CN111848809A - CAR molecule targeting Claudin18.2, its modified immune cells and use - Google Patents

Abstract

The present invention discloses a nucleic acid construct, characterized in that the nucleic acid construct has a structure as shown in the formula car-[(IRES)-f] _m , wherein IRES is an internal ribosome entry site sequence; f Coding a functional protein F, m is a natural number of 0 or non-0; car encodes a CAR and the CAR includes: (a) an extracellular binding domain that specifically recognizes CLDN18.2; (b) a hinge domain; (c) transmembrane domain; (d) co-stimulatory intracellular domain; (e) signaling domain; wherein the extracellular binding domain comprises a scFv, the amino acid sequence and function of which are as described below. The CAR molecule of the present invention has excellent safety and efficacy, and has a good effect of inhibiting tumors.

Description

CAR molecule targeting Claudin18.2, its modified immune cells and use

technical field

The invention belongs to the field of biomedicine, and in particular relates to a CAR molecule targeting Claudin18.2, its modified immune cells and uses.

Background technique

Cell therapy is an emerging disease treatment mode with remarkable curative effect. Those skilled in the art have been working on developing new cellular immunotherapy to improve the effect of cellular immunotherapy and reduce its side effects. Chimeric antigen receptor T cells (CART or CAR-T for short) are T cells isolated from patients, modified in vitro with chimeric antigen receptor (CAR) so that they can specifically recognize cancer cells, and then The transformed CART cells are expanded and reinfused into the patient to achieve the effect of treating tumors. Chimeric antigen receptor NK cells (CARNK or CAR-NK for short) are NK cells isolated from patients, modified with CAR in vitro to enable them to specifically recognize cancer cells, and then the modified CARNK cells are expanded. Increase and return to the patient's body to achieve the effect of tumor treatment.

The first-generation CART cells that only contain ITAM in the intracellular signal region can stimulate anti-tumor cytotoxic effects, but the secretion of cytokines is relatively low, and cannot stimulate lasting anti-tumor effects in vivo. The second generation of CART cells developed subsequently added the intracellular signaling region of CD28 or 4-1BB (also known as CD137), and the co-stimulation of B7/CD28 or 4-1BBL/4-1BB in the intracellular signaling region caused T lymphocytes It can continuously proliferate, and can increase the levels of cytokines such as IL-2 and IFN-γ secreted by T lymphocytes, and at the same time improve the survival cycle and anti-tumor effect of CART in vivo. The third generation of CART cells developed in recent years further improves the survival cycle of CART in vivo and its anti-tumor effect.

Cell junction claudin (Claudin or CLDN) is expressed in humans, mice and other species. It is an intercellular layer sealing-associated protein and plays an important role in controlling ion flow between cell layers, maintaining cell polarity and intercellular signal transduction. Claudin18.2 is a clinically proven target for the development of cancer therapy drugs. There are many antibody patents against Claudin18.2, such as WO2014146672A1 and CN201810610790.3, but no drugs have been listed yet. CN201410341504.X discloses a T lymphocyte targeting CLD18A2 (namely Claudin18.2) and its preparation method and application. There are also clinical data reports, but there is still much room for improvement in the efficacy and safety of CART.

SUMMARY OF THE INVENTION

In order to solve the problem of poor effect of immune cells targeting Claudin18.2 in the prior art, a CAR molecule targeting Claudin18.2, its modified immune cells and uses are provided.

When designing a CAR, the antibody gene targeting a specific antigen is a key choice. Given the complexity of gene expression in vivo and various uncontrollable factors, it is very difficult to select a suitable gene for CAR. In addition, for many tumor-specific antigens, it is difficult to find specific molecules that are suitable for the construction of CAR cells. After CAR is established, it is often impossible to obtain an active extracellular binding region, which is also a difficulty in developing CAR technology. In addition, although CAR cells have attractive prospects in tumor immunotherapy, their higher risks also need to be considered. For example, due to the low expression of specific antigens recognized by CAR in some normal tissues, CAR cells may damage normal tissues that express the corresponding antigens. In the prior art, the effect of targeting Claudin18.2 antibody specifically binding to Claudin18.2 is not good, and the effect of CAR cells targeting Claudin18.2 is not good enough. CAR cells targeting Claudin18.2 are extremely difficult. The inventor unexpectedly found through experiments that the antibody binding activity/affinity in the CAR of the present invention is high, which means that under the same process (same virus titer, transfection efficiency), the CAR of the present invention can better bind to target cells , the specificity is better, and the side effects caused by non-target cell binding are reduced. Using the CAR of the present invention, even a small dose can play a role, and has extremely high application value. The antigen-binding part (antibody) of the CAR cell against CLDN18.2 in the present invention is a humanized sequence, which can reduce the risk of immunogenicity caused by CAR treatment. Through the design of a new generation of CAR, specifically, cytokines, cytokines and their receptor binding complexes that can effectively kill tumors are introduced at the C-terminus of CAR, and these factors and/or complexes can secrete extracellular, tumor cells region to achieve better tumor suppression effect.

In order to solve the above-mentioned technical problems, one of the technical solutions of the present invention is to provide a nucleic acid construct having a structure as shown in formula car-[(IRES)-f] _m , wherein IRES is an internal Ribosomal entry site sequence; f encodes a functional protein F, and m is a natural number other than 0; car encodes a CAR and the CAR includes: (a) an extracellular binding domain scFv that specifically recognizes CLDN18.2; (b) ) hinge domain; (c) transmembrane domain; (d) costimulatory intracellular domain; (e) signaling domain;

Wherein, the extracellular binding domain includes a light chain variable region and a heavy chain variable region, and the light chain variable region includes at least one complementarity determining region (CDR) selected from the following: as SEQ ID NO: 11 Or CDR1 shown in SEQ ID NO:12 amino acid sequence or its mutation; CDR2 shown as SEQ ID NO:13 amino acid sequence or its mutation; CDR3 shown as SEQ ID NO:14 amino acid sequence or its mutation; said The heavy chain variable region comprises at least one CDR selected from the group consisting of: CDR1 as shown in the amino acid sequence of SEQ ID NO: 15 or a mutation thereof; CDR2 as shown in the amino acid sequence of SEQ ID NO: 16 or a mutation thereof; as shown in SEQ ID NO: 16 : CDR3 shown by the 17 amino acid sequence or a mutation thereof; the mutation maintains or improves the binding of the antibody or antigen-binding fragment to the CLDN18.2 antigen. In the present invention, "-" can be a chemical bond such as a peptide bond, a glycosidic bond, or a linker such as (GGGGS) [herein abbreviated as (G ₄ S) _n ]; it can also be only for dividing different groups, domains, groups An artificial means of grouping (including nucleotides or amino acids), in which case there are virtually no additional chemical bonds between the different groups, domains, groups of nucleotides or groups of amino acids.

In addition to the single-chain variable fragment (scFv), the above-mentioned extracellular binding domain that specifically recognizes CLDN18.2 can also be a Fab or a single-domain antibody (sdFv).

In a preferred embodiment, the structure of the scFv is light chain variable region (VL)-linker (Linker)-heavy chain variable region (VH) or VH-Linker-VL, - is a chemical bond, so The Linker is preferably (G ₄ S) _n ; the n is preferably an integer of 1 to 6, more preferably 3 and 4.

In a preferred embodiment, the light chain variable region comprises CDR1 as shown in the amino acid sequence of SEQ ID NO: 11 or a mutation thereof, CDR2 as shown in the amino acid sequence of SEQ ID NO: 13 or a mutation thereof, and , CDR3 as shown in the amino acid sequence of SEQ ID NO: 14 or a mutation thereof; or, CDR1 as shown in the amino acid sequence of SEQ ID NO: 12 or a mutation thereof, CDR2 as shown in the amino acid sequence of SEQ ID NO: 13 or a mutation thereof, and, CDR3 as shown in the amino acid sequence of SEQ ID NO: 14 or a mutation thereof; and/or, the heavy chain variable region comprises a CDR1 as shown in the amino acid sequence of SEQ ID NO: 15 or a mutation thereof, as shown in SEQ ID NO: CDR2 shown in the amino acid sequence of 16 or a mutation thereof, and, CDR3 shown in the amino acid sequence of SEQ ID NO: 17 or a mutation thereof.

Preferably, the light chain variable region comprises CDR1 shown in the amino acid sequence of SEQ ID NO: 11 or a mutation thereof, CDR2 shown in the amino acid sequence of SEQ ID NO: 13 or a mutation thereof and the amino acid sequence of SEQ ID NO: 14 or CDR3 as shown by a mutation thereof; and, the heavy chain variable region comprises CDR1 as shown in the amino acid sequence of SEQ ID NO: 15 or a mutation thereof, CDR2 as shown in the amino acid sequence of SEQ ID NO: 16 or a mutation thereof, and , CDR3 as shown in the amino acid sequence of SEQ ID NO: 17 or a mutation thereof; or, the light chain variable region comprises the amino acid sequence as shown in SEQ ID NO: 12 or a CDR1 of a mutation thereof, as shown in SEQ ID NO: 13 The amino acid sequence shown, or a mutated CDR2 thereof, and, the amino acid sequence shown in SEQ ID NO: 14, or a mutated CDR3 thereof; and, the heavy chain variable region comprises the amino acid shown in SEQ ID NO: 15 The sequence or a mutated CDR1 thereof, the amino acid sequence set forth in SEQ ID NO: 16 or a mutated CDR2 thereof, and, the amino acid sequence set forth in SEQ ID NO: 17 or a mutated CDR3 thereof.

In a preferred embodiment, the extracellular binding domain comprises a light chain variable region as shown in any one of the amino acid sequences of SEQ ID NOs: 29-33 or a mutant sequence thereof; and/or, as shown in SEQ ID The heavy chain variable region shown in any one of the amino acid sequences of NO: 34-37 or the mutated sequences thereof.

Preferably, the extracellular binding domain comprises a light chain variable region as set forth in the amino acid sequence of SEQ ID NO:29, a heavy chain variable region as set forth in the amino acid sequence of SEQ ID NO:34; or, as The light chain variable region shown in the amino acid sequence of SEQ ID NO:31, the heavy chain variable region shown in the amino acid sequence of SEQ ID NO:34; the light chain shown in the amino acid sequence of SEQ ID NO:7 can be A variable region, such as the heavy chain variable region shown in the amino acid sequence of SEQ ID NO:8.

In a preferred embodiment, in the nucleic acid construct:

(1) the hinge domain is selected from the hinge region of one or more of the following molecules: CD8α, CD28, CD152, PD1 and IgG1 heavy chain;

(2) The transmembrane domain is selected from the transmembrane region of one or more of the following molecules: α, β, ζ chains of TCR, CD3ε, CD3ζ, CD4, CD5, CD8α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, 4-1BB, CD152, CD154 and PD1;

(3) The co-stimulatory intracellular domain is selected from the intracellular region of one or more of the following molecules: CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54, CD83, OX40, CD134, 4-1BB , CD150, CD152, CD223, CD270, PD-L2, PD-L1, CD278, DAP10, NKD2C SLP76, TRIM, FcεRIγ and MyD88, preferably CD28 intracellular domain and/or 4-1BB intracellular domain; and/or,

(4) The signaling domain is selected from the intracellular regions of one or more of the following molecules: Igα, Igβ, TCRξ, FcR1γ, FcR1β, CD3γ, CD3δ, CD3ε, CD2, CD5, CD22, CD28, CD79a, CD79b , CD278, CD66d and CD3ζ. The signaling domain is preferably the CD3ζ intracellular domain.

In a preferred embodiment, the hinge domain is a CD8α hinge region, the transmembrane domain is a CD8α transmembrane domain, and the co-stimulatory intracellular domain is CD28 intracellular region and/or 4-1BB Intracellular region, the signal transduction domain is CD3ζ intracellular region (abbreviated as CD3ζ in the general formula of CAR in Example 4).

Preferably, the CD8α hinge region is a human CD8α hinge region, and its amino acid sequence is shown in SEQ ID NO: 27; the CD8α transmembrane region is a human CD8α transmembrane region, and its amino acid sequence is shown in SEQ ID NO: 18; The amino acid sequence of the CD28 intracellular region is shown in SEQ ID NO: 42; the amino acid sequence of the 4-1BB intracellular region is shown in SEQ ID NO: 38; and/or the amino acid sequence of the CD3ζ intracellular region As shown in SEQ ID NO:40.

More preferably, the nucleotide sequence encoding the human CD8α hinge region is shown in SEQ ID NO: 28; the nucleotide sequence encoding the human CD8α transmembrane region is shown in SEQ ID NO: 48; The nucleotide sequence of the CD28 intracellular region is shown in SEQ ID NO: 43; the nucleotide sequence encoding the 4-1BB intracellular region is shown in SEQ ID NO: 39; and/or, the CD3ζ cell encoding The nucleotide sequence of the inner region is shown in SEQ ID NO:41.

In a preferred embodiment, the nucleic acid construct is characterized in that, when m is a non-zero natural number, preferably 1, the functional protein F comprises:

(1) cytokines or active fragments thereof, preferably IL10 or IL15 or active fragments thereof; more preferably, the amino acid sequence of IL10 is shown in SEQ ID NO: 3, and the amino acid sequence of IL15 is shown in SEQ ID NO: 20 As shown, the amino acid sequence of the IL15 active fragment is shown in SEQ ID NO: 21 or SEQ ID NO: 22;

(2) a cytokine receptor or an active fragment thereof, preferably IL15Rα or a fragment thereof or an IL15Rα fragment (sushi) or an IL15Rα fragment (sushi+); more preferably, the amino acid sequence of the IL15Rα is shown in SEQ ID NO: 23; The amino acid sequence of the IL15Rα fragment (sushi) is shown in SEQ ID NO: 26; the amino acid sequence of the IL15Rα fragment (sushi+) is shown in SEQ ID NO: 25; or,

(3) fusion protein of cytokine receptor or its active fragment and cytokine, preferably IL15Rα or its fragment or IL15Rα(sushi) or fusion fragment of IL15Rα(sushi+) and IL15; more preferably, encoding the IL15Rα(sushi+) The amino acid sequence of the fusion fragment with the active fragment of IL15 is shown in SEQ ID NO:46.

In a preferred embodiment, the structure of the nucleic acid construct is:

(1) scFv-human CD8α hinge region-human CD8α transmembrane region-human 4-1BB intracellular region-human CD3ζ intracellular region; preferably, the encoded amino acid sequence is shown in SEQ ID NO:44; more preferably , whose nucleotide sequence is shown in SEQ ID NO: 45; the preferred one-nucleotide construct here is named CAR1a hereinafter;

(2) scFv-human CD8α hinge region-human CD8α transmembrane region-human 4-1BB intracellular region-human CD3ζ intracellular region-(IRES)-IL15; preferably, the nucleotide sequence of the IRES sequence is as shown in SEQ ID NO: 49; and/or, the amino acid sequence encoded by the nucleotide sequence of IL10 is shown in SEQ ID NO: 3; the preferred one-nucleotide construct is hereinafter named CAR3ab;

(3) scFv-human CD8α hinge region-human CD8α transmembrane region-human 4-1BB intracellular region-human CD3ζ intracellular region-(IRES)-IL10; preferably, the nucleotide sequence of the IRES sequence is as shown in SEQ ID NO: 49; and/or, the amino acid sequence encoded by the nucleotide sequence of IL10 is shown in SEQ ID NO: 3; the preferred one-nucleotide construct here is named CAR3ab10 hereinafter; or ,

(4) scFv-human CD8α hinge region-human CD8α transmembrane region-human 4-1BB intracellular region-human CD3ζ intracellular region-(IRES)-IL15Rα(sushi+)-IL15; preferably, the core of the IRES sequence The nucleotide sequence is shown in SEQ ID NO: 49; and/or, the amino acid sequence encoded by the nucleotide sequence of IL15Rα(sushi+)-IL15 is shown in SEQ ID NO: 46; the preferred nucleotide sequence here is The acid construct is hereinafter designated CAR4a.

Those skilled in the art need to know that the above-mentioned construct is a nucleic acid. For the sake of brevity, the expression "the nucleotide sequence encoding ..." is omitted for the four structures of the construct. For example, the scFv in the above construct structure is actually "the nucleotide sequence encoding the scFv", the human CD8α hinge region is actually "the nucleotide sequence encoding the human CD8α hinge region", and so on. In addition, IL10 and IL15 in the above structure may be wild-type IL10 and IL15, mutants of IL10 and IL15 or active fragments thereof.

In the above-mentioned nucleic acid construct, CD27T cell memory and survival signal transduction domains may be further included, and/or, self-destructing domains such as nucleotide sequences encoding inducible caspase (iCasp) or encoding pure Nucleotide sequence of herpes virus thymidine kinase (HSV-TK). The self-destructible domain can regulate the antigen recognition signaling pathway, minimize the damage of CAR cells to normal tissues, and reduce off-target effects. When adverse reactions occur, under the stimulation of non-toxic prodrugs, suicide genes are activated, CAR cell apoptosis is induced, and treatment is terminated.

In order to solve the above technical problems, the second technical solution of the present invention is to provide a CAR molecule encoded by the nucleic acid construct as described above or a composition containing the CAR molecule.

In order to solve the above-mentioned technical problem, the third technical solution of the present invention is to provide an expression vector, which comprises the above-mentioned nucleic acid construct.

In a preferred embodiment, the expression vector is selected from one or more of retroviral vectors, lentiviral vectors, adenoviral vectors and adeno-associated viral vectors. A lentiviral vector is preferred, and its backbone plasmid can be pBABEpuro.

In order to solve the above-mentioned technical problem, the fourth technical solution of the present invention is to provide a virus, and the virus comprises the above-mentioned expression vector.

In order to solve the above technical problem, the fifth technical solution of the present invention is to provide the use of the above nucleic acid construct, or the above expression vector, or the above virus in preparing genetically modified cells targeting CLDN18.2.

In order to solve the above-mentioned technical problem, the sixth technical solution of the present invention is to provide a genetically modified cell, which is transfected with the above-mentioned nucleic acid construct, the above-mentioned expression vector, or the above-mentioned virus.

In a preferred embodiment, the genetically modified cells are eukaryotic cells, preferably isolated human cells; more preferably immune cells such as T cells, or NK cells such as NK92 cell line.

In order to solve the above technical problem, the seventh technical solution of the present invention is to provide the use of the gene-modified cells in the preparation of a tumor-inhibiting drug; preferably, the tumor is a CLDN18.2 positive tumor.

In order to solve the above technical problem, the eighth technical solution of the present invention is to provide a pharmaceutical composition, the composition contains a pharmaceutically acceptable carrier and the above genetically modified cell or the above CAR molecule or contains the CAR composition of molecules.

In order to solve the above-mentioned technical problems, the ninth technical solution of the present invention is to provide a method for preparing genetically modified cells, the method comprising the steps of: adding the above-mentioned nucleic acid construct, or the above-mentioned expression vector, or the above-mentioned virus Into the cells to be modified to obtain genetically modified cells;

Preferably, the genetically modified cells are eukaryotic cells, preferably isolated human cells; more preferably immune cells such as T cells, or NK cells such as NK92 cell line.

