WO2022011531A1 - Anti-cld18a2 single-domain antibody - Google Patents

Abstract

An anti-CLD18A2 single-domain antibody. A complementarity determining region of the anti-CLD18A2 single-domain antibody comprises CDR-H1 represented by one of the amino acid sequences, for example, SEQ ID Nos. 4-11, CDR-H2 represented by one of the amino acid sequences, for example, SEQ ID Nos. 19-26, and CDR-H3 represented by one of the amino acid sequences, for example, SEQ ID Nos. 33-39. The anti-CLD18A2 single-domain antibody has good affinity to Claudin18.2, and can be used for further constructing a bispecific antibody, an antibody-drug conjugate, a chimeric antigen receptor (CAR), etc., and the constructed bispecific antibody, antibody-drug conjugate, and CAR-T cells, etc., have good targeting and a good kill effect for targeted cells.

Description

An anti-CLD18A2 single domain antibody technical field

The present invention relates to the field of biotechnology, in particular to an anti-CLD18A2 single-domain antibody.

Background technique

Claudin 18 (Claudin 18, CLD18) is a transmembrane protein with a molecular weight of about 28 kD, located in the tight junctions of the epithelium and the endothelium, which are tightly junctions between adjacent cells. In normal epithelial tissues, the claudin on the cell surface is difficult to be contacted due to the tight intercellular space, while the intercellular space of tumor cells is relatively loose. Therefore, claudin on tumor cells becomes a potential target for extracellular antibodies and immunotherapy. CLD18 has four hydrophobic regions that act as transmembrane domains to form two ectodomains, wherein hydrophobic region 1 and hydrophobic region 2 surround to form ectodomain 1, and hydrophobic region 3 and hydrophobic region 4 surround to form ectodomain 2. Due to different splicing of genes, CLD18 forms two spliceosomes: CLD18A1 (Claudin18.1) and CLD18A2 (Claudin18.2). CLD18A1 is selectively expressed in the epithelium of normal lung and stomach, whereas CLD18A2 is expressed only in gastric cells; more importantly, CLD18A2 is localized in differentiated gastric epithelium short-lived cells but absent in gastric stem cells (Niimi T. , et al. Biol. 2001;21(21):7380–7390.). These properties suggest that CLD18A2 is a clinically valuable therapeutic target for the treatment of gastric cancer and other CLD18A2-positive tumors.

Since the clinical effect of a single target is often limited, it is more inclined to adopt the form of dual targets, such as CD3-based bispecific antibody (T-cell-engaging bispecific antibody, TCB), or ADC, etc. However, there is still no safe and effective CLD18A2-based bispecific antibody or derivatives such as ADC.

SUMMARY OF THE INVENTION

In view of the above-mentioned shortcomings of the prior art, the purpose of the present invention is to provide an anti-CLD18A2 single-domain antibody for solving the problems in the prior art.

In order to achieve the above purpose and other related purposes, one aspect of the present invention provides an anti-CLD18A2 single-domain antibody, the complementarity determining region of the anti-CLD18A2 single-domain antibody comprises an amino acid sequence as shown in one of SEQ ID No. 4-11. CDR-H1 shown in one of the amino acid sequence, CDR-H2 shown in one of SEQ ID No. 19-26, and CDR-H3 whose amino acid sequence is shown in one of SEQ ID No. 33-39.

Another aspect of the present invention provides a bispecific antibody, the bispecific antibody comprises the above-mentioned anti-CLD18A2 single domain antibody and an anti-CD3 domain.

Another aspect of the present invention provides an isolated polynucleotide encoding the above-mentioned anti-CLD18A2 single-domain antibody, or the above-mentioned bispecific antibody.

Another aspect of the present invention provides a construct comprising the isolated polynucleotide described above.

Another aspect of the present invention provides an antibody expression system, the expression system comprising the above-mentioned construct or the above-mentioned exogenous polynucleotide integrated into the genome.

Another aspect of the present invention provides a method for preparing the above-mentioned anti-CLD18A2 single-domain antibody or the above-mentioned bispecific antibody, comprising the steps of: culturing the above-mentioned antibody expression system under conditions suitable for expressing the antibody, thereby expressing The antibody is obtained, and the antibody is purified and isolated.

Another aspect of the present invention provides the use of the above-mentioned anti-CLD18A2 single-domain antibody or the above-mentioned bispecific antibody in the preparation of a medicament for treating a tumor.

Another aspect of the present invention provides an antibody-drug complex comprising the above-mentioned anti-CLD18A2 single domain antibody and a cytotoxic drug.

Another aspect of the present invention provides a cell comprising a membrane-bound chimeric antigen receptor, the chimeric antigen receptor comprising a transmembrane domain, an intracellular domain and an extracellular domain, the extracellular domain comprising the above-mentioned anti- Single domain antibody to CLD18A2.

Description of drawings

FIG. 1 is a schematic diagram showing the results of the activation test of the T cell fluorescent reporter system in Example 3 of the present invention.

FIG. 2 is a schematic diagram showing the killing effect of anti-CLD18A2/anti-CD3 bispecific antibody on NUGC-4-Claudin18.2 in vitro in Example 3 of the present invention.

Figure 3 shows a schematic diagram of the in vivo tumor inhibition test of the anti-CLD18A2/anti-CD3 bispecific antibody in Example 3 of the present invention.

FIG. 4 is a schematic diagram showing the results of endocytosis mediated by anti-CLDN18A2 single-domain antibody fusion protein in Example 5 of the present invention.

FIG. 5 is a schematic diagram showing the results of the in vitro cytotoxicity test in Example 8 of the present invention.

FIG. 6 is a schematic diagram showing the results of the in vivo toxicity test in Example 9 of the present invention.

Fig. 7 is a schematic diagram showing the results of in vitro cytokine release content detection after aC18.2-CAR-T cells act on target cells in Example 11 of the present invention.

Figure 8 is a schematic diagram showing the inhibitory effect of aC18.2-CAR-T cells on tumor growth in mice in Example 11 of the present invention.

detailed description

In order to make the invention purpose, technical solution and beneficial technical effect of the present invention clearer, the present invention will be described in further detail below in conjunction with the embodiments. Those who are familiar with this technology can easily understand other advantages and other advantages of the present invention from the content disclosed in this specification. effect.

After a lot of exploratory research, the inventors of the present invention unexpectedly discovered an anti-CLD18A2 single-domain antibody. The anti-CLD18A2 single-domain antibody has good affinity for Claudin18.2, and can be used to further construct bispecific antibodies, Antibody-drug complexes, T cell chimeric antigen receptors, etc., can be used to prepare drugs with good targeting and therapeutic effects.

The first aspect of the present invention provides an anti-CLD18A2 single-domain antibody. The single-domain antibody generally refers to a type of antibody molecule that lacks the light chain of the antibody and only has the variable region of the heavy chain. Because of its small molecular weight, it is also commonly referred to as for nanobodies. The complementarity determining region (CDR, complementarity determining region) of the single-domain antibody of described anti-CLD18A2 includes amino acid sequence such as CDR-H1 shown in one of SEQ ID No.4～11, amino acid sequence such as SEQ ID No.19～26 CDR-H2 shown in one of them, and CDR-H3 whose amino acid sequence is shown in one of SEQ ID Nos. 33 to 39.

In a specific embodiment of the present invention, the complementarity determining region of the anti-CLD18A2 single domain antibody comprises CDR-H1 whose amino acid sequence is shown in SEQ ID No. 4, and CDR-H1 whose amino acid sequence is shown in SEQ ID No. 19 H2, and CDR-H3 whose amino acid sequence is shown in SEQ ID No.33.

In another specific embodiment of the present invention, the complementarity determining region of the anti-CLD18A2 single domain antibody includes CDR-H1 whose amino acid sequence is shown in SEQ ID No.5, and CDR-H1 whose amino acid sequence is shown in SEQ ID No.20 -H2, and CDR-H3 whose amino acid sequence is shown in SEQ ID No.34.

In another specific embodiment of the present invention, the complementarity determining region of the anti-CLD18A2 single-domain antibody includes CDR-H1 whose amino acid sequence is shown in SEQ ID No. 6, and CDR-H1 whose amino acid sequence is shown in SEQ ID No. 21 -H2, and CDR-H3 whose amino acid sequence is shown in SEQ ID No.35.

In another specific embodiment of the present invention, the complementarity determining region of the anti-CLD18A2 single-domain antibody comprises CDR-H1 whose amino acid sequence is shown in SEQ ID No.7, and CDR-H1 whose amino acid sequence is shown in SEQ ID No.22 -H2, and CDR-H3 whose amino acid sequence is shown in SEQ ID No.36.

In another specific embodiment of the present invention, the complementarity determining region of the anti-CLD18A2 single-domain antibody includes CDR-H1 whose amino acid sequence is shown in SEQ ID No. 8, and CDR-H1 whose amino acid sequence is shown in SEQ ID No. 23 -H2, and CDR-H3 whose amino acid sequence is shown in SEQ ID No.37.

In another specific embodiment of the present invention, the complementarity determining region of the anti-CLD18A2 single-domain antibody includes CDR-H1 whose amino acid sequence is shown in SEQ ID No. 9, and CDR-H1 whose amino acid sequence is shown in SEQ ID No. 24 -H2, and CDR-H3 whose amino acid sequence is shown in SEQ ID No.38.

In another specific embodiment of the present invention, the complementarity determining region of the anti-CLD18A2 single domain antibody comprises CDR-H1 whose amino acid sequence is shown in SEQ ID No.10, and CDR-H1 whose amino acid sequence is shown in SEQ ID No.25 -H2, and CDR-H3 whose amino acid sequence is shown in SEQ ID No.36.

In another specific embodiment of the present invention, the complementarity determining region of the anti-CLD18A2 single-domain antibody comprises CDR-H1 whose amino acid sequence is shown in SEQ ID No.11, and CDR-H1 whose amino acid sequence is shown in SEQ ID No.26 -H2, and CDR-H3 whose amino acid sequence is shown in SEQ ID No.39.

In the anti-CLD18A2 single-domain antibody provided by the present invention, the anti-CLD18A2 single-domain antibody further includes a framework region (FR, framework region), and the framework region FR includes an amino acid sequence such as one of SEQ ID No. 1-3 FR1 shown in one, FR2 whose amino acid sequence is shown in one of SEQ ID No. 12 to 18, FR3 whose amino acid sequence is shown in one of SEQ ID No. 27 to 32, and FR3 whose amino acid sequence is shown in SEQ ID No. 27 to 32. FR4 shown in one of 40 to 41.

In a specific embodiment of the present invention, the framework region FR includes FR1 whose amino acid sequence is shown in SEQ ID No.1, FR2 whose amino acid sequence is shown in SEQ ID No.12, and whose amino acid sequence is shown in SEQ ID No.27 FR3 shown, and FR4 whose amino acid sequence is shown in SEQ ID No. 40.

In another specific embodiment of the present invention, the framework region FR includes FR1 whose amino acid sequence is shown in SEQ ID No. 2, FR2 whose amino acid sequence is shown in SEQ ID No. 13, and whose amino acid sequence is shown in SEQ ID No. 28 The FR3 shown, and the amino acid sequence of FR4 shown in SEQ ID No. 41.

In another specific embodiment of the present invention, the framework region FR includes FR1 whose amino acid sequence is shown in SEQ ID No.3, FR2 whose amino acid sequence is shown in SEQ ID No.14, and FR2 whose amino acid sequence is shown in SEQ ID No.29 The FR3 shown, and the amino acid sequence of FR4 shown in SEQ ID No. 41.

In another specific embodiment of the present invention, the framework region FR includes FR1 whose amino acid sequence is shown in SEQ ID No. 1, FR2 whose amino acid sequence is shown in SEQ ID No. 15, and FR2 whose amino acid sequence is shown in SEQ ID No. 30 The FR3 shown, and the amino acid sequence of FR4 shown in SEQ ID No. 41.

In another specific embodiment of the present invention, the framework region FR includes FR1 whose amino acid sequence is shown in SEQ ID No.2, FR2 whose amino acid sequence is shown in SEQ ID No.16, and whose amino acid sequence is shown in SEQ ID No.31 The FR3 shown, and the amino acid sequence of FR4 shown in SEQ ID No. 41.

In another specific embodiment of the present invention, the FR in the framework region includes FR1 whose amino acid sequence is shown in SEQ ID No.2, FR2 whose amino acid sequence is shown in SEQ ID No.13, and whose amino acid sequence is shown in SEQ ID No.31 The FR3 shown, and the amino acid sequence of FR4 shown in SEQ ID No. 41.

In another specific embodiment of the present invention, the framework region FR includes FR1 whose amino acid sequence is shown in SEQ ID No.1, FR2 whose amino acid sequence is shown in SEQ ID No.17, and FR2 whose amino acid sequence is shown in SEQ ID No.30 The FR3 shown, and the amino acid sequence of FR4 shown in SEQ ID No. 41.

