser Met Lys Gly Arg Phe Thr Ile Ala Arg Asp Asn Ala Lys 85 90 95 Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Asn Phe Glu Asp Thr Ala 100 105 110 Val Tyr Tyr Cys Arg Asn Phe Ile Arg Gly Arg Asp Tyr Trp Gly Gln 115 120 125 Gly Thr Gln Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro 130 135 140 Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro 145 150 155 160 Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu 165 170 175 Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys 180 185 190 Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Arg Phe Ser 195 200 205 Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro 210 215 220 Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys 225 230 235 240 Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe 245 250 255 Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu 260 265 270 Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp 275 280 285 Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys 290 295 300 Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala 305 310 315 320 Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys 325 330 335 Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr 340 345 350 Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 355 360 <210> 33 <211> 364 <212> PRT <213> Artificial sequence <400> 33 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Glu Val Gln Val Val Glu Ser Gly Gly Gly Leu 20 25 30 Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Ser 35 40 45 Ser Asp Ser Met Asn Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys 50 55 60 Gln Arg Glu Thr Val Ala Val Ser Thr Ser Val Gly Thr Ala Ile Tyr 65 70 75 80 Gly Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys 85 90 95 Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Asn Phe Glu Asp Thr Ala 100 105 110 Val Tyr Tyr Cys Arg Ile Phe Ser Arg Gly Arg Asp Tyr Trp Gly Gln 115 120 125 Gly Thr Gln Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro 130 135 140 Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro 145 150 155 160 Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu 165 170 175 Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys 180 185 190 Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Arg Phe Ser 195 200 205 Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro 210 215 220 Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys 225 230 235 240 Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe 245 250 255 Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu 260 265 270 Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp 275 280 285 Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys 290 295 300 Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala 305 310 315 320 Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys 325 330 335 Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr 340 345 350 Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 355 360 <210> 34 <211> 380 <212> PRT <213> Artificial sequence <400> 34 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Glu Val Gln Leu Val Glu Thr Gly Gly Gly Leu 20 25 30 Val Gln Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe 35 40 45 Thr Leu Asp