To solve the above technical problem, a tenth technical solution of the present invention is to provide a method for inhibiting tumor growth in a subject in need, the method comprising administering to the subject an effective amount of the above-mentioned genetically modified cells. Preferably, the tumor is a CLDN18.2 positive tumor, preferably gastric cancer, esophageal cancer, lung cancer, melanoma, kidney cancer, breast cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, glioma or leukemia .

The method according to the above, wherein the genetically modified cells are autologous or allogeneic to the subject being treated; according to the method above, wherein the tumor is a CLDN18.2 positive tumor; according to The method, wherein the subject is a human.

In order to solve the above-mentioned technical problem, the eleventh technical solution of the present invention is to provide the above-mentioned nucleic acid construct, or the above-mentioned expression vector, or the above-mentioned virus, or the above-mentioned genetically modified cell in the preparation of a medicine for treating tumors. Applications. Preferably, the tumor is a CLDN18.2 positive tumor, preferably gastric cancer, esophageal cancer, lung cancer, melanoma, kidney cancer, breast cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, glioma or leukemia .

On the basis of conforming to common knowledge in the art, the above preferred conditions can be combined arbitrarily to obtain preferred examples of the present invention.

The reagents and raw materials used in the present invention are all commercially available.

The positive progressive effect of the present invention is:

1. Provides a new CAR molecule targeting Claudin18.2, which has higher activity and affinity and can better target tumor cells; it does not bind to Claudin18.1, has excellent specificity, and reduces off-target Side effects caused by cell binding; the antigen-binding sequence of the CAR molecule is preferably humanized to reduce the risk of immunogenicity.

2. CAR molecules are used together with cytokines or cytokine receptors, and the therapeutic effect is better.

3. The therapeutic effect of immune cells containing the CAR molecule of the present invention is better, especially in a preferred embodiment, the CAR molecule of the present invention is close to 100% of the tumor suppressor effect.

To sum up, the CAR molecule of the present invention has excellent safety and efficacy, and produces better specific binding at the same dose compared with the CAR molecule of the prior art, and produces the same effect at a small dose. CAR molecules containing humanized antigen-binding sequences reduce the risk of immunogenicity and have a good tumor suppressor effect.

Description of drawings

Figure 1 shows the positive rate of Ab10 antibody scFv expressed by CART cells.

Figure 2 shows the specific killing rate (FACS) of CART cells of the present invention to target cells, the left picture is the result of negative control CART cells, and the right picture is the result of CAR1a cells.

Figure 3 shows the in vivo efficacy of CART cells designed against CLDN18.2, which shows the changes in tumor volume in mice injected with CART (empty vector), CART1a, CART3ab, CART3ab10 and CART4a cells.

Detailed ways

After extensive and in-depth research, extensive screening and optimization, the inventor unexpectedly discovered chimeric antigen receptors that specifically bind to CLDN18.2. These chimeric antigen receptors can be used in the preparation of targeted anti-tumor drugs and diagnostics. cancer drugs. The present invention has been completed on this basis.

definition

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice of the tests of the present invention, the preferred materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

As used herein, the terms "comprising" or "comprising" are intended to mean that the compositions and methods include the stated elements but do not exclude other elements. "Consisting essentially of," when used to define compositions and methods, shall mean the exclusion of other elements that have any substantial effect on the combination for the intended use. For example, a composition consisting essentially of this element, as defined herein, will not exclude trace contaminants from isolation and purification methods and pharmaceutically acceptable carriers (eg, phosphate buffered saline, preservatives, etc.). "Consisting of" shall mean the exclusion of other ingredients than trace elements and substantial method steps for applying the compositions disclosed herein. Aspects defined by each of these transitional terms are within the scope of the invention.

The term "chimeric antigen receptor" or "CAR" as used herein refers to an extracellular domain capable of binding an antigen (extracellular binding domain), a hinge domain, a transmembrane domain (transmembrane region), and a cytoplasmic A polypeptide that signals to a domain (ie, an intracellular signaling domain). The hinge domain can be thought of as part of providing flexibility to the extracellular antigen binding region. Intracellular signaling domains refer to proteins that transmit information into cells to regulate cellular activity by generating second messengers via defined signaling pathways, or proteins that function as effectors by corresponding to such messengers, producing CARs that can promote Signals of immune effector function of cells such as CART cells. Intracellular signaling domains include signaling domains and may also include co-stimulatory intracellular domains derived from co-stimulatory molecules.

The term "signaling domain" refers to the portion of a CAR that transduces effector function signals and directs cells to perform their specialized functions. Examples of signaling domains include, but are not limited to, the zeta chain of the T cell receptor complex, or any homolog thereof (eg, n chain, FcεR1γ and β chains, MB1 (Igα) chain, B29 (Igβ) chain, etc.), human CD3ζ chain, CD3 polypeptides (Δ, δ, and ε), syk family tyrosine kinases (Syk, ZAP 70, etc.), src family tyrosine kinases (Lck, Fyn, Lyn, etc.) and those involved in T cell transduction Other molecules, such as CD2, CD5, CD22, CD28, CD79a, CD79b, CD278 and CD66d, etc.

The term "CD3ζ" is defined herein as the protein provided by GenBanK Accession No. BAG36664.1, or the equivalent residues from non-human species such as mouse, rodent, monkey, ape, and the like. The "CD3 zeta intracellular domain" is defined as the amino acid residues from the cytoplasmic domain of the zeta chain sufficient to functionally transmit the initial signals required for T cell activation. In one aspect, the CD3ζ intracellular region comprises residues 52 to 164 of GenBank Accession No. BAG36664.1, functional homologues thereof - equivalent residues from non-human species such as mouse, rodent, monkey, ape, and the like. As used herein, the term "CD3ζ signaling domain" or "CD3ζ intracellular domain" refers to the specific protein fragment associated with that designation and having at least 80% of the CD3ζ intracellular domain amino acid sequence set forth herein, or Alternatively any other molecule with a similar biological function that is at least 90% identical, preferably at least about 95%, more preferably at least about 97%, more preferably at least about 98%, most preferably at least about 99% identical.

The term "costimulatory intracellular domain" refers to the intracellular region of a costimulatory molecule, which is a cognate binding partner on a T cell that specifically binds a costimulatory ligand, thereby mediating a costimulatory response of an immune cell, e.g. But not limited to proliferation. Costimulatory molecules are cell surface molecules other than antigen receptors or their ligands that are required for an effective immune response. Costimulatory molecules include, but are not limited to, the intracellular domains of molecules such as MHC class I molecules, BTLA and Toll ligand receptors, and OX40, CD2, CD27, CD28, CDS, ICAM-1, LFA-1 (CD11a/CD18) and 4-1BB(CD137).

The term "4-1BB" herein refers to a member of the TNFR superfamily having the amino acid sequence of GenBank Accession No. AAA62478.2, or an equivalent residue from a non-human species such as mouse, rodent, monkey, ape, etc.; The "4-1BB" co-stimulatory intracellular domain is defined as amino acid sequence 214-255 of GenBank Accession No. AAA62478.2, the sequence provided in SEQ ID NO: 38 of the present application, or from non-classified species such as mouse, rodent Animal, monkey, simian, etc. equivalent residues, or have at least 80%, or alternatively at least 90% amino acid sequence identity, preferably 95% sequence identity, with the 4-1BB costimulatory domain sequence shown herein, More preferably any other molecule with a similar biological function of at least 97, 98 or 99% sequence identity.

As used herein, the term "CD28" costimulatory domain is the sequence provided in SEQ ID NO: 42 of the present application, or refers to the specific protein fragment associated with that name, and to the CD28 costimulatory structure shown in the present application Any other molecule with a similar biological function having a domain sequence of at least 80%, or alternatively at least 90% amino acid sequence identity, preferably 95% sequence identity, more preferably at least 97, 98 or 99% sequence identity.

In one aspect of the present invention, the CAR comprises a chimeric fusion protein comprising an extracellular antigen recognition domain, a transmembrane domain, a costimulatory domain and a signaling domain. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular binding domain, a transmembrane domain, a costimulatory domain, and a signaling domain that recognize an extracellular antigen. In one aspect, the CAR comprises a chimeric fusion protein comprising an extracellular binding domain, a costimulatory domain and a signaling domain comprising at least two derived from one or more costimulatory molecules Functional signaling domains. In one aspect, the CAR comprises an optional leader sequence (or signal peptide) at the N-terminus of the CAR fusion protein. In one aspect, the CAR further comprises a leader sequence at the N-terminus of the extracellular antigen recognition domain, wherein the leader sequence is optionally cleaved from the N-terminus of the antigen recognition domain (eg, scFv) during cellular processing and localization of the CAR to the cell membrane Down.

The term "antibody" as used herein refers to an immunoglobulin molecule that specifically binds to an antigen. Antibodies can be intact immunoglobulins derived from natural sources or from recombinant sources, and can be immunoreactive portions of intact immunoglobulins. Antibodies are usually tetramers of immunoglobulin molecules. Antibodies of the present invention can exist in a variety of forms including, for example, polyclonal, monoclonal, Fv, Fab and F(ab)2, as well as single chain, human and humanized antibodies (Harlow et al., 1999 in : Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989 in: Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85: 5879-5883 ; Bird et al., 1988, Science 242:423-426).

The term "immunoglobulin" or "Ig" as used herein defines a class of proteins that function as antibodies. Antibodies expressed by B cells are sometimes referred to as BCR (B cell receptor) or antigen receptors. The five members included in this class of proteins are IgA, IgG, IgM, IgD and IgE. IgA is the primary antibody present in bodily secretions such as saliva, tears, breast milk, gastrointestinal secretions, and mucous secretions of the respiratory and urogenital tracts. IgG is the most common circulating antibody. IgM is the major immunoglobulin produced in the primary immune response in most subjects. It is the most potent immunoglobulin in agglutination, complement fixation and other antibody responses, and is important in defense against bacteria and viruses. IgDs are immunoglobulins that do not have known antibody functions, but can act as antigen receptors. IgE is an immunoglobulin that mediates immediate hypersensitivity responses by causing mediator release from mast cells and basophils upon exposure to allergens.

The term "antigen-binding fragment" refers to a portion of an intact antibody, and refers to the antigenic determining variable regions of the intact antibody. Examples of antigen-binding fragments include, but are not limited to, Fab, Fab', F(ab') ₂ , and Fv fragments, linear antibodies, single-chain binding fragments (eg, scFv), and multispecific antibodies formed from antibody fragments.

The term "synthetic antibody" as used herein includes antibodies produced using recombinant DNA techniques, such as antibodies expressed by phage or cells as described herein. The term should also be interpreted to mean an antibody produced by synthesizing an antibody-encoding DNA molecule expressing the antibody protein or specifying the amino acid sequence of the antibody, obtained using synthetic DNA or amino acid sequence techniques available and well known in the art the DNA or amino acid sequence.

The term "antigen" as used herein is defined as a molecule that elicits an immune response. This immune response may involve antibody production, or activation of specific immunocompetent cells, or both. The skilled artisan will understand that any macromolecule (including virtually any protein or peptide) can serve as an antigen. In addition, antigens can be derived from recombinant or genomic DNA. Thus, the skilled artisan will understand that any DNA comprising a nucleotide sequence or part of a nucleotide sequence encoding a protein that elicits an immune response encodes the term "antigen" as used herein. Furthermore, those skilled in the art will understand that the antigen need not be encoded solely by the full-length nucleotide sequence of the gene. Obviously, the present invention includes, but is not limited to, the use of partial nucleotide sequences of more than one gene, and that these nucleotide sequences are arranged in various combinations to elicit a desired immune response. Furthermore, those skilled in the art will appreciate that the antigen need not be encoded by a "gene" at all. Obviously, the antigen can be produced synthetically or can be derived from a biological sample. Such biological samples may include, but are not limited to, tissue samples, tumor samples, cells, or biological fluids.

As used herein, "antibody heavy chain" refers to the larger of the two types of polypeptide chains present in all antibody molecules in its naturally occurring conformation. "Antibody light chain" as used herein refers to the smaller of the two types of polypeptide chains present in all antibody molecules in its naturally occurring conformation, kappa and lambda light chains refer to the two major antibody light chains isotype.

In certain aspects of any of the embodiments disclosed herein, the extracellular antigen binding region, eg, scFv, can comprise light chain CDRs specific for the antigen. The light chain CDRs can be the complementarity determining regions of the scFv light chain of an antigen binding unit such as a CAR. A light chain CDR can comprise a contiguous sequence of amino acid residues, or two or more contiguous sequences of amino acid residues separated by non-complementarity determining regions (eg, framework regions). In some cases, a light chain CDR may comprise two or more light chain CDRs, which may be referred to as light chain CDR1, CDR2, etc. In some cases, the light chain CDRs may comprise three light chain CDRs, which may be referred to as light chain CDR1, light chain CDR2, and light chain CDR3, respectively. In some instances, a set of CDRs present on a common light chain may be collectively referred to as light chain CDRs.

In certain aspects of any of the embodiments disclosed herein, the extracellular antigen binding region, eg, scFv, can comprise heavy chain CDRs specific for the antigen. The heavy chain CDRs may be the heavy chain complementarity determining regions of an antigen binding unit such as an scFv. A heavy chain CDR may comprise a contiguous sequence of amino acid residues, or a contiguous sequence of two or more amino acid residues separated by non-complementarity determining regions (eg, framework regions). In some cases, a heavy chain CDR may comprise two or more heavy chain CDRs, which may be referred to as heavy chain CDR1, CDR2, etc. In some cases, the heavy chain CDRs may comprise three heavy chain CDRs, which may be referred to as heavy chain CDR1, heavy chain CDR2, and heavy chain CDR3, respectively. In some cases, a set of CDRs present on a common heavy chain may be collectively referred to as heavy chain CDRs.

Through the use of genetic engineering, the extracellular antigen binding region can be modified in various ways. In some cases, the extracellular antigen binding region can be mutated so that the extracellular antigen binding region can be selected for higher affinity for its target. In some cases, the affinity of the extracellular antigen binding region to its target can be optimized for a target that is expressed at low levels on normal tissues. Optimizations can be made to minimize potential toxicity. In other cases, clones of extracellular antigen-binding regions with higher affinity for the membrane-bound form of the target may outperform their soluble counterparts.

As used herein, the term "mutation" includes amino acid sequences that differ from the parent protein by virtue of at least one amino acid modification compared to the parent. In specific embodiments, the mutant antibody sequences herein have at least about 80%, preferably at least about 90%, more preferably at least about 95%, more preferably at least about 97%, more preferably at least about 98%, Most preferably at least about 99% amino acid sequence identity. An antibody mutation may refer to the antibody itself, a composition comprising the parent antibody, or the amino acid sequence encoding it. The term "amino acid modification" includes amino acid substitutions, additions and/or deletions, "amino acid substitution" means replacing an amino acid at a particular position in the parent polypeptide sequence with another amino acid. "Amino acid insertion" means the addition of an amino acid at a specific position in the parent polypeptide sequence. "Amino acid deletion" or "deletion" means the removal of an amino acid at a particular position in the parent polypeptide sequence.

The term "sequence" includes amino acid sequences or nucleotide sequences. When used in reference to a nucleotide sequence, the term "sequence" and other grammatical forms thereof may include DNA or RNA, and may be single-stranded or double-stranded. Nucleotide sequences can be mutated and can be of any length.

The term "expression" as used herein is defined as the transcription and/or translation of a specific nucleotide sequence driven by its promoter.

"Lentivirus" as used herein refers to a genus of the Retroviridae family. Lentiviruses are unique among retroviruses in that they are capable of infecting non-dividing cells; they can deliver significant amounts of genetic information into the DNA of host cells, making them one of the most efficient methods of gene delivery vehicles. HIV, SIV and FIV are all examples of lentiviruses. Vectors derived from lentiviruses provide the means to achieve significant horizontal gene transfer in vivo.

The term "vector" as used herein is a composition that contains an isolated nucleic acid and can be used to deliver the isolated nucleic acid to the interior of a cell. Numerous vectors are known in the art, including, but not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Thus, the term "vector" includes autonomously replicating plasmids or viruses. The term should also be construed to include non-plasmid and non-viral compounds that facilitate transfer of nucleic acids into cells, such as polylysine compounds, liposomes, and the like. Examples of viral vectors include, but are not limited to, adenoviral vectors, adeno-associated viral vectors, retroviral vectors, and the like.

The term sequence "identity" as used herein determines percent identity by comparing two best-matched sequences over a comparison window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may contain additions or deletions (ie, gaps) , eg, a gap of 20% or less (eg, 5 to 15%, or 10 to 12%) for the best matched two sequences compared to the reference sequence (which contains no additions or deletions). Percentages are usually calculated by determining the number of positions in the two sequences at which identical nucleic acid bases or amino acid residues occur to yield the number of correctly matched positions, dividing the number of correctly matched positions by the total number of positions in the reference sequence ( i.e. window size) and multiply the result by 100 to yield the percent sequence identity.

The terms "IL10", "interleukin-10" and "interleukin-10" are used interchangeably and have the same meaning. Likewise, the terms "IL15", "interleukin-15" and "interleukin-15" are used interchangeably and have the same meaning.

The term "IL10" is an immunomodulatory factor with multiple biological activities, for example, the term "IL10" can refer to human IL10 as defined in the amino acid sequence of SEQ ID NO: 3 or an active fragment thereof, or other Species of IL10 or an active fragment thereof. Likewise, the term "IL15" may refer to human IL15 as defined in the amino acid sequence of SEQ ID NO: 20 or an active fragment thereof, and may also refer to IL15 of other species or an active fragment thereof. In some embodiments, the IL10 or IL15 can be naturally occurring, such as it can be isolated or purified from mammals; it can also be artificially prepared, such as recombinant IL10 or IL15 can be produced according to conventional genetic engineering recombination techniques. IL15. Preferably, the present invention can use recombinant wild-type IL10 or IL15.

Appropriate substitution of amino acids is a technique well known in the art that can be easily implemented and ensure that the biological activity of the resulting molecule is not altered. These techniques have enabled those in the art to recognize that, in general, altering a single amino acid in a non-essential region of a polypeptide does not substantially alter biological activity. The active fragments of IL10 or IL15 can all be used in the present invention. Here, the meaning of the biologically active fragment refers to a polypeptide, which, as a part of the full-length polypeptide, can still maintain all or part of the functions of the full-length polypeptide. Typically, the biologically active fragment retains at least 50% of the activity of the full-length polypeptide. Under more preferred conditions, the active fragment retains 80%, 90%, 95%, 97%, 98%, 99%, or 100% of the activity of the full-length polypeptide. Therefore, on the basis of the IL10 active fragment, amino acid sequences formed by substitution, deletion or addition of one or more amino acid residues are also included in the present invention. Preferably, an active fragment of IL15 (SEQ ID NO: 22) or a mutant thereof (SEQ ID NO: 21) can be used in the present invention.

