WO2022247795A1 - Nanobody targeting claudin18.2 and use thereof - Google Patents

Abstract

The present invention provides a nanobody targeting Claudin18.2 and an encoding nucleic acid sequence thereof. The present invention also provides multi-specific antibodies, chimeric antigen receptors and antibody conjugates comprising the Claudin18.2 nanobody, a pharmaceutical composition and a kit comprising same, and a use thereof in the diagnosis/treatment/prevention of diseases associated with Claudin18.2 expression.

Description

Nanobodies targeting Claudin18.2 and uses thereof technical field

The invention belongs to the field of immunotherapy. More specifically, the present invention relates to Nanobodies targeting Claudin18.2 and their use in the prevention and/or treatment and/or diagnosis of diseases.

Background technique

The human CLDN18 gene can express two different splice forms, Claudin18.1 and Claudin18.2, which are very similar in structure, but the expression levels in tumors are quite different. In normal tissues, Claudin18.1 is only expressed in the lung, while Claudin18.2 is limitedly expressed in the stomach; The expression is up-regulated in esophageal cancer, pancreatic cancer and other cancer types. In addition, the Claudin18.2 gene will also be abnormally activated, highly selectively and stably expressed in specific tumor tissues, and participate in the proliferation, differentiation and migration of tumor cells, which makes it an effective molecular target for potential anti-tumor drugs.

The present invention aims to provide a humanized nanobody specifically binding to Claudin18.2, and its use in preventing and/or treating and/or diagnosing diseases related to the expression of Claudin18.2.

Contents of the invention

In one aspect, the present invention provides a humanized Nanobody specifically binding to Claudin18.2, which consists of three complementarity determining regions CDR1, CDR2 and CDR3 and four framework regions FR1, FR2, FR3 and FR4, wherein CDR1 As shown in SEQ ID NO: 1, CDR2 as shown in SEQ ID NO: 2, CDR3 as shown in SEQ ID NO: 3, FR1 is selected from SEQ ID NO: 4, 8 or variants thereof, FR2 is selected from SEQ ID NO : 5, 9 or variants thereof, FR 3 is selected from SEQ ID NO: 6, 11, 12, 13 or variants thereof, FR4 is selected from SEQ ID NO: 7, 10 or variants thereof, the variants are selected from Conservative substitutions of up to 3 amino acids are included in the FRs.

In one embodiment, a humanized Nanobody of the invention comprises:

(1) FR1 as shown in SEQ ID NO: 4, FR2 as shown in SEQ ID NO: 5, FR3 as shown in SEQ ID NO: 6, FR4 as shown in SEQ ID NO: 7, or its variant A variant comprising a conservative substitution of up to 3 amino acids in said FR;

(2) FR1 as shown in SEQ ID NO: 8, FR2 as shown in SEQ ID NO: 9, FR3 as shown in SEQ ID NO: 6, FR4 as shown in SEQ ID NO: 10, or its variant A variant comprising a conservative substitution of up to 3 amino acids in said FR; or

(3) FR1 as shown in SEQ ID NO: 4, FR2 as shown in SEQ ID NO: 5, FR3 as shown in SEQ ID NO: 11, 12 or 13, FR4 as shown in SEQ ID NO: 10 , or a variant thereof comprising a conservative substitution of up to 3 amino acids in said FR.

In one embodiment, the humanized Nanobody of the invention has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity, and can specifically bind Claudin18.2 antigen.

The invention also provides nucleic acid molecules encoding the humanized Nanobodies of the invention. In one embodiment, the nucleic acid molecule has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the nucleotide sequence selected from SEQ ID NO: 20-23 , 98%, 99%, 100% sequence identity.

The invention also provides a multispecific antibody comprising a humanized Nanobody of the invention, and one or more second antibodies or antigen-binding portions thereof that specifically bind to other antigens. In one embodiment, the second antibody or antigen-binding portion thereof is selected from a full-length antibody, Fab, Fab', (Fab') ₂ , Fv, scFv, scFv-scFv, minibody, diabody, nanobody or sdAb.

The invention also provides a vector comprising a nucleic acid molecule encoding a humanized Nanobody or multispecific antibody of the invention.

The invention also provides a host cell expressing the humanized Nanobody or multispecific antibody of the invention.

The invention also provides a chimeric antigen receptor comprising a humanized Nanobody or multispecific antibody of the invention, a transmembrane domain and an intracellular signaling domain.

In one embodiment, the transmembrane domain is selected from the transmembrane domains of the following proteins: TCRα chain, TCRβ chain, TCRγ chain, TCRδ chain, CD3ζ subunit, CD3ε subunit, CD3γ subunit, CD3δ subunit, CD45, CD4, CD5, CD8α, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, and CD154.

In one embodiment, the intracellular signaling domain is selected from the intracellular regions of FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b and CD66d.

In one embodiment, the chimeric antigen receptor of the invention further comprises one or more co-stimulatory domains selected from the co-stimulatory signaling domains of the following proteins: CD94, LTB, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CLAUDIN18.2, CD8, CD18, CD27, CD28, CD30, CD40, CD54, CD83, CD134(OX40), CD137(4-1BB), CD270( HVEM), CD272(BTLA), CD276(B7-H3), CD278(ICOS), CD357(GITR), DAP10, LAT, NKG2C, SLP76, PD-1, LIGHT, TRIM, and ZAP70.

The present invention also provides engineered immune cells comprising the above-mentioned chimeric antigen receptor. In one embodiment, the engineered immune cells are selected from T cells, NK cells, NKT cells, macrophages, dendritic cells. In one embodiment, the engineered immune cells further comprise a second chimeric antigen receptor targeting other tumor antigens. In one embodiment, the engineered immune cells further comprise cytokines selected from IL7, XCL1, XCL2 or combinations thereof.

The present invention also provides an antibody conjugate comprising the humanized Nanobody or multispecific antibody of the present invention and a second functional structure, wherein the second functional structure is selected from the group consisting of Fc, radioactive isotopes, extended half-life structural moieties, detectable markers and drugs. In one embodiment, the half-life-prolonging structural moiety is selected from the group consisting of albumin binding structure, transferrin binding structure, polyethylene glycol molecule, recombinant polyethylene glycol molecule, human serum albumin, human serum albumin Fragments of proteins and albumin-binding human serum albumin; the detectable label is selected from the group consisting of fluorophores, chemiluminescence compounds, bioluminescence compounds, enzymes, antibiotic resistance genes and contrast agents; the drug is selected from the group consisting of cytotoxins and Immunomodulators.

The present invention also provides a detection kit, which comprises the humanized nanobody, multispecific antibody, chimeric antigen receptor, antibody conjugate or engineered immune cell of the present invention.

The present invention also provides a pharmaceutical composition comprising the humanized nanobody, multispecific antibody, chimeric antigen receptor, antibody conjugate or engineered immune cell of the present invention, and one or more pharmaceutically acceptable excipients.

The present invention also provides a method for treating and/or preventing and/or diagnosing diseases associated with CLAUDIN18.2 expression, comprising administering the humanized Nanobodies, multispecific antibodies, chimeric antigen receptors, antibody conjugates of the present invention Conjugates, engineered immune cells or pharmaceutical compositions. In one embodiment, the disease associated with CLAUDIN18.2 expression is selected from esophageal cancer, gastrointestinal cancer, pancreatic cancer, thyroid cancer, colorectal cancer, kidney cancer, lung cancer (such as non-small cell lung cancer), liver cancer, gastric cancer , gastroesophageal junction (GEJ) adenocarcinoma, head and neck cancer, bladder cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, testicular cancer, germ cell cancer, bone cancer, skin cancer, thymus cancer, bile duct cancer , gallbladder cancer, melanoma, mesothelioma, lymphoma, myeloma (such as multiple myeloma), sarcoma, glioblastoma, leukemia, teratoma, neuroblastoma, glioma, Rectal cancer, endometrial cancer, adrenal cancer, brain cancer, colon cancer, head and neck cancer, lymph node cancer, ear nose throat (ENT) cancer, preferably selected from gastric cancer, gastroesophageal junction (GEJ) adenocarcinoma, esophageal cancer, gastrointestinal cancer, pancreatic cancer, lung cancer.

Detailed description of the invention

Unless otherwise specified, all scientific and technical terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Claudin18.2 Humanized Nanobody

As used herein, the term "Nanobody" refers to a single immunoglobulin variable domain ( _VH , _VHH or _VL ) polypeptide having three complementarity determining regions (CDRs) that specifically binds an antigen. They do not require the presence of the corresponding CDR-containing light chain/heavy chain partners or other parts of intact antibodies to be able to bind antigen. Nanobodies derived from camelid heavy-chain-only antibodies that naturally lack light chains have been reported, as well as Nanobodies with human heavy-chain domains (Muyldermans 2001, Holliger 2005), as well as murine antibodies amplified from genomic DNA from the spleen of immunized mice. A single _VH domain identified in a _VH gene library (Ward et al., 1989, Nature 341:544-546). Nanobodies typically have the following structure from N-terminus to C-terminus: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4, where FR1 to FR4 refer to framework regions 1 to 4, respectively, and CDR1 to CDR3 refer to complementarity determining regions 1 to 3 .