Tyr Tyr Ala Ile Gly Trp Phe Arg Gln Ala Pro Gly Lys 50 55 60 Glu Arg Glu Gly Val Ser Cys Ile Thr Ser Ser Asp Asp Asn Thr Tyr 65 70 75 80 Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala 85 90 95 Lys Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr 100 105 110 Ala Val Tyr Tyr Cys Ala Thr Asp Ala Leu Ser Ile Arg Arg Ser Ser 115 120 125 Cys Gln Leu Gly Pro Thr Ala Tyr Phe Gly Met Asp Tyr Trp Gly Lys 130 135 140 Gly Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro 145 150 155 160 Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro 165 170 175 Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu 180 185 190 Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys 195 200 205 Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Arg Phe Ser 210 215 220 Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro 225 230 235 240 Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys 245 250 255 Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe 260 265 270 Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu 275 280 285 Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp 290 295 300 Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys 305 310 315 320 Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala 325 330 335 Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys 340 345 350 Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr 355 360 365 Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg 370 375 380 <210> 35 <211> 372 <212> PRT <213> Artificial sequence <400> 35 Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu 20 25 30 Val Gln Ala Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Phe Gly Asn 35 40 45 Lys Tyr Asp Phe Asn Thr Met Gly Trp Tyr Arg Gln Gly Pro Gly Lys 50 55 60 Gln Arg Glu Leu Val Ala Thr Ile Ala Ala Gly Gly Arg Thr Asn Tyr 65 70 75 80 Ala Asp Ser Ala Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys 85 90 95 Asn Thr Val Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala 100 105 110 Val Tyr Tyr Cys Ser Ala Glu Arg Val Lys Asn His Tyr Ala Asp Tyr 115 120 125 Ala Leu Asp Asp Tyr Trp Gly Gln Gly Ile Gln Val Thr Val Ser Ser 130 135 140 Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala 145 150 155 160 Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly 165 170 175 Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile 180 185 190 Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val 195 200 205 Ile Thr Leu Tyr Cys Arg Phe Ser Val Val Lys Arg Gly Arg Lys Lys 210 215 220 Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr 225 230 235 240 Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly 245 250 255 Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala 260 265 270 Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg 275 280 285 Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu 290 295 300 Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn 305 310 315 320 Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met 325 330 335 Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly 340 345 350 Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala 355 360 365 Leu Pro Pro Arg 370 <210> 36 <211> 1095 <212> DNA <213> Artificial sequence <400> 36 