In another specific embodiment of the present invention, the framework region FR includes FR1 whose amino acid sequence is shown in SEQ ID No. 2, FR2 whose amino acid sequence is shown in SEQ ID No. 18, and FR2 whose amino acid sequence is shown in SEQ ID No. 32 The shown FR3, and amino acid sequences are FR4 shown in SEQ ID No. 41.

In the anti-CLD18A2 single-domain antibody provided by the present invention, the amino acid sequence of the anti-CLD18A2 single-domain antibody may include: a) the amino acid sequence shown in one of SEQ ID Nos. 42 to 49; or, b) An amino acid sequence that has more than 80% sequence identity with the amino acid sequence shown in one of SEQ ID Nos. 42 to 49, and has the function of the amino acid sequence defined in a). Specifically, the amino acid sequence in b) specifically refers to: the amino acid sequence shown in one of SEQ ID No. 42 to 49 is substituted, deleted or added one or more (specifically, 1-50, 1- 30, 1-20, 1-10, 1-5, or 1-3) amino acids, or by adding one or more (specifically, 1) to the N-terminal and/or C-terminal -50, 1-30, 1-20, 1-10, 1-5, or 1-3) amino acids, and have amino acids as one of SEQ ID No. 42-49 Polypeptide fragments with the functions of the indicated polypeptide fragments, for example, may have specific binding ability to CLD18A2, so that they can specifically bind to cells expressing CLD18A2, but not to cells expressing only CLD18A1, that is, only recognize CLD18A2 and not Identify CLD18A1. The amino acid sequence in b) can be more than 80%, 85%, 90%, 93%, 95%, 97%, or 99% identical to one of SEQ ID Nos. 42-49.

As used herein, sequence identity refers to the percentage of identical residues in the sequences participating in the alignment. Sequence identity of two or more entry sequences can be calculated using computational software well known in the art, such software available from NCBI, for example.

The anti-CLD18A2 single-domain antibody provided by the present invention can be derived from alpaca (Vicugna pacos), and its overall molecular weight can be about half that of ScFv single-chain antibody, so it can effectively reduce the molecular weight of the overall structure, thereby enhancing its organization. Penetration, reaching target tissues and organs more effectively, improving the therapeutic effect, and this structure is more convenient to prepare than the structure with two ScFv in series.

The anti-CLD18A2 single domain antibody provided by the present invention can usually be a humanized antibody. The amino acid sequence of the anti-CLD18A2 single-domain antibody can include: c) the amino acid sequence shown in one of SEQ ID No. 67-90; or, d) and the amino acid sequence shown in one of SEQ ID No. 67-90 The amino acid sequence has more than 80% sequence identity and has the amino acid sequence function defined by c). Specifically, the amino acid sequence in d) specifically refers to: the amino acid sequence shown in one of SEQ ID No. 67-90 is substituted, deleted or added with one or more (specifically, 1-50, 1- 30, 1-20, 1-10, 1-5, or 1-3) amino acids, or by adding one or more (specifically, 1) to the N-terminal and/or C-terminal -50, 1-30, 1-20, 1-10, 1-5, or 1-3) amino acids, and have amino acids as one of SEQ ID No. 67-90 Polypeptide fragments that exhibit the function of the polypeptide fragments shown, for example, may be of a relatively higher degree of humanization. The humanization of the anti-CLD18.2 single-domain antibody of the present invention is mainly aimed at transforming the framework region, for example, by adopting the method of CDR transplantation, the FR region of a suitable human-derived antibody is replaced with the original sequence, while the CDR region of the original antibody is retained. The sequences SEQ ID No. 75-90 are obtained by humanizing FR1, FR2, FR3, and FR4 based on the original sequences SEQ ID No. 42-49; the sequences SEQ ID No. 67-74 are obtained from the original sequences Based on SEQ ID Nos. 42 to 49, the corresponding FR1, FR3, and FR4 are obtained by humanization transformation, while FR2 retains the corresponding original sequence. The amino acid sequence in d) can be more than 80%, 85%, 90%, 93%, 95%, 97%, or 99% identical to one of SEQ ID Nos. 67-90.

The second aspect of the present invention provides a bispecific antibody, and the bispecific antibody includes the anti-CLD18A2 single domain antibody and the anti-CD3 domain provided in the first aspect of the present invention. The bispecific antibody can simultaneously target the cell surface antigen CLD18A2 and the T cell surface triggering molecule CD3, thereby activating the T cells, resulting in the effect of killing the target cells.

In the bispecific antibody provided by the present invention, an anti-CD3 domain may be included. The structural form of the anti-CD3 domain can include, but is not limited to, single-chain antibody (scFv), antibody Fab fragments, and the like. The anti-CD3 domain can specifically bind to the triggering molecule CD3 on the surface of T cells, so that T cells can be activated to enhance the effect of killing target cells. The amino acid sequence of the heavy chain variable region of the anti-CD3 domain may include: e) as SEQ ID NO.91, SEQ ID NO.93, SEQ ID NO.95, SEQ ID NO.97, SEQ ID NO.99 , or the amino acid sequence shown in one of SEQ ID NO.101; or, f) with SEQ ID NO.91, SEQ ID NO.93, SEQ ID NO.95, SEQ ID NO.97, SEQ ID NO.99 , or the amino acid sequence shown in one of SEQ ID NO.101 has more than 80% sequence identity, and has the amino acid sequence function of the amino acid sequence defined in e). Specifically, the amino acid sequence in the f) specifically refers to: such as SEQ ID NO.91, SEQ ID NO.93, SEQ ID NO.95, SEQ ID NO.97, SEQ ID NO.99, or SEQ ID NO.99. The amino acid sequence shown in one of 101 has undergone substitution, deletion or addition of one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, or 1- 3) amino acids, or adding one or more (specifically 1-50, 1-30, 1-20, 1-10, 1- 5, or 1-3) amino acids, and have amino acids such as SEQ ID NO.91, SEQ ID NO.93, SEQ ID NO.95, SEQ ID NO.97, SEQ ID NO.99, or SEQ ID NO.99 A functional polypeptide fragment of one of the polypeptide fragments shown in ID NO. 101. The amino acid sequence in said f) can have one of SEQ ID NO.91, SEQ ID NO.93, SEQ ID NO.95, SEQ ID NO.97, SEQ ID NO.99, or SEQ ID NO.101 Concordance above 80%, 85%, 90%, 93%, 95%, 97%, or 99%. The amino acid sequence of the light chain variable region of the anti-CD3 domain may include: g) as SEQ ID NO.92, SEQ ID NO.94, SEQ ID NO.96, SEQ ID NO.98, SEQ ID NO.100 and, or the amino acid sequence shown in one of SEQ ID NO.102; or, h) with SEQ ID NO.92, SEQ ID NO.94, SEQ ID NO.96, SEQ ID NO.98, SEQ ID NO. 100, or the amino acid sequence of one of the amino acid sequences shown in SEQ ID NO. 102 with more than 80% sequence identity and having the amino acid sequence function defined in g). Specifically, the amino acid sequence in the h) specifically refers to: such as SEQ ID NO.92, SEQ ID NO.94, SEQ ID NO.96, SEQ ID NO.98, SEQ ID NO.100, or SEQ ID NO. The amino acid sequence shown in one of 102 has been substituted, deleted or added one or more (specifically can be 1-50, 1-30, 1-20, 1-10, 1-5, or 1- 3) amino acids, or adding one or more (specifically 1-50, 1-30, 1-20, 1-10, 1- 5, or 1-3) amino acids, and have amino acids such as SEQ ID NO.92, SEQ ID NO.94, SEQ ID NO.96, SEQ ID NO.98, SEQ ID NO.100, or SEQ ID NO. A functional polypeptide fragment of one of the polypeptide fragments shown in ID NO. 102. The amino acid sequence in h) can have one of SEQ ID NO.92, SEQ ID NO.94, SEQ ID NO.96, SEQ ID NO.98, SEQ ID NO.100, or SEQ ID NO.102 Concordance above 80%, 85%, 90%, 93%, 95%, 97%, or 99%. In a specific embodiment of the present invention, the heavy chain variable region sequence of the anti-CD3 structural domain can be SEQ ID NO.95, and the light chain variable region sequence of the anti-CD3 structural domain can be SEQ ID NO.96. The sequence of the ScFv form is shown in SEQ ID NO.103, or SEQ ID NO.104. In another specific embodiment of the present invention, the sequence of the heavy chain variable region of the anti-CD3 domain can be SEQ ID NO.93, and the sequence of the light chain variable region of the anti-CD3 domain can be SEQ ID NO.94. The sequence of the ScFv form is shown in SEQ ID NO.105, or SEQ ID NO.106. The CD3 may be of human origin, non-human primate (eg, monkey) origin, and/or murine origin.

Among the bispecific antibodies provided by the present invention, the bispecific antibodies may also include a domain for prolonging serum half-life. The domain for prolonging serum half-life can be selected from the Fc domain of mammalian IgG, albumin, albumin binding domain (ABD) or polyethylene glycol (PEG) and the like. The domain for prolonging serum half-life can specifically include: i) a combination of amino acid sequences shown in SEQ ID No. 107, SEQ ID No. 108, or SEQ ID No. 156; or, j) and SEQ ID No. 107. The amino acid sequence shown in one of SEQ ID No. 108, or SEQ ID No. 156 has more than 80% sequence identity and has the amino acid sequence function as defined in i). Specifically, the amino acid sequence in j) specifically refers to: the amino acid sequence shown in one of SEQ ID No. 107, SEQ ID No. 108, or SEQ ID No. 156 is substituted, deleted, or added with one or more (specifically can be 1-50, 1-30, 1-20, 1-10, 1-5, or 1-3) amino acids, or at the N-terminal and/or C- It is obtained by adding one or more (specifically, 1-50, 1-30, 1-20, 1-10, 1-5, or 1-3) amino acids to the end, and has amino acids such as The functional polypeptide fragment of one of SEQ ID No. 107, SEQ ID No. 108, or SEQ ID No. 156 may, for example, be capable of extending the serum half-life of a bispecific antibody. The amino acid sequence in said j) can have 80%, 85%, 90%, 93%, 95%, 97%, 80%, 85%, 90%, 93%, 95%, 97%, SEQ ID No. or more than 99% consistency.

In the bispecific antibody provided by the present invention, the anti-CLD18A2 single-domain antibody, the anti-CD3 domain and the domain for prolonging serum half-life further include a linker peptide. In the bispecific antibody, a plurality of linker peptide fragments can be included, for example, an anti-CLD18A2 single-domain antibody, a linker peptide can be included between the anti-CD3 domains, for example, an anti-CD3 domain, a domain for prolonging serum half-life Linking peptides may be included in between. The connecting peptide fragment can usually be a flexible polypeptide of suitable length composed of glycine (G) and/or serine (S) and/or alanine (A) and/or threonine (T), which can maintain the bispecific antibody Correct folding of each domain of the molecule, and mutual flexibility, for example, the amino acid sequence of the linker peptide fragment may include, for example, (GS)n, (GGS)n, (GGSG)n, (GGGS)nA, (GGGGS) Sequences of nA, (GGGGA)nA, (GGGGG)nA, etc., wherein n is selected from an integer between 1-10. In a specific embodiment of the present invention, the amino acid sequence of the connecting peptide may include: GGGGSGGGS (SEQ ID NO. 157), GGGGGGSGGSGGSGGSGG (SEQ ID NO. 158), GGGGSGGGGSGGGGS (SEQ ID NO. 159) and the like.

The bispecific antibody provided by the present invention, its structure can be one of the following:

(a) S1 structure: the C-terminus of anti-CLD18A2 single-domain antibody (V _HH/CLD18A2 ) was linked to anti-CD3 single-chain antibody (scFv _CD3 ) through a linking peptide. For example, the bispecific antibody can include, from N-terminal to C-terminal, an anti-CLD18A2 single domain antibody, a linking peptide, an anti-CD3 single-chain antibody (ie _{, the structure of VHH/CLD18A2} -linking peptide-scFv _CD3 ), and, for example, anti-CD3 From the N-terminal to the C-terminal, the single-chain antibody may sequentially include its heavy chain variable region fragment, connecting peptide, light chain variable region fragment (ie _{, the structure of VH} -connecting peptide- _VL ), or sequentially include its light chain. variable region fragment, connecting peptide, a heavy chain variable region fragment (i.e., V _L - linker peptide structure -V _H).

(b) S2 structure: the C-terminus of the anti-CLD18A2 single-domain antibody ( _VHH/CLD18A2 ) was linked to the albumin-binding domain (ABD) through a linker peptide, and then to the anti-CD3 single-chain antibody (scFv _CD3 ) through the linker peptide. For example, a bispecific antibody can include, from N-terminal to C-terminal, anti-CLD18A2 single domain antibody, linker peptide, ABD, linker peptide, anti-CD3 single chain antibody (ie _VHH/CLD18A2 -linker peptide-ABD-linker peptide-scFv _{The structure of CD3} ), for another example, the anti-CD3 single-chain antibody can sequentially include its heavy chain variable region fragment, connecting peptide, light chain variable region fragment (ie, _VH -connecting peptide- _VL ) from the N-terminus to the C-terminus. structure), or sequentially including its light chain variable region fragment, connecting peptide, and heavy chain variable region fragment (ie, the structure of _VL -connecting peptide- _VH).