The term "complementarity determining region" or "CDR" is well known to those skilled in the art and used interchangeably, and refers to the non-contiguous sequence of amino acids within the variable region of an antibody that confers antigen specificity and/or binding affinity. The term "framework region" or "FR" is also known in the art and refers to the non-CDR portion of the antibody variable region, the sequence of which is generally conserved.

The precise amino acid sequence boundaries for a given CDR or FR can be readily determined using a number of numbering schemes well known in the art, including: Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th ed. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme); Al-Lazikani et al., (1997) JMB 273, 927-948 ("Chothia" numbering scheme); MacCallum et al., J.Mol.Biol .262:732-745 (1996), "Antibody-antigen interactions: Contact analysis and binding sitetopography," J. Mol. Biol. 262, 732-745" ("Contact" numbering scheme); Lefranc MP et al., "IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains,” Dev Comp Immunol, 2003 Jan;27(1):55-77 (“IMGT” numbering scheme); Honegger A and Plückthun A,” Yet another numbering scheme for immunoglobulin variable domains: an automatic modeling and analysis tool,” JMol Biol, 2001 Jun 8; 309(3):657-70 (“Aho” numbering scheme); and Martin et al., “Modeling antibody hypervariable loops: a combined algorithm," PNAS, 1989, 86(23): 9268-9272 ("AbM" numbering scheme).

The boundaries of a given CDR or FR may vary depending on the protocol used for identification. For example, the Kabat scheme is based on structural alignments, while the Chothia scheme is based on structural information. Both the Kabat and Chothia schemes numbering are based on the sequence lengths of the most common antibody regions where insertions are provided by caret letters (eg "30a") and deletions occur in some antibodies. These two schemes place certain insertions and deletions ("indels") at different positions, resulting in different numbering. The Contact scheme is based on the analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme. The AbM scheme is a compromise between the Kabat and Chothia definitions and is based on the scheme used by Oxford Molecular's AbM antibody modeling software.

Thus, unless otherwise specified, a "CDR" of a given antibody or region thereof (eg, variable region thereof) is understood to encompass the CDRs defined by any of the above schemes or other known schemes. For example, where it is specified that a particular CDR (eg, CDR3) contains a given amino acid sequence, it is understood that such a CDR may also have the sequence of the corresponding CDR (eg, CDR3) as defined by any of the above schemes or other known schemes. Likewise, unless otherwise specified, FRs for a given antibody or region thereof (eg, variable region thereof) are understood to encompass FRs as defined by any of the above schemes or other known schemes.

As used herein, a "humanized" antibody refers to an antibody in which all or substantially all CDR amino acid residues are derived from non-human CDRs and all or substantially all FR amino acid residues are derived from human FRs. "Humanized forms" of non-human antibodies refer to variants of such non-human antibodies that have been humanized to generally reduce immunogenicity in humans, while retaining the specificity and affinity of the parent non-human antibody. In some embodiments, some FR residues in a humanized antibody are substituted with corresponding residues from a non-human antibody (eg, an antibody from which the CDR residues are derived), eg, to restore or improve antibody specificity or affinity.

Humanized antibodies and methods for their preparation are well known to those skilled in the art, see eg Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008). Human framework regions that can be used for humanization include, but are not limited to: framework regions selected using a "best fit" approach; framework regions derived from the consensus sequences of human antibodies of a particular subgroup of light or heavy chain variable regions ; human mature (somatically mutated) framework regions or human germline framework regions; and framework regions obtained from screening FR libraries.

In some embodiments, a Nanobody against Claudin18.2 is modified, eg humanized, without reducing its natural affinity for the antigen, while reducing its immunogenicity towards heterologous species. For example, the amino acid residues of the antibody variable domain ( _VHH ) of a llama antibody can be determined and, for example, one or more camelid amino acids in the framework regions replaced by their human counterparts. Humanization does not significantly affect the antigen-binding ability of the resulting polypeptide. Humanization of camelid nanobodies requires mutagenesis of only a limited number of amino acids in a single polypeptide chain. This is in contrast to the humanization of scFv, Fab', (Fab') ₂ and IgG, which requires the introduction of amino acid changes in both chains, light and heavy, and ensures the pairing capabilities of the two chains.

Thus, in one aspect, the invention provides a humanized Nanobody specifically binding to Claudin18.2, which consists of three complementarity determining regions CDR1, CDR2 and CDR3 and four framework regions FR1, FR2, FR3 and FR4, Wherein CDR1 is shown in SEQ ID NO: 1, CDR2 is shown in SEQ ID NO: 2, CDR3 is shown in SEQ ID NO: 3, FR1 is selected from SEQ ID NO: 4, 8 or variants thereof, FR2 is selected from SEQ ID NO: ID NO: 5, 9 or variant thereof, FR 3 is selected from SEQ ID NO: 6, 11, 12, 13 or variant thereof, FR4 is selected from SEQ ID NO: 7, 10 or variant thereof, said variant Conservative substitutions of up to 3 amino acids are included in the FRs.

In one embodiment, a humanized Nanobody of the invention comprises:

(1) FR1 as shown in SEQ ID NO: 4, FR2 as shown in SEQ ID NO: 5, FR3 as shown in SEQ ID NO: 6, FR4 as shown in SEQ ID NO: 7, or its variant A variant comprising a conservative substitution of up to 3 amino acids in said FR;

(2) FR1 as shown in SEQ ID NO: 8, FR2 as shown in SEQ ID NO: 9, FR3 as shown in SEQ ID NO: 6, FR4 as shown in SEQ ID NO: 10, or its variant A variant comprising a conservative substitution of up to 3 amino acids in said FR; or

(3) FR1 as shown in SEQ ID NO: 4, FR2 as shown in SEQ ID NO: 5, FR3 as shown in SEQ ID NO: 11, 12 or 13, FR4 as shown in SEQ ID NO: 10 , or a variant thereof comprising a conservative substitution of up to 3 amino acids in said FR.

In one embodiment, said anti-Claudin18.2 humanized Nanobody has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity, and can specifically bind Claudin18.2 antigen. Preferably, the amino acid sequence of the anti-Claudin18.2 humanized nanobody is shown in SEQ ID NO: 15-18.

As used herein, the term "conservative substitution" refers to an amino acid substitution that does not significantly affect or alter the binding characteristics of an antibody or antibody fragment comprising the amino acid sequence. Amino acid substitutions can be introduced into the antibodies of the invention by standard techniques known in the art, such as site-directed mutagenesis and PCR-mediated mutagenesis. A conservative amino acid substitution is one in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acid residues with similar side chains have been defined in the art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid, ), uncharged polar side chains (e.g. glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g. alanine, valine acid, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g. threonine, valine, isoleucine) and aromatic side chains (eg, tyrosine, phenylalanine, tryptophan, histidine). Conservative modifications can be selected, for example, on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved.

As used herein, the term "sequence identity" means the degree to which two (nucleotide or amino acid) sequences in an alignment have the same residue at the same position, and is usually expressed as a percentage. Preferably, identity is determined over the entire length of the sequences being compared. Therefore, two copies of the exact same sequence have 100% identity. Those skilled in the art know that several algorithms can be used to determine sequence identity, such as Blast (Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402), Blast2 (Altschul et al. (1990) J. Mol. Biol. 215: 403-410), Smith-Waterman (Smith et al. (1981) J. Mol. Biol. 147:195-197) and Clustal W.

As used herein, the term "variant" or "functional fragment" has at most 10 (1, 2, 3, 4, 5, 6, 7, 8 , 9 or 10) amino acid substitutions, or 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity with the parental amino acid sequence properties, and retain the biological activity of the parent amino acid, such as binding activity.

Examples of Nanobodies include, but are not limited to, heavy chain variable domains from heavy chain antibodies, binding molecules naturally devoid of light chains, single domains (such as _VH or _VL ) derived from conventional four-chain antibodies, humanized Heavy chain antibodies, human Nanobodies produced by transgenic mice or rats expressing human heavy chain fragments, and the like. Nanobodies can be from any species including, but not limited to, mouse, rat, human, camel, llama, lamprey, fish, shark, goat, rabbit and cow.

In one embodiment, Nanobodies are single domain antigen binding molecules derived from naturally occurring heavy chain antibodies (also known as HCAbs). For example, Nanobodies may be derived from species of the family Camelidae, such as camels, llamas, vicunas, dromedaries, alpacas and llamas. Nanobodies derived from camelids, also known as V _HH , have a molecular weight of approximately 15kD and are considered to be the smallest functional antigen-binding fragment.

In some embodiments, Nanobodies are derived from the variable regions of immunoglobulins found in cartilaginous fish. For example, nanobodies can be derived from an immunoglobulin isotype called neoantigen receptor (NAR) found in shark serum.

In some embodiments, the Nanobody is a human Nanobody produced by a transgenic mouse or rat expressing a human heavy chain fragment. See eg US20090307787A1, US Patent No. 8,754,287, US20150289489A1, US20100122358A1 and WO2004049794.