atggctctgc ctgtgaccgc cctgctgctg cctctggctc tgctgctgca cgccgctcgg 60 cctcaagtgc agctggtgga gtctggggga ggcttggtgc aggctggggg gtctctgaga 120 ctctcctgtg cagcctctgg aagcatcttc agtggcagtg ccatgggctg gtaccgccag 180 gctccaggga agcagcgcga gttggtcgtt gtagagtcta gtggtggtag cacaaactac 240 gcagactccg tgaagggccg attcaccatc tccagagaca acgccaagaa cacggtgtat 300 ctgcaaatga acagcctgaa acctgaggac acagccgtct attactgtag gggcttttac 360 tacggcatgg gctactgggg caaagggacc caggtcaccg tctcctcaac tacaactcca 420 gcacccagac cccctacacc tgctccaact atcgcaagtc agcccctgtc actgcgccct 480 gaagcctgtc gccctgctgc cgggggagct gtgcatactc ggggactgga ctttgcctgt 540 gatatctaca tctgggcgcc cttggccggg acttgtgggg tccttctcct gtcactggtt 600 atcacccttt actgcaggtt cagtgtcgtg aagagaggcc ggaagaagct gctgtacatc 660 ttcaagcagc ctttcatgag gcccgtgcag actacccagg aggaagatgg atgcagctgt 720 agattccctg aagaggagga aggaggctgt gagctgagag tgaagttctc ccgaagcgca 780 gatgccccag cctatcagca gggacagaat cagctgtaca acgagctgaa cctgggaaga 840 cgggaggaat acgatgtgct ggacaaaagg cggggcagag atcctgagat gggcggcaaa 900 ccaagacgga agaaccccca ggaaggtctg tataatgagc tgcagaaaga caagatggct 960 gaggcctact cagaaatcgg gatgaagggc gaaagaagga gaggaaaagg ccacgacgga 1020 ctgtaccagg ggctgagtac agcaacaaaa gacacctatg acgctctgca catgcaggct 1080 ctgccaccaa gatag 1095 <210> 37 <211> 1131 <212> DNA <213> Artificial sequence <400> 37 atggctctgc ctgtgaccgc cctgctgctg cctctggctc tgctgctgca cgccgctcgg 60 cctgaggtgc agctcgtgga gtctggggga ggcttggtgc agcctggggg gtctctgaga 120 ctctcctgtg cagcctctgg attcacttcg gattattatg ccataggctg gttccgccag 180 gccccaggga aggagcgcga gggggtctca tgtattaata atagtgatgg tagcacatac 240 tatgcagact ccgtgaaggg ccgattcacc atctccagag acaatgccaa gaacacggtg 300 tatctgcaaa tgaacagcct gaaacctgag gacacggccg tttattactg tgcagcagac 360 gatgacgtag tagctggctt ttcgtgtgcg agctacaggt atcttgaact ttggggccag 420 ggcaccctgg tcactgtctc ctcaactaca actccagcac ccagaccccc tacacctgct 480 ccaactatcg caagtcagcc cctgtcactg cgccctgaag cctgtcgccc tgctgccggg 540 ggagctgtgc atactcgggg actggacttt gcctgtgata tctacatctg ggcgcccttg 600 gccgggactt gtggggtcct tctcctgtca ctggttatca ccctttactg caggttcagt 660 gtcgtgaaga gaggccggaa gaagctgctg tacatcttca agcagccttt catgaggccc 720 gtgcagacta cccaggagga agatggatgc agctgtagat tccctgaaga ggaggaagga 780 ggctgtgagc tgagagtgaa gttctcccga agcgcagatg ccccagccta tcagcaggga 840 cagaatcagc tgtacaacga gctgaacctg ggaagacggg aggaatacga tgtgctggac 900 aaaaggcggg gcagagatcc tgagatgggc ggcaaaccaa gacggaagaa cccccaggaa 960 ggtctgtata atgagctgca gaaagacaag atggctgagg cctactcaga aatcgggatg 1020 aagggcgaaa gaaggagagg aaaaggccac gacggactgt accaggggct gagtacagca 1080 acaaaagaca cctatgacgc tctgcacatg caggctctgc caccaagata g 1131 <210> 38 <211> 1095 <212> DNA <213> Artificial sequence <400> 38 atggctctgc ctgtgaccgc cctgctgctg cctctggctc tgctgctgca cgccgctcgg 60 cctgaggtgc aggtggtgga gtctggggga ggcttggtgc aggctggggg gtctctgaga 120 ctgtcctgtg tagcctctgg aagtaacgat agtatgactg ccatgggctg gtaccgccag 180 gctccaggga agcagcgcga aatggtcgca gtgtctacta gtactggtac ggcaatctat 240 ggagactcca tgaagggccg attcaccatc gccagagaca acgccaaaaa cacggtgtat 300 ctgcaaatga acagcctgaa ctttgaggac acagccgtct attactgccg gaacttcatt 360 cgagggagag actactgggg ccaggggacc caggtcaccg