(c) S3 structure: anti CLD18A2 single domain antibodies (V _{HH / CLD18A2)} connected via the C-terminus of the peptide to anti-CD3 single-chain antibody (the scFv _CD3) is connected, and then connected binding domain (ABD) to albumin via a linker peptide. For example, a bispecific antibody may include, in order from N-terminus to C-terminus, an anti-CLD18A2 single domain antibody, a linker peptide, an anti-CD3 single chain antibody, a linker peptide, an ABD (ie, _VHH/CLD18A2 -linker peptide-scFv _CD3 -linker peptide- The structure of ABD), for another example, the anti-CD3 single-chain antibody can sequentially include its heavy chain variable region fragment, connecting peptide, and light chain variable region fragment from the N-terminus to the C-terminus (ie, the _VH -connecting peptide- _VL) . structure), or sequentially including its light chain variable region fragment, connecting peptide, and heavy chain variable region fragment (ie, the structure of _VL -connecting peptide- _VH).

(d) S4 structure: a heterodimer structure composed of anti-CLD18A2 single-domain antibody (V _HH/CLD18A2 ), anti-CD3 domain and IgG Fc fragment. The amino acid sequence of one of the IgG1 Fc variants in the S4 structure of the bispecific antibody may include SEQ ID NO. 107, and the amino acid sequence of the other may include SEQ ID NO. 108. The CH3 domains of the two variants are modified to form asymmetrical pestle-hole structure and form a stable heterodimeric structure. At the same time, Fc has been mutated, such as L234A and L235A mutations, the purpose is to reduce a biological effect of Fc, that is, to reduce the affinity of Fc with FcγRIII on NK cells, so as to avoid possible NK cells. kill.

In a specific embodiment of the present invention, the amino acid sequence of the bispecific antibody may include the sequence shown in one of SEQ ID NO. 109-118, wherein SEQ ID NO. Single domain antibody ABD (SEQ ID NO.156) structure to prolong drug half-life, while SEQ ID NO.115 and SEQ ID NO.116 form a heterodimer to prolong half-life through Fc, SEQ ID NO.117 and SEQ ID NO. 118 forms heterodimers, extending half-life by Fc.

The third aspect of the present invention provides an isolated polynucleotide encoding the anti-CLD18A2 single domain antibody provided by the first aspect of the present invention, or the bispecific antibody provided by the second aspect of the present invention.

The fourth aspect of the present invention provides a construct comprising the isolated polynucleotide provided by the third aspect of the present invention. The construct contains the isolated polynucleotide provided by the second aspect of the present invention. The construct can generally be constructed by inserting the isolated polynucleotide into a suitable vector, and those skilled in the art can select a suitable expression vector. For example, the type of vector may include, but is not limited to, plasmids, phagemids, phage derivatives, animal viruses, cosmids, and the like. For another example, the vector may be an expression vector or a cloning vector.

The fifth aspect of the present invention provides an antibody expression system, the expression system contains the construct provided by the fourth aspect of the present invention or the exogenous polynucleotide provided by the third aspect of the present invention is integrated into the genome, so that it can be Express the single-domain antibody or bispecific antibody of the anti-CLD18A2. The expression system can be a host cell, which can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; a filamentous fungal cell, or a higher eukaryotic cell, such as a mammalian cell . Representative examples are: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast, filamentous fungi, plant cells; insect cells of Drosophila S2 or Sf9; CHO, COS, 293 cells, or Bowes black melanoma cells of animal cells, etc. Methods for introducing constructs into host cells should be known to those skilled in the art, for example, microinjection, biolistic, electroporation, virus-mediated transformation, electron bombardment, calcium phosphate precipitation can be used method, etc.

In a specific embodiment of the present invention, the plasmid may be pPIC9K.

In another specific embodiment of the present invention, the expression system uses Pichia pastoris cells as a host. Yeast has unique advantages in expressing exogenous proteins. For example, yeast has a complete eukaryotic expression system, which can realize the correct folding and post-translational modification of antibody structure; yeast can also realize high-density fermentation to realize exogenous protein. high expression and obtain high unit yield.

In another specific embodiment of the present invention, the plasmid contains a DHFR (dihydrofolate reductase) gene or a GS (glutamine synthase) gene, which is both a selection marker and a gene co-amplification. The gene copy number of the introduced target gene in the host is increased, and the product expression level is also increased accordingly.

In another embodiment of the present invention, the selected mammalian host cells can be NSO, HEK293, PERC6 or CHO, and CHO cells are preferably used as the expression host of the bispecific antibody. CHO cells have a complete set of cellular machinery for synthesizing, assembling and secreting proteins. Therefore, the produced antibody molecules can maintain the correct protein conformation and post-translational glycosylation processing, become functional antibody molecules, and be secreted outside the cell, which is convenient for Isolation and Purification. CHO cells also have the characteristics of secreting and producing less self-proteins and proteases. At the same time, CHO cells can be grown in suspension after serum-free acclimation, which can achieve high-density culture. The target protein yield can reach a high yield of 2-10g/L. Industrial application, and therefore, most of the antibody drugs on the market in the past 30 years have used the CHO cell expression system.

The sixth aspect of the present invention provides a method for preparing the anti-CLD18A2 single domain antibody provided in the first aspect of the present invention, or the bispecific antibody provided in the second aspect of the present invention, comprising the following steps: under conditions suitable for expressing the antibody Next, the antibody expression system provided by the fifth aspect of the present invention is cultured to express the anti-CLD18A2 single domain antibody or bispecific antibody, and the antibody is purified and isolated.

The seventh aspect of the present invention provides an antibody-drug complex, the drug complex comprising the anti-CLD18A2 single domain antibody and the cytotoxic drug provided in the first aspect of the present invention.

The structure of the antibody-drug complex provided by the present invention can be shown in formula 1:

V _HH/CLD18A2 -Linking Peptide-Z-[LD]n I

Wherein, _VHH/CLD18A2 is a single domain antibody against CLD18A2;

Z is an accessory functional region, and Z is selected from a domain and/or a drug-conjugating domain for prolonging serum half-life, or is absent;

D is a cytotoxic drug molecule;

L is the connection chain;

n represents the average number of D coupled, and 0<n≤10, preferably 2≤n≤7; more preferably 3≤n≤6; most preferably 4.

In the antibody-drug complex provided by the present invention, in formula I, the VHH/CLD18A2-connecting peptide-Z part can be a fusion protein, and _{the C-terminus of the anti-CLD18A2 single-domain antibody (VHH/CLD18A2} ) is connected with the connecting peptide through the connecting peptide. Accessory functional domains are linked, or linked to linker chains, and cytotoxic drug molecules. For example, when Z is not present, it can include anti-CLD18A2 single domain antibody and linker peptide in order from N-terminal to C-terminal, and for example, when Z is present, it can include anti-CLD18A2 single-domain antibody from N-terminal to C-terminal in order , linker peptides, domains for extending serum half-life and/or drug-conjugation domains.

In the antibody-drug complex provided by the present invention, _VHH/CLD18A2 is a single-domain antibody against CLD18A2, and the _VHH/CLD18A2 can be monovalent, that is, it includes an antigen-binding site, and the _VHH/CLD18A2 can also be To be multivalent, that is, it may include two or more antigen-binding sites of the same or different sequences, and these antigen-binding sites may be in a tandem structure.

In the antibody-drug complex provided by the present invention, an accessory functional region (Z) may be included. The accessory functional region may include, but is not limited to, an immunoglobulin Fc region, a serum albumin fragment, a polyethylene glycol fragment (PEG), a serum albumin binding domain (ABD), a polypeptide chain, an antibody or a gelatin-like unit A combination of one or more of the above, as well as derivatives of the above structures, including mutants and fusion proteins, and the like.

In a specific embodiment of the present invention, Z is ABD-(GGC) _n , wherein n is an integer greater than or equal to 1, preferably n is 4, 5, 6, 7, 8, 9, or 10. ABD is a single-domain antibody that binds human serum albumin, and (GGC) _n provides a drug-conjugated cysteine group.

In a specific embodiment of the present invention, Z is (PAEC)n, wherein, n is an integer greater than or equal to 1, preferably n is 4, 5, 6, 7, 8, 9, 10, PAEC is composed of proline (P) , alanine (A) and glutamic acid (E) and cysteine (C). More specifically, the amino acid sequence of Z can include the sequence shown in SEQ ID NO.124. This sequence can significantly prolong the half-life in vivo and increase the conjugation site of the drug.

In a specific embodiment of the present invention, Z is an immunoglobulin Fc region.

In another specific embodiment of the present invention, the human immunoglobulin Fc region includes a mutation for altering Fc-mediated effector function, the effector function including one of CDC activity, ADCC activity, ADCP activity or various combinations.

In another specific embodiment of the present invention, the immunoglobulin is selected from a combination of one or more of IgG, IgA1, IgA2, IgD, IgE, and IgM, and the IgG is selected from IgG1, IgG2, IgG3 or IgG4 A combination of one or more of the subtypes.

In another specific embodiment of the present invention, the amino acid sequence of the immunoglobulin Fc region may include the sequence shown in one of SEQ ID NO. 119-123.

In another specific embodiment of the present invention, the accessory functional region is selected from the Fc portion of the human immunoglobulin IgG1 constant region, and the sequence comprises the hinge region and CH2 and CH3, such that the antibody-drug complex forms the dimerization of formula 1 body structure, and maintain the CDC and ADCC roles of IgG1 constant region Fc. In another specific embodiment of the present invention, the accessory functional region is selected from a single domain antibody that specifically binds to human serum albumin.

In the antibody-drug complex provided by the present invention, the connecting peptide fragment can usually be a segment of suitable length composed of glycine (G) and/or serine (S) and/or alanine (A) and/or threonine ( T) is a flexible polypeptide that can maintain the correct folding of each domain of the bispecific antibody molecule and the flexibility of each other. For example, the amino acid sequence of the connecting peptide fragment can include such as (GS)n, (GGS)n, ( GGSG)n, (GGGS)nA, (GGGGS)nA, (GGGGA)nA, (GGGGG)nA and other sequences, wherein n is selected from an integer between 1-10. In a specific embodiment of the present invention, the amino acid sequence of the linking peptide may include: GGGGSGGGS (SEQ ID NO. 160).

In the antibody-drug complex provided by the present invention, a linker chain may be included. A linker can usually be used to connect the cytotoxic small molecule drug and the anti-CLD18A2 single domain antibody fusion protein, and a linker that can bind the reactive functional group of the drug molecule and the amino acid side chain can usually be used. Linking chains can generally be divided into two categories: degradable linking chains and non-degradable linking chains. The degradable linking chain may include: hydrazones, cis-aconiyl, hydrazide bonds, disulfide bonds, peptide bonds, β-glucuronic acid-based bonds, etc.; Linking chains of the non-degradable type may include succinimide-thioether linkages and the like. Specifically, for example, N-succinimidyl 4-(2-pyridyldithio)valerate (SPP), N-succinimidyl(4-iodoacetyl)aminobenzoate ( SIAB), N-succinimidyl 4-(maleimidomethyl)cyclohexanecarboxylate (SMCC), 6-maleimidohexanoyl (MC), maleimidopropyl Acyl (MP), valine-citrulline (VC), alanine-phenylalanine (ala-phe), p-aminobenzyloxycarbonyl (PAB), etc. and any combination of the above linkers. These linking chains can be used alone or in combination with each other, for example, MC-VC-PAB, etc. In a specific implementation of the present invention, the chemical structural formula of the linking chain of L can be as follows:

-B _b -S _s -A _a -S` <sub>s`</sub> - II

Wherein, B is a covalent binding unit covalently bound to the amino acid on the anti-CLD18A2 single-domain antibody fusion protein, and b is 0 or 1;

S and S` are interval units, and s and s` are each independently an integer from 0 to 3;

A is an amino acid unit; a is an integer of 0-12.

In the linking chain, a covalent binding unit may be included. For example, when b is 1, a group with electrophilic properties on the linking chain can react with nucleophilic cysteine or selenocysteine on the anti-CLD18A2 single domain antibody fusion protein to form a co- price key. Groups of electrophilic nature include, but are not limited to, maleimide and haloacetamide groups. The group with electrophilic properties on the intermediate of the drug-linking chain can also react with the nucleophilic cysteine or selenocysteine on the anti-CLD18A2 single domain antibody fusion protein to form a covalent bond. The linker chain can be a branched structure for covalently binding more than one drug moiety to the hydrophilic polypeptide bound to the targeting ligand. Branched linker chains can increase the molar ratio of drug to antibody, ie, loading.