In some embodiments, Nanobodies can also be obtained from (natural or immune) libraries of Camelidae _VHH sequences. Such methods include, for example, screening such libraries using the corresponding antigen or fragment, antigenic determinant or epitope, etc., by screening techniques known in the art. Alternatively, improved synthetic or semi-synthetic libraries can be obtained from natural _VHH libraries by random mutagenesis and/or CDR shuffling.

multispecific antibody

In one aspect, the present invention also provides a multispecific antibody (preferably a bispecific antibody or a trispecific antibody) comprising the Claudin18.2 humanized Nanobody as described above, which further comprises one or more antibodies specific for other antigens. Bound secondary antibody or antigen-binding portion thereof.

As used herein, the term "multispecific" means that the antigen binding protein has polyepitopic specificity (i.e., is capable of specifically binding two, three or more different epitopes on a biomolecule or is capable of specifically binding binds epitopes on two, three or more different biomolecules). As used herein, the term "bispecific" means that an antigen binding protein has two different antigen binding specificities.

In one embodiment, the second antibody or antigen-binding portion thereof may be in any antibody or antibody fragment form, such as a full-length antibody, Fab, Fab', (Fab') ₂ , Fv, scFv, scFv-scFv, minibody, Diabodies, Nanobodies or sdAbs.

Thus, in one embodiment, the second antibody, or antigen binding portion thereof, targets an antigen selected from the group consisting of: CD2, CD3, CD4, CD5, CD7, CD8, CD14, CD15, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD30, CD33, CD37, CD38, CD40, CD40L, CD44, CD46, CD47, CD52, CD54, CD56, CD70, CD73, CD80, CD97, CD123, CD126, CD138, CD171, CD 179a, DR4 , DR5, TAC, TEM1/CD248, VEGF, GUCY2C, EGP40, EGP-2, EGP-4, CD133, IFNAR1, DLL3, kappa light chain, TIM3, TSHR, CD19, BAFF-R, CLL-1, EGFRvIII, tEGFR , GD2, GD3, BCMA, Tn antigen, PSMA, ROR1, FLT3, FAP, TAG72, CD44v6, CEA, EPCAM, B7H3, KIT, IL-13Ra2, IL-llRa, IL-22Ra, IL-2, mesothelin, PSCA, PRSS21, VEGFR2, LewisY, PDGFR-β, SSEA-4, AFP, Folate receptor α, ErbB2(Her2/neu), ErbB3, ErbB4, MUC1, MUC16, EGFR, CS1, NCAM, Claudin18.2, c- Met, Prostase, PAP, ELF2M, Ephrin B2, IGF-I receptor, CAIX, LMP2, gpl00, bcr-abl, tyrosinase, EphA2, Fucosyl GMl, sLe, GM3, TGS5, HMWMAA, o-acetyl- GD2, Folate receptor β, TEM7R, CLDN6, GPRC5D, CXORF61, ALK, polysialic acid, PLAC1, GloboH, NY-BR-1, UPK2, HAVCR1, ADRB3, PANX3, GPR20, LY6K, OR51E2, TARP, WT1, NY-ESO-1, LAGE-la, MAGE-A1, MAGE-A3, MAGE-A6, pod protein, HPV E6, E7, ETV6-AML, sperm protein 17, XAGE1, Tie 2, MAD-CT-1, MAD -CT-2, Fos-related antigen 1, p53, p53 mutant, PSA, survivin and telomerase, PCTA-1/Galectin 8, MelanA/MART1, Ras mutant, hTERT, sarcoma translocation breakpoint, ML- IAP, TMPR SS2ETS fusion gene, NA17, PAX3, androgen receptor, progesterone receptor, Cyclin Bl, MYCN, RhoC, TRP-2, CYP1B 1, BORIS, SART3, PAX5, OY-TES 1, LCK, AKAP-4, SSX2 , RAGE-1, human telomerase reverse transcriptase, RU1, RU2, intestinal carboxylesterase, mut hsp70-2, CD79a, CD79b, CD72, LAIR1, FCAR, LILRA2, CD300LF, CLEC12A, BST2, EMR2, LY75 , GPC3, FCRL5, IGLL1, PD1, PDL1, PDL2, TGFβ, APRIL, NKG2D, NKG2D ligands, and/or pathogen-specific antigens, biotinylated molecules, molecules expressed by HIV, HCV, HBV, and/or other pathogens and/or neo-epitopes or neoantigens.

nucleic acid, vector, host cell

In another aspect, the invention relates to a nucleic acid molecule encoding a Claudin 18.2 Nanobody or multispecific antibody of the invention. A nucleic acid of the invention may be RNA, DNA or cDNA. According to one embodiment of the invention, the nucleic acid of the invention is a substantially isolated nucleic acid.

In one embodiment, the nucleic acid molecule encoding said Claudin18.2 Nanobody has at least 90%, 91%, 92%, 93%, 94%, 95% of the nucleotide sequence selected from SEQ ID NO: 20-23. %, 96%, 97%, 98%, 99%, 100% sequence identity, and can specifically bind Claudin18.2 antigen. Preferably, the nucleic acid molecule encoding the Claudin18.2 Nanobody is shown in SEQ ID NO: 20-23.

A nucleic acid of the invention may also be in the form of, may be present in and/or may be part of a vector, such as a plasmid, cosmid or YAC. The vector may especially be an expression vector, ie a vector providing for expression of the Claudin 18.2 Nanobody in vitro and/or in vivo (ie in a suitable host cell, host organism and/or expression system). The expression vector typically comprises at least one nucleic acid molecule of the invention operably linked to one or more suitable expression control elements (eg, promoters, enhancers, terminators, etc.). Selection of such regulatory elements and their sequences for expression in a particular host is well known to those skilled in the art. Specific examples of regulatory and other elements useful or necessary for the expression of the Claudin18.2 Nanobodies of the invention include, but are not limited to, promoters, enhancers, terminators, integrators, selectable markers, leader sequences, reporter genes.

In another aspect, the present invention also provides host cells expressing the Claudin18.2 Nanobody, the multispecific antibody of the present invention and/or containing the nucleic acid or vector of the present invention. Preferred host cells of the invention are bacterial cells, fungal cells or mammalian cells.

Suitable bacterial cells include Gram-negative bacterial strains (such as Escherichia coli strains, Proteus strains, and Pseudomonas strains) and Gram-positive bacterial strains (such as Bacillus Bacillus strains, Streptomyces strains, Staphylococcus strains and Lactococcus strains).

Suitable fungal cells include cells of species of Trichoderma, Neurospora, and Aspergillus; or Saccharomyces (e.g., Saccharomyces cerevisiae), fission Schizosaccharomyces (such as Schizosaccharomyces pombe), Pichia (such as Pichia pastoris and Pichia methanolica) and Hansen A cell of a species of Saccharomyces (Hansenula).

Suitable mammalian cells include, for example, HEK293 cells, CHO cells, BHK cells, HeLa cells, COS cells, and the like.

However, amphibian cells, insect cells, plant cells, and any other cells known in the art for expressing heterologous proteins can also be used in the present invention.

chimeric antigen receptor

In another aspect, the present invention also provides a recombinant receptor, such as a recombinant TCR receptor or a chimeric antigen receptor, comprising the Claudin 18.2 Nanobody as described above. Preferably, the present invention also provides a chimeric antigen receptor comprising a Claudin 18.2 Nanobody as described above.

As used herein, the term "chimeric antigen receptor" or "CAR" refers to an artificially constructed hybrid polypeptide that generally includes a ligand-binding domain (such as an antigen-binding portion of an antibody), a transmembrane domain, Optional co-stimulatory domain and intracellular signaling domain, each domain is connected by a linker. CARs are able to exploit the antigen-binding properties of antibodies to redirect the specificity and reactivity of T cells and other immune cells to a target of choice in a non-MHC-restricted manner.

In one embodiment, the invention provides a chimeric antigen receptor comprising a Claudin18.2 humanized Nanobody as described above or a multispecific antibody comprising said Claudin18.2 Nanobody, a transmembrane domain and intracellular signaling domains.

As used herein, the term "transmembrane domain" refers to a polypeptide capable of expressing a chimeric antigen receptor on the surface of an immune cell (such as a lymphocyte, NK cell or NKT cell) and directing a cellular response of the immune cell against a target cell structure. Transmembrane domains can be natural or synthetic and can be derived from any membrane-bound or transmembrane protein. The transmembrane domain is capable of signaling when the chimeric antigen receptor binds to the target antigen. Transmembrane domains particularly suitable for use in the present invention may be derived from, for example, TCRα chain, TCRβ chain, TCRγ chain, TCRδ chain, CD3ζ subunit, CD3ε subunit, CD3γ subunit, CD3δ subunit, CD45, CD4, CD5, CD8α , CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, CD154 and functional fragments thereof. Alternatively, the transmembrane domain may be synthetic and may comprise predominantly hydrophobic residues such as leucine and valine. Preferably, the transmembrane domain is derived from CD8α or CD28, which has at least 70%, preferably at least 80%, more preferably at least 90%, 95% of the amino acid sequence shown in SEQ ID NO:24 or SEQ ID NO:26. %, 97% or 99% or 100% sequence identity, or its coding sequence has at least 70%, preferably at least 80%, more preferably at least 90% with the nucleic acid molecule shown in SEQ ID NO: 25 or SEQ ID NO: 27 %, 95%, 97% or 99% or 100% sequence identity.