tctcctcaac tacaactcca 420 gcacccagac cccctacacc tgctccaact atcgcaagtc agcccctgtc actgcgccct 480 gaagcctgtc gccctgctgc cgggggagct gtgcatactc ggggactgga ctttgcctgt 540 gatatctaca tctgggcgcc cttggccggg acttgtgggg tccttctcct gtcactggtt 600 atcacccttt actgcaggtt cagtgtcgtg aagagaggcc ggaagaagct gctgtacatc 660 ttcaagcagc ctttcatgag gcccgtgcag actacccagg aggaagatgg atgcagctgt 720 agattccctg aagaggagga aggaggctgt gagctgagag tgaagttctc ccgaagcgca 780 gatgccccag cctatcagca gggacagaat cagctgtaca acgagctgaa cctgggaaga 840 cgggaggaat acgatgtgct ggacaaaagg cggggcagag atcctgagat gggcggcaaa 900 ccaagacgga agaaccccca ggaaggtctg tataatgagc tgcagaaaga caagatggct 960 gaggcctact cagaaatcgg gatgaagggc gaaagaagga gaggaaaagg ccacgacgga 1020 ctgtaccagg ggctgagtac agcaacaaaa gacacctatg acgctctgca catgcaggct 1080 ctgccaccaa gatag 1095 <210> 39 <211> 1095 <212> DNA <213> Artificial sequence <400> 39 atggctctgc ctgtgaccgc cctgctgctg cctctggctc tgctgctgca cgccgctcgg 60 cctgaggtgc aggtggtgga gtctggggga ggcttggtgc aggctggggg gtctctgaga 120 ctgtcctgtg tagcctctgg aagtagcgac agtatgaatg ccatgggctg gtaccgccag 180 gctccaggga agcagcgcga aacggtcgca gtgtctacta gtgttggtac ggcgatctat 240 ggagactccg tgaagggccg attcaccatc tccagagaca acgccaaaaa cacggtgtat 300 ctgcaaatga acagcctgaa ctttgaggac acagccgtct attactgccg gatcttctct 360 cgagggagag actactgggg ccaggggacc caggtcaccg tctcctcaac tacaactcca 420 gcacccagac cccctacacc tgctccaact atcgcaagtc agcccctgtc actgcgccct 480 gaagcctgtc gccctgctgc cgggggagct gtgcatactc ggggactgga ctttgcctgt 540 gatatctaca tctgggcgcc cttggccggg acttgtgggg tccttctcct gtcactggtt 600 atcacccttt actgcaggtt cagtgtcgtg aagagaggcc ggaagaagct gctgtacatc 660 ttcaagcagc ctttcatgag gcccgtgcag actacccagg aggaagatgg atgcagctgt 720 agattccctg aagaggagga aggaggctgt gagctgagag tgaagttctc ccgaagcgca 780 gatgccccag cctatcagca gggacagaat cagctgtaca acgagctgaa cctgggaaga 840 cgggaggaat acgatgtgct ggacaaaagg cggggcagag atcctgagat gggcggcaaa 900 ccaagacgga agaaccccca ggaaggtctg tataatgagc tgcagaaaga caagatggct 960 gaggcctact cagaaatcgg gatgaagggc gaaagaagga gaggaaaagg ccacgacgga 1020 ctgtaccagg ggctgagtac agcaacaaaa gacacctatg acgctctgca catgcaggct 1080 ctgccaccaa gatag 1095 <210> 40 <211> 1143 <212> DNA <213> Artificial sequence <400> 40 atggctctgc ctgtgaccgc cctgctgctg cctctggctc tgctgctgca cgccgctcgg 60 cctgaggtgc agctcgtgga gactggggga ggcttggtgc agcctggggg gtctctgaga 120 ctctcctgtg cagcctctgg attcactttg gattattatg ccataggctg gttccgccag 180 gccccaggga aggagcgcga gggggtctca tgtattacaa gtagtgatga taacacatac 240 tatgcagact ccgtgaaggg ccgattcacc atctccagag acaatgccaa gaacacggtg 300 tatctgcaaa tgaacagcct gaaacctgag gacacggccg tttattactg tgcgacggac 360 gccctaagta ttcggcgtag tagctgccag ctggggccca cggcttactt tggcatggac 420 tactggggca aagggaccct ggtcactgtc tcctcaacta caactccagc acccagaccc 480 cctacacctg ctccaactat cgcaagtcag cccctgtcac tgcgccctga agcctgtcgc 540 cctgctgccg ggggagctgt gcatactcgg ggactggact ttgcctgtga tatctacatc 600 tgggcgccct tggccgggac ttgtggggtc cttctcctgt cactggttat caccctttac 660 tgcaggttca gtgtcgtgaa gagaggccgg aagaagctgc tgtacatctt caagcagcct 720 ttcatgaggc ccgtgcagac tacccaggag gaagatggat gcagctgtag attccctgaa 780 gaggaggaag gaggctgtga gctgagagtg aagttctccc gaagcgcaga