When the anti-CLD18A2 single-domain antibody fusion protein sequence contains an unnatural amino acid, the linking chain may have a reactive functional group that has the ability to react with an electrophilic group present on the anti-CLD18A2 single-domain antibody fusion protein Nucleophilic group. Useful electrophilic groups on anti-CLD18A2 single domain antibody fusion proteins include, but are not limited to, aldehyde and ketone carbonyl groups. The heteroatoms of the nucleophilic group linking the chain can react with the electrophilic group on the anti-CLD18A2 single domain antibody fusion protein and form a covalent bond. Useful nucleophilic groups on the linking chain include, but are not limited to, hydrazides, oximes, amino groups, hydrazines, thiosemicarbazones, hydrazine carboxylates, and aryl hydrazides. The electrophilic group on the anti-CLD18A2 single domain antibody fusion protein provides a convenient location for binding (attaching) the linker chain.

The bis-maleimide reagent enables the sulfhydryl group on the Nanobody derivative to bind to the sulfhydryl group-containing drug moiety or linker intermediate in a sequential or simultaneous manner.

In the linking chain, amino acid units may be included, and the amino acid units usually include amino acid residues. In formula II, A may be a dipeptide, tripeptide, tetrapeptide, pentapeptide, hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide, undecapeptide or dodecapeptide unit. Amino acid residues comprising amino acid units include those naturally occurring as well as minimal amino acids and non-naturally occurring amino acid analogs, such as citrulline. The amino acid unit can be enzymatically cleaved with one or more enzymes, including tumor-associated proteases, to release the drug moiety (-D).

In the connecting chain, a spacer unit (S or S') may be included. The spacer unit may typically link the amino acid unit (-A-) to the drug moiety (D) in the presence of the spacer unit; alternatively, the spacer unit may link the Bb unit of formula II to the drug moiety in the absence of the amino acid unit. The spacer unit also links the drug moiety to the targeting ligand derivative unit when neither the amino acid unit nor the B _{b unit is present.} Spacer units are generally of two types: self-immolative and non-self-eliminating. A non-self-eliminating spacer unit is one in which some or all of the spacer unit remains bound to the drug moiety after cleavage from the ligand derivative-drug complex, especially after enzymatic cleavage of the amino acid unit.

The antibody-drug complexes provided by the present invention may include cytotoxic drug molecules. The cytotoxic molecule is generally any compound or group that has a cytotoxic or cytostatic effect. These cytotoxic molecules can include: (i) chemotherapeutic agents that can act as tubulin inhibitors, mitotic inhibitors, topoisomerase inhibitors, or DNA intercalators; (ii) protein toxins that can act enzymatically ; and (iii) radioisotopes.

Typical cytotoxic molecules may include, but are not limited to, maytansinoids, auristatin, dolastatin, trichothecene, CC1065, calicheamicin ( Calicheamicin and other enediyne antibiotics, taxanes, anthracyclines and their stereoisomers, isosteres, analogs or derivatives or combinations thereof. More specifically, the monomethyl auristatin can be monomethyl auristatin E (MMAE) or monomethyl auristatin F (MMAF), and the maytansinoids can be N2'-deacetyl -N2'-(3-mercapto-1-oxopropyl)-maytansine (DM1), N2'-deacetyl-N2'-(4-mercapto-1-oxopentyl)-maytansine (DM3) and N2'-deacetyl-N2'-(4-mercapto-4-methyl-1-oxopentyl)-maytansine (DM4).

In the antibody-drug complex shown in formula I, the drug moiety (D) can also include a camptothecin derivative 7-ethyl-10-hydroxycamptothecin (SN38), which is based on topoisomerase as an action target Anticancer drugs that inhibit DNA synthesis.

In the antibody-drug complex shown in formula I, protein toxins include but are not limited to: diphtheria toxin A chain, non-binding active fragment of diphtheria toxin, exotoxin A chain, ricin A chain, acacia soybean protein A chain ( abrin A chain), modeccin A chain, alpha-sarcin, Aleurites fordii protein, dianthin proteins, curcin , crotonin (crotin), gelonin (gelonin), mitogellin (mitogellin), restricted aspergillin (restrictocin), phenomycin (phenomycin), enomycin (enomycin) and so on.

In the antibody-drug complex of formula I, therapeutic radionuclides include, but are not limited to: radioisotopes of 32P, 33P, 90Y, 125I, 131I, 131In, 153Sm, 186Re, 188Re, 211At, 212Bi, 212Pb and Lu.

The eighth aspect of the present invention provides the preparation method of the antibody-drug complex provided by the seventh aspect of the present invention, including: on the basis of the anti-CLD18A2 single-domain antibody, it can be formed by increasing the tandem connection of the single-domain antibody and the setting of the accessory functional region. A fusion protein of an anti-CLD18A2 single domain antibody, the fusion protein is cross-linked with a cytotoxic molecule to provide the antibody-drug complex. The multivalent tandem of anti-CLD18A2 single-domain antibody can increase the binding ability to human CLD18A2, and the accessory functional region can be designed according to different purposes, which can prolong the half-life in vivo, or improve the hydrophilicity, or increase the coupling of toxic chemical groups site.

In a specific embodiment of the present invention, two anti-CLD18A2 single-domain antibodies of the same sequence are connected in series, and a six-repeat structure of three amino acids of human serum albumin binding sequence (ABD) and GGC (SEQ ID NO. 125), after the formed single-domain antibody fusion protein is cross-linked with linker-MMAE through sulfhydryl groups, a single-domain antibody-drug complex with a DAR of 4.42 is obtained.

In another specific embodiment of the present invention, an anti-CLD18A2 single-domain antibody of the same sequence is connected in series at the C-terminus of the anti-CLD18A2 single-domain antibody, and a highly hydrophilic polypeptide is fused at the C-terminus, and 6 Cys amino acids are introduced to form The single-domain antibody derivative (SEQ ID NO. 126) was cross-linked with linker-MMAE through sulfhydryl groups to obtain an antibody-drug complex with a DAR of 4.10.

In another specific embodiment of the present invention, a single-domain antibody derivative (SEQ ID NO. 147) formed by fusing human IgG1FC to the C-terminus of anti-CLD18A2 single-domain antibody, after cross-linking with linker-MMAE through interchain sulfhydryl groups , an antibody-drug complex with a DAR of 4.21 was obtained.

In the preparation method of the antibody-drug complex provided by the present invention, those skilled in the art can select various well-known organic chemical reactions, conditions and reagents to provide the antibody-drug complex provided by the seventh aspect of the present invention. For example, when the cross-linking amino acid is cysteine, it can include: (1) The cysteine group on the single-domain antibody derivative reacts with the linking chain reagent, thereby forming a ligand derivative-linking through a covalent bond chain intermediate, which is subsequently reacted with the activated drug moiety D; or, (2) the nucleophilic group of the drug moiety is reacted with the linking chain reagent, thereby forming a drug-linking chain intermediate by covalent bonding, which is subsequently reacted with the single domain antibody The cysteine group on the derivative reacts. Coupling methods (1) and (2) can be used for a variety of targeting ligands, drug moieties and linkers to prepare ligand derivative-drug complexes of formula I. Cysteine sulfhydryl groups are nucleophilic and can react with electrophilic groups on linker reagents and drug-linker intermediates to form covalent bonds, said drug-linker intermediates including: (i) Active esters such as NHS esters, HOBt esters, haloformates and acid halides; (ii) alkyl and benzyl halides such as haloacetamides; (iii) aldehydes, ketones, Carboxyl and maleimide groups; and (iv) disulfides exchanged by sulfide, including pyridyl disulfides. Nucleophilic groups on the drug moiety can include, but are not limited to, amine, sulfhydryl, hydroxyl, hydrazide, oxime, hydrazine, thiosemicarbazide, hydrazine carboxylate, and arylhydrazide groups, which can interact with the linker moiety and The electrophilic groups on the linker reagent react to form a covalent bond.

Under certain conditions, such as cell-expressed full-length antibodies, intra- and inter-chain disulfide bonds exist within the molecule. ) or TCEP (tris (2-carboxyethyl) phosphine hydrochloride treatment to reduce the interchain disulfide bond of the antibody, and then react with the linking chain reagent.

When the cross-linking amino acid is selenocysteine, refer to the scheme of Xiuling Li et al. (Bioconjug Chem. 201518; 26(11): 2243-8.), when the cross-linking amino acid is an unnatural amino acid, refer to Han The protocol of Xiao et al. (Angew Chem Int Ed Engl. 2013 Dec 23;52(52):14080-3).

The ninth aspect of the present invention provides a pharmaceutical composition, comprising the anti-CLD18A2 single domain antibody provided by the first aspect of the present invention, the bispecific antibody provided by the second aspect of the present invention, and the antibody provided by the fifth aspect of the present invention The culture of the expression system, or the antibody-drug complex provided by the seventh aspect of the present invention. In the pharmaceutical composition, the content of the anti-CLD18A2 single domain antibody, bispecific antibody, culture, or antibody-drug complex is usually a therapeutically effective amount. The pharmaceutical composition may also include a pharmaceutically acceptable carrier. The carrier may include various excipients and diluents which are not themselves necessary for the active ingredient and which are not unduly toxic after administration. Suitable carriers will be well known to those skilled in the art, for example, a thorough discussion of pharmaceutically acceptable carriers can be found in Remington's Pharmaceutical Sciences (Mack Pub. Co., N.J., 1991). In a preferred embodiment of the present invention, the pharmaceutical composition can be administered by injection route, especially intravitreal injection, so the pharmaceutical composition is preferably a powder for injection (such as freeze-dried powder for injection) and a liquid preparation .

A tenth aspect of the present invention provides a cell comprising a membrane-bound chimeric antigen receptor, wherein the chimeric antigen receptor includes a transmembrane domain, an intracellular domain and an extracellular domain, and the extracellular domain includes the first In one aspect the anti-CLD18A2 single domain antibodies are provided. The surface of the cells provided by the present invention can express the anti-CLD18A2 single-domain antibody, and the cells can usually bind to their corresponding antigens through the extracellular domain, and more specifically can bind to the anti-CLD18A2 single-domain antibody through the extracellular domain. Claudin18.2 antigen, when the polypeptide binds to its corresponding antigen, the cells can be activated and/or stimulated to proliferate and kill the corresponding target cells. Specifically, the cells can be T lymphocytes, NK cells, macrophages and the like.

In the cells provided by the present invention, the chimeric antigen receptor may include an extracellular domain, and the extracellular domain may generally include an antibody targeting the target antigen, that is, the above-mentioned anti-CLD18A2 single domain antibody.

In the cells provided by the present invention, the chimeric antigen receptor can also include a transmembrane domain, and the transmembrane domain can mainly fix the chimeric antigen receptor on the cell membrane of T cells. The transmembrane domain may include sequences of CD4, CD8, CD8b, CD28 transmembrane domains, subunits of T cell receptors such as alpha, beta, gamma or delta, subunits of IL-2 receptors (alpha chain), low Affinity Nerve growth factor receptor (LNGFR or p75) subunit (beta chain or gamma chain), or the transmembrane domain of the subunit chain of Fc receptors. In a specific embodiment of the present invention, the transmembrane domain may comprise the amino acid sequence shown in SEQ ID NO:128. In another specific embodiment of the present invention, the transmembrane domain may comprise the amino acid sequence shown in SEQ ID NO:129.

In the cells provided by the present invention, the chimeric antigen receptor may also include an intracellular domain. The intracellular domain may be the cytoplasmic sequences of native T cell receptors and coreceptors that cooperate to initiate signal transduction upon antigen binding, as well as any derivatives or variants of these sequences, and any synthetic functional equivalents sequence. The intracellular domains can generally be divided into two broad categories, and can include, for example, costimulatory domains and/or signaling domains. The co-stimulatory domain can typically provide a secondary or co-stimulatory signal for full cell activation in an antigen-independent manner, which can bind to a cognate co-stimulatory ligand on an antigen-presenting cell to enhance T cell responses, for example by increasing Proliferation activation, differentiation, etc. The costimulatory domain may include CD28, CD27, 4-1BB (CD137), OX40 (CD134), ICOS (CD278), CD30, CD40, PD-1 (CD279), CD2, CD7, NKG2C (CD94), B7 - Intracellular domain of H3 (CD276). In a specific embodiment of the present invention, the costimulatory domain may include CD28 and/or CD137, and the amino acid sequence thereof may include the amino acid sequences shown in SEQ ID NO: 130 and SEQ ID NO: 131. The signaling domain generally refers to a region capable of transducing a signal into the cell when the above-mentioned antibody recognizes an antigen on the surface of the target cell. The signaling domain may be an immunoreceptor tyrosine-based activation motif (ITAM), which is a well-defined signaling motif typically present in the intracytoplasmic tail of various receptors and used as syk/ Binding site for zap70-like tyrosine kinases. Specifically, the signaling domains may include the signaling domains of CD3ζ, FcRγ, FcRβ, FcRε, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d. In a specific embodiment of the present invention, the signaling domain includes the signaling domain of CD3ζ, and its amino acid sequence may include the amino acid sequence shown in SEQ ID NO: 132. Natural TCRs contain the CD3ζ signaling molecule, so the use of this effector domain is closest to the TCR constructs that occur in nature.