In one embodiment, the chimeric antigen receptors of the invention may further comprise a hinge region located between the antibody and the transmembrane domain. As used herein, the term "hinge region" generally refers to any oligopeptide or polypeptide that functions to link the transmembrane domain to the ligand binding domain. Specifically, the hinge region serves to provide greater flexibility and accessibility to the ligand binding domain. The hinge region may comprise up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids. The hinge region may be derived in whole or in part from a natural molecule, such as in whole or in part from the extracellular region of CD8, CD4 or CD28, or in whole or in part from an antibody constant region. Alternatively, the hinge region may be a synthetic sequence corresponding to a naturally occurring hinge sequence, or may be an entirely synthetic hinge sequence. In a preferred embodiment, the hinge region comprises a CD8α, CD28, FcγRIIIα receptor, IgG4 or IgG1 hinge region portion, more preferably a CD8α, CD28 or IgG4 hinge, which is identical to that shown in SEQ ID NO: 40, 42 or 44 The amino acid sequence has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity, or its coding sequence and SEQ ID NO: 41, 43 or 45 The indicated nucleotide sequences have at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity.

As used herein, the term "intracellular signaling domain" refers to the portion of a protein that transduces effector function signals and directs the cell to perform a given function. In one embodiment, the intracellular signaling domains comprised by the chimeric antigen receptors of the present invention may be intracellular domain sequences of T cell receptors and co-receptors, which act together to elicit Signal transduction, and any derivatives or variants of these sequences and any synthetic sequences having the same or similar function. The intracellular signaling domain can contain many immunoreceptor tyrosine-based activation motifs (Immunoreceptor Tyrosine-based Activation Motifs, ITAM). Non-limiting examples of intracellular signaling domains of the present invention include, but are not limited to, intracellular regions of FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b, and CD66d, among others. In a preferred embodiment, the signaling domain of the CAR of the present invention may comprise a CD3ζ intracellular region, which has at least 70%, preferably at least 80%, of the amino acid sequence shown in SEQ ID NO: 32 or SEQ ID NO: 34, More preferably at least 90%, 95%, 97% or 99% or 100% sequence identity, or its coding sequence has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity.

In one embodiment, the chimeric antigen receptor may also comprise one or more co-stimulatory domains. A co-stimulatory domain may be an intracellular functional signaling domain from a co-stimulatory molecule comprising the entire intracellular portion of said co-stimulatory molecule, or a functional fragment thereof. A "costimulatory molecule" refers to a cognate binding partner that specifically binds to a costimulatory ligand on a T cell, thereby mediating a costimulatory response (eg, proliferation) of the T cell. Costimulatory molecules include, but are not limited to, MHC class 1 molecules, BTLA, and Toll ligand receptors. Non-limiting examples of co-stimulatory domains of the invention include, but are not limited to, co-stimulatory signaling domains derived from the following proteins: CD94, LTB, TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9 , TLR10, CARD11, CD2, CD7, CD8, CD18, CD27, CD28, CD30, CD40, CD54, CD83, CD134(OX40), CD137(4-1BB), CD270(HVEM), CD272(BTLA), CD276(B7 -H3), CD278(ICOS), CD357(GITR), DAP10, LAT, NKG2C, SLP76, PD-1, LIGHT, TRIM, and ZAP70. Preferably, the costimulatory domain of the CAR of the present invention is from 4-1BB, CD28 or 4-1BB+CD28. In one embodiment, the 4-1BB co-stimulatory domain has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% of the amino acid sequence shown in SEQ ID NO:30 % sequence identity, or its coding sequence has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence with the nucleic acid molecule shown in SEQ ID NO:31 identity. In one embodiment, the CD28 co-stimulatory domain has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% of the amino acid sequence shown in SEQ ID NO:28. Sequence identity, or its coding sequence has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity with the nucleic acid molecule shown in SEQ ID NO:29 .

In one embodiment, the CAR of the invention may also comprise a signal peptide such that when it is expressed in a cell such as a T cell, the nascent protein is directed to the endoplasmic reticulum and subsequently to the cell surface. The core of the signal peptide may contain a long stretch of hydrophobic amino acids with a propensity to form a single α-helix. At the end of the signal peptide, there is usually a stretch of amino acids that is recognized and cleaved by the signal peptidase. The signal peptidase can cleave during translocation or after completion to generate a free signal peptide and mature protein. Then, the free signal peptide is digested by specific proteases. Signal peptides that can be used in the present invention are well known to those skilled in the art, such as signal peptides derived from B2M, CD8α, IgG1, GM-CSFRα, and the like. In one embodiment, the signal peptide that can be used in the present invention is from CD8α or B2M, and it has at least 70%, preferably at least 80%, more preferably at least 90% of the amino acid sequence shown in SEQ ID NO:36 or SEQ ID NO:38 %, 95%, 97% or 99% or 100% sequence identity, or its coding sequence has at least 70%, preferably at least 80%, more A sequence identity of at least 90%, 95%, 97% or 99% or 100% is preferred.

In one embodiment, the CAR comprises a Claudin18.2 humanized Nanobody as provided herein or a multispecific antibody comprising said Claudin18.2 Nanobody, CD8α transmembrane region, CD28 and/or 4-1BB costimulatory domain, and CD3ζ intracellular signaling domain. In this embodiment, the CAR may further comprise a signal peptide from B2M, CD8α, IgG1 or GM-CSFRα.

The present invention also provides a nucleic acid molecule encoding a Claudin18.2-targeting chimeric antigen receptor as defined above, and a vector comprising said nucleic acid molecule.

As used herein, the term "vector" is a nucleic acid molecule used as a vehicle for the transfer of (exogenous) genetic material into a host cell where it can eg be replicated and/or expressed. Vectors generally include targeting vectors and expression vectors. A "targeting vector" is a medium that delivers an isolated nucleic acid to the interior of a cell by, for example, homologous recombination or a hybrid recombinase using a sequence at a specific targeting site. An "expression vector" is a vector used for the transcription of heterologous nucleic acid sequences, such as those encoding chimeric antigen receptor polypeptides of the invention, and the translation of their mRNA in a suitable host cell. Suitable vectors for use in the present invention are known in the art and many are commercially available. In one embodiment, vectors of the present invention include, but are not limited to, plasmids, viruses (such as retroviruses, lentiviruses, adenoviruses, vaccinia virus, Rous sarcoma virus (RSV, polyoma virus, and adeno-associated virus (AAV), etc. ), phages, phagemids, cosmids, and artificial chromosomes (including BACs and YACs). The vector itself is usually a nucleic acid molecule, usually a DNA sequence containing an insert (transgene) and a larger sequence that acts as the "backbone" of the vector. Engineering An L vector also usually contains an origin of autonomous replication in the host cell (if stable expression of the polynucleotide is desired), a selectable marker, and a restriction enzyme cleavage site (such as a multiple cloning site, MCS). The vector may additionally contain a promoter, a multiple PolyA tail (polyA), 3'UTR, enhancer, terminator, insulator, operator, selectable marker, reporter gene, targeting sequence and/or protein purification tag and other elements. In a specific embodiment, The vector is an in vitro transcribed vector.

engineered immune cells

In one aspect, the present invention also provides engineered immune cells expressing the CAR of the present invention.

As used herein, the term "immune cell" refers to any cell of the immune system that has one or more effector functions (eg, cytotoxic cell killing activity, secretion of cytokines, induction of ADCC and/or CDC). For example, immune cells can be T cells, macrophages, dendritic cells, monocytes, NK cells and/or NKT cells. In one embodiment, the immune cells are derived from stem cells, such as adult stem cells, embryonic stem cells, cord blood stem cells, progenitor cells, bone marrow stem cells, induced pluripotent stem cells, totipotent stem cells, or hematopoietic stem cells, among others. Preferably, the immune cells are T cells. The T cells may be any T cells, such as T cells cultured in vitro, such as primary T cells, or T cells from T cell lines cultured in vitro, such as Jurkat, SupT1, etc., or T cells obtained from a subject. Examples of subjects include humans, dogs, cats, mice, rats, and transgenic species thereof. T cells can be obtained from a variety of sources, including peripheral blood mononuclear cells, bone marrow, lymph node tissue, cord blood, thymus tissue, tissue from a site of infection, ascites, pleural effusion, spleen tissue, and tumors. T cells can also be enriched or purified. T cells can be at any developmental stage, including, but not limited to, CD4+/CD8+ T cells, CD4+ helper T cells (e.g., Th1 and Th2 cells), CD8+ T cells (e.g., cytotoxic T cells), tumor infiltrating cells, memory T cells, naive T cells, CD4-CD8-T cells, regulatory T cells, γδ-T cells, αβ-T cells, etc. In a preferred embodiment, the immune cells are human T cells. T cells can be obtained from the blood of a subject using a variety of techniques known to those of skill in the art, such as Ficoll separation.