tgccccagcc 840 tatcagcagg gacagaatca gctgtacaac gagctgaacc tgggaagacg ggaggaatac 900 gatgtgctgg acaaaaggcg gggcagagat cctgagatgg gcggcaaacc aagacggaag 960 aacccccagg aaggtctgta taatgagctg cagaaagaca agatggctga ggcctactca 1020 gaaatcggga tgaagggcga aagaaggaga ggaaaaggcc acgacggact gtaccagggg 1080 ctgagtacag caacaaaaga cacctatgac gctctgcaca tgcaggctct gccaccaaga 1140 tag 1143 <210> 41 <211> 1119 <212> DNA <213> Artificial sequence <400> 41 atggctctgc ctgtgaccgc cctgctgctg cctctggctc tgctgctgca cgccgctcgg 60 cctcaggtgc agctggtaga gtctggggga ggcttggtgc aggctggggg gtctctcaga 120 ctctcctgtg cagcctttgg aaataagtac gatttcaata ccatgggctg gtaccgccag 180 ggtccaggga agcagcgcga gttggtcgca acgatagctg ctggtggtcg cacaaactat 240 gcagactccg cgaagggccg attcaccatc tccagagaca acgccaagaa cacggtgtat 300 ctgcaaatga acagcctgaa acctgaggac acagccgtct attactgtag tgccgagagg 360 gtcaagaacc actatgccga ctatgccctc gatgactact ggggccaggg gatccaggtc 420 accgtctcct caactacaac tccagcaccc agacccccta cacctgctcc aactatcgca 480 agtcagcccc tgtcactgcg ccctgaagcc tgtcgccctg ctgccggggg agctgtgcat 540 actcggggac tggactttgc ctgtgatatc tacatctggg cgcccttggc cgggacttgt 600 ggggtccttc tcctgtcact ggttatcacc ctttactgca ggttcagtgt cgtgaagaga 660 ggccggaaga agctgctgta catcttcaag cagcctttca tgaggcccgt gcagactacc 720 caggaggaag atggatgcag ctgtagattc cctgaagagg aggaaggagg ctgtgagctg 780 agagtgaagt tctcccgaag cgcagatgcc ccagcctatc agcagggaca gaatcagctg 840 tacaacgagc tgaacctggg aagacgggag gaatacgatg tgctggacaa aaggcggggc 900 agagatcctg agatgggcgg caaaccaaga cggaagaacc cccaggaagg tctgtataat 960 gagctgcaga aagacaagat ggctgaggcc tactcagaaa tcgggatgaa gggcgaaaga 1020 aggagaggaa aaggccacga cggactgtac caggggctga gtacagcaac aaaagacacc 1080 tatgacgctc tgcacatgca ggctctgcca ccaagatag 1119 <210> 42 <211> 661 <212> PRT <213> Artificial sequence <400> 42 Phe Glu Ile Ala Leu Pro Arg Glu Ser Asn Ile Thr Val Leu Ile Lys 1 5 10 15 Leu Gly Thr Pro Thr Leu Leu Ala Lys Pro Cys Tyr Ile Val Ile Ser 20 25 30 Lys Arg His Ile Thr Met Leu Ser Ile Lys Ser Gly Glu Arg Ile Val 35 40 45 Phe Thr Phe Ser Cys Gln Ser Pro Glu Asn His Phe Val Ile Glu Ile 50 55 60 Gln Lys Asn Ile Asp Cys Met Ser Gly Pro Cys Pro Phe Gly Glu Val 65 70 75 80 Gln Leu Gln Pro Ser Thr Ser Leu Leu Pro Thr Leu Asn Arg Thr Phe 85 90 95 Ile Trp Asp Val Lys Ala His Lys Ser Ile Gly Leu Glu Leu Gln Phe 100 105 110 Ser Ile Pro Arg Leu Arg Gln Ile Gly Pro Gly Glu Ser Cys Pro Asp 115 120 125 Gly Val Thr His Ser Ile Ser Gly Arg Ile Asp Ala Thr Val Val Arg 130 135 140 Ile Gly Thr Phe Cys Ser Asn Gly Thr Val Ser Arg Ile Lys Met Gln 145 150 155 160 Glu Gly Val Lys Met Ala Leu His Leu Pro Trp Phe His Pro Arg Asn 165 170 175 Val Ser Gly Phe Ser Ile Ala Asn Arg Ser Ser Ile Lys Arg Leu Cys 180 185 190 Ile Ile Glu Ser Val Phe Glu Gly Glu Gly Ser Ala Thr Leu Met Ser 195 200 205 Ala Asn Tyr Pro Glu Gly Phe Pro Glu Asp Glu Leu Met Thr Trp Gln 210 215 220 Phe Val Val Pro Ala His Leu Arg Ala Ser Val Ser Phe Leu Asn Phe 225 230 235 240 Asn Leu Ser Asn Cys Glu Arg Lys Glu Glu Arg Val Glu Tyr Tyr Ile 245 250 255 Pro Gly Ser Thr Thr Asn Pro Glu Val Phe Lys Leu Glu Asp Lys Gln 260 265 270 Pro Gly Asn Met Ala Gly Asn Phe Asn Leu Ser Leu Gln Gly Cys Asp 275 280 285 Gln Asp Ala Gln Ser