In the cells provided by the present invention, the chimeric antigen receptor may also include a hinge region, and the hinge region includes the CD8 dumpling region or the CH2 and/or CH3 domains of IgG1 or IgG4, preferably the dumpling chain of CD8b Area. In a specific embodiment of the present invention, the hinge region may comprise the amino acid sequence shown in SEQ ID NO:127.

In the cells provided by the present invention, the chimeric antigen receptor generally includes an anti-CLD18A2 single domain antibody, a transmembrane domain (TM) and an intracellular domain in sequence from the N-terminal to the C-terminal, and the intracellular domain starts from the N-terminal To the C-terminus, it includes a costimulatory domain (ITAM) and a signaling domain (ζ). A hinge region may also be included, and the hinge region may typically be located between the anti-CLD18A2 single domain antibody, transmembrane domains.

In a specific embodiment of the present invention, the chimeric antigen receptor may include anti-CLD18A2 single domain antibody, CD8bhinge, CD8bTM, CD28ITAM, and CD3ζ in sequence from the N-terminus to the C-terminus.

In another specific embodiment of the present invention, the chimeric antigen receptor may include anti-CLD18A2 single domain antibody, CD8bhinge, CD8bTM, CD137ITAM, CD3ζ in sequence from the N-terminus to the C-terminus.

In another specific embodiment of the present invention, the chimeric antigen receptor may include anti-CLD18A2 single domain antibody, CD8bhinge, CD28TM, CD28ITAM, CD137ITAM, and CD3ζ in sequence from the N-terminus to the C-terminus.

In the cells provided by the present invention, the chimeric antigen receptor may further comprise a signal peptide, and the signal peptide is mainly used to express the chimeric antigen receptor on the cell membrane. In a specific embodiment of the present invention, the amino acid sequence shown in SEQ ID NO: 136.

In another specific embodiment of the present invention, the amino acid sequence of the chimeric antigen receptor includes the sequence shown in one of SEQ ID NO. 137-140.

The eleventh aspect of the present invention provides a culture of the anti-CLD18A2 single domain antibody provided by the first aspect of the present invention, the bispecific antibody provided by the second aspect of the present invention, and the expression system of the antibody provided by the fifth aspect of the present invention , or the use of the antibody-drug complex provided by the seventh aspect of the present invention, the pharmaceutical composition provided by the ninth aspect of the present invention, or the cell provided by the tenth aspect of the present invention in preparing a medicine. Specifically, the drug may be a drug for treating tumors. The tumor may be a solid tumor or a hematological tumor, more specifically, colon cancer, lung cancer, liver cancer, breast cancer, esophageal cancer, head and neck cancer, skin cancer, kidney cancer, leukemia, coad (colon cancer), lihc (hepatocellular carcinoma) ), ov (ovarian serous cystadenocarcinoma), ucec (endometrial cancer), thca (thyroid cancer), skcm (skin melanoma), luad (lung adenocarcinoma), hnsc (head and neck squamous cell carcinoma), gbm (glioma multiforme), prad (prostate cancer), thym (thymic carcinoma), lgg (brain low-grade glioma), read (rectal adenocarcinoma), pcpg (pheochromocytoma and paraganglioma) ), esca (esophageal cancer), kirc (renal clear cell carcinoma), csc (cervical squamous and adenocarcinoma), blca (bladder urothelial carcinoma), kirp (renal papillary cell carcinoma), paad (pancreatic carcinoma), stad (gastric cancer), kich (renal chromophobe carcinoma), brca (breast invasive carcinoma), lusc (lung squamous cell carcinoma), sarc (sarcoma), LAML (acute myeloid leukemia), etc.

In the use provided by the present invention, the above-mentioned substances can be a single medicinal component, or can be combined with other active components to jointly treat tumors.

The twelfth aspect of the present invention provides a treatment method, comprising administering to an individual a therapeutically effective amount of the anti-CLD18A2 single domain antibody provided in the first aspect, the bispecific antibody provided in the second aspect of the present invention, and the fifth aspect of the present invention. The culture of the antibody expression system provided by the aspect, or the antibody-drug complex provided by the seventh aspect of the present invention, the pharmaceutical composition provided by the ninth aspect of the present invention, or the cell provided by the tenth aspect of the present invention. The treatment methods provided by the present invention can be used to treat indications including but not limited to tumors. The "individual" generally includes mammals, which may be rodents, artiodactyls, odd ungulates, lagomorphs, non-human primates, primates, etc., which may be be monkeys, apes or humans, such as mammals, dogs, cats, horses, sheep, pigs, cattle, etc., which may benefit from treatment with the T lymphocytes, or compositions. The "therapeutically effective amount" generally refers to an amount which, after an appropriate period of administration, is effective in treating the diseases listed above. Selection of a preferred therapeutically effective amount can be determined by one of ordinary skill in the art based on a variety of factors (eg, through clinical trials), such as tumor growth, proliferation, recurrence and/or metastasis when the above-mentioned substances are used in the individual to which they are administered Can be inhibited, more specifically, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% of tumor growth, proliferation, recurrence and/or metastasis or 99% partially suppressed.

The anti-CLD18A2 single domain antibody provided by the present invention has good affinity for Claudin18.2, and can be used to further construct bispecific antibodies, antibody-drug complexes, chimeric antigen receptors, etc. Antibodies, antibody-drug complexes, CAR-T cells, etc. have good targeting and killing effects on target cells, and have good industrialization prospects.

The invention of the present application is further illustrated by the following examples, but the scope of the present application is not limited thereby.

Unless otherwise specified, the experimental methods, detection methods and preparation methods disclosed in the present invention all adopt the conventional molecular biology, biochemistry, chromatin structure and analysis, analytical chemistry, cell culture, recombinant DNA technology and related fields in the technical field. conventional technology. These techniques have been well described in the existing literature. For details, please refer to Sambrook et al. MOLECULAR CLONING: A LABORATORY MANUAL, Second edition, Cold Spring Harbor Laboratory Press, 1989 and Third edition, 2001; Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, 1987 and periodic updates; the series METHODS IN ENZYMOLOGY, Academic Press, San Diego; Wolfe, CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998; METHODS IN ENZYMOLOGY, Vol.304, Chromatin (PMWassarman and APWolffe, eds.), Academic Press, San Diego, 1999; and METHODS IN MOLECULAR BIOLOGY, Vol. 119, Chromatin Protocols (PBBecker, ed.) Humana Press, Totowa, 1999, et al.

Example 1: Preparation of anti-CLD18A2 single domain antibody

The human Claudin18.2 full-length gene expression vector pCDNA3.1/CLDN18A2 was constructed and electrotransfected into CHO-S cells, and then positive clones were screened by G418 to obtain the CHO-S cell line CHO-S-Claudin18 expressing Claudin18.2. 2. CHO-S-Claudin18.2 cells were subcutaneously immunized with alpaca at multiple points and supplemented with adjuvant. After 3 to 4 times of immunization, blood was drawn to check that the antibody titer met the requirements, and then blood was drawn to extract PBMC, and total RNA was extracted and reverse transcribed. cDNA, and then two rounds of PCR were used to obtain the gene sequence against the single-domain antibody VHH, which was then ligated with a phage display vector, transformed into the display vector pcomb3xss (Addgene plasmad#63890; RRID:Addgene_63890), and ligated by T4 ligase (Takara). The ligation product was transformed into electrotransformation competent cells ER2738 to construct an anti-Claudin18.2 single domain antibody library. The constructed Claudin18.2 single domain antibody library was packaged with helper phage M13KO7 (NEB). The packaged phage was subjected to subtractive hybridization between CHO-S and CHO-S expressing Claudin18.2, as well as multiple rounds of panning, and finally multiple high-affinity single-domain antibodies that specifically bind to Claudin18.2 were obtained. Finally, a group of humanized high-affinity single-domain antibodies that specifically bind to Claudin18.2 are obtained, as shown in the sequences of SEQ ID NOs: 67-90.

The high-fidelity enzyme GVP8 (General Biosystems (Anhui) Co., Ltd.) was used for PCR amplification with the synthetic humanized sequence as a template, a signal peptide sequence was introduced at the 5' end of the sequence, and a 6xHis histidine tag code was introduced at the 3' end. Sequence, PCR product electrophoresis and gel cutting to recover a band of about 500 bp, and the recovered PCR product was recombined with the pET32a+ vector (Novagen) digested with endonuclease NdeI and EcoRI with a recombination kit (Nearshore Protein Technology Co., Ltd.) Connected, constructed E. coli expression plasmid, transformed into E. coli competent Top10F', coated with ampicillin resistance plate, and cultivated overnight in an incubator at 37°C. The clones on the ampicillin-resistant plate were respectively picked, and the plasmids were extracted and sequenced to confirm the correct insertion of the sequence on the pET32a+ vector.

The E. coli expression plasmid determined by sequencing was transformed into the E. coli expression host Rosetta (DE3) to construct an E. coli expression strain. Recombinant clones were picked on ampicillin-resistant plates, cultured, and induced to express overnight with 1 mM IPTG at 30°C. The bacterial liquid induced to express overnight was sonicated, centrifuged at 12,000g at 4°C for 10 minutes, and the supernatant was taken and purified with a Ni column (Borgron Biotechnology Co., Ltd.), and the final protein purity reached more than 90%.

Affinities determined by cellular ELISA are shown in Table 1. Specifically, CHO-S-CLD18A2 ^{was plated in a 96-well plate at 5×10 5} /ml cells per well, and blocked with 3% BSA for 1 hour at room temperature. The purified histidine tag-fused CLD18A2 nanobody was used 1% BSA gradient dilution was added to the blocked cells, and incubated at room temperature for 1 hour. After washing, 100 μl/well of mouse anti his tag antibody (R&D Systems, Inc) diluted at 1:5000 was added and incubated for 1 hour at room temperature. After washing, 1:10000 diluted HRP-Goat anti mouse IgG antibody (Thermo Scientific) was added, 100 μl per well, and incubated at room temperature for 1 hour. After washing, TMB was added for color development, and the OD value was detected at 450 nm. The software GraphPad Prism v5.0 was used for data processing and graph analysis, and the EC50 value of Anti-CLD18A2 humanized nanobody to CLD18A2 in cells was obtained to reflect the affinity of the antibody to CLD18A2.

Table 1

Example 2: Preparation of anti-CLD18A2/anti-CD3 bispecific antibody and reference substance

Table 2

According to the relevant sequences shown in Table 2, the S4-14 bispecific antibody converted the amino acid sequence into each base sequence according to the codon preference of CHO cells, and obtained the full-length DNA by gene synthesis (Nanjing GenScript Biotechnology Co., Ltd.). Take each DNA as a template, carry out PCR amplification with high-fidelity enzyme GVP8 (Anhui General Biotechnology Co., Ltd.), insert pCDNA3.1 vector after HindIII/EcoRI digestion, and recombine and connect to construct two expression plasmids (expressing SEQ ID NO. : 115 and SEQ ID NO: 116 sequence), the two plasmids were extracted with the endotoxin-removing plasmid large extraction kit (Biomiga) and mixed 1:1, and the mixed plasmid was mixed with the transfection reagent PEI (Polysciences, Inc.) 1: 3 Mix well and let it stand for 30 min, then add it to HEK293F cells for co-transfection, _{culture in a 37°C, 5% CO 2} shaker incubator for 7 days, and then centrifuge to get the supernatant.

Prepare samples: take 1L of culture medium, centrifuge at 5000rcf for 20 minutes, separate the precipitate, and take the supernatant. Then, filter the cell debris with a 0.22 μm microporous membrane, and check that the turbidity of the sample is below 20 NTU, and retain 1 ml of the sample;

Affinity chromatography: 50ml Protein A chromatography column (Borgron, AA0273) was treated with 0.1M NaOH at a flow rate of 10ml/min for 30min to remove residual protein on the column. It was then equilibrated with 150 mM NaCl 20 mM PB (pH 6.5) for at least 3 column volumes until the pH reached 6.5 and the conductance was around 15 mS/cm. The culture filtrate was passed through a Protein A chromatography column at a flow rate of 10 ml/min with a residence time of about 5 min on the column, and then equilibrated with 150 mM NaCl 20 mM PB (pH 6.5) for at least 3 column volumes. Equilibrate 3 column volumes with 500 mM NaCl 20 mM PB (pH 6.5) followed by 3 column volumes with 100 mM Arg-HAc (pH 5.0). Elute with 100 mM Gly-HCl (pH 3.2), collect the elution peaks according to the absorption value at 280 nm, add 1.0 M NaCit-HCit (pH 6.5) at a volume of 10% to the obtained sample, and mix gently.