In one embodiment, the engineered immune cells of the present invention further express cytokines selected from IL7, XCL1, XCL2 or a combination thereof. In one embodiment, the engineered immune cells of the invention further express XCL1 and/or XCL2, more preferably in combination with IL7 (ie, XCL1+IL7, XCL2+IL7 or XCL1+XCL2+IL7). In one embodiment, the cytokine used in the present invention is a wild type or a variant thereof, and the variant has the same or similar or even better function than the wild type. In one embodiment, the IL7 used in the present invention has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% of the amino acid sequence shown in SEQ ID NO: 47 or 51 % sequence identity, or the coding gene of IL7 has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% with the nucleic acid sequence shown in SEQ ID NO: 46 or 50 % sequence identity.

Both XCL1 and XCL2 belong to the C-type chemokine family and are mainly produced by CD8+ T cells and natural killer cells. The nucleic acid sequences of XCL2 and XCL1 have 97% identity, and the amino acid sequences differ only by two residues. Studies have found that XCL2 and XCL1 are very similar in expression profile, structure and function. For example, like XCL1, XCL2 also has two interconvertible protein spatial conformations, monomeric and dimeric. After activating XCR1, the dimer conformation has a higher affinity for the hairpin structure in glycosaminoglycans (GAG). XCR1, the receptor of XCL1 and XCL2, is selectively expressed on DC (cDC1) cells with antigen-presenting ability. In one embodiment, the XCL1 used in the present invention has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% of the amino acid sequence shown in SEQ ID NO: 49 or 53 % sequence identity, or the coding sequence of XCL1 has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% with the nucleotide sequence shown in SEQ ID NO: 48 or 52 % sequence identity. In one embodiment, the XCL2 used in the present invention has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% of the amino acid sequence shown in SEQ ID NO: 55. Sequence identity, or the coding sequence of XCL2 has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 97% or 99% or 100% sequence identity with the nucleic acid sequence shown in SEQ ID NO: 54 sex.

Nucleic acid sequences encoding chimeric antigen receptors can be introduced into immune cells by conventional methods known in the art (eg, by transduction, transfection, transformation, etc.). "Transfection" is the process of introducing nucleic acid molecules or polynucleotides, including vectors, into target cells. An example is RNA transfection, the process of introducing RNA (such as in vitro transcribed RNA, ivtRNA) into a host cell. The term is mainly used for non-viral methods in eukaryotic cells. The term "transduction" is generally used to describe the virus-mediated transfer of nucleic acid molecules or polynucleotides. Transfection of animal cells typically involves opening transient pores or "holes" in the cell membrane to allow uptake of material. Transfection can be performed using calcium phosphate, by electroporation, by cell extrusion, or by mixing cationic lipids with the material to generate liposomes that fuse with cell membranes and deposit their cargo inside. Exemplary techniques for transfecting eukaryotic host cells include lipid vesicle-mediated uptake, heat shock-mediated uptake, calcium phosphate-mediated transfection (calcium phosphate/DNA co-precipitation), microinjection, and electroporation. perforation. The term "transformation" is used to describe the non-viral transfer of nucleic acid molecules or polynucleotides, including vectors, into bacteria, but also into non-animal eukaryotic cells, including plant cells. Thus, transformation is the genetic alteration of a bacterial or non-animal eukaryotic cell resulting from the direct uptake from its surroundings through the cell membrane and subsequent incorporation of exogenous genetic material (nucleic acid molecules). Transformation can be achieved by manual means. In order for transformation to occur, the cells or bacteria must be in a competent state. For prokaryotic transformation, techniques can include heat shock-mediated uptake, fusion of bacterial protoplasts with intact cells, microinjection, and electroporation. After the nucleic acid or vector is introduced into the immune cells, those skilled in the art can amplify and activate the obtained immune cells by conventional techniques.

In one embodiment, in order to reduce the risk of graft-versus-host disease, the engineered immune cells further comprise suppressed or silenced expression of at least one gene selected from: CD52, GR, dCK, TCR/CD3 gene ( Such as TRAC, TRBC, CD3γ, CD3δ, CD3ε, CD3ζ), MHC related genes (HLA-A, HLA-B, HLA-C, B2M, HLA-DPA, HLA-DQ, HLA-DRA, TAP1, TAP2, LMP2, LMP7, RFX5, RFXAP, RFXANK, CIITA) and immune checkpoint genes such as PD1, LAG3, TIM3, CTLA4, PPP2CA, PPP2CB, PTPN6, PTPN22, PDCD1, HAVCR2, BTLA, CD160, TIGIT, CD96, CRTAM, TNFRSF10B, TNFRSF10A , CASP8, CASP10, CASP3, CASP6, CASP7, FADD, FAS, TGFBRII, TGFRBRI, SMAD2, SMAD3, SMAD4, SMAD10, SKI, SKIL, TGIF1, IL10RA, IL10RB, HMOX2, IL6R, IL6ST, EIF2AK4, CSK, PAG1, SIT , FOXP3, PRDM1, BATF, GUCY1A2, GUCY1A3, GUCY1B2, and GUCY1B3. Preferably, the engineered immune cells further comprise suppressed or silenced expression of at least one gene selected from: TRAC, TRBC, HLA-A, HLA-B, HLA-C, B2M, RFX5, RFXAP, RFXANK, CIITA, PD1, LAG3, TIM3, CTLA4, more preferably TRAC, TRBC, HLA-A, HLA-B, HLA-C, B2M, RFX5, RFXAP, RFXANK, CIITA.

Methods of inhibiting gene expression or silencing genes are well known to those skilled in the art. For example, antisense RNA, RNA decoys, RNA aptamers, siRNA, shRNA/miRNA, trans dominant negative protein (TNP), chimeric/antibody conjugates, chemokine ligands, anti-infective cellular proteins can be used , intracellular antibodies (sFv), nucleoside analogs (NRTI), non-nucleoside analogs (NNRTI), integrase inhibitors (oligonucleotides, dinucleotides, and chemical agents) and protease inhibitors to inhibit gene expression Express. Alternatively, genes can also be silenced by mediated DNA fragmentation, for example, by meganucleases, zinc finger nucleases, TALE nucleases, or Cas enzymes in CRISPR systems.

In one embodiment, a plurality of immune cells is provided, each immune cell engineered to express one or more chimeric antigen receptors. For example, in some embodiments, an immune cell is engineered to express a chimeric antigen receptor that binds and/or targets Claudin18.2 (e.g., a CAR comprising a Claudin18.2 Nanobody of the invention), and is further A cell engineered to express chimeric antigen receptors that bind and/or target other antigens. In one embodiment, immune cells may also express multispecific chimeric antigen receptors that target one or more antigens including Claudin 18.2. For example, this multispecific chimeric antigen receptor may comprise a multispecific antibody targeting Claudin18.2, or simultaneously comprise the Claudin18.2 nanobody of the present invention and antibodies targeting other antigens. In such embodiments, the plurality of engineered immune cells may be administered together or separately. In one embodiment, the plurality of immune cells can be in the same composition or in different compositions. Exemplary compositions of cells include those described in the following sections of this application.

antibody conjugate

In one aspect, the invention provides an antibody conjugate comprising a Claudin18.2 Nanobody as defined in the invention and a second functional structure, wherein the second functional structure is selected from the group consisting of Fc, radioisotope, half-life extension structural moieties, detectable markers and drugs.

In one embodiment, the present invention provides an antibody conjugate comprising a Claudin 18.2 Nanobody as defined in the present invention and Fc. As used herein, the term "Fc" is used to define the C-terminal region of an immunoglobulin heavy chain, which includes native Fc and variant Fc. "Native Fc" refers to a molecule or sequence comprising a non-antigen-binding fragment, whether monomeric or multimeric, produced by digestion of an intact antibody. The source of immunoglobulin from which native Fc is produced is preferably of human origin. Native Fc fragments are composed of monomeric polypeptides that can be linked in dimeric or multimeric form by covalent linkages (eg, disulfide bonds) and non-covalent linkages. Natural Fc molecules have 1-4 intermolecular disulfides between monomeric subunits depending on class (e.g. IgG, IgA, IgE, IgD, IgM) or subtype (e.g. IgG1, IgG2, IgG3, IgA1, IgGA2) key. An example of a native Fc is a disulfide-linked dimer produced by papain digestion of IgG (see Ellison et al. (1982), Nucleic Acids Res. 10:4071-9). The term "native Fc" as used herein generally refers to monomeric, dimeric and multimeric forms. A "variant Fc" refers to an amino acid sequence that differs from that of a "native" or "wild-type" Fc by virtue of at least one "amino acid modification" as defined herein, also referred to as an "Fc variant". Thus, "Fc" also includes single-chain Fc (scFc), ie, a single-chain Fc consisting of two Fc monomers linked by a polypeptide linker, which is capable of naturally folding into a functional dimeric Fc region. In one embodiment, the Fc is preferably the Fc of a human immunoglobulin, more preferably the Fc of a human IgG1.

In one embodiment, the present invention provides an antibody conjugate comprising a Claudin 18.2 Nanobody as defined in the present invention and a radioisotope. Examples of radioisotopes useful in the present invention include, but are not limited to, At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² , Pb ²¹² , ^99m Tc, ¹²³ I, ¹⁸ F and ⁶⁸ Ga.

In one embodiment, the present invention provides an antibody conjugate comprising a Claudin18.2 Nanobody as defined in the present invention and a half-life extending moiety selected from the group consisting of albumin binding structures, transgenic Binding structures of ferritin, polyethylene glycol molecules, recombinant polyethylene glycol molecules, human serum albumin, fragments of human serum albumin and albumin binding human serum albumin (including antibodies).