Based on the IL10 or IL10 polypeptide sequence, modified or improved polypeptides may also be used in the present invention, for example, modified or modified polypeptides may be employed to enhance their half-life, effectiveness, metabolism, and/or polypeptide potency. improved peptides. That is, any variation that does not affect the biological activity of the polypeptide can be used in the present invention.

It is well known to those skilled in the art that IL15 binds to the IL15 receptor to exert its biological function. The IL15 receptor has three subunits, IL15 receptor alpha (IL15Ralpha, SEQ ID NO: 23), IL15Rbeta (CD122) and gamma (also known as CD132). The extracellular domain of IL15Rα is the part that binds to IL15, and the sushi domain (SEQ ID NO: 26) in the region binds to IL15 to exert the biological function of IL15. In the present invention, "sushi+" (SEQ ID NO: 25) means that in addition to the sushi fragment, other polypeptide fragments are also included. The combination of IL15 and IL15Rα, in addition to the activation of the own cells, can also transmit signals to another cell to activate cell activity because of the mediation of IL15Rα. These activities include selective expansion of CD8+ T cells, NK cells, etc., and do not activate regulatory T cells like IL2, which may play a different role in antitumor immune responses.

The term "engineered" and other grammatical forms thereof can refer to one or more alterations of a nucleic acid, such as a nucleic acid within an organism's genome. The term "engineering" can refer to changes, additions and/or deletions of genes. Engineered cells can also refer to cells containing added, deleted and/or altered genes.

The terms "cell" or "engineered cell" and other grammatical forms thereof can refer to cells of human or non-human animal origin. Engineered cells can also refer to CAR-expressing cells.

The term "transfection" refers to the introduction of exogenous nucleic acid into a eukaryotic cell. Transfection can be achieved by various means known in the art, including calcium phosphate-DNA co-precipitation, DEAE-dextran-mediated transfection, polybrene-mediated transfection, electroporation, microinjection, Liposome fusion, lipofection, protoplast fusion, retroviral infection and biolistics.

As used herein, the terms "encoding nucleic acid molecule", "encoding nucleic acid sequence" refer to the sequence or sequence of deoxyribonucleotides along a deoxyribonucleic acid chain that determines the sequence along which a polypeptide (protein ) chain of amino acids. Thus, a nucleotide sequence encodes an amino acid sequence.

As used herein, the terms "peptide", "polypeptide" and "protein" are used interchangeably and refer to a compound composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least three amino acids, and there is no limit to the maximum number of amino acids that can comprise a protein or peptide sequence. Polypeptides include any peptide or protein comprising two or more amino acids linked to each other by peptide bonds. As used herein, the term refers to both short chains (which are also commonly referred to in the art as eg peptides, oligopeptides and oligomers), and longer chains (which are commonly referred to in the art as proteins), wherein There are many types. "Polypeptide" includes, for example, biologically active fragments, substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, polypeptide variants, modified polypeptides, derivatives, analogs, fusion proteins, and the like. Polypeptides include natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

The term "immune cell" refers to a cell that can elicit an immune response, and "immune cell" and other grammatical forms thereof can refer to immune cells of any origin. "Immune cells" include, for example, white blood cells (leukocytes), lymphocytes (T cells, B cells, natural killer (NK) cells, and bone marrow-derived cells (neutrophils) derived from hematopoietic stem cells (HSCs) produced in the bone marrow , eosinophils, basophils, monocytes, macrophages, dendritic cells). The term "immune cells" may also be human or non-human.

As used herein, the term "T cell" refers to a type of lymphocyte that matures in the thymus. T cells play an important role in cell-mediated immunity and differ from other lymphocytes, such as B cells, by the presence of T cell receptors on the cell surface. "T cells" include all types of immune cells that express CD3, including T helper cells (CD4+ cells), cytotoxic T cells (CD8+ cells), natural killer T cells, T regulatory cells (Treg), and gamma-delta T cells. "Cytotoxic cells" include CD8+ T cells, natural killer (NK) cells, and neutrophils, which are capable of mediating cytotoxic responses. As used herein, the term "NK cells" refers to a class of lymphocytes that originate in the bone marrow and play an important role in the innate immune system. NK cells provide a rapid immune response against virus-infected cells, tumor cells, or other stressed cells, even in the absence of antibodies and major histocompatibility complexes on the cell surface.

For example, immune cells can be blood-derived, such as autologous T cells, allogeneic T cells, autologous NK cells, allogeneic NK cells, or can be derived from cell lines, such as NK cell lines prepared by EBV infection, from embryonic stem cells and iPSC induced differentiated NK cells and NK92 cell lines.

The antibodies, immunoconjugates comprising the antibodies, and genetically modified immune cells of the present invention can be used to prepare "pharmaceutical compositions" or diagnostic reagents. In addition to comprising an effective amount of the antibody, immunoconjugate or immune cell, the pharmaceutical composition may also comprise a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" means that the molecular entities and compositions do not produce adverse, allergic or other adverse reactions when properly administered to animals or humans.

Specific examples of some substances that may be pharmaceutically acceptable carriers or components thereof are sugars such as lactose, glucose and sucrose; starches; polyols such as propylene glycol, glycerol, sorbitol, mannitol and polyethylene glycols ; alginic acid; emulsifiers such as Tween; wetting agents such as sodium lauryl sulfate; colorants; flavors; tableting agents, stabilizers; antioxidants; preservatives; pyrogen-free water; isotonic saline solutions; and phosphoric acid salt buffer, etc.

The pharmaceutical composition of the present invention can be prepared into various dosage forms according to needs, and can be administered by physicians according to factors such as the type, age, weight and general disease state of the patient, administration mode and other factors that are beneficial to the patient. The mode of administration can be, for example, by injection or other treatment.

The term "tumor" refers to a disease characterized by the pathological proliferation of cells or tissues and their subsequent migration or invasion of other tissues or organs. Tumor growth is usually uncontrolled and progressive, and does not induce or inhibit normal cell proliferation. Tumors can affect a variety of cells, tissues or organs, including but not limited to those selected from the group consisting of bladder, breast, esophagus, intestine, kidney, liver, lung, lymph nodes, nerve tissue, ovary, pancreas, prostate, skeletal muscle, skin, spinal cord, spleen , stomach, uterine organs, or tissues or corresponding cells. The tumor described in the present invention is a CLDN18.2 positive tumor, which may include, but is not limited to, gastric cancer, esophageal cancer, lung cancer, melanoma, kidney cancer, breast cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, neuroglial cancer tumor or leukemia. Preferably, the "tumor" includes but is not limited to: gastric cancer, lung cancer, liver cancer, and breast cancer.

The term "tumor antigen" as used herein refers to an antigen associated with cancer cells. Examples of tumor antigens include, but are not limited to, CD19, CD20, CD22, CD33/IL3Ra, c-Met, PSMA, EGFRvIII, GD-2, and CLDN18.2.

As used herein, the term "anti-tumor effect" refers to a biological effect that can be manifested as a reduction in tumor volume, a reduction in tumor cell number, a reduction in the number of metastases, an increase in life expectancy, or an improvement in multiple factors associated with cancerous conditions. physical symptoms. An "anti-tumor effect" can also be expressed as the ability of the peptides, polynucleotides, cells and antibodies of the invention to prevent tumorigenesis in the first place.

As used herein, the term "specifically binds" with respect to an antibody means an antibody that recognizes a specific antigen but does not substantially recognize or bind to other molecules in a sample. For example, an antibody that specifically binds an antigen from one species can also bind that antigen from one or more species. However, this interspecies cross-reactivity does not in itself alter the classification of antibodies according to specificity. In another example, an antibody that specifically binds an antigen can also bind to different allelic forms of the antigen. However, this cross-reactivity by itself does not alter the classification of antibodies according to specificity. In some cases, the terms "specifically binds" or "specifically binds" may be used to refer to the interaction of an antibody, protein or peptide with a second chemical, meaning that the interaction depends on a specific structure on the chemical (eg, antigenic determinants or epitopes); for example, antibodies generally recognize and bind to specific protein structures, not proteins. If the antibody is specific for epitope "A", in a reaction containing labeled "A" and the antibody, the presence of a molecule containing epitope A (or free, unlabeled A) will reduce binding to the antibody The amount of marked A.

List of Abbreviations

CAR: chimeric antigen receptor; HLA: histocompatibility lymphocyte antigen; Ip: intraperitoneal; IRES: internal ribosome entry site; MFI: mean fluorescence intensity; MOI: multiplicity of infection; PBMC: peripheral blood mononuclear cells ; PBS: phosphate buffered saline; scFv: single chain variable fragment.

The present invention is further described below by way of examples, but the present invention is not limited to the scope of the described examples. The experimental methods that do not specify specific conditions in the following examples are selected according to conventional methods and conditions, or according to the product description. The following Examples 1-3 relate to the preparation and detection of the CLDN18.2 antibody of the present invention, see patent application CN201811295845.2.

Example 1 Cloning, expression and purification of recombinant proteins and antibodies

The recombinant proteins involved in the present invention, including the expression and purification of monoclonal antibodies, are obtained by the cloning, expression and purification of the present invention. The vector used for expression cloning was pTT5 vector (Biovector, Cat#: 102762). All protein expressions (including antibody light and heavy chains) were purified by transient transfection of HEK 293F cells (Life Technologies Cat. No. 11625019) with pTT5 vector.

293F cells were expanded in Gibco FreeStyle 293Expression Medium (Gibco, Cat#12338018). Before the transient start, adjust the cell concentration to 6-8×10 ⁵ cells/ml, 1% FBS (Aus Gene X FBSExcellent supplier: AusGeneX, China, Cat#FBSSA 500-S), 37°C 8% CO ₂ shaker After culturing for 24 hours, the viability was again >95% and the cell concentration was 1.2×10 ⁶ cells/ml. Prepare 300ml of culture system cells, dissolve 150μg of heavy chain and light chain plasmids into 15ml of Opti-MEM (Gibco, Cat#31985070) (if it is a recombinant protein, the amount of a single plasmid is 300μg), and filter and sterilize at 0.22μm. Then, 15 ml of Opti-MEM was dissolved in 600 μl of 1 mg/ml PEI (Polysciences, Inc, Cat#23966-2), and then allowed to stand for 5 min. Slowly add PEI to the plasmid, incubate at room temperature for 10 min, slowly drop the plasmid PEI mixed solution while shaking the culture flask, culture at 37°C with 8% CO ₂ shaker for 5 days, collect the sample, and centrifuge at 3300G for 10 min to take the supernatant for purification. The samples were centrifuged at high speed to remove impurities, equilibrated with PBS (pH 7.4) on a gravity column (Sangon Biotechnology, Cat#F506606-0001) containing Protein A (Mabselect, GE HealthcareLife Science, Cat#71-5020-91AE), 2- 5 column volume washes. Pass the sample through the column. The column was rinsed with 5-10 column volumes of PBS (Sanggong Bio, Cat#B548117-0500). The target protein was eluted with 0.1M acetic acid with a pH of 3.5, and then adjusted to neutrality with Tris-HCl with a pH of 8.0, and the concentration was measured by a microplate reader, and then packed and stored for later use.

Example 2 Human CLDN18.2 binding ELISA experiment

The human CLDN18.2 high expression cell line used in the present invention is completed by the company's stable cell line construction platform. Specific steps are as follows:

On the first day of the experiment, 293T cells (Cat#GNHu17, cell bank of the Type Culture Collection, Chinese Academy of Sciences) were seeded in two 6cm dishes, and the number of cells in each dish reached 7.5×10 ⁵ . On the second day, the encapsulated plasmid (BioVector such as pGag-pol, pVSV-G, pBabe, etc., plasmid vector strain cell gene collection center) and cloned human CLDN18.1 gene (NCBI Reference Sequence: NP_057453.1), human CLDN18.2 gene (NCBIReference Sequence: NP_001002026.1), murine CLDN18.1 (NP_062789.1) or murine CLDN18.2 (NP_001181850.1) gene plasmid pBabe 4 μg each was added to OPTI-MEM (Thermofisher Scientific Cat#31985070), so that the final volume was 200μl, prepare another 200μl OPTI-MEM, add 36μl transfection reagent fectin (Cat#F210 of Shanghai Yuanpei Biotechnology Co., Ltd.), mix the two, leave at room temperature for 5min, then add the mixture (200μl per dish) dropwise to the cultured 293T cells. On the third day, the 293T cell culture medium was changed to 4 ml of DMEM high-glucose medium (Shanghai Yuanpei Biotechnology Co., Ltd./Yuanpei Bio: Cat#: 310KJ). On the 4th day, CHO-K1 cells (Cat#SCSP-507, a cell bank of the Type Culture Collection, Chinese Academy of Sciences) were inoculated in a 10 cm dish to make the number of cells reach 5×10 ⁵ . On the 5th day, the supernatant (virus) of 293T cells was collected, filtered with a 0.45 μm filter to the cultured CHO-K1 cells, and 10 μg/ml polybrene (Shanghai Yisheng Biotechnology Co., Ltd. Cat#40804ES76) was added at the same time, and then placed in culture after mixing. After 3 to 4 hours, the medium was changed to DMEM/F12 10% FBS medium (Original Biology, Cat#L310KJ). CHO-K1 cells were passaged on day 7, and 10 μg/ml puromycin was added to the passaged cells on day 8 for screening (Yuanbi Bio, Cat#S250J0). After 2-3 days, a large number of cells die, and the culture medium is changed to continue the culture. When the cells are no longer dead, the cells are expanded in large quantities. The monoclonal cell lines are screened, expanded and cryopreserved.

Cell binding ELISA: Take the above-mentioned monoclonal cell lines with high expression of human CLDN18.1 and human CLDN18.2 after expansion, spread 96-well plates at 5×10 ⁴ cells/well, incubate overnight in a 37°C incubator and remove the supernatant , fixed with 100 μl/well of immunostaining fixative (Shanghai Biyuntian Biotechnology Co., Ltd. Cat#P0098) for half an hour at room temperature. After washing once with PBS (Cat#B320), the cells were blocked with 5% milk at 37° C. for 2 hours, and washed with PBST for 3 times. Add the sample to be tested. Incubate at 37°C for 1 hour, and then wash three times with PBST. Add Anti-human HRP 1:2500 50 μl/well and incubate at 37°C for 1 hour, then wash with PBST for 3 times, TMB (Surmodic Cat#TTMB-1000-01) to develop color, and add 50 μl/well of 1M H ₂ SO ₄ to stop the reaction. Microplate reader (MultiskanGO Thermo model 51119200) read, Graphpad prism 5 for data analysis.

Example 3 Discovery and effect identification of anti-human CLDN18.2 antibody

1. Discovery of anti-human CLDN18.2 antibody

The anti-human CLDN18.2 monoclonal antibody of the present invention is a mouse immunized with the human CLDN18.2 high-expressing cell line (hCLDN18.2+cell) obtained in Example 2, and the spleen of the immunized mouse is taken for hybridoma fusion. Screened and optimized from millions of hybridoma clones.

Experimental mice, female, 4 weeks old (SJL mice were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., animal production license number: SCXK (Jing) 2016-0011; Balb/c mice were purchased from Shanghai West Poole-Bike Laboratory Animal Co., Ltd.). After purchase, they were reared in a laboratory environment for 1 week, with daytime light/night dark cycle adjustment, temperature 20-25°C, and humidity 40-60%. Mice were divided into 3 mice/group/cage.

CRL-8287^TM)进行融合得到杂交瘤细胞铺96孔板。The human CLDN18.2 high-expressing cell line (human CLDN18.2+ cells) constructed in Example 2 was cultured, digested with trypsin, washed with DMEM medium (Original Biology, Cat#L310KJ), and resuspended in DMEM medium . The mice were immunized by intraperitoneal injection at 100 μl/1×10 ⁷ cells/mouse. For the first immunization, Titermax (Sigma-Aldrich, T2684) was used to mix 1:1 with cells. Then immunized once a week. After 10 immunizations, using the ELISA method of Example 2, human CLDN18.1+ cells and human CLDN18.2+ cells were plated at the same time, and the serum titers of the immunized mice were detected. The plated ELISA values were used as background, and the immune titers (titers) of mouse serum were calculated. After 12-15 immunizations, mice with high serum titers and plateau titers were selected for splenocyte fusion. Before fusion, mice were immunized with 200 μl/2×10 ⁷ cells/mouse. Mouse spleen lymphocytes and myeloma cells Sp2/0 cells (

CRL-8287 ^™ ) was fused to obtain hybridoma cells plated in 96-well plates.

The supernatant of the hybridoma cells in the 96-well plate was taken and plated with human CLDN18.1+cell and human CLDN18.2+cell to detect the binding of antibodies produced by the hybridoma cells. Table 1a shows the detection results of some of the hybridoma supernatants.

Table 1a Hybridoma fusion clone and human CLDN18.2+cell, human CLDN18.1+cell binding activity detection

Because human CLDN18.2 and CLDN18.1 have up to 92% homology (240/261), and the protein is a transmembrane protein, only a small part of the peptide is extracellular (such as 51 amino acid ECL1), immune The originality is very low, and the possibility of producing specific antibodies is very small. Therefore, in the above screening, there are not only few hybridomas that can secrete and recognize CLDN18, but among the few hybridomas, most of the antibodies in the supernatants of hybridomas are antibodies that can bind both human CLDN18.2 and CLDN18.1. .

Unexpectedly, the present invention found a hybridoma clone, the supernatant secreted by it only binds to human CLDN18.2+cell, and does not bind to human CLDN18.1+cell, see mab5 in Table 1a, the initial hybridoma Clone number C13C1. The data in Table 1a shows that under the same screening conditions, the supernatant of this clone only binds to human CLDN18.2+cell, and the detection value is 1.41, while it hardly binds to human CLDN18.1+cell, and the binding activity read value is only 0.09.

It was further confirmed that the hybridoma cell line C13C1 unexpectedly discovered by the present invention can secrete a unique anti-human CLDN18.2 antibody. The C13C1 hybridoma cells were subjected to several limiting dilutions, and the monoclonal cells after each dilution were carefully and finely screened. Finally, a monoclonal cell line that secreted a unique anti-human CLDN18.2 antibody was found. The results are shown in Table 1b .

Table 1b Hybridoma monoclonal cell lines found by optimized screening of hybridoma C13C1

It can be seen from the results in Table 1b that the antibodies secreted by the monoclonal cell lines C13C1F1D3G6 and C13C1F1D3H5 obtained through the fine and optimized screening of the initial hybridoma clone C13C1 obtained by the present invention all retained binding to human CLDN18.2 cells, and the readings were respectively are 0.8895 and 0.8778. However, it did not bind to human CLDN18.1 cells, with read values of 0.0859 and 0.0756, respectively. This reading is close to the 0.081 background of this ELISA. The initial hybridoma clone F2A4 was screened simultaneously to obtain monoclonal cell lines F2A4F6F3E4 and F2A4F6F3H7. These monoclonal cell lines were expected to have the same binding activity as human CLDN18.2 cells and human CLDN18.1 cells, and the data are shown in Table 1b. These results demonstrate that the present inventors have discovered that monoclonal cell lines, such as C13C1F1D3G6, are capable of secreting unique antibodies that, unexpectedly, only bind to human CLDN18.2, but not to human CLDN18.1.