Anion exchange chromatography: 50ml Q FF chromatography column (Borgron, AI0024) was treated with 0.5M NaOH at a flow rate of 10ml/min for 30min to remove residual protein on the column. It was then equilibrated with 20 mM PB (pH 6.5) for at least 5 column volumes until the pH reached 6.5. The sample obtained by affinity chromatography was passed through the Q FF chromatography column at a flow rate of 10ml/min, the residence time on the column was about 5min, and the flow-through sample was collected and stored. It was then washed with 500 mM NaCl 20 mM PB (pH 6.5) to remove impurities bound to the column and identified.

Cation exchange chromatography: 50ml SP mustang chromatography column (Borgron, AI0192) was treated with 0.5M NaOH at a flow rate of 10ml/min for 30min to remove residual protein on the column. It was then equilibrated with 20 mM PB (pH 6.5) for at least 5 column volumes until the pH reached 6.5. The flow-through sample obtained by anion exchange chromatography was passed through the SP mustang chromatography column at a flow rate of 10ml/min, and the residence time on the column was about 5min. It was then eluted with 200 mM NaCl 20 mM PB (pH 6.5) and eluted samples were collected.

Preservation of samples: The eluted samples of cation exchange chromatography were sterile filtered at 0.22 μm and stored at 4°C.

According to the related sequences shown in Table 2, the S2-12 bispecific antibody, and the negative controls Anti-C18.2-hu19V3 and Anti-CD3 were prepared according to the vector construction, expression and purification methods provided in Example 1.

All 4 samples prepared above were tested for purity using SEC-HPLC-UV analysis. Detector: Agilent 1100LC; Detection wavelength: 214nm; Mobile phase: 150mM pH7.0PB+5% isopropanol; Chromatographic column: Superdex 200Increase 5/150GL; Running time: 15 minutes; Column temperature 25°C. Purity is greater than 95%.

Example 3: Identification of the function of anti-CLD18A2/anti-CD3 bispecific antibodies

3.1 Cellular activity of anti-CLD18A2/anti-CD3 bispecific antibodies

Construction of luciferase detection cells: Jurkat cells were transformed into NFAT-responsive luciferase system, and the detection cell line Jurkat-PB-NFAT-luc2p was obtained.

Target cell plating: Take an appropriate amount of CHO-S-Claudin18.2 cells cultured in suspension, centrifuge at 800 rpm for 5 min, discard the supernatant, add RPMI1640 medium (containing FBS) to resuspend, count, take the required cells and dilute to the specified concentration, Add to 96-well cell culture plates.

Protein dilution: S2-12 and S4-14 bispecific antibodies prepared by gradient dilution in RPMI1640 medium (containing FBS), as well as negative controls Anti-C18.2-hu19V3 and Anti-CD3 to the specified concentration, and then add an appropriate volume In a 96-well plate, target cells were incubated with antibody protein for 30 min at 37°C, and Jurkat-PB-NFAT-luc2p cells were added.

Jurkat-PB-NFAT-luc2p plating: Dilute an appropriate amount of Jurkat-PB-NFAT-luc2p cells to the specified concentration and add them to a 96-well cell culture plate. Incubate for about 20h for detection.

Photometric value detection: add 10 μl/well of the detection solution provided by the luciferase detection kit (Promega, E2620), shake for 2 min, transfer 60 μl of the solution to a 96-well white plate, and detect in a microplate reader. The software GraphPad Prism v5.0 was used for data processing and graph analysis, and the activation curves and EC50 values of various anti-CLD18A2/anti-CD3 bispecific antibodies for the detection system were obtained, as shown in Figure 1, S2-12, S4- 14 Two bispecific antibodies with different structures produced similar activation ability of the in vitro T cell system, while the negative controls Anti-C18.2-hu19V3 and Anti-CD3 did not have the ability to activate the detection system. It shows that anti-CLD18A2 antibody and anti-CD3 antibody can activate the Claudin18.2 antigen target cells and T cell effector system only when bispecific antibodies are formed, but neither antibody alone has this function.

3.2 In vitro cell killing assay of anti-CLD18A2/anti-CD3 bispecific antibodies

In order to evaluate the cell killing effect of the anti-CLD18A2/anti-CD3 bispecific antibody, the present invention uses T cells (Miaotong Biotechnology) as effector cells to conduct a cytotoxicity test.

Anti-S2-12, S4-14 bispecific antibodies, as well as negative controls Anti-C18.2-hu19V3 and Anti-CD3 were serially diluted, and 50 μl was added to each well. Claudin18.2 and Claudin18.1 stably transfected cells NUGC-4-Claudin18.2 and NUGC-4-Claudin18.1 were washed and resuspended in 5% FBS 1640 medium (Gibco), respectively, to a concentration of about 2×10 ⁵ /ml For cell density, add 50 μl per well to the corresponding 96-well plate. Human T lymphocytes from healthy donors were resuspended in 5% FBS 1640 medium, and 1 × 10 ⁵ cells were added to each well to make the effect-target ratio 10:1. After incubating at 37°C for 4 hours, press The LDH detection kit (Dongren Chemical Technology (Shanghai) Co., Ltd.) detected the release of LDH and evaluated the in vitro cell killing effect of anti-Claudin18.2/anti-CD3 bispecific antibodies.

In the in vitro cytotoxicity experiments, the anti-CLD18A2/anti-CD3 bispecific antibodies S2-12 and S4-14 had a very significant killing effect on NUGC-4-Claudin18.2 with high Claudin18.2 expression, with EC50 values of 24.65pM and 21.15pM, respectively. ; For NUGC-4-Claudin18.1 cells, anti-CLD18A2/anti-CD3 bispecific antibodies had no obvious killing effect (as shown in Figure 2). It shows that the anti-S2-12 and S4-14 bispecific antibodies have specific killing effect on NUGC-4-Claudin18.2 cells in the in vitro experiments with the participation of T lymphocytes, and have no toxicity to cells that do not express Claudin18.2 , reflecting a high degree of killing specificity.

3.3 Tumor inhibitory activity of anti-CLD18A2/anti-CD3 bispecific antibodies

In the present invention, tumor-bearing NSG mice of a xenograft tumor model (patientl derived xenograft, PDX) established by gastric cancer tissue derived from patients are used to analyze the tumor suppressive effect of anti-Claudin18.2/anti-CD3 bispecific antibody. When the tumor grew to ^{about 100mm 3} , the tumor-bearing mice were randomly assigned to 5 mice in each group, and 2×10 ⁷ healthy human PBMC cells were injected intraperitoneally. One day later, tumor-bearing mice were intraperitoneally injected with 5 μg (25 μg/ml, 200 μl PBS), bispecific antibody S2-12, once every two days for 4 weeks, and the tumor volume was recorded twice a week. As well as injection of S4-14, 10 μg (25 μg/ml, 200 μl PBS) once a week for 4 weeks, tumor volume was recorded twice a week.

It can be seen from the experimental results in Figure 3 that the tumor volume of the experimental group gradually decreased over time, and both bispecific antibodies S2-12 and S4-14 had obvious growth inhibitory effects on the transplanted tumors.

Example 4: Preparation of anti-CLD18A2 single-domain antibody fusion protein and control antibody

4.1 Preparation of anti-CLD18A2 single-domain antibody fusion protein

The specific positive sequences and humanized sequences obtained by screening were used as templates, the upstream primer 5'-gtgctgctgctgtgggtgccaggatccaccgggcaggtgcagctcgtggagtc-3' (SEQ ID NO.141) and the downstream primer 5'-gcaggacttgggctcagaagacacggtgaccagggtcccctggcc-3' (SEQ ID NO.142) , use high-fidelity enzyme GVP8 (Anhui General Biotechnology Co., Ltd.) to carry out PCR amplification, the PCR product is electrophoresed and cut into the gel to recover a band of about 400bp, and the recovered PCR product is mixed with a signal peptide and human IgG1Fc sequence (amino acid sequence SEQ NO.119) pCDNA3.1 vector was recombined to construct a cell expression plasmid fused with Anti-CLD18A2 single domain antibody and human IgG1Fc, and the anti-CLD18A2 nanobody human IgG1Fc was extracted with endotoxin removal plasmid kit (Biomiga). The fused cells express the plasmid, mix the plasmid with the transfection reagent PEI (Polysciences, Inc.) 1:3 and let stand for 30 min, then add it to HEK293F cells, and culture at 37°C, 5% CO _{2 in a} shaking incubator for 7 Days later, the supernatant was collected by centrifugation. The supernatant was adjusted to pH 7.0 and loaded onto a ProteinA affinity chromatography column (Borgron Biotechnology Co., Ltd.), eluted with 100% 0.1M Gly-HCl (pH 3.0); the eluent was pre-added with 10% 1M Tris-HCl (pH 8.5). The 100% eluate was diluted to a conductivity of 4ms/cm, adjusted to pH 5.5, centrifuged (8000rpm, 4°C, 10min), and the supernatant was adjusted to pH 5.0 and loaded onto a DSP column (Borgron Biotechnology Co., Ltd. Company), 0-60% eluent (20 mM NaAc, 0.5 M NaCl, pH 5.0) linear elution, flow rate 2 ml/min, 15 min.

4.2 Expression and purification of positive control antibody ch-175D10

The chimeric antibody (named ch-175D10 in the US9751934B2 patent) composed of the heavy chain of SEQ ID NO: 118 and the light chain of SEQ ID NO: 125 in US9751934B2 was used as a control antibody, and the corresponding polynucleotide sequence of its amino acid sequence was compared with that of pCDNA3. 1. The vector was recombined and ligated, and the transient transfection, expression and purification of HEK293F cells were carried out by the same method as in Example 2.

Example 5: Detection of endocytic activity of anti-CLD18A2 single-domain antibody fusion protein

The full-length gene of CLD18A2 (SEQ ID NO. 143) and the gfp gene were linked with an internal ribosome entry site (IRES) sequence and constructed in pCDNA3.1 vector (Life Technologies) to achieve co-expression of CLD18A2 and gfp. The expression plasmid was extracted, and Lipofectamine 3000 (Invitrogen, L3000001) transfection reagent was used to transfect CHO-K1 cells according to the instructions. The next day, the transfected CHO-K1 cells were trypsinized and then seeded in a 96-well plate ^{at a final concentration of 2×10 6 /ml cells.} Conjugate pH-sensitive fluorescent dyes (<pH7 can be excited to fluoresce) with antibodies. When the labeled antibody is endocytosed through receptors, etc., it can be excited to generate fluorescence in an acidic environment, and the endocytosis can be judged by the intensity of fluorescence generated. efficient. On the third day, the culture supernatant of the 96-well plate was removed, and pH-sensitive fluorescein-labeled Anti-C18.2-Fc fusion protein (FC fusion protein of humanized antibody V3 of all 8 Nanobodies) and control antibody were added (The steps were all performed according to the instructions in the pHAb Amine Reactive Dye kit) DMEM dilution, the final concentration of fluorescently labeled antibody was 10 μg/ml, incubated on ice for 1 hour, and washed three times with pre-cooled DMEM. The wells on one of the plates were kept on ice as samples for endocytosis for 0 hours, and the rest were incubated in a 37°C incubator for 3 hours, respectively, and pre-cooled on ice to stop endocytosis. All samples were subjected to fluorescence detection. The results are shown in Figure 4 and Table 3:

table 3

SEQID NO. sample name Mean fluorescence density fold increase 144 Anti-C18.2-hu6V3-Fc 10.5 145 Anti-C18.2-hu7V3-Fc 10.3 146 Anti-C18.2-hu15V3-Fc 9.8 147 Anti-C18.2-hu19V3-Fc 10.9 148 Anti-C18.2-hu20V3-Fc 9.7 149 Anti-C18.2-hu28V3-Fc 8.9 150 Anti-C18.2-hu32V3-Fc 8.5 151 Anti-C18.2-hu69V3-Fc 9.6 152, 153 ch-175D10 7.5

154 isotype control antibody 0.1

The above results show that the humanized antibody selected in the present invention is an endocytic antibody, which can specifically bind to CLD18A2 and mediate endocytosis, while the isotype control antibody has no obvious endocytosis.