In one embodiment, the present invention provides an antibody conjugate comprising a Claudin18.2 Nanobody as defined in the present invention and a detectable label. The term "detectable label" means herein a compound that produces a detectable signal. For example, the detectable marker may be an MRI contrast agent, a scintigraphy contrast agent, an X-ray imaging contrast agent, an ultrasound contrast agent, an optical imaging contrast agent. Examples of detectable labels include fluorophores (such as fluorescein, Alexa, or cyanine), chemiluminescent compounds (such as luminol), bioluminescent compounds (such as luciferase or alkaline phosphatase), enzymes (such as paprika root peroxidase, glucose-6-phosphatase, β-galactosidase), antibiotics (such as kanamycin, ampicillin, chloramphenicol, tetracycline, etc.) resistance genes and contrast agents (such as nanoparticles or gadolinium). Those skilled in the art can select an appropriate detectable label according to the detection system used.

In one embodiment, the present invention provides an antibody conjugate comprising a Claudin18.2 Nanobody as defined in the present invention and a drug conjugated to said Claudin18.2 Nanobody, such as a cytotoxin or an immunomodulator ( ie, antibody drug conjugates). Usually the drug is covalently linked to the antibody, and usually relies on a linker. In one embodiment, the drug is a cytotoxin. In another embodiment, the drug is an immunomodulator. Examples of cytotoxics include, but are not limited to, methotrexate, aminopterin, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, dacarbazine, nitrogen mustard, thiotepa, phentermine Nitrogen mustard, melphalan, carmustine (BSNU), lomustine (CCNU), 1-methylnitrosourea, cyclophosphamide, nitrogen mustard, busulfan, dibromomannitol, chain Zuocin, mitomycin, cis-dichlorodiamine platinum (II) (DDP), cisplatin, carboplatin, zorubicin, doxorubicin, detorubicin, caraminomycin, i Darubicin, epirubicin, mitoxantrone, actinomycin D, bleomycin, calicheamicin, mithromycin, antramycin (AMC), vincristine, vinblastine, Paclitaxel, ricin, Pseudomonas exotoxin, gemcitabine, cytochalasin B, gramicidin D, ethidium bromide, emetine, etoposide, teniposide, colchicine, dihydroxyanthracene Ketones, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, procarbazine, hydroxyurea, asparaginase, corticosteroids, rice Totan (O,P'-(DDD)), interferon, and combinations thereof. Examples of immunomodulators include, but are not limited to, ganciclovir, etanercept, tacrolimus, sirolimus, vorcyclosporine, cyclosporine, rapamycin, cyclophosphamide, azathioprine , mycophenolate mofetil, methotrexate, glucocorticoids and their analogs, cytokines, stem cell growth factors, lymphotoxins, tumor necrosis factor (TNF), hematopoietic factors, interleukins (such as IL-1, IL-2, IL-3, IL-6, IL-10, IL-12, IL-18 and IL-21), colony-stimulating factors (such as G-CSF and (GM-CSF), interferon (such as interferon-α, interferon interferon-beta and interferon-gamma), stem cell growth factors designated "S1 factor", erythropoietin and thrombopoietin, or combinations thereof.

Kits and pharmaceutical compositions

In another aspect, the present invention also provides a detection kit, which comprises the nanobody, multispecific antibody, antibody conjugate or chimeric antigen receptor described in the present invention.

In another aspect, the present invention also provides a pharmaceutical composition comprising the Nanobody, chimeric antigen receptor, multispecific antibody or antibody conjugate described in the present invention, and one or more pharmaceutically acceptable Accepted excipients.

As used herein, the term "pharmaceutically acceptable excipient" means pharmacologically and/or physiologically compatible with the subject and the active ingredient (i.e., capable of eliciting the desired therapeutic effect without causing any adverse desired local or systemic effect), which are well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995). Examples of pharmaceutically acceptable excipients include, but are not limited to, fillers, binders, disintegrants, coating agents, adsorbents, anti-adhesive agents, glidants, antioxidants, flavoring agents, coloring agents, Sweeteners, solvents, co-solvents, buffers, chelating agents, surfactants, diluents, wetting agents, preservatives, emulsifiers, coating agents, isotonic agents, absorption delaying agents, stabilizers and tonicity regulators . The selection of suitable excipients is known to those skilled in the art for the preparation of the desired pharmaceutical compositions of the present invention. Exemplary excipients for use in pharmaceutical compositions of the invention include saline, buffered saline, dextrose and water. In general, the selection of suitable excipients depends inter alia on the active agent used, the disease to be treated and the desired dosage form of the pharmaceutical composition.

The pharmaceutical compositions according to the present invention are suitable for various routes of administration. Typically, administration is accomplished parenterally. Parenteral delivery methods include topical, intraarterial, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, intrauterine, intravaginal, sublingual or intranasal administration.

The pharmaceutical composition according to the present invention can also be prepared in various forms, such as solid, liquid, gaseous or lyophilized forms, especially ointments, creams, transdermal patches, gels, powders, tablets, solutions, gaseous In the form of aerosols, granules, pills, suspensions, emulsions, capsules, syrups, elixirs, extracts, tinctures or liquid extracts, or in a form especially adapted to the desired method of administration. Processes known in the present invention for the production of medicaments may include, for example, conventional mixing, dissolving, granulating, dragee-making, milling, emulsifying, encapsulating, entrapping or lyophilizing processes. Pharmaceutical compositions comprising immune cells such as those described herein are typically provided in solution, and preferably comprise a pharmaceutically acceptable buffer.

The pharmaceutical composition according to the invention can also be administered in combination with one or more other agents suitable for the treatment and/or prophylaxis of the disease to be treated. Preferred examples of agents suitable for combination include known anticancer drugs such as cisplatin, maytansine derivatives, rachelmycin, calicheamicin, docetaxel, etoposide , gemcitabine, ifosfamide, irinotecan, melphalan, mitoxantrone, sorfimer sodium photofrin II, temozolomide, topotecan, trimetreate glucuronate, Auristatin E, vincristine, and doxorubicin; peptide cytotoxins, such as ricin, diphtheria toxin, Pseudomonas bacterial exotoxin A, DNase, and RNase; radionuclides, such as iodine 131, rhenium 186, indium 111, iridium 90, bismuth 210 and 213, actinium 225 and astatine 213; prodrugs, such as antibody-directed enzyme prodrugs; immunostimulants, such as platelet factor 4, melanoma growth stimulating protein, etc.; antibodies or fragments thereof, such as anti-CD3 antibodies or fragments thereof, complement activators, heterologous protein domains, homologous protein domains, viral/bacterial protein domains and viral/bacterial peptides. In addition, the pharmaceutical composition of the present invention can also be used in combination with one or more other treatment methods, such as chemotherapy and radiotherapy.

Therapeutic/preventive/diagnostic use

In another aspect, the present invention also provides a method for treating and/or preventing and/or diagnosing a disease associated with Claudin18.2 expression, comprising administering to a subject a humanized Nanobody, a chimeric antigen as described above Receptors, multispecific antibodies, antibody conjugates, engineered immune cells or pharmaceutical compositions.

In one embodiment, diseases associated with Claudin18.2 expression include but are not limited to esophageal cancer, gastrointestinal cancer, pancreatic cancer, thyroid cancer, colorectal cancer, kidney cancer, lung cancer (such as non-small cell lung cancer), liver cancer, gastric cancer , gastroesophageal junction (GEJ) adenocarcinoma, head and neck cancer, bladder cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, testicular cancer, germ cell cancer, bone cancer, skin cancer, thymus cancer, bile duct cancer , gallbladder cancer, melanoma, mesothelioma, lymphoma, myeloma (such as multiple myeloma), sarcoma, glioblastoma, leukemia, teratoma, neuroblastoma, glioma, Rectal cancer, endometrial cancer, adrenal cancer, brain cancer, colon cancer, head and neck cancer, lymph node cancer, ear nose throat (ENT) cancer, and metastatic, recurrent or refractory lesions of these cancers.

In a preferred embodiment, the disease associated with the expression of Claudin18.2 is selected from gastric cancer, gastroesophageal junction (GEJ) adenocarcinoma, esophageal cancer, gastrointestinal cancer, pancreatic cancer, lung cancer.

Description of drawings

Figure 1: Shows the expression levels of the Claudin18.2 humanized Nanobody in CAR-T cells.

Figure 2: Shows the killing effect of CAR-T cells on NUGC4-18.2 target cells under different effect-to-target ratios.

Figure 3: Shows the release levels of cytokines IL-2 (A) and IFN-γ (B) after CAR-T cells were co-cultured with target cells NUGC4-18.2 or non-target cells K562.

Figure 4: The killing effect of CAR-T cells expressing XCL1 on target cells.

Figure 5. In vivo tumor suppression effect of XCL1-expressing CAR-T cells.

Figure 6: The killing effect of CAR-T cells expressing XCL1 and IL7 on target cells.

Figure 7. In vivo anti-tumor effect of CAR-T cells expressing XCL1 and IL7.