Pro Gly Ile Leu Arg Leu Gln Phe Gln Val Leu 290 295 300 Val Gln His Pro Gln Asn Glu Ser Asn Lys Ile Tyr Val Val Asp Leu 305 310 315 320 Ser Asn Glu Arg Ala Met Ser Leu Thr Ile Glu Pro Arg Pro Val Lys 325 330 335 Gln Ser Arg Lys Phe Val Pro Gly Cys Phe Val Cys Leu Glu Ser Arg 340 345 350 Thr Cys Ser Ser Asn Leu Thr Leu Thr Ser Gly Ser Lys His Lys Ile 355 360 365 Ser Phe Leu Cys Asp Asp Leu Thr Arg Leu Trp Met Asn Val Glu Lys 370 375 380 Thr Ile Ser Cys Thr Asp His Arg Tyr Cys Gln Arg Lys Ser Tyr Ser 385 390 395 400 Leu Gln Val Pro Ser Asp Ile Leu His Leu Pro Val Glu Leu His Asp 405 410 415 Phe Ser Trp Lys Leu Leu Val Pro Lys Asp Arg Leu Ser Leu Val Leu 420 425 430 Val Pro Ala Gln Lys Leu Gln Gln His Thr His Glu Lys Pro Cys Asn 435 440 445 Thr Ser Phe Ser Tyr Leu Val Ala Ser Ala Ile Pro Ser Gln Asp Leu 450 455 460 Tyr Phe Gly Ser Phe Cys Pro Gly Gly Ser Ile Lys Gln Ile Gln Val 465 470 475 480 Lys Gln Asn Ile Ser Val Thr Leu Arg Thr Phe Ala Pro Ser Phe Gln 485 490 495 Gln Glu Ala Ser Arg Gln Gly Leu Thr Val Ser Phe Ile Pro Tyr Phe 500 505 510 Lys Glu Glu Gly Val Phe Thr Val Thr Pro Asp Thr Lys Ser Lys Val 515 520 525 Tyr Leu Arg Thr Pro Asn Trp Asp Arg Gly Leu Pro Ser Leu Thr Ser 530 535 540 Val Ser Trp Asn Ile Ser Val Pro Arg Asp Gln Val Ala Cys Leu Thr 545 550 555 560 Phe Phe Lys Glu Arg Ser Gly Val Val Cys Gln Thr Gly Arg Ala Phe 565 570 575 Met Ile Ile Gln Glu Gln Arg Thr Arg Ala Glu Glu Ile Phe Ser Leu 580 585 590 Asp Glu Asp Val Leu Pro Lys Pro Ser Phe His His His Ser Phe Trp 595 600 605 Val Asn Ile Ser Asn Cys Ser Pro Thr Ser Gly Lys Gln Leu Asp Leu 610 615 620 Leu Phe Ser Val Thr Leu Thr Pro Arg Thr Val Asp Leu Thr Ala Ser 625 630 635 640 Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu His 645 650 655 His His His His His 660 <210> 43 <211> 1983 <212> DNA <213> Artificial sequence <400> 43 ttcgagatcg ccctgccccg cgagagcaac atcaccgtgc tgatcaagct gggcaccccc 60 accctgctgg ccaagccctg ctacatcgtg atcagcaagc gccacatcac catgctgagc 120 atcaagagcg gcgagcgcat cgtgttcacc ttcagctgcc agagccccga gaacccttc 180 gtgatcgaga tccagaagaa catcgactgc atgagcggcc cctgcccctt cggcgaggtg 240 cagctgcagc ccagcaccag cctgctgccc accctgaacc gcaccttcat ctgggacgtg 300 aaggcccaca agagcatcgg cctggagctg cagttcagca tcccccgcct gcgccagatc 360 ggccccggcg agagctgccc cgacggcgtg acccacagca tcagcggccg catcgacgcc 420 accgtggtgc gcatcggcac cttctgcagc aacggcaccg tgagccgcat caagatgcag 480 gagggcgtga agatggccct gcacctgccc tggttccacc cccgcaacgt gagcggcttc 540 agcatcgcca accgcagcag catcaagcgc ctgtgcatca tcgagagcgt gttcgagggc 600 gagggcagcg ccaccctgat gagcgccaac taccccgagg gcttccccga ggacgagctg 660 atgacctggc agttcgtggt gcccgcccac ctgcgcgcca gcgtgagctt cctgaacttc 720 aacctgagca actgcgagcg caaggaggag cgcgtggagt actacatccc cggcagcacc 780 accaaccccg aggtgttcaa gctggaggac aagcagcccg gcaacatggc cggcaacttc 840 aacctgagcc tgcagggctg cgaccaggac gcccagagcc ccggcatcct gcgcctgcag900 ttccaggtgc tggtgcagca cccccagaac gagagcaaca agatctacgt ggtggacctg agcaacgagc gcgccatgag cctgaccatc gagccccgcc ccgtgaagca gagccgcaag ttcgtgcccg gctgcttcgt gtgcctggag agccgcacct gcagcagcaa cctgaccctg 1080 accagcggca gcaagcacaa gatcagcttc ctgtgcgacg acctgacccg cctgtggatg aacgtggaga agaccatcag ctgcaccgac caccgctact