Example 6 Preparation of anti-CLDN18A2 single domain antibody-drug complex

6.1 Preparation of anti-CLDN18A2 single-domain antibody fusion protein

hu19V3-hu19V3-ABD-(GGC) ₆ (Ab1)

The design was based on the humanized sequence Anti-C18.2-hu19V3 of the specific positive sequence Anti-C18.2-19 obtained by screening, and the Anti-C18.2-hu19V3 was concatenated twice, and the C-terminal A six-repeat structure of three amino acids of human serum albumin binding sequence (ABD) and GGC is introduced, and the complete sequence is shown in SEQ ID NO.125. The complete base sequence encoding the amino acid sequence shown in SEQ ID NO.125 was synthesized in Universal Biosystems (Anhui) Co., Ltd., and constructed in the pET32a vector, and the expression plasmid was transformed into E. coli expression host Rosetta (DE3) to construct E. coli expression strains. Recombinant clones were picked on ampicillin-resistant plates, cultured, and induced to express overnight with 1 mM IPTG at 30°C. The bacterial liquid induced to express overnight was sonicated, centrifuged at 12,000g at 4°C for 10 minutes, and the supernatant was taken and purified with a Ni column (Borgron Biotechnology Co., Ltd.), and the final protein purity reached more than 90%.

hu19V3-hu19V3-PAEC6(Ab2)

The design was based on the humanized sequence Anti-C18.2-hu19V3 of the specific positive sequence Anti-C18.2-19 obtained by screening, and the Anti-C18.2-hu19V3 was concatenated twice. (Anhui) Co., Ltd. synthesized the base sequence encoding the amino acid sequence shown in SEQ ID NO.155, and at the same time synthesized the PAEC sequence encoding the amino acid sequence shown in SEQ ID NO.124, and constructed the PAEC sequence with 6 repeats in series (PAEC6) , connect PAEC6 to the C-terminus of Anti-C18.2-hu19V3 sequence, construct a complete amino acid base sequence as shown in SEQ ID NO.126, and construct the complete sequence in pET32a vector, and transform the expression plasmid into Escherichia coli The expression host Rosetta (DE3) was used to construct an E. coli expression strain. Recombinant clones were picked on ampicillin-resistant plates, cultured, and induced to express overnight with 1 mM IPTG at 30°C. The bacterial liquid induced to express overnight was sonicated, centrifuged at 12,000g at 4°C for 10 minutes, and the supernatant was taken and purified with a Ni column (Borgron Biotechnology Co., Ltd.), and the final protein purity reached more than 90%.

Anti-C18.2-hu19V3-Fc(Ab3)

The sequence of SEQ ID NO. 147 was prepared as shown in Example 4.

6.2 Preparation of antibody-drug complexes

The purified anti-CLDN18A2 single-domain antibody fusion protein and ch-175D10 were dissolved in PBS solution, and 10-fold excess TCEP was added to reduce the interchain disulfide bonds at 25°C, and the reducing agent was removed by dialysis. Re-formation of interchain disulfide bonds can be obtained by adding 2 times the molar concentration of TCEP to CuSO4 at 25 °C. Then, MC-VC-PAB-MMAE with a molar concentration of 10 times the antibody was added and reacted at 25°C for 1 hour to form the antibody derivative Ab-MC-VC-PAB-MMAE complex. The complex was dialyzed or ultrafiltered to remove unbound MC-VC-PAB-MMAE. The DAR of the final product obtained was determined by LC/MS.

The final concentration of the obtained Ab1-MC-VC-PAB-MMAE was 1.2 mg/ml. The LC-MS method was used to detect and analyze, and it was proved that there were no free toxin small molecules in the obtained sample. The absorption peaks of A252 and A280 were detected by a spectrophotometer (UV method), and the ratio DAR of the obtained toxin and antibody was determined, that is, y=4.42. Store in aliquots at 4°C.

The final concentration of the obtained Ab2-MC-VC-PAB-MMAE was 2.5 mg/ml. The LC-MS method was used to detect and analyze, and it was proved that there were no free toxin small molecules in the obtained sample. The absorption peaks of A252 and A280 were detected by spectrophotometer (UV method), and the ratio DAR of the obtained toxin and antibody was determined, that is, y=4.10. Store in aliquots at 4°C.

The final concentration of the obtained Ab3-MC-VC-PAB-MMAE was 5.4 mg/ml. The LC-MS method was used for detection and analysis, which proved that there were no free toxin small molecules in the obtained sample. The absorption peaks of A252 and A280 were detected by spectrophotometer (UV method), and the ratio DAR of the obtained toxin and antibody was determined, that is, y=4.21. Store in aliquots at 4°C.

The final concentration of the obtained ch175D10-MC-VC-PAB-MMAE was 4.4 mg/ml. The LC-MS method was used to detect and analyze, and it was proved that there were no free toxin small molecules in the obtained sample. The absorption peaks of A252 and A280 were detected by a spectrophotometer (UV method), and the ratio DAR of the obtained toxin and antibody was determined, that is, y=4.31. Store in aliquots at 4°C.

Example 7 Detection of binding activity of anti-CLDN18A2 single-domain antibody-drug complexes

ELISA binding activity detection of the prepared single-domain antibody-drug complexes: CLD18A2 protein (Kactus Biosystems) was coated on a 96-well microtiter plate, 100 ng per well, overnight at 4°C, 10 mM Na ₂ CO ₃ -NaHCO ₃ ( After washing at pH 10.0), 5% nonfat milk powder was coated for 1 hour, and after washing again, the single-domain antibody-drug complex was added; after incubation at 37°C for 2 hours, biotin-labeled anti-single-domain antibody rabbit polyclonal antibody (self-made) was added. ), and then added Strep-HRP to incubate at 37°C for 1 h. After washing, TMB developed color. The results are shown in Table 4. The binding activity of Ab3 before and after drug conjugation was not affected, and the remaining two structures, Ab1 and Ab2, still had good binding activity after drug conjugation.

Table 4

sample EC50nM

Ab1-MC-VC-PAB-MMAE 0.8 Ab2-MC-VC-PAB-MMAE 1.2 Ab3-MC-VC-PAB-MMAE 0.43 Ab3 0.4 ch175D10-MC-VC-PAB-MMAE 2.7

Example 8 In vitro cytotoxicity test

In vitro cytotoxicity assays were performed using NUGC-4 cells overexpressing human CLD18A2 (NUGC-4-CLD18A2) and original NUGC-4: cells grown to 90% confluence were cultured, washed with PBS, and trypsinized , after termination of digestion, cells were collected and seeded in 96-well plates, 2x10 ⁴ /well, DMEM medium with 10% fetal bovine serum, 2 mM glutamate, and cultured overnight _{in a 5% CO 2 incubator.} The next day, the medium was removed and replaced with fresh medium containing different concentrations of anti-CLD18A2 single-domain antibody-drug complexes and their corresponding single-domain antibody fusion proteins, 50 μl per well, three replicate wells per concentration; 37 Incubate for 72 hours at ℃, 5% CO ₂ , remove the supernatant and use LDH kit (purchased from Dongren Chemical Technology (Shanghai) Co., Ltd., product number CK12) to detect the release of LDH. The detection method is carried out according to the instruction manual. Cell killing percentage (%)=100*(OD490 test sample-OD490 control well)/(OD490 cells were completely lysed-OD490 control well). The experimental results are shown in Figure 5 and Table 5.

table 5

  IC50(nM) Ab1-MC-VC-PAB-MMAE 3 Ab2-MC-VC-PAB-MMAE 1.86 Ab3-MC-VC-PAB-MMAE 1.32 Ab3 - ch175D10-MC-VC-PAB-MMAE 9.54

The above results show that the antibody-drug complex described in the present invention can specifically target human CLD18A2, and the positive cytotoxicity is strong. cell killing. The unconjugated single domain antibody fusion protein Ab3 had no obvious cytotoxic effect.

Example 9 In vivo toxicity test

In this experiment, the tumor-bearing mice of the xenograft model (patientl derived xenograft, PDX) established by patient-derived gastric cancer tissue were used to determine the anti-tumor effects of antibody-drug compounds. Similar to the method described in Example 7.4, ^{tumor-bearing mice with a size of about 100 mm 3} were randomly divided into groups, with 4-6 mice in each experimental group. 15 days after tumor transplantation, intravenous injection of different antibody-drug complexes at a dose of 6 mg/kg was given. During the administration period, the tumor volume of mice in each group was monitored. The administration frequency was a single administration, and the monitoring frequency was once every 3 days. Continuous monitoring for 6 weeks. Determination of tumor volume: The maximum long axis (L) and the maximum width axis (W) of the tumor were measured with vernier calipers, and the tumor volume was calculated according to the following formula: V=L×W ² /2.

The experimental results are shown in Figure 6. Over time, the tumor volume of mice inoculated with the antibody-drug complex was well controlled relative to the PBS control group, compared with the positive control group ch175D10-MC-VC. -PAB-MMAE also has better tumor suppressive effect. Although Ab1-MC-VC-PAB-MMAE and Ab2-MC-VC-PAB-MMAE rebounded to a certain extent in the later stage, they eventually maintained a lower tumor volume without further significant increase, indicating that there was a significant increase in tumor volume. tumor suppressor effect.

Example 10 Anti-CLD18A2 Single Domain Antibody for Chimeric Antigen Receptor

The anti-CLD18A2 single-domain antibody of the present invention is used for the construction of chimeric antigen receptors. Table 6 lists the constructed chimeric antigen receptors comprising signal peptides and their structures (signal peptide-antigen recognition domain-hinge region-trans membrane domain - intracellular signaling domain).

Table 6

1. Construction of lentiviral plasmid vector for expressing specific anti-CLD18A2 single-domain antibody

As an example construction, the present invention uses the third-generation self-inactivating lentiviral vector system, which has four plasmids, namely, the enveloped plasmid pLP/VSVG (purchased from Addgene) encoding the VSV-G protein; Packaging plasmid pLP1; packaging plasmid pLP2 (purchased from Addgene) encoding Rev protein and recombinant expression vector encoding the target gene CAR constructed based on the empty vector pLVX-IRES-ZsGreen1 (purchased from Addgene).

Synthetic structural sequences (SEQ ID NO: 137, SEQ ID NO: 138, SEQ ID NO: 139, SEQ ID NO: 140), the synthesized sequences pass through the EcoRI and BamHI restriction sites at both ends, and the same restriction vector pLVX -IRES-ZsGreen1 (Addgene) was ligated by T4 ligase (Takara). The ligation product was transformed into Top10F', coated with ampicillin-resistant plate, cloned, cultured and sequenced to confirm, and the expression vectors expressing the above CAR were obtained: pLVX-aC18.2-hu6V3-28Z, pLVX-aC18.2-hu6V3-28-137Z , pLVX-aC18.2-hu19V3-28Z and pLVX-aC18.2-hu19V3-28-137Z,

2. Plasmid transfection of 293T packaging lentivirus

Lentiviral packaging follows the conventional methods, as follows: 5 × 10 ⁶ cell density cultivation of HEK-293T cells (ATCC) cells in 10cm dishes, 37 ℃, 5% CO ₂ incubator overnight, the medium containing 10% fetal bovine Serum (Gibco) in DMEM (Gibco). Change the culture medium to serum-free DMEM about 2 hours before transfection. When transfecting cells, in addition to using CAR-expressing lentiviral plasmids, plasmids (providing viral membrane proteins and structural proteins) pLP/VSVG, pLP1, and pLP2 are also required. The lentiviral plasmid expressing the target sequence CAR or empty vector used 5 μg, pLP1 used 2.5 μg, pLP2 used 2.5 μg, and pLP/VSVG used 1.25 μg. During transfection, the mixture of the above four plasmids was added to 500 μl MEM medium, and 25 μL Lipofectamine 2000 transfection reagent (thermo fisher) was added to 500 μL MEM medium in another microcentrifuge tube. Add the transfection reagent on top of the diluted plasmid, mix well, stand at room temperature for 20 minutes, add the mixture of plasmid and transfection reagent into a 10cm petri dish, shake, mix well, put it in a 37°C incubator, and replace it after 6 hours into DMEM medium with 10% fetal bovine serum. After 3 days of cell transfection, the virus can be harvested, and the virus-containing culture supernatant is transferred to a centrifuge tube, centrifuged at 4°C, 1500 rpm for 5 minutes to remove the cells, and then the virus-containing medium is filtered and aliquoted and frozen at -80°C . HEK-293T cells were seeded in a 96-well culture plate in DMEM with 10% fetal bovine serum at a ^{cell density of 1×10 5} /mL at a cell density of 100 μL/well, and cultured overnight at _{37°C, 5% CO 2 .} The next day, discard 50 μL/well of the culture supernatant, and add 50 μL/well of fresh culture medium containing polybrene with a final concentration of 6 μg/mL, incubate at 37°C, 5% CO ₂ for 30 min. Add 10 μL/well of virus stock solution, incubate at 37°C, 5% CO ₂ . 48h after infection, GFP was detected by flow cytometer, and the number of cells with a positive rate of 5-20% was appropriate, and the calculated titer was about 2×10 ⁶ U/mL.

Example 11: CAR-T cells specifically targeting Claudin18.2

1. Preparation of aC18.2-CAR-T

Human peripheral blood mononuclear cells (Shanghai Miaotong) were obtained from healthy human peripheral blood by density gradient centrifugation, and sorted by CD3 MicroBeads (Miltenyi Biotec GmbH) according to the instructions. Quantum007 lymphocyte culture medium (purchased from PAA Laboratories GmbH) was added at a density of about 1 × 10 ⁶ /mL, and DynabeadsTM Human T-Activator CD3/CD28 (thermofisher) was added at a cell:magnetic bead ratio of 1:1 and the final cells were added. Recombinant human IL-2 (Shanghai nearshore) at a concentration of 100 U/mL was stimulated and cultured for 24 h. T cells were then infected with the recombinant lentivirus described above (Example 10.3) at MOI ≈ 5. The infected cells ^{were passaged at a density of 5×10 5} /mL every other day, and the lymphocyte culture medium was supplemented with recombinant human IL-2 at a final concentration of 100 U/mL. On the 8th day of culture, by flow cytometry, due to the co-expression of GFP and CAR, the positive cells detected by GFP were regarded as positive cells expressing the chimeric antigen receptor. Uninfected T cells were used as negative control, and the positive rate of virus-infected T cells expressing different chimeric antigen receptors was about 64.2%.