Detailed ways

Example 1. Preparation of Claudin18.2 Humanized Nanobody

The humanized antibody was prepared based on the Claudin18.2 camel-derived nanobody (SEQ ID NO: 14), and the specific method was as follows: using the VHH humanized general framework transplantation method established by Vincke C et al., the universality of the design was completed according to the sequence homology Humanized VHH framework, replacing the corresponding CDR region with the CDR region of the camel-derived Claudin18.2 Nanobody, and further optimizing the individual amino acid positions in the FR2 region to obtain 4 humanized variants, the amino acid sequences of which Respectively as shown in SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18.

Example 2. Preparation of CAR-T cells comprising Claudin18.2 humanized nanobody

Sequences encoding the following proteins were synthesized and cloned into pLVX vector (Public Protein/Plasmid Library (PPL), Cat. No.: PPL00157-4a): CD8α signal peptide (SEQ ID NO: 38), claudin18.2 humanized Nanobody (selected from SEQ ID NO: 15-18), CD8α hinge region (SEQ ID NO: 40), CD8α transmembrane region (SEQ ID NO: 24), 4-1BB intracellular region (SEQ ID NO: 30), CD3ζ Intracellular signaling domain (SEQ ID NO: 32), and the correct insertion of the target sequence was confirmed by sequencing to obtain 4 hCAR plasmids. Wherein, the amino acid sequence of the anti-Claudin18.2 humanized nanobody contained in the hCAR-1 plasmid is shown in SEQ ID NO: 15; the amino acid sequence of the anti-Claudin18.2 humanized nanobody contained in the hCAR-2 plasmid is shown in SEQ ID Shown in NO: 16; the amino acid sequence of the anti-Claudin18.2 humanized nanobody contained in the hCAR-3 plasmid is shown in SEQ ID NO: 17; the amino acid sequence of the anti-Claudin18.2 humanized nanobody contained in the hCAR-4 plasmid The sequence is shown in SEQ ID NO:18.

Add 3ml Opti-MEM (Gibco, Cat. No. 31985-070) to a sterile tube to dilute the above plasmid, and then add the packaging vector psPAX2 (Addgene, Cat. No. 12260) and the envelope vector pMD2.G (Addgene, Cat. No. 12259). Then, add 120ul X-treme GENE HP DNA Transfection Reagent (Roche, Cat. No. 06366236001), mix immediately, incubate at room temperature for 15min, and then add the plasmid/vector/transfection reagent mixture dropwise to the culture flask of 293T cells . Viruses were collected at 24 hours and 48 hours, combined, and ultracentrifuged (25000g, 4°C, 2.5 hours) to obtain concentrated lentiviruses.

T cells were activated with DynaBeads CD3/CD28CTS ^™ (Gibco, Cat. No. 40203D) and cultured for 1 day at 37°C and 5% CO2. Then, the concentrated lentivirus was added, and after continuous culture for 3 days, four kinds of CAR T cells expressing different claudin18.2 humanized nanobodies were obtained, namely hCAR-1 T cells, hCAR-2 T cells, hCAR-3 T cells and hCAR-4 T cells. Unmodified wild-type T cells were used as negative controls (NT).

Use Biotin-SP (long spacer) AffiniPure Goat Anti-human IgG, F(ab') ₂ Fragment Specific (min X Hu, Bov, Hrs Sr Prot) (jackson immunoresearch, Cat. No. 109-065-097) as primary antibody, APC Streptavidin (BD Pharmingen, Cat. No. 554067) or PE Streptavidin (BD Pharmingen, Cat. No. 554061) was used as the secondary antibody to detect the expression levels of scFv on the four CAR T cells by flow cytometry, and the results are shown in Figure 1.

It can be seen that all claudin18.2 humanized nanobodies in the CAR T cells prepared by the present invention can be effectively expressed.

Example 3: The killing effect and cytokine release of CAR T cells on target cells

3.1 The killing effect of CAR-T cells on target cells

When T cells kill target cells, the number of target cells decreases. After co-culturing T cells with target cells expressing luciferase, the number of target cells decreases and the secretion of luciferase decreases accordingly. Luciferase can catalyze the conversion of luciferin to oxidized luciferin, and during this oxidation process, bioluminescence will be generated, and the intensity of this luminescence will depend on the level of luciferase expressed by the target cells. Therefore, the detected fluorescence intensity can reflect the ability of T cells to kill target cells.

In order to detect the killing ability of CAR-T cells on target cells, NUGC4-18.2 target cells carrying the luciferin gene were first spread into 96-well plates at 1x10 ⁴ /well, and then 10:1, 5:1 and 2.5:1 The effect-to-target ratio (that is, the ratio of effector T cells to target cells) CAR T cells and NT cells were plated in a 96-well plate for co-culture, and the fluorescence value was measured with a microplate reader after 16-18 hours. According to the calculation formula: (average fluorescence value of target cells-average fluorescence value of samples)/average fluorescence value of target cells×100%, the killing efficiency was calculated, and the results are shown in FIG. 2 .

It can be seen that compared with NT, the CAR T cells of the present invention can specifically kill target cells.

3.2 Cytokine release of CAR-T cells

When T cells kill target cells, the number of target cells decreases and cytokines are released at the same time. According to the following steps, enzyme-linked immunosorbent assay (ELISA) was used to measure the release levels of cytokines IL2 and IFNγ when the CAR T cells of the present invention kill target cells.

(1) Collect the cell co-culture supernatant

Spread target cells in 96-well plate at 1x10 ⁵ /well, then co-culture CAR T and NT cells (negative control) with target cells NUGC4-18.2 or non-target cells K562 at a ratio of 1:1 for 18-24 hours Afterwards, the cell co-culture supernatant was collected.

(2) ELISA detection of the secretion of IL-2 and IFN-γ in the supernatant

Use capture antibody Purified anti-human IL2 Antibody (Biolegend, Cat. No. 500302) or Purified anti-human IFN-γAntibody (Biolegend, Cat. No. 506502) to coat the 96-well plate and incubate overnight at 4°C, then remove the antibody solution, add 250 μL containing 2% A solution of BSA (sigma, product number V900933-1kg) in PBST (1XPBS containing 0.1% Tween) was incubated at 37° C. for 2 hours. Plates were then washed 3 times with 250 [mu]L PBST (1XPBS with 0.1% Tween). Add 50 μL of cell co-culture supernatant or standard to each well and incubate at 37° C. for 1 hour, then wash the plate 3 times with 250 μL PBST (1XPBS containing 0.1% Tween). Then add 50 μL detection antibody Anti-Interferon gamma antibody [MD-1] (Biotin) (abcam, product number ab25017) to each well, incubate at 37°C for 1 hour, wash with 250 μL PBST (1XPBS containing 0.1% Tween) Plate 3 times. Then add HRP Streptavidin (Biolegend, Cat. No. 405210), incubate at 37°C for 30 minutes, discard the supernatant, add 250 μL PBST (1XPBS containing 0.1% Tween), and wash 5 times. Add 50 μL of TMB substrate solution to each well. The reaction was allowed to occur at room temperature in the dark _for 30 minutes, after which 50 μL of 1 mol/L _H2SO4 was added to each well to stop the reaction. Within 30 minutes of stopping the reaction, use a microplate reader to detect the absorbance at 450nm, and calculate the cytokine content according to the standard curve (drawn according to the reading value and concentration of the standard), and the results are shown in Figure 3.

It can be seen that after the CAR T cells of the present invention are co-cultured with the non-target cell K562, the release of cytokines is almost undetectable, while the level of cytokine release after co-culture with the target cell NUGC4-18.2 is significantly higher than that of the control NT cells , indicating that the killing of the target cells by the CAR T cells of the present invention is specific.

Example 4. Preparation of CAR-T cells co-expressing cytokines and verification of their functions

According to the method described in Example 2, CAR-T cells co-expressing cytokines were prepared, wherein XCL1 (SEQ ID NO: 48) linked by 2A peptide was further included in the hCAR-3 plasmid, which was named 18.2-CAR- XCL1 T cells. The killing activity of the CAR-T cells on target cells was detected according to the method described in Example 3.1, and the results are shown in Figure 4 (wherein 18.2-CAR is hCAR-3). It can be seen that the killing activity of 18.2-CAR-XCL1 T cells and 18.2-CAR T cells alone on target cells is comparable, and both are significantly higher than those of control NT cells.

In addition, the tumor inhibitory effect of CAR-T cells in vivo was tested according to the following method: first, 5×10 ⁶ NUGC4-18.2 gastric cancer cells were subcutaneously inoculated on D0 in the axilla of the left forelimb of NCG mice. Then on D3, 5× ¹⁰⁶ PBMC cells were injected into each mouse through the tail vein to obtain a gastric cancer NCG mouse model with a humanized immune system. When the tumor grew to D10, 5×10 ⁵ NT cells, 18.2-CAR T cells or 18.2-CAR-XCL1 T cells were injected into each mouse through the tail vein. The tumor volume changes of the mice were monitored until the end of the experiment, and the results are shown in Figure 5. It can be seen that at a low dose of 5× ¹⁰⁵ cells per mouse, 18.2-CAR T cells did not exhibit significant tumor-suppressive effects compared with NT cells. Unexpectedly, 18.2-CAR-XCL1 T cells controlled the tumor volume at a lower level from D30 and maintained it until the end of the experiment without recurrence, showing a significant tumor suppressive effect. It can be seen that the additionally expressed XCL1 can significantly enhance the tumor suppressive effect of CAR-T cells.