This means that the antibody unexpectedly discovered in the present invention can effectively recognize only human CLDN18.2 protein, and has the potential to be used as a monoclonal antibody, or used for CAR-T (CART), CAR-NK (CARNK) cells to treat tumor patients, especially It is the treatment of cancer patients with overexpression of human CLDN18.2 protein, including but not limited to pancreatic cancer, gastric cancer, esophageal cancer, lung cancer and the like. Because it does not bind to human CLDN18.1 protein at all, it is expected to avoid the toxic and side effects caused by non-specific binding of therapeutic antibodies, CAR-T, CAR-NK cell therapy and human CLDN18.1 proteins.

2. Screening and identification of mouse anti-human CLDN18.2 antibody

The antibody sequence secreted by the cell line was extracted from the above hybridoma monoclonal cell line C13C1F1D3G6 (Table 1b) to obtain the murine antibody mab5b sequence of the present invention. The process of extracting antibody sequences from the preferred monoclonal cell lines of hybridomas is a method well known and commonly used by those skilled in the art.

Specifically, in the present invention, the hybridoma monoclonal cell C13C1F1D3G6 found in the above example is amplified and cultured, 1×10 ⁶ cells are collected, and RNA is extracted with Trizol (Invitrogen, 15596-018) (according to the kit instructions), and the The extracted RNA was reverse transcribed into cDNA, and the reverse transcription kit was purchased from Sangon Biotechnology (Shanghai) Co., Ltd., Cat#B532435. Using the cDNA obtained by reverse transcription as a template, after PCR amplification, the amplified product is sequenced to obtain the variable region sequence of the antibody light and heavy chain of mab5b. The primers used were referred to the manual published by Novagen (TB326Rev.C0308).

The nucleotide sequence of the light chain of the anti-human CLDN18.2 (anti-hCLDN18.2) monoclonal antibody obtained from the preferred hybridoma monoclonal cell line C13C1F1D3G6 of the present invention is as follows, wherein the underlined part is the nucleotide encoding the variable region sequence:

taatgggcttcaagatgaagtcacagtttctggtcctcatgtccctgctgttctgggtatctggtacctgtggg gacattgtgatgacacagtctccatcctccctgactgtgacagcaggagagaaggtcactatgagttgca agtccagtcagagtctgttaaacagtggaaatcaaaagaactacttgacctggtaccagcagaaaccagggcagcc tcctaaactgttgatctactgggcatccactagggaatctggggtccctgatcgcttcacaggcagtggatctgga acacatttcactctcaccatcagcagtgtgcaggctgaagacctggcagtttattactgtcagaatgattattttt atccattcacgttcggctcggggacaaagttggaaaaaaaa cgggctgatgctgcaccaactgtatccatcttcccaccatccagtgagcagttaacatctggaggtgcctcagtcgtgtgcttctgaacaactctaccccaaagaccatccatgccc(SEQ ID NO:5)

The nucleotide sequence of the heavy chain of the anti-human CLDN18.2 (anti-hCLDN18.2) monoclonal antibody obtained from the preferred hybridoma monoclonal cell line C13C1F1D3G6 of the present invention is as follows, wherein the underlined part is the nucleoside encoding the variable region Acid sequence:

taatgggatggaccgggatctttatctttctcctgtcagtaactgcaggtgttcactcc caggtccag ctgcagcagtctggagctgagctgataggacctgggacttcagtgaaggtgtcctgcaaggcctctggatacgcct tcagtaattacttgatagaatgggtaaaacagaggcctgaacagggccttgagtggattggtttgattaatcctgg aagtggtggcactaactacaatgagaagttcaagggcaaggcaacactgactgcagacaaatcctccagcactgcc tacatgcaactcagcagcctgacatctgatgactctgcggtctacttctgtgcaagggtctactatggtaactcct ttgcttactggggccaagggactctggtcactgtctctgcag ccaaaacgacacccccatctgtctatccactggcccctggatctgctgcccaaactaactccatggtgaccctgggatgcctggtcaagggctattaccgagcaagaaatgtcg(SEQ ID NO:6)

The amino acid sequence of the murine anti-human CLDN18.2 (anti-hCLDN18.2) monoclonal antibody mab5b light chain variable region extracted from the hybridoma monoclonal cell line found in the present invention is obtained by translating the above-mentioned light chain base sequence:

DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTHFTLTISSVQAEDLAVYYCQNDYFYPFTFGSGTKLEKK(SEQ ID NO:7)

The amino acid sequence of the murine anti-human CLDN18.2 (anti-hCLDN18.2) monoclonal antibody mab5b heavy chain variable region extracted from the hybridoma monoclonal cell line found in the present invention is obtained from the above heavy chain base sequence:

QVQLQQSGAELIGPGTSVKVSCKASGYAFSNYLIEWVKQRPEQGLEWIGLINPGSGGTNYNEKFKGKATLTADKSSSTAYMQLSSLTSDDSAVYFCARVYYGNSFAYWGQGTLVTVSA (SEQ ID NO: 8)

The above-mentioned antibody mab5b extracted from the hybridoma monoclonal cell line found in the present invention was cloned (sequence as follows) for recombinant expression of the light and heavy chain variable regions and constant regions of the antibody by the method described in Example 1. After purification, and The control antibody Ref simultaneously detected the binding activity to human hCLDN18.1, hCLDN18.2, mouse mCLDN18.1 and mCLDN18.2. The results are shown in Table 2a and Table 2b below.

The amino acid sequence of the anti-hCLDN18.2 antibody mab5b light chain (L Chain) of the present invention is:

DIVMTQSPSSLTVTAGEKVTMSCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFTGSGSGTHFTLTISSVQAEDLAVYYCQNDYFYPFTFGSGTKLEKKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC)(SEQ

The amino acid sequence of the anti-hCLDN18.2 antibody mab5b heavy chain (H Chain) of the present invention is:

QVQLQQSGAELIGPGTSVKVSCKASGYAFSNYLIEWVKQRPEQGLEWIGLINPGSGGTNYNEKFKGKATLTADKSSSTAYMQLSSLTSDDSAVYFCARVYYGNSFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYI2VNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK(SEQ ID NO:10)

Table 2a Binding activity of anti-hCLDN18.2 murine antibody mab5b of the present invention and human CLDN18+cell

Table 2b Binding activity of anti-hCLDN18.2 murine antibody mab5b of the present invention and murine CLDN18+cell

The results in Table 2a show that the anti-hCLDN18.2 murine antibody mab5b found in the present invention and the control antibody Ref (sequence derived from WO2014146672_24 (light chain), WO2014146672_17 (heavy chain)) do not bind to hCLDN18.1+cell, EC50 detection Less than (ND), no binding activity can be detected even at a high concentration of 200 nM, and the binding value Emax (referring to the binding value when the sample concentration increases and the binding reaches a plateau, that is, the maximum specific binding value) is still the background, that is Background values. Both antibodies have good binding activity to hCLDN18.2+cell. Unexpectedly, the binding activity of the antibody mab5b of the present invention was more than 1-fold better than that of Ref (EC50 was 0.115nM vs 0.249nM). More surprisingly, the maximum binding value _Emax that mab5b can achieve is more than 36% higher than that of Ref [(1.92-1.41)/1.41].

The results in Table 2b show that neither the anti-hCLDN18.2 murine antibody mab5b found in the present invention nor the control antibody (Ref) binds to murine CLDN18.1+cell, and the EC50 cannot be detected (ND), even at a high concentration of 200nM Still no binding activity was detected and the binding value Emax was still the background, ie the background value. The two antibodies have good binding activity to mouse CLDN18.2+cell. Unexpectedly, the binding activity of the antibody mab5b of the present invention was more than 4 times better than that of Ref (EC50 was 0.182nM vs 1.04nM). What is even more surprising is that the maximum binding value Emax that mab5b can achieve is more than 1 times higher than that of Ref [2.21vs1.0].

The above results show that the new molecule mab5b discovered by the present invention has better binding activity (EC50 and Emax) than the control molecule. And neither hCLDN18.1 nor mCLDN18.1 has any binding activity, indicating that mab5b not only has better binding activity, but also has excellent specificity. This indicates that mab5b can provide better efficacy and safety advantages for tumor treatment product development. And it also has better binding with murine CLDN18.2, providing a more convenient non-primate choice for preclinical research in mice.

3. Humanization of antibody mab5b

The activity of the antibody mab5b found in the present invention is better than that of Ref, indicating that the antibody can be used for the development of tumor therapeutic drugs. In order to reduce the risk of immunogenicity and other aspects in the process of drug development, for example, humanization is completed by mouse-derived antibodies, and the molecular properties after humanization are optimized to facilitate drug development. The present invention conducts humanization screening of mab5b , and sequence optimization work. The specific process is described as follows.

CDR definitions of antibodies There are many different methods in the art, and these methods of labeling CDRs can be summarized in Table 2c below.

Table 2c Summary of different methods for defining antibody CDRs in the art*

Loop CCG Definition Definition of Kabat Definition of AbM Definition of Chothia Contact Definition light chain CDR1 L24-L34 L24-L34 L24-L34 L24-L34 L30-L36 light chain CDR2 L50-L56 L50-L56 L50-L56 L50-L56 L45-L55 light chain CDR3 L89-L97 L89-L97 L89-L97 L89-L97 L89-L96 heavy chain CDR1 H26-35 H31-35 H26-35 H26-32 H30-35 heavy chain CDR2 H50-65 H50-65 H50-58 H52-56 H47-H58 heavy chain CDR3 H95-H102 H95-H102 H95-H102 H95-H102 H93-H101

*More information can be found on the website: http://www.bioinf.org.uk/abs/#cdrdef

The murine anti-human CLDN18.2 antibody mab5b obtained above was defined in accordance with the CCG method in Table 2c, and its CDR sequences were marked/annotated as follows in Table 2d.

Table 2d Anti-human CLDN18.2 antibody mab5b CDR sequences defined by CCG

For the above-mentioned CDR analysis of the anti-hCLDN18.2 antibody (mab5b) of the present invention, according to the antibody labeling system, the CDR regions (as above) of the light and heavy chains of the antibody are labeled, and the variable region sequences of the light and heavy chains of the murine antibody mab5b are respectively Compare with the human antibody germline database (v-base) to find out the light and heavy chain germlines of human antibodies with high homology. , back-mutate key sites and combinations, and screen out humanized antibody molecules with preferred activity.

Specifically, through the comparative analysis of sequence homology, it was found that the human antibody germline with better homology to the light chain of mab5b contains IGKV4-1*01(F), IGKV2-28*01(F), IGKV2D-28* 01(F), IGKV1-27*01(F), IGKV1-39*01(F), IGKV1D-39*01(F), IGKV2-40*01(F), IGKV2D-29*01(F), IGKV2D-40*01(F), IGKV3-15*01(F). For further comparison and analysis, the human antibody germline light chain IGKV4-1*01(F) is preferred. In particular, the CDR2 sequence of the selected human germline light chain IGKV4-1*01(F) is WASTRES, which is exactly the same as the CDR2 sequence of the murine antibody mab5b light chain discovered in the present invention. The sequence alignment found that the J gene region of the light chain of mab5b has high homology with the human antibody germlines hJK1, hJK2.1, hJK2.2, hJK2.3 and hJK2.4. For further comparison and analysis, hJK2.1 is preferred for mab5b Light chain humanized human antibody germline J region, humanized design, screening and sequence optimization.

Through the comparative analysis of sequence homology, it was found that the human antibody germline with better homology to the heavy chain of mab5b contains IGHV1-69*02(F), IGHV1-69*06(F), IGHV1-69*08(F) ), IGHV1-69*09(F), IGHV1-69*10(F), IGHV1-69*04(F), IGHV1-69*14(F), IGHV1/OR15-2*02(P), IGHV1 -69*01(F), IGHV1-69*11(F), for further comparison and analysis, the sequence of human germline heavy chain IGHV1-69*01(F) is preferred for humanization of the antibody of the present invention. The sequence alignment found that the heavy chain J gene region of mab5b has high homology with the human antibody germline heavy chain J gene hJh4.1, hJh4.2, hJh4.3, hJh1, hJh2, hJh3.1, hJh3.2, and further comparison , analysis, preferably hJh4.1 is used in the germline J region of the mab5b heavy chain humanized human antibody of the present invention, and humanized design, screening and sequence optimization are carried out.

The mab5b CDR region of the antibody of the present invention (see CDR definition above) is grafted onto the selected humanized light and heavy chain human antibody germline template, and then recombined with the IgG light and heavy chain constant regions. Then, based on the three-dimensional structure of the murine antibody, backmutation was performed on the embedded residues, residues that have direct interactions with the CDR regions, and residues that have important effects on the conformation of VL and VH, and screened for these mutations and Mutation combination, see the impact on antibody activity, and optimize the chemically unstable amino acid residues in the CDR region to obtain antibody molecular sequences with optimized structure and activity, that is, the humanized series of the anti-human CLDN18.2 mouse antibody mab5b of the present invention Optimize antibody molecules.

Specifically, in the analysis of the light chain of the antibody mab5b of the present invention, it is found that the sequence KSSQSLLNSGNQKNYLT WYQQKPGQPPKLLIY WASTRES (SEQ ID NO. :47) and the humanized preferably human germline light chain IGKV4-1*01 (F) of the present invention has a high sequence homology. Among them, L24-L34CDR1 differs in only 5 amino acids, which are L29, L30A, L30C, L30E and L34. The L50-L56-bit CDR2 is exactly the same. In the present invention, five positions (L29, L30A, L30C, L30E and L34 positions) of mab5b CDR1 were first mutated to the amino acids of the corresponding positions of adult germline IGKV4-1*01 (F). The combination design is shown in the following table.

Table 2e The sequence of the murine anti-hCLDN18.2 antibody mab5b of the present invention is based on the amino acids at positions L24-L34 (CDR1, namely SEQ ID NO: 11) defined by Kabat and humanized

The above-mentioned humanized design molecules Var1, Var2, Var3, Var4, Var5, Var6, Var7, Var8 and mab5b were cloned, expressed and purified according to the aforementioned Example 1, and the binding activity to human CLDN18.2+ cells was detected according to Example 2. The results are as follows Table 2f.

Table 2f Humanization optimization of antibody light chain CDR1 sequence of the present invention

Antibody EC50(nM) Emax mab5b 0.215 1.93 Var1 0.461 1.83 Var2 0.221 1.7 Var3 0.143 1.46 Var4 0.434 1.7 Var5 0.656 1.37 Var6 7.67 1.14 Var7 9.51 1.02 Var8 0.20 1.79

Further, using the above-mentioned humanization method, the mab5b is designed to be humanized, and the preferred sequence of the humanized light chain variable region of the mab5b antibody of the present invention is preferably obtained as follows:

L14:

DIVMTQSPDSLAVSLGERATISCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYFYPFTFGQGTKLEIK (SEQ ID NO: 29)

L11:

DIVMTQSPDSLAVSLGERATISCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTHFTLTISSLQAEDVAVYYCQNDYFYPFTFGQGTKLEIK (SEQ ID NO: 30)

L12:

DIVMTQSPDSLAVSLGERATISCKSSQSLLNSGNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYFYPFTFGQGTKLEIK(SEQ ID NO:31)

L13:

DIVMTQSPDSLAVSLGERATINCKSSQSLLNSGNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYFYPFTFGQGTKLEIK(SEQ ID NO:32)

L15:

DIVMTQSPDSLAVSLGERATISCKSSQSLLNSGNNKNYLAWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTHFTLTISSLQAEDVAVYYCQNDYFYPFTFGQGTKLEIK(SEQ ID NO:33)

The preferred sequence of the humanized heavy chain variable region of the present invention obtained by the method described above is as follows:

H51:

QVQLVQSGAEVKKPGSSVKVSCKASGYAFSNYLIEWVKQAPGQGLEWIGLINPGSGGTNYNEKFKGKATITADKSTSTAYMELSSLRSEDTAVYYCARVYYGNSFAYWGQGTLVTVSS (SEQ ID NO: 34)

H52:

QVQLVQSGAEVKKPGSSVKVSCKASGYAFSNYLIEWVKQAPGQGLEWIGLINPGSGGTNYNEKFKGKATLTADKSTSTAYMELSSLRSEDTAVYFCARVYYGNSFAYWGQGTLVTVSS (SEQ ID NO: 35)

H53:

QVQLVQSGAEVKKPGSSVKVSCKASGYAFSNYLIEWVRQAPGQGLEWMGLINPGSGGTNYNEKFKGRVTITADESTSTAYMELSSLRSEDTAVYYCARVYYGNSFAYWGQGTLVTVSS (SEQ ID NO: 36)

H54:

QVQLVQSGAEVKKPGSSVKVSCKASGYAFSNYLIEWVRQAPGQGLEWMGLINPGSGGTNYNEKFKGKVTITADKSTSTAYMELSSLRSEDTAVYYCARVYYGNSFAYWGQGTLVTVSS (SEQ ID NO: 37)

The above-mentioned light chain variable region sequence, including listed as L14, L11, L12, L13, L15 and any sequence shown not listed, combined with human antibody light chain κ type or λ type light chain constant region to obtain the inventors Antibody light chain sequences. The above-mentioned heavy chain variable region sequence, including listed as H51, H52, H53, L54 and unlisted shown heavy chain variable region sequence and hIgG1, hIgG2, hIgG3, hIgG4 and other different subtypes of constant region sequence combination Obtain the heavy chain sequence of the antibody of the present invention. Any combination of the light chain and heavy chain can obtain the humanized antibody of the present invention, and the partial humanized antibody sequence is shown in Table 2g below.

Table 2g Preferred sequences of humanized antibody parts of the present invention

Humanized Ab10 antibody amino acid sequence:

Light chain: the sequence is the same as SEQ ID NO:38 of CN201811295845.2;

Heavy chain: the sequence is the same as SEQ ID NO:39 of CN201811295845.2;

Humanized Ab6 antibody amino acid sequence:

Light chain: the sequence is the same as SEQ ID NO:42 of CN201811295845.2;

Heavy chain: the sequence is the same as SEQ ID NO:39 of CN201811295845.2.

The recombinant antibody was cloned, expressed and purified by the method of Example 1 of the present invention, and the binding activity of the above-mentioned humanized antibody to hCLDN18.2+cell and hCLDN18.1+cell was detected and screened by the ELISA method described in Example 2. The results are shown in the following table 2h.

Table 2h Binding activity of the humanized anti-hCLDN18.2 antibody of the present invention and hCLDN18.2+cell, hCLDN18.1+cell

NA: not applicable; ND: not detected

The results in Table 2h show that after humanization of the antibody mab5b found in the present invention, the advantage of the binding activity of the murine antibody is higher than that of the control antibody Ref, and the EC 50 of the humanized antibody Ab10 is 2 times better than that of Ref (0.117vs 0.345). Not only that, the binding maxima Emax of these humanized optimized molecules were 39.8%-54.1% higher than the control antibody Ref. It is more superior to the control antibody than the murine antibody mab5b.