gccagcgcaa gagctacagc ctgcaggtgc ccagcgacat cctgcacctg cccgtggagc tgcacgactt cagctggaag ctgctggtgc ccaaggaccg cctgagcctg gtgctggtgc ccgcccagaa gctgcagcag 1380. cacacccacg agaagccctg cacacccagc ttcagctacc tggtggccag cgccatcccc agccaggacc tgtacttcgg cagcttctgc cccggcggca gcatcaagca gatccaggtg aagcagaaca tcagcgtgac cctgcgcacc ttcgccccca gcttccagca ggaggccagc cgccagggcc tgaccgtgag cttcatcccc tacttcaagg aggagggcgt gttcaccgtg 1560 acccccgaca ccaagagcaa ggtgtacctg cgcaccccca actgggaccg cggcctgccc 1620 agcctgacca gcgtgagctg gaacatcagc gtgccccgcg accaggtggc ctgcctgacc 1680 ttcttcaagg agcgcagcgg cgtggtgtgc cagaccggcc gcgccttcat gatcatccag 1740 gagcagcgca cccgcgccga ggagatcttc agcctggacg aggacgtgct gcccaagccc 1800 agcttccacc accacagctt ctgggtgaac atcagcaact gcagccccac cagcggcaag 1860 cagctggacc tgctgttcag cgtgaccctg accccccgca ccgtggacct gaccgctagc 1920 ggtctgaacg acatcttcga ggctcagaaa atcgaatggc acgaacatca tcaccatcac 1980 cat 1983

Claims

1. An anti-CD318 nanobody, characterized in that, The anti-CD318 nanobody comprises CDR1 with the sequence shown in SEQ ID NO:16, CDR2 with the sequence shown in SEQ ID NO:17, and CDR3 with the sequence shown in SEQ ID NO:18, and the amino acid sequence of the heavy chain variable region of the anti-CD318 nanobody is shown in SEQ ID NO:

24.

2. A chimeric antigen receptor, characterized in that, The chimeric antigen receptor comprises a signal peptide, the anti-CD318 nanobody of claim 1, a hinge region, a transmembrane region, and an intracellular region, wherein the intracellular region includes an intracellular co-stimulatory domain and an intracellular signaling domain; from the N-terminus to the C-terminus, the chimeric antigen receptor sequentially comprises the signal peptide, the anti-CD318 nanobody of claim 1, the hinge region, the transmembrane region, the intracellular co-stimulatory domain, and the intracellular signaling domain.

3. A nucleic acid molecule encoding the anti-CD318 nanobody of claim 1 or the chimeric antigen receptor of claim 2.

4. A nucleic acid construct comprising the nucleic acid molecule of claim 3.

5. A host cell, characterized in that... The host cell meets one of the following conditions: (1) Expressing the anti-CD318 nanobody of claim 1 or the chimeric antigen receptor of claim 2; (2) Contains the nucleic acid molecule as described in claim 3; (3) It includes the nucleic acid construct of claim 4.

6. A method for producing the anti-CD318 nanobody of claim 1 or the chimeric antigen receptor of claim 2, characterized in that... include: The host cells of claim 5 are cultured under conditions suitable for producing the anti-CD318 nanobody or the chimeric antigen receptor.

7. A pharmaceutical composition comprising the anti-CD318 nanobody 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, and pharmaceutically acceptable excipients.

8. A kit for detecting CD318, the kit comprising the anti-CD318 nanobody of claim 1, the nucleic acid molecule of claim 3, the nucleic acid construct of claim 4, or the host cell of claim 5.

9. Use of the anti-CD318 nanobody of claim 1 in the preparation of a kit for detecting CD318 in a sample.

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