Example 5. Preparation of CAR-T cells co-expressing cytokines and verification of their functions

According to the method described in Example 2, CAR-T cells co-expressing cytokines were prepared, wherein XCL1 (SEQ ID NO: 48) and IL7 (SEQ ID NO: 46) linked by 2A peptide were further included in the hCAR-3 plasmid , named it 18.2-CAR-XCL1-IL7 T cells. The killing activity of the CAR-T cells on target cells was detected according to the method described in Example 3.1, and the results are shown in Figure 6 (wherein 18.2-CAR is hCAR-3). It can be seen that the killing activity of 18.2-CAR-XCL1-IL7 T cells and 18.2-CAR T cells alone on target cells is comparable, and both are significantly higher than that of control NT cells.

In addition, the in vivo tumor suppressive effect of CAR-T cells was tested according to the method described in Example 4, and the results are shown in FIG. 7 . It can be seen that compared with 18.2-CAR T cells, 18.2-CAR-XCL1-IL7 T cells exhibited a significant tumor suppressive effect. It can be seen that the combination of additionally expressed XCL1+IL7 can significantly enhance the tumor suppressive effect of CAR-T cells.

It should be noted that the above are only preferred embodiments of the present invention, and are not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and changes. Those skilled in the art understand that any modification, equivalent replacement, improvement, etc. made within the spirit and principles of the present invention shall be included within the protection scope of the present invention.

Claims (24)

A humanized nanobody that binds to Claudin18.2, which consists of three complementarity determining regions CDR1, CDR2 and CDR3 and four framework regions FR1, FR2, FR3 and FR4, wherein CDR1 is shown in SEQ ID NO: 1, CDR2 is shown in SEQ ID NO: 2, CDR3 is shown in SEQ ID NO: 3, FR1 is selected from SEQ ID NO: 4, 8 or variants thereof, FR2 is selected from SEQ ID NO: 5, 9 or variants thereof, FR 3 is selected from SEQ ID NO: 6, 11, 12, 13 or variants thereof, FR4 is selected from SEQ ID NO: 7, 10 or variants thereof comprising at most 3 amino acids in said FR Conservative substitutions. The Nanobody of claim 1, comprising: (1) FR1 as shown in SEQ ID NO: 4, FR2 as shown in SEQ ID NO: 5, FR3 as shown in SEQ ID NO: 6, FR4 as shown in SEQ ID NO: 7, or its variant A variant comprising a conservative substitution of up to 3 amino acids in said FR; (2) FR1 as shown in SEQ ID NO: 8, FR2 as shown in SEQ ID NO: 9, FR3 as shown in SEQ ID NO: 6, FR4 as shown in SEQ ID NO: 10, or its variant A variant comprising a conservative substitution of up to 3 amino acids in said FR; or (3) FR1 as shown in SEQ ID NO: 4, FR2 as shown in SEQ ID NO: 5, FR3 as shown in SEQ ID NO: 11, 12 or 13, FR4 as shown in SEQ ID NO: 10 , or a variant thereof comprising a conservative substitution of up to 3 amino acids in said FR. The Nanobody of claim 1, wherein the Nanobody has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% sequence identity, and can specifically bind Claudin18.2 antigen. A nucleic acid molecule encoding the Nanobody of any one of claims 1-3. The nucleic acid molecule of claim 4, which has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97% of the nucleotide sequence selected from SEQ ID NO: 20-23 , 98%, 99%, 100% sequence identity. A multispecific antibody comprising the Nanobody of any one of claims 1-5, and one or more second antibodies or antigen-binding portions thereof that specifically bind to other antigens. The multispecific antibody of claim 6, wherein the second antibody or antigen-binding portion thereof is selected from the group consisting of full length antibody, Fab, Fab', (Fab') ₂ , Fv, scFv, scFv-scFv, minibody, Diabodies, Nanobodies or sdAbs. A vector comprising a nucleic acid molecule encoding the Nanobody of any one of claims 1-5 or the multispecific antibody of claim 6 or 7. A host cell expressing the Nanobody of any one of claims 1-5 or the multispecific antibody of claim 6 or 7. A chimeric antigen receptor comprising the Nanobody of any one of claims 1-5 or the multispecific antibody of claim 6 or 7, a transmembrane domain and an intracellular signaling domain. The chimeric antigen receptor according to claim 10, wherein the transmembrane domain is selected from the transmembrane domain of the following proteins: TCRα chain, TCRβ chain, TCRγ chain, TCRδ chain, CD3ζ subunit, CD3ε subunit, CD3γ subunit CD3δ subunit, CD45, CD4, CD5, CD8α, CD9, CD16, CD22, CD33, CD28, CD37, CD64, CD80, CD86, CD134, CD137, and CD154. The chimeric antigen receptor of claim 10, wherein the intracellular signaling domain is selected from the intracellular region of the following proteins: FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD3ζ, CD22, CD79a, CD79b and CD66d. The chimeric antigen receptor of claim 10, further comprising one or more co-stimulatory domains selected from the co-stimulatory signaling domains of the following proteins: CD94, LTB, TLR1, TLR2 , TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, CARD11, CD2, CLAUDIN18.2, CD8, CD18, CD27, CD28, CD30, CD40, CD54, CD83, CD134(OX40), CD137(4- 1BB), CD270(HVEM), CD272(BTLA), CD276(B7-H3), CD278(ICOS), CD357(GITR), DAP10, LAT, NKG2C, SLP76, PD-1, LIGHT, TRIM, and ZAP70. An engineered immune cell comprising the chimeric antigen receptor according to any one of claims 10-13. The engineered immune cell according to claim 14, which is selected from T cells, NK cells, NKT cells, macrophages, and dendritic cells. The engineered immune cell of claim 15, wherein the T cells are selected from CD4+/CD8+T cells, CD4+ helper T cells, CD8+T cells, tumor infiltrating cells, memory T cells, naive T cells, CD4-CD8 - T cells, regulatory T cells, γδ-T cells, αβ-T cells. The engineered immune cell of claim 14, further comprising a second chimeric antigen receptor targeting other tumor antigens. The engineered immune cell according to any one of claims 14-17, which further expresses a cytokine selected from IL7, XCL1, XCL2 or a combination thereof. An antibody conjugate comprising the nanobody of any one of claims 1-5 or the multispecific antibody of claim 6 or 7 and a second functional structure, wherein the second functional structure selected from Fc, radioisotopes, half-life extending moieties, detectable labels and drugs. The antibody conjugate according to claim 19, wherein the structural moiety extending the half-life is selected from the group consisting of albumin binding structure, transferrin binding structure, polyethylene glycol molecule, recombinant polyethylene glycol molecule, human serum albumin, fragments of human serum albumin, and albumin-binding albumin polypeptides; said detectable marker is selected from the group consisting of fluorophores, chemiluminescent compounds, bioluminescent compounds, enzymes, antibiotic resistance genes, and contrast agents; said Drugs are selected from cytotoxins and immunomodulators. A detection kit, which comprises the nanobody described in any one of claims 1-5, the multispecific antibody described in claim 6 or 7, the chimeric antigen receptor described in any one of claims 10-13 body, the engineered immune cell according to any one of claims 14-18, or the antibody conjugate according to claim 19 or 20. A pharmaceutical composition comprising the nanobody of any one of claims 1-5, the multispecific antibody of claim 6 or 7, the chimeric antigen receptor of any one of claims 10-13 The body, the engineered immune cell of any one of claims 14-18, or the antibody conjugate of claim 19 or 20, and one or more pharmaceutically acceptable excipients. The nanobody of any one of claims 1-5, the multispecific antibody of claim 6 or 7, the chimeric antigen receptor of any one of claims 10-13, the chimeric antigen receptor of any one of claims 14-18, The engineered immune cell described in one item, the antibody conjugate described in claim 19 or 20, or the pharmaceutical composition described in claim 22 is used for treatment and/or prevention and/or diagnosis and Claudin18.2 expression Use in medicine for related diseases. The use according to claim 23, wherein the disease associated with the expression of Claudin18.2 is selected from esophageal cancer, gastrointestinal cancer, pancreatic cancer, thyroid cancer, colorectal cancer, kidney cancer, lung cancer (such as non-small cell lung cancer), Liver cancer, gastric cancer, gastroesophageal junction (GEJ) adenocarcinoma, head and neck cancer, bladder cancer, breast cancer, uterine cancer, cervical cancer, ovarian cancer, prostate cancer, testicular cancer, germ cell cancer, bone cancer, skin cancer, thymus cancer , cholangiocarcinoma, gallbladder cancer, melanoma, mesothelioma, lymphoma, myeloma (such as multiple myeloma), sarcoma, glioblastoma, leukemia, teratoma, neuroblastoma, glia Tumor, rectal cancer, endometrial cancer, adrenal gland cancer, brain cancer, colon cancer, head and neck cancer, lymph node cancer, ear nose throat (ENT) cancer.

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