In order to further optimize the humanized molecule of the present invention, it is preferred that the final sequence and the light and heavy chains of the human antibody germline be as consistent as possible to reduce the immunogenicity that may be caused by a small amount of sequences in the mouse antibody, and it is preferred to optimize the light chain CDR1 sequence by humanization Var3 (see Table 2e) designed a series of humanized antibodies and screened for specific binding activity. The results are shown in Table 2i below.

Table 2i The binding activity of the optimized antibody of the humanized anti-hCLDN18.2 antibody of the present invention to hCLDN18.2+cell and hCLDN18.1+cell

ND: not detected

The results in Table 2i above show that in the humanized antibodies obtained by further optimization of the present invention, the number of back-mutated amino acids of these humanized antibodies is different. 5 heavy chain); there are 6 back mutations, and the light chain CDR1 is optimized for humanization, such as Ab6 (1 light chain, 5 heavy chain); there are 2 back mutations such as Ab14 (0 light chain, heavy chain) There are only 1 back mutation such as Ab11 (1 light chain, 0 heavy chain), and no back mutation at all, such as Ab13. The activities of these optimized humanized molecules, including EC50 and Emax activities, maintained the same level as Ab10 (the optimized mab5b humanized molecule in Table 2i) and neither bound to hCLDN18.1+ cells.

The above results show that the antibody molecule obtained by humanization and optimization of the murine antibody mab5b sequence of the present invention contains only humanized FR region, and the light chain CDR1 remains wild type (without mutation), such as Ab10; or human FR region In addition to the humanization, the light chain CDR1 was also humanized with the optimized sequence Var3, and the resulting Ab6, Ab11-15, etc., all maintained their binding activity and were all better than the control molecule, and the EC ₅₀ was 1 times stronger than that of the control molecule. , _Emax was 30%-50% higher than that of the control molecule, and all were not bound to hCLDN18.1+ cells.

Taking Ab10 as an example, the affinity of the antibody of the present invention and human CLDN18.2 expressed on the cell surface was evaluated by KinExA method. The method was carried out with reference to the KinExA 4000 instrument manual, that is, the test antibody Ref and Ab10 were used as Constant binding Partner (CBP) respectively, and hCLDN18.2+ cells were used as Titrant. The cells (Titrant) were serially diluted with a fixed concentration of antibody (CBP). After incubation, the unbound free antibody (freeCBP) was captured by an anti-human IgG Fc column, and the signal value was obtained with anti-human Fc Alexa Flour 647. Antibody affinity was calculated with software. Specifically, firstly, according to the estimated affinity, a reasonable concentration was calculated to conduct a signal test, and it was determined that 500 μl of 120pM Ref antibody and 100pM Ab10 antibody were used as Signal 100%, and a satisfactory net signal value was obtained at this concentration, and the PBS blank was used as the negative signal value (NSB ). The 120 pM Ref antibody and 100 pM Ab10 antibody concentrations were determined as CBP concentrations. In the following equilibration experiment, two tubes of hCLDN18.2+ cells were collected by centrifugation at 300g for 10 minutes, and the number of cells in each tube was 5×10 ⁸ (the positive rate was 100% detected by FACS). The cells were washed once with PBS, centrifuged at 300g for 10 minutes, and the cells were collected into 15ml centrifuge tubes. Prepare 15ml of 120pM Ref and 100pM Ab10 antibody solutions, respectively. Add 120pM Ref or Ab10 antibody solution to 2ml to ^5x108 cells, and dilute cells 2-fold with 120pM Ref or Ab10 antibody solution as buffer, starting concentration ^2.5x108 cells/ml, 18 gradients, 0.6ml per gradient. The cells were incubated with the antibody suspension for 2 h at room temperature with shaking. After incubation, centrifuge at 450g for 10 minutes, and take the supernatant. Prepare 1 μg/ml anti human Fc AlexaFlour 647 solution. Place the sample in the corresponding position in the tube rack. Signal values were detected with a KinExA 3200 instrument and affinity data were obtained. Results: The affinity of Ab10 (13.3 pM) was more than 10 times higher than that of the Ref antibody (144 pM).

Taking Ab10 as an example, its PK in mice was evaluated, and it was found that the T1/2 of Ab10 in mice was 170 hours. Under the same conditions, Ref T1/2 is 139 hours.

The efficacy evaluation of mouse tumor model showed that in the hCLDN18.2+ cell line Xenograft model, the tumor inhibition rate of Ab10 and Ab6 was over 90%. In the gastric cancer cell line NUGC4 cell Xenograft model, on the 21st day of administration (antibody), Ab10 and Ab6 could see a 20% tumor inhibition rate. Under the same conditions, Ref did not see the tumor suppressing effect.

Example 4 CAR molecular design for CLDN18.2

The present invention refers to the previously published patent CN106755107A for the design of a new CAR molecule for CLDN18.2.

Specifically, the general formula of the nucleic acid construct of the CAR molecule designed in the present invention is CAR-[(IRES)-f] _m . In this general formula, CAR represents a chimeric antigen receptor, including, scFv-H-TM-S-CD3ζ, where scFv (single chain Fv) is a single-chain variable fragment that specifically targets the CLDN18.2 antigen, or called Single-chain antibodies, single-chain variable regions. Its sequence is composed of the variable region sequence of the anti-CLDN18.2 antibody found in the present invention (see the preceding examples). Its structure is VL-Linker-VH or VH-Linker-VL, the Linker is preferably (G ₄ S) _n , and n is 0, 1, 2, 3, 4; preferably n=3 or n=4. H is the hinge domain, TM is the transmembrane domain, and S is the costimulatory signaling region. The co-stimulatory signaling region includes co-stimulatory molecules derived from CD28, and/or co-stimulatory molecules derived from 4-1BB. In the general formula, CD3ζ is a cytoplasmic signaling sequence (intracellular domain) derived from CD3ζ.

IRES represents the internal ribosome entry site sequence (Internal ribosome entry site, IRES); f represents the coding of functional protein F, and m is a natural number of 0 or non-0. The functional protein includes cytokines IL10, IL15 or active fragments thereof, and/or receptors of the cytokines such as IL15 receptors or active fragments thereof, and/or cytokines such as IL10, IL15 or active fragments thereof and IL15 Fusion fragment of acceptor sushi+ fragment (SEQ ID NO: 25).

If there is no f part in the designed CAR molecule structure, the CAR molecule also has no IRES sequence. In addition, IRES and (IRES) represent the same meaning, and when IRES contains "()", it means that when a nucleotide containing an IRES sequence is used to encode a protein, the IRES sequence does not encode the corresponding protein.

In the above CAR or CAR-(IRES)-f molecule, the sequence of the scFv designed according to the variable region of the anti-CLDN18.2 antibody sequence is the novel antibody sequence of the present invention, see the above example. Fab or single domain antibody (sdFv) structures can also be present at the scFV. Other sequences (sequences other than scFv) can be searched from the US National Library of Medicine website http://www.pubmed.com, GenBank database, including human CD8α signal peptide (SEQ ID NO: 4), human CD8α hinge region ( SEQ ID NO:27), CD8α transmembrane region (SEQ ID NO:18), human CD28 intracellular region (SEQ ID NO:42), human 4-1BB intracellular region (SEQ ID NO:38), human CD3ζ cell Inner region (SEQ ID NO:40), internal ribosomal entry elements (IRESelements, SEQ ID NO:49), human IL15 (SEQ ID NO:20), human IL15 receptor alpha (IL15Rα, SEQ ID NO:23) wild type and mutation/sushi portion (US2014/01314; WO2007/046006, SEQ ID NO: 25), human IL10 (SEQ ID NO: 3) protein sequence, etc. All base sequences constructed into clones are codon-optimized according to the protein sequence to ensure that the encoded amino acid sequence is more suitable for human cell expression.

Among them, the nucleotide sequence of IRES is:

GCCCCTCTCCCTCCCCCCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTACACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATGATAATATGGCCACA(SEQ ID NO:49)

The nucleotide sequence of IL10, eg, the sequence shown in GenBank Accession No. NM_000572, can encode the amino acid sequence shown in SEQ ID NO:3.

Specifically, the construction of a representative CAR molecule of the present invention refers to the method described in the patent application with publication number CN106755107A. In Example 3 of the patent, the c-Met antibody scFv encoding the c-Met antibody in the JX005 plasmid (derived from pBABEpuro) was replaced with the Ab10scFv of the present invention, and the plasmid JX1a of the new CAR molecule CAR1a of the present invention was obtained. The amino acid sequence of CAR1a encoded by plasmid JX1a is:

DIVMTQSPDSLAVSLGERATISCKSSQSLLNSGNQKNYLTWYQQKPGQPPKLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQNDYFYPFTFGQGTKLEIKGGGGSGGGGSGGGGSQVQLVQSGAEVKKPGSSVKVSCKASGYAFSNYLIEWVKQAPGQGLEWIGLINPGSGGTNYNEKFKGKATITADKSTSTAYMELSSLRSEDTAVYYCARVYYGNSFAYWGQGTLVTVSS TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIY IWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYKQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR(SEQ ID NO:44)

Among them, the 1-246th position is the scFv coding sequence that binds Ab10; the 247-293rd position is the human CD8α hinge region coding sequence (underlined part); the 294th-315th position is the human CD8α transmembrane region coding sequence; The 316-357th position is 4-1BB intracellular region coding sequence; positions 358-469 are CD3zeta (ζ) intracellular signal region coding sequence.

The nucleotide sequence encoding CAR1a is as follows:

atggccctgcccgtgaccgccctgctgctgcccctggccctgctgctgcacgccgccagaccc (SEQ ID NO: 45),

Among them, the 1st to 63rd nucleotides (underlined part) are the signal peptide coding region. Positions 64-801 are the scFv coding sequences of the Ab10 antibody binding to CLDN18.2; positions 802-942 are the coding sequences of the hinge region of human CD8α; positions 943-1008 are the coding sequences of the transmembrane region of human CD8α; positions 1009-1134 are the coding sequences of the human CD8α transmembrane region 4-1BB intracellular region coding sequence; 1135-1470th is the CD3zeta (ζ) intracellular signal region coding sequence.

Replace the scFv encoding c-Met antibody scFv in the JX007 plasmid constructed in Example 5 of the patent application with publication number CN106755107A with Ab10scFv of the present invention (the method is the same as the construction method of JX1a of the present invention), that is, the plasmid used for the new CAR molecule CAR3ab of the present invention is obtained JX3ab. The amino acid sequence encoded by plasmid JX3ab is the same as that of JX1a above, and in addition, it also encodes the active fragment of cytokine IL15 (wild type) (SEQ ID NO. 22). The nucleotide sequence of the IL15 active fragment can be any sequence used in the prior art to encode SEQ ID NO. 22, eg, nucleotide sequence 699-1040 of SEQ ID NO: 31 of CN106755107A.

The amino acid sequence of the above-mentioned IL15 active fragment (wild type) is as follows:

NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO. 22)

The sequence encoding IL15 in the plasmid JX3ab constructed above (for example, the nucleotide sequence at positions 699-1040 of SEQ ID NO: 31 of CN106755107A) was replaced with the sequence encoding IL10 (for example, the sequence shown in GenBank accession number NM_000572), The plasmid JX3ab10 of the new CAR molecule CAR3ab10 of the present invention is obtained. The nucleotide sequence encoding IL10 is as follows (the underlined part is the signal peptide coding region):

ATGCACAGCAGCGCCCTGCTGTGCTGCCTGGTGCTGCTGACCGGCGTGAGAGCC AGCCCCGGCCAGGGCACCCAGAGCGAGAACAGCTGCACCCACTTCCCCGGCAACCTGCCCAACATGCTGAGAGACCTGAGAGACGCCTTCAGCAGAGTGAAGACCTTCTTCCAGATGAAGGACCAGCTGGACAACCTGCTGCTGAAGGAGAGCCTGCTGGAGGACTTCAAGGGCTACCTGGGCTGCCAGGCCCTGAGCGAGATGATCCAGTTCTACCTGGAGGAGGTGATGCCCCAGGCCGAGAACCAGGACCCCGACATCAAGGCCCACGTGAACAGCCTGGGCGAGAACCTGAAGACCCTGAGACTGAGACTGAGAAGATGCCACAGATTCCTGCCCTGCGAGAACAAGAGCAAGGCCGTGGAGCAGGTGAAGAACGCCTTCAACAAGCTGCAGGAGAAGGGCATCTACAAGGCCATGAGCGAGTTCGACATCTTCATCAACTACATCGAGGCCTACATGACCATGAAGATCAGAAACTGA(SEQ ID NO:19)

The above-mentioned plasmid JX3ab10 encodes the following IL10 protein sequence:

SPGQGTQSENSCTHFPGNLPNMLRDLRDAFSRVKTFFQMKDQLDNLLLKESLLEDFKGYLGCQALSEMIQFYLEEVMPQAENQDPDIKAHVNSLGENLKTLRLRLRRCHRFLPCENKSKAVEQVKNAFNKLQEKGIYKAMSEFDIFINYIEAYMTMKIRN (SEQ ID NO: 3)

The c-Met antibody scFv encoded in the JX008 plasmid constructed in Example 6 of the patent CN106755107A was replaced with the Ab10scFv of the present invention (the method is the same as the construction method of JX1a of the present invention), and the plasmid JX4a used for the new CAR molecule CAR4a of the present invention was obtained. In addition to the coding sequence of CAR1a, the amino acid sequence encoded by JX4a also encodes the cytokine IL15 active fragment (mutant type) (SEQ ID NO: 22) and IL15Rα (sushi+) (SEQ ID NO: 25; sushi+ means that in addition to the sushi fragment In addition, the fusion protein of other polypeptide fragments) is also included, and the amino acid sequence of the fusion protein is as follows:

ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSGGSGGGGSGGGSGGGGSLQNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANDSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS(SEQ ID NO:46)

Using the same method as above, the scFv of the Ab6 antibody sequence was replaced by the scFv of the Ab10 antibody to construct the new CAR molecules CAR1a.2, CAR3ab.2, CAR3ab10.2 and CAR4a.2 corresponding plasmids JX1a.2, JX3ab.2, JX3ab10. 2 and JX4a.2.

Example 5 Identification of CAR molecules designed against CLDN18.2

Virus packaging, preparation and concentration: The virus preparation method refers to the method used in the patent CN106755107A, using the three-plasmid virus packaging system pGag-Pol, pVSVG and each new CAR molecule expression plasmid pBABEpuro of the present invention (all purchased from Youbao Bio), such as JX1a, JX3ab , JX3ab10 or JX4a were co-transfected into 293 cells to obtain virus supernatant, and concentrated by ultracentrifugation to obtain concentrated virus.

Specifically, take 6 μg of each of the packaging plasmids pGag-Pol, pVSVG and expression plasmids JX1a, JX3ab, JX3ab10 or JX4a, 36 μg of PEI (Polysciences, Inc, Cat#: 23966-2), mix well, stand at room temperature for 5 min, add 293 ( Chinese Academy of Sciences Type Culture Collection Cell Bank). After culturing for 48h, the first supernatant was collected; the next day, culturing for 72h, the second supernatant was collected. Combine the two supernatants, add 3 ml of 20% sucrose solution, carefully spread the virus supernatant on top of the sucrose solution, centrifuge at 125,000g for 1.5 hours, resuspend the pellet in PBS at low temperature, aliquot, and freeze at -80°C. The viruses were denoted as 1a, 3ab, 3ab10, and 4a, respectively. Ultracentrifuge model Beckman Coulter Optima XPN-100; rotor model SW32i, ultracentrifuge tube Beckman 344058.

Virus titer detection: 293 cells were infected with serially diluted virus, and the virus titer was determined by the method of protein L staining to determine the positive rate of cells expressing scFv after 48 hours. Specifically, 20 μl of viruses (1a, 3ab, 3ab10, 4a) were taken respectively, and RPMI 1640 medium (Peiyuan Bio, Cat#L210KJ) containing 10% FBS (Gibco, Cat#: 10099141), 0.8 μg polybrene (Shanghai Yi) was used. Sheng Biotechnology Co., Ltd. Cat#: 40804ES76) 5-fold gradient dilution, the final system is 250 μl. Added to pre-plated 293, the total culture system was 500 μl/well, 293 5×10 ⁴ cells/well. After 48 hours, the cells were collected and labeled with biotin-protein L (GenScript, Cat. No. M00097) 293, 1 μl/sample. After 20 min incubation at room temperature, 1 ml of FACS buffer was added, and the cells were washed by centrifugation. Resuspend in 100 μl of FACS buffer, add 0.4 μl/sample of PE-labeled Strepavidin (eBioscience, Cat. No. 12-4317-87), incubate at room temperature for 20 min, add 1 ml of FACS buffer, and wash the cells by centrifugation. The proportion of positive cells was detected by FACS, and the samples with a positive proportion of 10% were selected to calculate the virus titer. The results showed that the titers of 1a, 3ab, 3ab10 and 4a viruses obtained this time were 9.2, 1.3, 3.2 and 1.5×10 ⁶ IU/ml, respectively.

Preparation of CART cells: Fresh peripheral blood of healthy volunteers was taken, and peripheral blood mononuclear cells (PBMC) were isolated by the same method as in patent CN106755107A. PBMCs were activated with magnetic beads (Gibco, Cat. No. 11131D) coupled with anti-human CD3 and CD28 antibodies for 40-48 h and then added viruses 1a, 3a, 3ab10, 4a (MOI in the range of 0.05-5), polybrene (final concentration 8μg/ml) ). After infection for 3 hours, the medium was replenished to 1 ml, and the medium was changed overnight. The medium was RPMI1640 containing 10% FBS and 500 IU/mL IL2 (Beijing Sihuan Bio-Pharmaceutical Co., Ltd., Cat#: S20040007). Change the medium every other day and expand the culture volume 1:2 until enough cells are obtained for in vitro and in vivo experiments. The T cells (CART cells) of each CAR molecule after infection were denoted as CART1a, CART3ab, CART3ab10 and CART4a cells, respectively; uninfected T cells (empty vector) were used as negative cells (control).

Identification of the scFv expression of the CART molecule Ab10 antibody of the present invention: each of the above CART cells was stained with Protein L to determine the expression of the CART molecule scFv and the positive rate of infected cells. Specifically, 2×10 ⁵ infected T cells (CART cells) were collected, and the cells were labeled with biotin-protein L (Nanjing GenScript Biotechnology Co., Ltd., product number M00097) (the labeling method was the same as the aforementioned virus titer detection). The proportion of CART positive cells was detected by FACS, and negative cells were used as control. Positive rate = FACS (%) of CART cells - FACS (%) of negative cells (control), the results are shown in Table 3a and FIG. 1 below.

CART1a.2, CART3ab.2, CART3ab10.2 and CART4a.2 were prepared in the same manner as described above. The positive rate of detecting its Ab6 antibody scFv was close to that of CART1a, CART3ab, CART3ab10 and CART4a, as shown in Table 3b.

Table 3a Positive rate of Ab10 antibody scFv expressed by CART cells of the present invention

CART cells Positive rate (%) CART1a 47.1 CART3ab 24.1 CART3ab10 28.6 CART4a 25.7

Table 3b Positive rate of Ab6 antibody scFv expressed by CART cells of the present invention

CART cells Positive rate (%) CART1a.2 34.2 CART3ab.2 23.3 CART3ab10.2 27.5 CART4a.2 20.9

The above results show that the positive rates of the four CART cells (including Ab10 and Ab6 scFv) in this experiment are 20%-47%, confirming that the obtained CART cells all express the scFv of Ab10 or Ab6 antibody normally. The following uses CART1a, CART3ab, CART3ab10 and CART4a as examples to evaluate the activity and function of the CAR cells of the present invention.

CART cells designed by the present invention express and secrete cytokine identification: the new CART cells designed by the present invention CART3ab and CART4a express and secrete cytokines IL15 and IL15/IL15R; CART3ab10 expresses and secretes cytokine IL10. In order to identify the expression and secretion of the factors by the CART cells, the culture supernatant of the CART cells was taken, and ELISA kits (Beijing Yiqiao Shenzhou Biotechnology Co., Ltd., product numbers SEKA10947, SEK10360) were used to detect the expressions of IL10 and IL15, respectively. The results showed that only 2256pg/ml and 844pg/ml could be detected in CART3ab10 supernatant (repeated preparation of CART cells infected with PBMC-derived T cells from different donors), and the background value of CART1a supernatant prepared at the same time was only 13.7pg/ml. and 5.3 pg/ml (repeated preparation of CART cells obtained by infecting T cells derived from PBMCs from different donors). This data indicates that CART3ab10 designed by the present invention specifically expresses and secretes the cytokine IL10. ELISA method could not detect the expression and secretion of IL15 and IL15/IL15Rα in the supernatant of CART3ab and CART4a, indicating that the amount of cytokines IL15 and IL15/IL15Rα secreted by cultured CART3ab and CART4a was low.

Since the expression of IL15 and IL15/IL15Rα could not be detected in the cell culture supernatant, the produced 4 CART cells were injected into mice respectively. Balb/c nude mice were taken, and 1×10 ⁶ CART cells were administered intravenously. Serum was collected on the 7th and 14th days after injection, and the levels of IL10 and IL15 in the serum were detected by ELISA. The results are shown in the table below.

Table 4 Evaluation of cytokine expression in CART cell mouse serum of the present invention

*: Not suitable

The above results showed that the IL10 expression level of CART3ab10 reached 5154.7 pg/ml on the 7th day in mice. On the 14th day, IL10 expression decreased to 350.7 pg/ml. CART1a, CART (empty) as control samples did not detect the expression of IL10, and the detection values of 14, 152.7 and 128.7 pg/ml were all background values. CART3ab detected the expression of IL15 active fragment on the 14th day, and the expression amount was 118.8pg/ml. CART4a detected the expression of IL15/IL15Rα on the 7th day, and the expression level was 206.4pg/ml. Neither CART1a nor CART (null) as control samples detected IL15 expression with a read value of 0 (background).

The above results show that the designed CAR-transfected T cells CART3ab and CART4a cells express and secrete the IL15 active fragment. CART3ab10 cells express and secrete IL10.

Example 6 CAR cell activity designed against CLDN18.2

In order to evaluate the in vitro activity of the CART cells of the present invention, human CLDN18.2 high-expressing cells (hCLDN18.2+ cells) were used as target cells, and human CLDN18.1 high-expressing cells (hCLDN18.1+ cells) were used as control negative target cells to compare the CART The specific in vitro killing activity of CART cells was evaluated by the survival ratio of target cells after killing two kinds of cells. Specifically, hCLDN18.2+ cells were labeled with CFSE (Biolegend, Cat. No. 423801). To prepare a target cell suspension, hCLDN18.1+ cells and CFSE-labeled hCLDN18.2+ cells were mixed in equal proportions, each at 1.5×10 ⁵ cells/ml. Target cell killing experiments were carried out in a 24-well plate, and the target cell suspension was 100 μl per well. CART1a, CART3ab, CART3ab10, CART4a cells and negative cells (empty vector) were diluted with the same medium to form different ratios of CART cells and target cells, 20:1, 10:1, 3:1 and 1:1, respectively . An unkilled group was also set up, that is, no CART cells, and only the above-mentioned target cell group. After the CART cells were co-cultured with the target cells for 16 hours, the supernatant was discarded, the residual CART cells and the killed target cells were gently washed with PBS, and the adherent target cells were digested with trypsin and collected with 7AAD (Biolegend: 420404). ), the ratio of 7AAD-negative CFSE-labeled hCLDN18.2+/hCLDN18.1+ cells was detected by FACS.

Target cell specific lysis (killing) rate=1-[CART cells 7AAD negative hCLDN18.2+/hCLN18.1+]/[negative control (empty vector) cells 7AAD negative hCLDN18.2+/hCLN18.1+]. The specific lysis (killing) rate of target cells is high, that is, the specific killing effect of CART cells is strong. Figure 2 shows the calculation results of the killing rate of CART1a when the ratio of CART cells and target cells is 10:1. The picture on the left is the result of the negative control CART cells. The data shows that the CLDN18.1:CLDN18.2 ratio of viable cells is 50.5% vs 48.6%, which is close to 1:1, indicating that the negative control CART cells have no killing effect on both non-target cells and target cells. The right picture shows the results of CAR1a cells, the data shows that the viable cells CLDN18.1:CLDN18.2 is 57.5% vs 37.5%, indicating that CART1a cells have no killing effect on non-target cells (close to 50%), but on target cells (CLDN18.2) Has a killing effect (reduced from 50% to 37.5%). The calculation method of killing rate (%) is: 1-[(37.5%/57.5%)/(48.6%/50.5%)]=32.2%. The results are shown in the table below.

Table 5 In vitro cell activity of CART cells of the present invention (target cell specific killing rate, %)

The above results (the experiments were repeated more than three times, and the negative control CART cells in each experiment had no killing effect on non-target cells and target cells) showed that the four CART cells of the present invention all showed killing effects on target cells at a ratio of 20:1. , the kill rate is 19.8%-34.8%. As the ratio of CART cells:target cells decreased, the killing effect on target cells was weakened, and the fastest weakening was CART4a, CART3ab10 and CART3a. When these three CART cells are in the ratio of 3:1 and 1:1, the killing effect on the target cells can hardly be seen. This shows that the CART design of the present invention is related to cell function, and the resulting effects show unexpected effects with different designs.

Example 7 In vivo efficacy of CART cells designed for CLDN18.2 in animals

Balb/c nude mice were taken and subcutaneously inoculated with hCLDN18.2+ cells to establish a tumor model. CART cells were injected intravenously, and tumor volume (TV) and body weight (BW) were measured to evaluate the anti-tumor effect and safety of CART cells. Specifically, hCLDN18.2+ cells were cultured in DMEM/F12 medium containing 10% fetal bovine serum, and were continuously cultured in a cell incubator at 37°C containing 5% CO2 to logarithmic growth phase (confluence at 80% -90%), trypsinize, collect cells, wash cells twice with serum-free DMEM/F12, resuspend in PBS, count, and adjust the cell concentration to 1×10 ⁸ /ml. Balb/c nude mice, each inoculated with 100ul of hCLDN18.2+ cell suspension, subcutaneously in the right flank. Three weeks later, mice with a tumor volume of 80-130 mm ³ were selected and grouped (2 mice/group) and injected intravenously with 2×10 ⁶ / mice of CART cells. The day of dosing was Day 0. After that, the tumor volume was measured twice a week, the body weight was weighed, and the data were recorded.

Tumor size calculation formula: tumor volume TV (mm ³ )=0.5×(tumor long diameter×tumor short diameter ² ); relative tumor volume (RTV)=TV/TV0, where TV0 is the tumor volume at the beginning (Day 0) , set to 1. TV is the tumor volume at the time of detection. Relative tumor volume (RTV) was the fold increase of tumor volume at each detection time point. Relative tumor growth rate (T/C%)=100%*(T-T0)/(C-C0); tumor inhibition rate (TGI)=(1-T/C)*100%. Among them, T0 and T are the tumor volumes of the sample group at the beginning and the end of the experiment, respectively; C0 and C are the tumor volumes at the beginning and the end of the control group, respectively.

The results showed that after two weeks of CART injection, there was no significant change in the body weight of each mouse, indicating that there is no significant safety issue with CART cells. In terms of efficacy, from day 13 to day 19 of CART (empty vector) injected mice, the tumor volume continued to increase, and the increase fold (relative tumor volume) continued from 14 to 39 times (Figure 3). The tumor volume of mice injected with CART1a, CART3ab, CART3ab10 and CART4a cells increased by less than 10 times (tumor inhibition rate was 40%-90%). In particular, mice injected with CART3ab10 and CART4a cells maintained almost no growth in tumor volume from day 13 to day 17, achieving sustained suppression (near 100% suppression). This result shows that CART cells produced by different CAR molecules designed with the anti-CLND18.2 antibody of the present invention show unexpected pharmacodynamic effects in animals.

Example 8 CARNK cell activity obtained by transfecting NK cells with CAR designed for CLDN18.2

Virus 1a, 3a, 3ab10, 4a (MOI in the range of 0.5-5), polybrene (final concentration 8μg/ mL). After infection for 3 hours, the liquid was replenished to 1 mL, and the medium was changed overnight. Change the medium every other day and expand the culture volume 1:2 until enough cells are obtained for in vitro and in vivo experiments. Infected NK92 cells were designated as CARNK1a, CARNK3a, CARNK3ab10, and CARNK4a cells, respectively; uninfected NK92 cells were designated as CARNK (empty vector) as a negative control. Seven days after infection, the expression on the surface of CARNK cells was detected by the same method as in Example 5 above, and the results are shown in the following table.

Table 6 The positive rate of the anti-CLDN18.2 antibody of the present invention to construct CARNK cells expressing antibody scFv

CARNK cells Positive rate (%) CARNK1a twenty one CARNK3a twenty two CARNK3ab10 30 CARNK4a 41

The above results show that the newly designed CAR of the present invention can also well express the scFv of antibody Ab10 in NK cells and recognize human hCLDN18.2.

Example 9 In vivo efficacy of CARNK cells designed against CLDN18.2 in animals

The same animal model as in Example 7 above was used to evaluate the animal efficacy of the CARNK cells of the present invention. Take the prepared CARNK (empty vector) control, CARNK1a, CARNK3ab, CARNK3ab10, CARNK4a cells 2×10 ⁵ /cell, 2 cells in each group, and inject once on Day 0 and Day 3 each. The tumor size was measured twice a week after that, and the results are shown in Table 7 below.

Table 7 In vivo efficacy of the anti-CLDN18.2 antibody of the present invention to construct CARNK cells

The results in Table 7 show that the CARNK cells constructed with the anti-CLDN18.2 antibody of the present invention continued to exhibit tumor-inhibiting effects on the 11th and 13th days in vivo. The tumor inhibition rate was in the range of 16.5%-60.7%. Among them, CARNK3ab10 showed the best efficacy in animals.

All documents mentioned herein are incorporated by reference in this application as if each document were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

SEQUENCE LISTING

<110> Shanghai Jianxin Biomedical Technology Co., Ltd.

<120> CAR molecule targeting Claudin18.2, its modified immune cells and use

<130> P19010199C

<160> 49

<170> PatentIn version 3.5

<210> 1

<211> 261

<212> PRT

<213> Artificial Sequence

<220>

<223> Human CLDN18.1 Sequence

<400> 1

Met Ser Thr Thr Thr Cys Gln Val Val Ala Phe Leu Leu Ser Ile Leu

1 5 10 15

Gly Leu Ala Gly Cys Ile Ala Ala Thr Gly Met Asp Met Trp Ser Thr

20 25 30

Gln Asp Leu Tyr Asp Asn Pro Val Thr Ser Val Phe Gln Tyr Glu Gly

35 40 45

Leu Trp Arg Ser Cys Val Arg Gln Ser Ser Gly Phe Thr Glu Cys Arg

50 55 60

Pro Tyr Phe Thr Ile Leu Gly Leu Pro Ala Met Leu Gln Ala Val Arg

65 70 75 80

Ala Leu Met Ile Val Gly Ile Val Leu Gly Ala Ile Gly Leu Leu Val

85 90 95

Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Glu Asp Ser

100 105 110

Ala Lys Ala Asn Met Thr Leu Thr Ser Gly Ile Met Phe Ile Val Ser

115 120 125

Gly Leu Cys Ala Ile Ala Gly Val Ser Val Phe Ala Asn Met Leu Val

130 135 140

Thr Asn Phe Trp Met Ser Thr Ala Asn Met Tyr Thr Gly Met Gly Gly

145 150 155 160

Met Val Gln Thr Val Gln Thr Arg Tyr Thr Phe Gly Ala Ala Leu Phe

165 170 175

Val Gly Trp Val Ala Gly Gly Leu Thr Leu Ile Gly Gly Val Met Met

180 185 190

Cys Ile Ala Cys Arg Gly Leu Ala Pro Glu Glu Thr Asn Tyr Lys Ala

195 200 205

Val Ser Tyr His Ala Ser Gly His Ser Val Ala Tyr Lys Pro Gly Gly

210 215 220

Phe Lys Ala Ser Thr Gly Phe Gly Ser Asn Thr Lys Asn Lys Lys Ile

225 230 235 240

Tyr Asp Gly Gly Ala Arg Thr Glu Asp Glu Val Gln Ser Tyr Pro Ser

245 250 255

Lys His Asp Tyr Val

260

<210> 2

<211> 261

<212> PRT

<213> Artificial Sequence

<220>

<223> Human CLDN18.2 sequence

<400> 2

Met Ala Val Thr Ala Cys Gln Gly Leu Gly Phe Val Val Ser Leu Ile

1 5 10 15

Gly Ile Ala Gly Ile Ile Ala Ala Thr Cys Met Asp Gln Trp Ser Thr

20 25 30

Gln Asp Leu Tyr Asn Asn Pro Val Thr Ala Val Phe Asn Tyr Gln Gly

35 40 45

Leu Trp Arg Ser Cys Val Arg Glu Ser Ser Gly Phe Thr Glu Cys Arg

50 55 60

Gly Tyr Phe Thr Leu Leu Gly Leu Pro Ala Met Leu Gln Ala Val Arg

65 70 75 80

Ala Leu Met Ile Val Gly Ile Val Leu Gly Ala Ile Gly Leu Leu Val

85 90 95

Ser Ile Phe Ala Leu Lys Cys Ile Arg Ile Gly Ser Met Glu Asp Ser

100 105 110

Ala Lys Ala Asn Met Thr Leu Thr Ser Gly Ile Met Phe Ile Val Ser

115 120 125

Gly Leu Cys Ala Ile Ala Gly Val Ser Val Phe Ala Asn Met Leu Val

130 135 140

Thr Asn Phe Trp Met Ser Thr Ala Asn Met Tyr Thr Gly Met Gly Gly

145 150 155 160

Met Val Gln Thr Val Gln Thr Arg Tyr Thr Phe Gly Ala Ala Leu Phe

165 170 175

Val Gly Trp Val Ala Gly Gly Leu Thr Leu Ile Gly Gly Val Met Met

180 185 190

Cys Ile Ala Cys Arg Gly Leu Ala Pro Glu Glu Thr Asn Tyr Lys Ala

195 200 205

Val Ser Tyr His Ala Ser Gly His Ser Val Ala Tyr Lys Pro Gly Gly

210 215 220

Phe Lys Ala Ser Thr Gly Phe Gly Ser Asn Thr Lys Asn Lys Lys Ile

225 230 235 240

Tyr Asp Gly Gly Ala Arg Thr Glu Asp Glu Val Gln Ser Tyr Pro Ser

245 250 255

Lys His Asp Tyr Val

260

<210> 3

<211> 160

<212> PRT

<213> Artificial Sequence

<220>

<223> IL10

<400> 3

Ser Pro Gly Gln Gly Thr Gln Ser Glu Asn Ser Cys Thr His Phe Pro

1 5 10 15

Gly Asn Leu Pro Asn Met Leu Arg Asp Leu Arg Asp Ala Phe Ser Arg

20 25 30

Val Lys Thr Phe Phe Gln Met Lys Asp Gln Leu Asp Asn Leu Leu Leu

35 40 45

Lys Glu Ser Leu Leu Glu Asp Phe Lys Gly Tyr Leu Gly Cys Gln Ala

50 55 60

Leu Ser Glu Met Ile Gln Phe Tyr Leu Glu Glu Val Met Pro Gln Ala

65 70 75 80

Glu Asn Gln Asp Pro Asp Ile Lys Ala His Val Asn Ser Leu Gly Glu

85 90 95

Asn Leu Lys Thr Leu Arg Leu Arg Leu Arg Arg Cys His Arg Phe Leu

100 105 110

Pro Cys Glu Asn Lys Ser Lys Ala Val Glu Gln Val Lys Asn Ala Phe

115 120 125

Asn Lys Leu Gln Glu Lys Gly Ile Tyr Lys Ala Met Ser Glu Phe Asp

130 135 140

Ile Phe Ile Asn Tyr Ile Glu Ala Tyr Met Thr Met Lys Ile Arg Asn

145 150 155 160

<210> 4

<211> 21

<212> PRT

<213> Artificial Sequence

<220>

<223> CD8α signal peptide

<400> 4

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> 5

<211> 530

<212> DNA

<213> Artificial Sequence

<220>

<223> Anti-hCLDN18.2 monoclonal antibody light chain nucleotide sequence

<400> 5

taatgggctt caagatgaag tcacagtttc tggtcctcat gtccctgctg ttctgggtat 60

ctggtacctg tggggacatt gtgatgacac agtctccatc ctccctgact gtgacagcag 120

gagagaaggt cactatgagt tgcaagtcca gtcagagtct gttaaacagt ggaaatcaaa 180

agaactactt gacctggtac cagcagaaac cagggcagcc tcctaaactg ttgatctact 240

gggcatccac tagggaatct ggggtccctg atcgcttcac aggcagtgga tctggaacac 300

atttcactct caccatcagc agtgtgcagg ctgaagacct ggcagtttat tactgtcaga 360

atgattattt ttatccattc acgttcggct cggggacaaa gttggaaaaa aaacgggctg 420

atgctgcacc aactgtatcc atcttcccac catccagtga gcagttaaca tctggaggtg 480

cctcagtcgt gtgcttctga acaactctac cccaaagacc atccatgccc 530

<210> 6

<211> 528

<212> DNA

<213> Artificial Sequence

<220>

<223> Anti-hCLDN18.2 monoclonal antibody heavy chain nucleotide sequence

<400> 6

taatgggatg gaccgggatc tttatctttc tcctgtcagt aactgcaggt gttcactccc 60

aggtccagct gcagcagtct ggagctgagc tgataggacc tgggacttca gtgaaggtgt 120

cctgcaaggc ctctggatac gccttcagta attacttgat agaatgggta aaacagaggc 180

ctgaacaggg ccttgagtgg attggtttga ttaatcctgg aagtggtggc actaactaca 240

atgagaagtt caagggcaag gcaacactga ctgcagacaa atcctccagc actgcctaca 300

tgcaactcag cagcctgaca tctgatgact ctgcggtcta cttctgtgca agggtctact 360

atggtaactc ctttgcttac tggggccaag ggactctggt cactgtctct gcagccaaaa 420

cgacaccccc atctgtctat ccactggccc ctggatctgc tgcccaaact aactccatgg 480

tgaccctggg atgcctggtc aagggctatt accgagcaag aaatgtcg 528

<210> 7

<211> 113

<212> PRT

<213> Artificial Sequence

<220>

<223> anti-hCLDN18.2 antibody mab5b light chain variable region

<400> 7

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr His Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn

85 90 95

Asp Tyr Phe Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Lys

100 105 110

Lys

<210> 8

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> anti-hCLDN18.2 antibody mab5b heavy chain variable region

<400> 8

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ile Gly Pro Gly Thr

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Asn Tyr

20 25 30

Leu Ile Glu Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile

35 40 45

Gly Leu Ile Asn Pro Gly Ser Gly Gly Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Val Tyr Tyr Gly Asn Ser Phe Ala Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ala

115

<210> 9

<211> 220

<212> PRT

<213> Artificial Sequence

<220>

<223> anti-hCLDN18.2 antibody mab5b light chain

<400> 9

Asp Ile Val Met Thr Gln Ser Pro Ser Ser Leu Thr Val Thr Ala Gly

1 5 10 15

Glu Lys Val Thr Met Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Thr Gly Ser Gly Ser Gly Thr His Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Val Gln Ala Glu Asp Leu Ala Val Tyr Tyr Cys Gln Asn

85 90 95

Asp Tyr Phe Tyr Pro Phe Thr Phe Gly Ser Gly Thr Lys Leu Glu Lys

100 105 110

Lys Arg Thr Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp

115 120 125

Glu Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn

130 135 140

Phe Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu

145 150 155 160

Gln Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser Lys Asp

165 170 175

Ser Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr

180 185 190

Glu Lys His Lys Val Tyr Ala Cys Glu Val Thr His Gln Gly Leu Ser

195 200 205

Ser Pro Val Thr Lys Ser Phe Asn Arg Gly Glu Cys

210 215 220

<210> 10

<211> 448

<212> PRT

<213> Artificial Sequence

<220>

<223> anti-hCLDN18.2 antibody mab5b heavy chain

<400> 10

Gln Val Gln Leu Gln Gln Ser Gly Ala Glu Leu Ile Gly Pro Gly Thr

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Asn Tyr

20 25 30

Leu Ile Glu Trp Val Lys Gln Arg Pro Glu Gln Gly Leu Glu Trp Ile

35 40 45

Gly Leu Ile Asn Pro Gly Ser Gly Gly Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr

65 70 75 80

Met Gln Leu Ser Ser Leu Thr Ser Asp Asp Ser Ala Val Tyr Phe Cys

85 90 95

Ala Arg Val Tyr Tyr Gly Asn Ser Phe Ala Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ala Ala Ser Thr Lys Gly Pro Ser Val Phe Pro

115 120 125

Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu Gly

130 135 140

Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp Asn

145 150 155 160

Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu Gln

165 170 175

Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser Ser

180 185 190

Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro Ser

195 200 205

Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys Thr

210 215 220

His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser

225 230 235 240

Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg

245 250 255

Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro

260 265 270

Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala

275 280 285

Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val

290 295 300

Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr

305 310 315 320

Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr

325 330 335

Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu

340 345 350

Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys

355 360 365

Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser

370 375 380

Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp

385 390 395 400

Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser

405 410 415

Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala

420 425 430

Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

435 440 445

<210> 11

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Light chain CDR1

<400> 11

Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu

1 5 10 15

Thr

<210> 12

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Light chain CDR1 humanized sequence

<400> 12

Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Asn Lys Asn Tyr Leu

1 5 10 15

Ala

<210> 13

<211> 7

<212> PRT

<213> Artificial Sequence

<220>

<223> Light chain CDR2

<400> 13

Trp Ala Ser Thr Arg Glu Ser

1 5

<210> 14

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Light chain CDR3

<400> 14

Gln Asn Asp Tyr Phe Tyr Pro Phe Thr

1 5

<210> 15

<211> 10

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy chain CDR1

<400> 15

Gly Tyr Ala Phe Ser Asn Tyr Leu Ile Glu

1 5 10

<210> 16

<211> 17

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy chain CDR2

<400> 16

Leu Ile Asn Pro Gly Ser Gly Gly Thr Asn Tyr Asn Glu Lys Phe Lys

1 5 10 15

Gly

<210> 17

<211> 9

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy chain CDR3

<400> 17

Val Tyr Tyr Gly Asn Ser Phe Ala Tyr

1 5

<210> 18

<211> 22

<212> PRT

<213> Artificial Sequence

<220>

<223> CD8a transmembrane domain

<400> 18

Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu

1 5 10 15

Val Ile Thr Leu Tyr Cys

20

<210> 19

<211> 537

<212> DNA

<213> Artificial Sequence

<220>

<223> Nucleotide sequence encoding IL10

<400> 19

atgcacagca gcgccctgct gtgctgcctg gtgctgctga ccggcgtgag agccagcccc 60

ggccagggca cccagagcga gaacagctgc acccacttcc ccggcaacct gcccaacatg 120

ctgagagacc tgagagacgc cttcagcaga gtgaagacct tcttccagat gaaggaccag 180

ctggacaacc tgctgctgaa ggagagcctg ctggaggact tcaagggcta cctgggctgc 240

caggccctga gcgagatgat ccagttctac ctggaggagg tgatgcccca ggccgagaac 300

caggaccccg acatcaaggc ccacgtgaac agcctgggcg agaacctgaa gaccctgaga 360

ctgagactga gaagatgcca cagattcctg ccctgcgaga acaagagcaa ggccgtggag 420

caggtgaaga acgccttcaa caagctgcag gagaagggca tctacaaggc catgagcgag 480

ttcgacatct tcatcaacta catcgaggcc tacatgacca tgaagatcag aaactga 537

<210> 20

<211> 162

<212> PRT

<213> Artificial Sequence

<220>

<223> IL-15

<400> 20

Met Arg Ile Ser Lys Pro His Leu Arg Ser Ile Ser Ile Gln Cys Tyr

1 5 10 15

Leu Cys Leu Leu Leu Asn Ser His Phe Leu Thr Glu Ala Gly Ile His

20 25 30

Val Phe Ile Leu Gly Cys Phe Ser Ala Gly Leu Pro Lys Thr Glu Ala

35 40 45

Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile

50 55 60

Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His

65 70 75 80

Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln

85 90 95

Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu

100 105 110

Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val

115 120 125

Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile

130 135 140

Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn

145 150 155 160

Thr Ser

<210> 21

<211> 114

<212> PRT

<213> Artificial Sequence

<220>

<223> IL-15 active fragment (mutant type)

<400> 21

Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile

1 5 10 15

Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His

20 25 30

Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln

35 40 45

Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu

50 55 60

Asn Leu Ile Ile Leu Ala Asn Asp Ser Leu Ser Ser Asn Gly Asn Val

65 70 75 80

Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile

85 90 95

Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn

100 105 110

Thr Ser

<210> 22

<211> 114

<212> PRT

<213> Artificial Sequence

<220>

<223> IL-15 active fragment (wild type)

<400> 22

Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu Ile

1 5 10 15

Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val His

20 25 30

Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu Gln

35 40 45

Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val Glu

50 55 60

Asn Leu Ile Ile Leu Ala Asn Asn Ser Leu Ser Ser Asn Gly Asn Val

65 70 75 80

Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn Ile

85 90 95

Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile Asn

100 105 110

Thr Ser

<210> 23

<211> 267

<212> PRT

<213> Artificial Sequence

<220>

<223> IL15Ra

<400> 23

Met Ala Pro Arg Arg Ala Arg Gly Cys Arg Thr Leu Gly Leu Pro Ala

1 5 10 15

Leu Leu Leu Leu Leu Leu Leu Arg Pro Pro Ala Thr Arg Gly Ile Thr

20 25 30

Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val Lys Ser

35 40 45

Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly Phe Lys

50 55 60

Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn Lys Ala

65 70 75 80

Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile Arg Asp

85 90 95

Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro Ser Thr Val Thr Thr

100 105 110

Ala Gly Val Thr Pro Gln Pro Glu Ser Leu Ser Pro Ser Gly Lys Glu

115 120 125

Pro Ala Ala Ser Ser Pro Ser Ser Asn Asn Thr Ala Ala Thr Thr Ala

130 135 140

Ala Ile Val Pro Gly Ser Gln Leu Met Pro Ser Lys Ser Pro Ser Thr

145 150 155 160

Gly Thr Thr Glu Ile Ser Ser His Glu Ser Ser His Gly Thr Pro Ser

165 170 175

Gln Thr Thr Ala Lys Asn Trp Glu Leu Thr Ala Ser Ala Ser His Gln

180 185 190

Pro Pro Gly Val Tyr Pro Gln Gly His Ser Asp Thr Thr Val Ala Ile

195 200 205

Ser Thr Ser Thr Val Leu Leu Cys Gly Leu Ser Ala Val Ser Leu Leu

210 215 220

Ala Cys Tyr Leu Lys Ser Arg Gln Thr Pro Pro Leu Ala Ser Val Glu

225 230 235 240

Met Glu Ala Met Glu Ala Leu Pro Val Thr Trp Gly Thr Ser Ser Arg

245 250 255

Asp Glu Asp Leu Glu Asn Cys Ser His His Leu

260 265

<210> 24

<211> 801

<212> DNA

<213> Artificial Sequence

<220>

<223> Nucleotides encoding IL15Ra

<400> 24

atggccccgc ggcgggcgcg cggctgccgg accctcggtc tcccggcgct gctactgctg 60

ctgctgctcc ggccgccggc gacgcggggc atcacgtgcc ctccccccat gtccgtggaa 120

cacgcagaca tctgggtcaa gagctacagc ttgtactcca gggagcggta catttgtaac 180

tctggtttca agcgtaaagc cggcacgtcc agcctgacgg agtgcgtgtt gaacaaggcc 240

acgaatgtcg cccactggac aacccccagt ctcaaatgca ttagagaccc tgccctggtt 300

caccaaaggc cagcgccacc ctccacagta acgacggcag gggtgacccc acagccagag 360

agcctctccc cttctggaaa agagcccgca gcttcatctc ccagctcaaa caacacagcg 420

gccacaacag cagctattgt cccgggctcc cagctgatgc cttcaaaatc accttccaca 480

ggaaccacag agataagcag tcatgagtcc tcccacggca ccccctctca gacaacagcc 540

aagaactggg aactcacagc atccgcctcc caccagccgc caggtgtgta tccacagggc 600

cacagcgaca ccactgtggc tatctccacg tccactgtcc tgctgtgtgg gctgagcgct 660

gtgtctctcc tggcatgcta cctcaagtca aggcaaactc ccccgctggc cagcgttgaa 720

atggaagcca tggaggctct gccggtgact tgggggacca gcagcagaga tgaagacttg 780

gaaaactgct ctcaccacct a 801

<210> 25

<211> 78

<212> PRT

<213> Artificial Sequence

<220>

<223> IL15Ra (sushi+)

<400> 25

Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val

1 5 10 15

Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly

20 25 30

Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn

35 40 45

Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile

50 55 60

Arg Asp Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro Ser

65 70 75

<210> 26

<211> 228

<212> DNA

<213> Artificial Sequence

<220>

<223> Nucleotide sequence encoding IL15Ra (sushi)

<400> 26

gccggcacgt ccagcctgac ggagtgcgtg ttgaacaagg ccacgaatgt cgcccactgg 60

acaaccccca gtctcaaatg cattagagac cctgccctgg ttcaccaaag gccagcgcca 120

ccctccacag taacgacggc aggggtgacc ccacagccag agagcctctc cccttctgga 180

aaagagcccg cagcttcatc tcccagctca aacaacacag cggccaca 228

<210> 27

<211> 47

<212> PRT

<213> Artificial Sequence

<220>

<223> Hinge region of CD8a

<400> 27

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 Ile Tyr

35 40 45

<210> 28

<211> 141

<212> DNA

<213> Artificial Sequence

<220>

<223> Nucleotide sequence encoding CD8a hinge region

<400> 28

actaccaccc ccgccccccg gccccccacc cccgccccca ccatcgcctc ccagcccctg 60

tccctgcggc ccgaggcctg ccggcccgcc gccggcggcg ccgtgcacac ccggggcctg 120

gacttcgcct gcgacatcta c 141

<210> 29

<211> 113

<212> PRT

<213> Artificial Sequence

<220>

<223> Light chain variable region sequence L14

<400> 29

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn

85 90 95

Asp Tyr Phe Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

100 105 110

Lys

<210> 30

<211> 113

<212> PRT

<213> Artificial Sequence

<220>

<223> Light chain variable region sequence L11

<400> 30

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr His Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn

85 90 95

Asp Tyr Phe Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

100 105 110

Lys

<210> 31

<211> 113

<212> PRT

<213> Artificial Sequence

<220>

<223> Light chain variable region sequence L12

<400> 31

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn

85 90 95

Asp Tyr Phe Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

100 105 110

Lys

<210> 32

<211> 113

<212> PRT

<213> Artificial Sequence

<220>

<223> Light chain variable region sequence L13

<400> 32

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn

85 90 95

Asp Tyr Phe Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

100 105 110

Lys

<210> 33

<211> 113

<212> PRT

<213> Artificial Sequence

<220>

<223> Light chain variable region sequence L15

<400> 33

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr His Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn

85 90 95

Asp Tyr Phe Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

100 105 110

Lys

<210> 34

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy chain variable region sequence H51

<400> 34

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Asn Tyr

20 25 30

Leu Ile Glu Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Leu Ile Asn Pro Gly Ser Gly Gly Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Val Tyr Tyr Gly Asn Ser Phe Ala Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 35

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy chain variable region sequence H52

<400> 35

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Asn Tyr

20 25 30

Leu Ile Glu Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

35 40 45

Gly Leu Ile Asn Pro Gly Ser Gly Gly Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Ala Thr Leu Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Phe Cys

85 90 95

Ala Arg Val Tyr Tyr Gly Asn Ser Phe Ala Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 36

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy chain variable region sequence H53

<400> 36

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Asn Tyr

20 25 30

Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Leu Ile Asn Pro Gly Ser Gly Gly Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Arg Val Thr Ile Thr Ala Asp Glu Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Val Tyr Tyr Gly Asn Ser Phe Ala Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 37

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> Heavy chain variable region sequence H54

<400> 37

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

1 5 10 15

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Asn Tyr

20 25 30

Leu Ile Glu Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met

35 40 45

Gly Leu Ile Asn Pro Gly Ser Gly Gly Thr Asn Tyr Asn Glu Lys Phe

50 55 60

Lys Gly Lys Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

65 70 75 80

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Val Tyr Tyr Gly Asn Ser Phe Ala Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 38

<211> 42

<212> PRT

<213> Artificial Sequence

<220>

<223> 4-1BB intracellular domain

<400> 38

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

1 5 10 15

Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

20 25 30

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu

35 40

<210> 39

<211> 126

<212> DNA

<213> Artificial Sequence

<220>

<223> Nucleotide sequence encoding the intracellular region of 4-1BB

<400> 39

aagcggggcc ggaagaagct gctgtacatc ttcaagcagc ccttcatgcg gcccgtgcag 60

accacccagg aggaggacgg ctgctcctgc cggttccccg aggaggagga gggcggctgc 120

gagctg 126

<210> 40

<211> 112

<212> PRT

<213> Artificial Sequence

<220>

<223> CD3ζ intracellular domain

<400> 40

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly

1 5 10 15

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

20 25 30

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

35 40 45

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

50 55 60

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

65 70 75 80

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

85 90 95

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 41

<211> 336

<212> DNA

<213> Artificial Sequence

<220>

<223> Nucleotide sequence encoding CD3ζ intracellular domain

<400> 41

cgggtgaagt tctcccggtc cgccgacgcc cccgcctaca agcagggcca gaaccagctg 60

tacaacgagc tgaacctggg ccggcgggag gagtacgacg tgctggacaa gcggcggggc 120

cgggaccccg agatgggcgg caagccccgg cggaagaacc cccaggaggg cctgtacaac 180

gagctgcaga aggacaagat ggccgaggcc tactccgaga tcggcatgaa gggcgagcgg 240

cggcggggca agggccacga cggcctgtac cagggcctgt ccaccgccac caaggacacc 300

tacgacgccc tgcacatgca ggccctgccc ccccgg 336

<210> 42

<211> 41

<212> PRT

<213> Artificial Sequence

<220>

<223> CD28 intracellular domain

<400> 42

Arg Ser Lys Arg Ser Arg Leu Leu His Ser Asp Tyr Met Asn Met Thr

1 5 10 15

Pro Arg Arg Pro Gly Pro Thr Arg Lys His Tyr Gln Pro Tyr Ala Pro

20 25 30

Pro Arg Asp Phe Ala Ala Tyr Arg Ser

35 40

<210> 43

<211> 123

<212> DNA

<213> Artificial Sequence

<220>

<223> Nucleotide sequence encoding the intracellular domain of CD28

<400> 43

cggtccaagc ggtcccggct gctgcactcc gactacatga acatgacccc ccggcggccc 60

ggccccaccc ggaagcacta ccagccctac gcccccccccc gggacttcgc cgcctaccgg 120

tcc 123

<210> 44

<211> 469

<212> PRT

<213> Artificial Sequence

<220>

<223>CAR1a

<400> 44

Asp Ile Val Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu Gly

1 5 10 15

Glu Arg Ala Thr Ile Ser Cys Lys Ser Ser Gln Ser Leu Leu Asn Ser

20 25 30

Gly Asn Gln Lys Asn Tyr Leu Thr Trp Tyr Gln Gln Lys Pro Gly Gln

35 40 45

Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly Val

50 55 60

Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr

65 70 75 80

Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln Asn

85 90 95

Asp Tyr Phe Tyr Pro Phe Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile

100 105 110

Lys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser

115 120 125

Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser

130 135 140

Ser Val Lys Val Ser Cys Lys Ala Ser Gly Tyr Ala Phe Ser Asn Tyr

145 150 155 160

Leu Ile Glu Trp Val Lys Gln Ala Pro Gly Gln Gly Leu Glu Trp Ile

165 170 175

Gly Leu Ile Asn Pro Gly Ser Gly Gly Thr Asn Tyr Asn Glu Lys Phe

180 185 190

Lys Gly Lys Ala Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr

195 200 205

Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys

210 215 220

Ala Arg Val Tyr Tyr Gly Asn Ser Phe Ala Tyr Trp Gly Gln Gly Thr

225 230 235 240

Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr

245 250 255

Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala

260 265 270

Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe

275 280 285

Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val

290 295 300

Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys

305 310 315 320

Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr

325 330 335

Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu

340 345 350

Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro

355 360 365

Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly

370 375 380

Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro

385 390 395 400

Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr

405 410 415

Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly

420 425 430

Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln

435 440 445

Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln

450 455 460

Ala Leu Pro Pro Arg

465

<210> 45

<211> 1470

<212> DNA

<213> Artificial Sequence

<220>

<223> Nucleotide sequence encoding CAR1a

<400> 45

atggccctgc ccgtgaccgc cctgctgctg cccctggccc tgctgctgca cgccgccaga 60

cccgacatcg tgatgaccca gagccccgac agcctggccg tgagcctggg cgagagagcc 120

accatcagct gcaagagcag ccagagcctg ctgaacagcg gcaaccagaa gaactacctg 180

acctggtacc agcagaagcc cggccagccc cccaagctgc tgatctactg ggccagcacc 240

agagagagcg gcgtgcccga cagattcagc ggcagcggca gcggcaccga cttcaccctg 300

accatcagca gcctgcaggc cgaggacgtg gccgtgtact actgccagaa cgactacttc 360

taccccttca ccttcggcca gggcaccaag ctggagatca agggcggcgg cggcagcggc 420

ggcggcggca gcggcggcgg cggcagccag gtgcagctgg tgcagagcgg cgccgaggtg 480

aagaagcccg gcagcagcgt gaaggtgagc tgcaaggcca gcggctacgc cttcagcaac 540

tacctgatcg agtgggtgaa gcaggccccc ggccagggcc tggagtggat cggcctgatc 600

aaccccggca gcggcggcac caactacaac gagaagttca agggcaaggc caccatcacc 660

gccgacaaga gcaccagcac cgcctacatg gagctgagca gcctgagaag cgaggacacc 720

gccgtgtact actgcgccag agtgtactac ggcaacagct tcgcctactg gggccagggc 780

accctggtga ccgtgagcag cactaccacc cccgcccccc ggccccccac ccccgccccc 840

accatcgcct cccagcccct gtccctgcgg cccgaggcct gccggcccgc cgccggcggc 900

gccgtgcaca cccggggcct ggacttcgcc tgcgacatct acatctgggc ccccctggcc 960

ggcacctgcg gcgtgctgct gctgtccctg gtgatcaccc tgtactgcaa gcggggccgg 1020

aagaagctgc tgtacatctt caagcagccc ttcatgcggc ccgtgcagac cacccaggag 1080

gaggacggct gctcctgccg gttccccgag gaggaggagg gcggctgcga gctgcgggtg 1140

aagttctccc ggtccgccga cgcccccgcc tacaagcagg gccagaacca gctgtacaac 1200

gagctgaacc tgggccggcg gaggagtac gacgtgctgg acaagcggcg gggccgggac 1260

cccgagatgg gcggcaagcc ccggcggaag aacccccagg agggcctgta caacgagctg 1320

cagaaggaca agatggccga ggcctactcc gagatcggca tgaagggcga gcggcggcgg 1380

ggcaagggcc acgacggcct gtaccagggc ctgtccaccg ccaccaagga cacctacgac 1440

gccctgcaca tgcaggccct gccccccccgg 1470

<210> 46

<211> 211

<212> PRT

<213> Artificial Sequence

<220>

<223> Fusion fragment of IL15Rα (sushi+) and IL15

<400> 46

Ile Thr Cys Pro Pro Pro Met Ser Val Glu His Ala Asp Ile Trp Val

1 5 10 15

Lys Ser Tyr Ser Leu Tyr Ser Arg Glu Arg Tyr Ile Cys Asn Ser Gly

20 25 30

Phe Lys Arg Lys Ala Gly Thr Ser Ser Leu Thr Glu Cys Val Leu Asn

35 40 45

Lys Ala Thr Asn Val Ala His Trp Thr Thr Pro Ser Leu Lys Cys Ile

50 55 60

Arg Asp Pro Ala Leu Val His Gln Arg Pro Ala Pro Pro Ser Gly Gly

65 70 75 80

Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Gly Gly Gly Gly Gly Ser Leu

85 90 95

Gln Asn Trp Val Asn Val Ile Ser Asp Leu Lys Lys Ile Glu Asp Leu

100 105 110

Ile Gln Ser Met His Ile Asp Ala Thr Leu Tyr Thr Glu Ser Asp Val

115 120 125

His Pro Ser Cys Lys Val Thr Ala Met Lys Cys Phe Leu Leu Glu Leu

130 135 140

Gln Val Ile Ser Leu Glu Ser Gly Asp Ala Ser Ile His Asp Thr Val

145 150 155 160

Glu Asn Leu Ile Ile Leu Ala Asn Asp Ser Leu Ser Ser Asn Gly Asn

165 170 175

Val Thr Glu Ser Gly Cys Lys Glu Cys Glu Glu Leu Glu Glu Lys Asn

180 185 190

Ile Lys Glu Phe Leu Gln Ser Phe Val His Ile Val Gln Met Phe Ile

195 200 205

Asn Thr Ser

210

<210> 47

<211> 39

<212> PRT

<213> Artificial Sequence

<220>

<223> mab5b light chain CDR1&2

<400> 47

Lys Ser Ser Gln Ser Leu Leu Asn Ser Gly Asn Gln Lys Asn Tyr Leu

1 5 10 15

Thr Trp Tyr Gln Gln Lys Pro Gly Gln Pro Pro Lys Leu Leu Ile Tyr

20 25 30

Trp Ala Ser Thr Arg Glu Ser

35

<210> 48

<211> 66

<212> DNA

<213> Artificial Sequence

<220>

<223> Nucleotide sequence encoding CD8a transmembrane region

<400> 48

atctggggcccc ccctggccgg cacctgcggc gtgctgctgc tgtccctggt gatcaccctg 60

tactgc 66

<210> 49

<211> 585

<212> DNA

<213> Artificial Sequence

<220>

<223> IRES

<400> 49

gcccctctcc ctcccccccc cctaacgtta ctggccgaag ccgcttggaa taaggccggt 60

gtgcgtttgt ctatatgtta ttttccacca tattgccgtc ttttggcaat gtgagggccc 120

ggaaacctgg ccctgtcttc ttgacgagca ttcctagggg tctttcccct ctcgccaaag 180

gaatgcaagg tctgttgaat gtcgtgaagg aagcagttcc tctggaagct tcttgaagac 240

aaacaacgtc tgtagcgacc ctttgcaggc agcggaaccc cccacctggc gacaggtgcc 300

tctgcggcca aaagccacgt gtataagata cacctgcaaa ggcggcacaa ccccagtgcc 360

acgttgtgag ttggatagtt gtggaaagag tcaaatggct ctcctcaagc gtattcaaca 420

aggggctgaa ggatgcccag aaggtacccc attgtatggg atctgatctg gggcctcggt 480

acacatgctt tacatgtgtt tagtcgaggt taaaaaaacg tctaggcccc ccgaaccacg 540

gggacgtggt tttcctttga aaaacacgat gataatatgg ccaca 585

Claims (19)

1. A nucleic acid construct of formula car- [ (IRES) -f]_mThe structure shown in the specification, wherein IRES is an internal ribosome entry site sequence; f codes for functional protein F, m is 0 or non-0 natural number; CAR encodes a CAR and the CAR comprises: (a) an extracellular binding domain scFv that specifically recognizes CLDN 18.2; (b) a hinge domain; (c) transmembrane knotA domain; (d) a costimulatory intracellular domain; (e) a signaling domain;

wherein the extracellular binding domain comprises a light chain variable region comprising at least 1 Complementarity Determining Region (CDR) selected from: CDR1 as set forth in SEQ ID NO 11 or SEQ ID NO 12 amino acid sequences or mutations thereof; CDR2 set forth in the amino acid sequence of SEQ ID NO. 13 or a mutation thereof; CDR3 set forth in SEQ ID NO. 14 amino acid sequence or a mutation thereof; the heavy chain variable region comprises at least 1 CDR selected from: CDR1 set forth in the amino acid sequence of SEQ ID NO. 15 or a mutation thereof; CDR2 set forth in the amino acid sequence of SEQ ID NO. 16 or a mutation thereof; 17 amino acid sequence or a mutation thereof, as shown in CDR 3; the mutation maintains or improves the binding of the antibody or antigen binding fragment to the CLDN18.2 antigen.

2. The nucleic acid construct of claim 1, wherein the scFv is structured as a light chain variable region-linker-heavy chain variable region or a heavy chain variable region-linker-light chain variable region, preferably the linker is (GGGGS)_n(ii) a n is preferably an integer of 1 to 6, more preferably 3 and 4.

3. The nucleic acid construct of claim 1 or 2, wherein the light chain variable region comprises CDR1 as set forth in the amino acid sequence of SEQ ID No. 11 or a mutation thereof, CDR2 as set forth in the amino acid sequence of SEQ ID No. 13 or a mutation thereof, and CDR3 as set forth in the amino acid sequence of SEQ ID No. 14 or a mutation thereof; or, CDR1 as set forth in the amino acid sequence of SEQ ID NO. 12 or a mutation thereof, CDR2 as set forth in the amino acid sequence of SEQ ID NO. 13 or a mutation thereof, and CDR3 as set forth in the amino acid sequence of SEQ ID NO. 14 or a mutation thereof;

and/or, the heavy chain variable region comprises the CDR1 as set forth in SEQ ID No. 15 amino acid sequence or mutation thereof, the CDR2 as set forth in SEQ ID No. 16 amino acid sequence or mutation thereof, and the CDR3 as set forth in SEQ ID No. 17 amino acid sequence or mutation thereof;

preferably, the light chain variable region comprises the amino acid sequence shown as SEQ ID NO. 11 or a mutation thereof shown as CDR1, the amino acid sequence shown as SEQ ID NO. 13 or a mutation thereof shown as CDR2, and the amino acid sequence shown as SEQ ID NO. 14 or a mutation thereof shown as CDR 3; and, the heavy chain variable region comprises the amino acid sequence of SEQ ID NO. 15 or a mutation thereof shown as CDR1, the amino acid sequence of SEQ ID NO. 16 or a mutation thereof shown as CDR2, and the amino acid sequence of SEQ ID NO. 17 or a mutation thereof shown as CDR 3; or the like, or, alternatively,

The light chain variable region comprises an amino acid sequence shown as SEQ ID NO. 12 or a mutated CDR1 thereof, an amino acid sequence shown as SEQ ID NO. 13 or a mutated CDR2 thereof, and an amino acid sequence shown as SEQ ID NO. 14 or a mutated CDR3 thereof; and, the heavy chain variable region comprises an amino acid sequence as set forth in SEQ ID NO. 15 or a mutated CDR1 thereof, an amino acid sequence as set forth in SEQ ID NO. 16 or a mutated CDR2 thereof, and an amino acid sequence as set forth in SEQ ID NO. 17 or a mutated CDR3 thereof.

4. The nucleic acid construct of any one of claims 1 to 3, wherein the extracellular binding domain comprises a light chain variable region as set forth in any one of amino acid sequences of SEQ ID Nos. 29 to 33 or a mutated sequence thereof; and/or, the heavy chain variable region as shown in any one of SEQ ID NO 34-37 or its mutant sequence;

preferably, the extracellular binding domain comprises the light chain variable region as represented by the amino acid sequence of SEQ ID NO. 29 and the heavy chain variable region as represented by the amino acid sequence of SEQ ID NO. 34; or, the light chain variable region as shown in the amino acid sequence of SEQ ID NO. 31 and the heavy chain variable region as shown in the amino acid sequence of SEQ ID NO. 34; the light chain variable region shown by the amino acid sequence of SEQ ID NO. 7 and the heavy chain variable region shown by the amino acid sequence of SEQ ID NO. 8.

5. The nucleic acid construct according to any one of claims 1 to 4, wherein, in the nucleic acid construct,

(1) the hinge domain is selected from the hinge regions of one or more of the following molecules: CD8 α, CD28, CD152, PD1, and IgG1 heavy chain;

(2) the transmembrane domain is selected from the transmembrane regions of one or more of the following molecules: α, β, ζ chain of TCR, CD3, CD3 ζ, CD4, CD5, CD8 α, CD9, CD16, CD22, CD27, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, 4-1BB, CD152, CD154 and PD 1;

(3) the co-stimulatory intracellular domain is selected from the intracellular domains of one or more of the following molecules: CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54, CD83, OX40, CD134, 4-1BB, CD150, CD152, CD223, CD270, PD-L2, PD-L1, CD278, DAP10, NKD2C SLP76, TRIM, FcRIgamma and MyD88, preferably CD28 intracellular domain and/or 4-1BB intracellular domain; and/or the presence of a gas in the gas,

(4) the signaling domain is selected from the intracellular regions of one or more of the following molecules: ig α, Ig β, TCR ξ, FcR1 γ, FcR1 β, CD3 γ, CD3, CD3, CD2, CD5, CD22, CD28, CD79a, CD79b, CD278, CD66d, and CD3 ζ; the intracellular domain of CD3 ζ is preferred.

6. The nucleic acid construct of any one of claims 1 to 5, wherein the hinge domain is a CD8 a hinge region, the transmembrane domain is a CD8 a transmembrane region, the costimulatory intracellular domain is a CD28 intracellular region and/or a 4-1BB intracellular region, and the signaling domain is a CD3 ζ intracellular region;

Preferably, the CD8 alpha hinge region is a human CD8 alpha hinge region, and the amino acid sequence of the hinge region is shown as SEQ ID NO. 27; the CD8 alpha transmembrane region is a human CD8 alpha transmembrane region, and the amino acid sequence of the CD8 alpha transmembrane region is shown as SEQ ID NO 18; the amino acid sequence of the CD28 intracellular domain is shown as SEQ ID NO: 42; the amino acid sequence of the 4-1BB intracellular domain is shown in SEQ ID NO: 38; and/or, the amino acid sequence of intracellular domain of CD3 ζ is shown in SEQ ID NO 40;

more preferably, the nucleotide sequence encoding the human CD8 a hinge region is set forth in SEQ ID NO 28; the nucleotide sequence of the human CD8 alpha transmembrane region is shown as SEQ ID NO. 48; the nucleotide sequence of the intracellular region of the CD28 is shown as SEQ ID NO. 43; the nucleotide sequence of the 4-1BB intracellular domain is shown as SEQ ID NO: 39; and/or, the nucleotide sequence of the intracellular domain encoding CD3 zeta is shown in SEQ ID NO: 41.

7. The nucleic acid construct according to any one of claims 1 to 6, wherein the functional protein F comprises, when m is a non-0 natural number, preferably 1:

(1) a cytokine or an active fragment thereof, preferably IL10 or IL15 or an active fragment thereof; more preferably, the amino acid sequence of the IL10 is shown as SEQ ID NO. 3, the amino acid sequence of the IL15 is shown as SEQ ID NO. 20, and the amino acid sequence of the IL15 active fragment is shown as SEQ ID NO. 21 or SEQ ID NO. 22;

(2) A cytokine receptor or an active fragment thereof, preferably IL15 ra or a fragment thereof or IL15 ra fragment (sushi) or IL15R a fragment (sushi +); more preferably, the amino acid sequence of the IL15R alpha is shown as SEQ ID NO. 23; the amino acid sequence of the IL15R alpha fragment (sushi) is shown as SEQ ID NO. 26; the amino acid sequence of the IL15R alpha fragment (sushi +) is shown as SEQ ID NO. 25; or the like, or, alternatively,

(3) a fusion protein of a cytokine receptor or an active fragment thereof and a cytokine, preferably IL15R alpha or a fragment thereof or a fusion fragment of IL15R alpha (sushi) or IL15R alpha (sushi +) and IL 15; more preferably, the amino acid sequence of the fusion fragment of IL15R alpha (sushi +) and IL15 active fragment is shown in SEQ ID NO: 46.

8. The nucleic acid construct of any one of claims 1-7, wherein the nucleic acid construct has the structure:

(1) scFv-human CD8 α hinge region-human CD8 α transmembrane region-human 4-1BB intracellular region-human CD3 ζ intracellular region; preferably, the coded amino acid sequence is shown as SEQ ID NO. 44; more preferably, the nucleotide sequence is shown as SEQ ID NO. 45;

(2) scFv-human CD8 α hinge region-human CD8 α transmembrane region-human 4-1BB intracellular region-human CD3 ζ intracellular region- (IRES) -IL 15; preferably, the nucleotide sequence of the IRES sequence is shown in SEQ ID NO. 49; and/or the nucleotide sequence of the IL15 codes an amino acid sequence shown as SEQ ID NO. 22;

(3) scFv-human CD8 α hinge region-human CD8 α transmembrane region-human 4-1BB intracellular region-human CD3 ζ intracellular region- (IRES) -IL 10; preferably, the nucleotide sequence of the IRES sequence is shown in SEQ ID NO. 49; and/or the amino acid sequence coded by the nucleotide sequence of the IL10 is shown as SEQ ID NO. 3; or the like, or, alternatively,

(4) scFv-human CD8 α hinge region-human CD8 α transmembrane region-human 4-1BB intracellular region-human CD3 ζ intracellular region- (IRES) -IL15R α (sushi +) -IL 15; preferably, the nucleotide sequence of the IRES sequence is shown in SEQ ID NO. 49; and/or the amino acid sequence coded by the nucleotide sequence of the IL15R alpha (sushi +) -IL15 is shown as SEQ ID NO: 46.

9. A CAR molecule encoded by the nucleic acid construct of any of claims 1-8 or a composition comprising said CAR molecule.

10. An expression vector comprising the nucleic acid construct of any one of claims 1-8.

11. The expression vector of claim 10, wherein the expression vector is selected from one or more of a retroviral vector, a lentiviral vector, an adenoviral vector, and an adeno-associated viral vector.

12. A virus comprising the expression vector of claim 10 or 11.

13. Use of the nucleic acid construct of any one of claims 1-8, or the expression vector of claim 10 or 11, or the virus of claim 12 for the preparation of a genetically modified cell targeting CLDN 18.2.

14. A genetically modified cell transfected with the nucleic acid construct of any one of claims 1 to 8, the expression vector of claim 10 or 11, or the virus of claim 12.

15. The genetically modified cell according to claim 14, which is a eukaryotic cell, preferably an isolated human cell; more preferably immune cells such as T cells, or NK cells such as NK92 cell line.

16. Use of the genetically modified cell of claim 14 or 15 for the preparation of a medicament for inhibiting a tumor; preferably, the tumor is a CLDN18.2 positive tumor.

17. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and the genetically modified cell of claim 14 or 15 or the CAR molecule of claim 9 or a composition comprising said CAR molecule.

18. A method of making a genetically modified cell, comprising the steps of: transferring the nucleic acid construct of any one of claims 1 to 8, or the expression vector of claim 10 or 11, or the virus of claim 12 into a cell to be modified, thereby obtaining a genetically modified cell;

Preferably, the genetically modified cell is a eukaryotic cell, preferably an isolated human cell; more preferably immune cells such as T cells or NK cells; even more preferably the NK92 cell line.

19. Use of a nucleic acid construct according to any one of claims 1 to 8, or an expression vector according to claim 10 or 11, or a virus according to claim 12, or a genetically modified cell according to claim 14 or 15, for the preparation of a medicament for the treatment of a tumour; preferably, the tumor is a CLDN18.2 positive tumor, preferably gastric cancer, esophageal cancer, lung cancer, melanoma, renal cancer, breast cancer, colorectal cancer, liver cancer, pancreatic cancer, bladder cancer, glioma or leukemia.

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