2. Killing experiments of aC18.2-CAR-T

We observed the killing effect of different aC18.2-CAR-T cells on NUGC-4-Claudin18.2 cells and CLD18A2 negative cell line NUGC-4-Claudin18.1 in vitro. The effector-target ratios were set as 3:1, 1:1 and 1:3, respectively, and the number of target cells was 10,000/well. Five duplicate wells were set in each group, and the average value of the five duplicate wells was taken. After co-cultivation for 16 hours, the LDH content of the supernatant was detected by LDH detection kit (Shanghai Dongren) to evaluate killing. Results Table 7 shows that when the effector-target ratio is 3:1, specific aC18.2-CAR-T cells can effectively kill Claudin18.2-positive cells, but almost no Claudin18.2-negative cells. The above results show that aC18.2-CAR-T can specifically kill Claudin18.2 positive cells, and the killing effect is positively correlated with the effector-target ratio.

Table 7

3. In vitro cytokine release

Claudin18.2-positive cells NUGC-4-Claudin18.2 and aC18.2-CAR-T cells were co-cultured at a ratio of 1:1, and the culture supernatant was collected after 24 hours of incubation, and IL-2 (R&D Systems, Inc.), TNF-α (R&D Systems, Inc.) and IFN-γ (R&D Systems, Inc.) were detected according to the kit instructions. Figure 7 shows that in NUGC-4-Claudin18.2, aC18.2-CAR-T cells aC18.2-hu19V3-28-137Z co-incubated the secretion of cytokines such as IL-2, TNF-α and IFN-γ significantly higher than negative cells NUGC-4-Claudin18.1.

4. In vivo pharmacodynamic study of aC18.2-CAR-T

With NUGC-4-Claudin18.2, a subcutaneous xenograft model was established. To 3 × 10 ⁶ th NUGC-4-Claudin18.2 inoculated subcutaneously NOD / SCID mice. When the average tumor volume in mice reached 100-150 mm ³ , 100 mg/kg of cyclophosphamide was injected intraperitoneally to clear the immune cells of NOD/SCID mice, so that the adoptively transferred transgenic T lymphocytes could play a better anti-tumor function. On the second day, 1.0×10 ⁷ aC18.2-CAR-T cells aC18.2-hu19V3-28-137Z were infused through the tail vein, and the Mock group expressing 28-137Z was used as a control to observe and measure the subcutaneous transplanted tumor. grow. Results Figure 8 shows that aC18.2-CAR-T cells can significantly inhibit the growth of NUGC-4-Claudin18.2 xenografts.

To sum up, the present invention effectively overcomes various shortcomings in the prior art and has high industrial utilization value.

The above-mentioned embodiments merely illustrate the principles and effects of the present invention, but are not intended to limit the present invention. Anyone skilled in the art can modify or change the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical idea disclosed in the present invention should still be covered by the claims of the present invention.

Claims (20)

An anti-CLD18A2 single-domain antibody, the complementarity determining region of the anti-CLD18A2 single-domain antibody comprises CDR-H1 whose amino acid sequence is shown in one of SEQ ID No. 4 to 11, and an amino acid sequence such as SEQ ID No. 19. CDR-H2 shown in one of ~26, and CDR-H3 whose amino acid sequence is shown in one of SEQ ID Nos. 33 to 39. The anti-CLD18A2 single-domain antibody according to claim 1, wherein the complementarity determining region of the anti-CLD18A2 single-domain antibody comprises CDR-H1 whose amino acid sequence is shown in SEQ ID No. 4, and whose amino acid sequence is SEQ ID No. 4. CDR-H2 shown in ID No. 19, and CDR-H3 whose amino acid sequence is shown in SEQ ID No. 33; or, The complementarity determining region of the single-domain antibody against CLD18A2 includes CDR-H1 with amino acid sequence shown in SEQ ID No.5, CDR-H2 with amino acid sequence shown in SEQ ID No.20, and amino acid sequence shown in SEQ ID No. 20. .34 CDR-H3; or, The complementarity determining region of the anti-CLD18A2 single-domain antibody comprises CDR-H1 with amino acid sequence as shown in SEQ ID No.6, CDR-H2 with amino acid sequence as shown in SEQ ID No.21, and amino acid sequence as shown in SEQ ID No. 21 .35 CDR-H3; or, The complementarity determining region of the single-domain antibody against CLD18A2 includes CDR-H1 whose amino acid sequence is shown in SEQ ID No. 7, CDR-H2 whose amino acid sequence is shown in SEQ ID No. 22, and amino acid sequence such as SEQ ID No. 22. .36 CDR-H3; or, The complementarity determining region of the single-domain antibody against CLD18A2 includes CDR-H1 whose amino acid sequence is shown in SEQ ID No. 8, CDR-H2 whose amino acid sequence is shown in SEQ ID No. 23, and amino acid sequence such as SEQ ID No. 23. .37 CDR-H3; or, The complementarity determining region of the single-domain antibody against CLD18A2 includes CDR-H1 whose amino acid sequence is shown in SEQ ID No. 9, CDR-H2 whose amino acid sequence is shown in SEQ ID No. 24, and amino acid sequence such as SEQ ID No. 24. .38 CDR-H3; or, The complementarity determining region of the single-domain antibody against CLD18A2 includes CDR-H1 whose amino acid sequence is shown in SEQ ID No. 10, CDR-H2 whose amino acid sequence is shown in SEQ ID No. 25, and amino acid sequence such as SEQ ID No. 25. .36 CDR-H3; or, The complementarity determining region of the single-domain antibody against CLD18A2 includes CDR-H1 whose amino acid sequence is shown in SEQ ID No. 11, CDR-H2 whose amino acid sequence is shown in SEQ ID No. 26, and amino acid sequence such as SEQ ID No. 26 CDR-H3 shown in .39. The anti-CLD18A2 single-domain antibody of claim 1, wherein the anti-CLD18A2 single-domain antibody further comprises a framework region, and the framework region FR comprises an amino acid sequence such as one of SEQ ID No. 1-3 The FR1 shown, the FR2 whose amino acid sequence is shown in one of SEQ ID No. 12 to 18, the FR3 whose amino acid sequence is shown in one of SEQ ID No. 27 to 32, and the amino acid sequence shown in SEQ ID No. 40 FR4 shown in one of ~41. The single-domain antibody against CLD18A2 according to claim 3, wherein the framework region FR comprises FR1 whose amino acid sequence is shown in SEQ ID No.1, FR2 whose amino acid sequence is shown in SEQ ID No.12, FR3 whose amino acid sequence is shown in SEQ ID No. 27, and FR4 whose amino acid sequence is shown in SEQ ID No. 40; or, The framework region FR includes FR1 whose amino acid sequence is shown in SEQ ID No.2, FR2 whose amino acid sequence is shown in SEQ ID No.13, FR3 whose amino acid sequence is shown in SEQ ID No.28, and FR3 whose amino acid sequence is shown in SEQ ID No.28. FR4 shown in ID No. 41; or, The framework region FR includes FR1 whose amino acid sequence is shown in SEQ ID No.3, FR2 whose amino acid sequence is shown in SEQ ID No.14, FR3 whose amino acid sequence is shown in SEQ ID No.29, and FR3 whose amino acid sequence is shown in SEQ ID No.29. FR4 shown in ID No. 41; or, The framework region FR includes FR1 whose amino acid sequence is shown in SEQ ID No.1, FR2 whose amino acid sequence is shown in SEQ ID No.15, FR3 whose amino acid sequence is shown in SEQ ID No.30, and FR3 whose amino acid sequence is shown in SEQ ID No.30. FR4 shown in ID No. 41; or, The framework region FR includes FR1 whose amino acid sequence is shown in SEQ ID No.2, FR2 whose amino acid sequence is shown in SEQ ID No.16, FR3 whose amino acid sequence is shown in SEQ ID No.31, and FR3 whose amino acid sequence is shown in SEQ ID No.31. FR4 shown in ID No. 41; or, The framework region FR includes FR1 whose amino acid sequence is shown in SEQ ID No.2, FR2 whose amino acid sequence is shown in SEQ ID No.13, FR3 whose amino acid sequence is shown in SEQ ID No.31, and FR3 whose amino acid sequence is shown in SEQ ID No.31. FR4 shown in ID No. 41; or, The framework region FR includes FR1 whose amino acid sequence is shown in SEQ ID No.1, FR2 whose amino acid sequence is shown in SEQ ID No.17, FR3 whose amino acid sequence is shown in SEQ ID No.30, and FR3 whose amino acid sequence is shown in SEQ ID No.30. FR4 shown in ID No. 41; or, The framework region FR includes FR1 whose amino acid sequence is shown in SEQ ID No.2, FR2 whose amino acid sequence is shown in SEQ ID No.18, FR3 whose amino acid sequence is shown in SEQ ID No.32, and FR3 whose amino acid sequence is shown in SEQ ID No.32. FR4 shown in ID No. 41. The anti-CLD18A2 single-domain antibody according to claim 1, wherein the amino acid sequence of the anti-CLD18A2 single-domain antibody comprises: a) an amino acid sequence as shown in one of SEQ ID No. 42 to 49; or, b) An amino acid sequence having a sequence identity of more than 80% with the amino acid sequence shown in one of SEQ ID Nos. 42 to 49, and having the function of the amino acid sequence defined in a). The anti-CLD18A2 single-domain antibody of claim 1, wherein the anti-CLD18A2 single-domain antibody is a humanized antibody. And/or, the anti-CLD18A2 single domain antibody is derived from alpaca. The anti-CLD18A2 single-domain antibody according to claim 1, wherein the amino acid sequence of the anti-CLD18A2 single-domain antibody comprises: c) an amino acid sequence as shown in one of SEQ ID No. 67-90; or, d) An amino acid sequence that has more than 80% sequence identity with the amino acid sequence shown in one of SEQ ID Nos. 67 to 90, and has the function of the amino acid sequence defined in c). A bispecific antibody comprising the anti-CLD18A2 single domain antibody and the anti-CD3 domain according to any one of claims 1 to 7. The bispecific antibody of claim 8, wherein the bispecific antibody further comprises a domain for extending serum half-life. The bispecific antibody of claim 8, wherein the anti-CLD18A2 single-domain antibody, the anti-CD3 domain and the domain for prolonging serum half-life further comprise a linking peptide. An isolated polynucleotide encoding an anti-CLD18A2 single domain antibody according to any one of claims 1-7, or a bispecific antibody according to any one of claims 8-10. A construct comprising the isolated polynucleotide of claim 11. An antibody expression system comprising the construct of claim 12 or the exogenous polynucleotide of claim 11 integrated into the genome. The preparation method of the anti-CLD18A2 single domain antibody according to any one of claims 1-7, or the preparation method of the bispecific antibody according to any one of claims 8-10, comprising the steps of: The antibody expression system according to claim 13 is cultured under the condition of the antibody, so that the antibody is expressed, and the antibody is purified and isolated. Use of the anti-CLD18A2 single domain antibody according to any one of claims 1 to 7, or the use of the bispecific antibody according to any one of claims 8 to 10 in the preparation of a medicament for use in Treat tumors. An antibody-drug complex comprising the anti-CLD18A2 single domain antibody according to any one of claims 1 to 7 and a cytotoxic drug. The antibody-drug complex of claim 11, wherein the cytotoxic drug is selected from small molecule drugs; And/or, the structure of the antibody-drug complex is shown in formula 1: V _HH/CLD18A2 -Linking Peptide-Z-[LD]n I Wherein, _VHH/CLD18A2 is a single domain antibody against CLD18A2; Z is an accessory functional region, and Z is selected from a domain and/or a drug-conjugating domain for prolonging serum half-life, or is absent; D is a cytotoxic drug molecule; L is the connection chain; n represents the average number of D coupled, and 0<n≤10, preferably 2≤n≤7; more preferably 3≤n≤6; most preferably 4. A cell comprising a membrane-bound chimeric antigen receptor comprising a transmembrane domain, an intracellular domain and an extracellular domain, the extracellular domain comprising any of claims 1-7 The single domain antibody described against CLD18A2 is required. The cell of claim 18, wherein the cell is a T lymphocyte, NK cell, or macrophage; And/or, the transmembrane domain comprises the sequence of CD4, CD8, CD28 transmembrane domain; and/or, the intracellular domain includes a costimulatory domain and/or a signaling domain; And/or, the chimeric antigen receptor also includes a hinge region, and the hinge region includes a CD8 dumpling chain region; And/or, costimulatory domains include CD28, CD27, 4-1BB (CD137), OX40 (CD134), ICOS (CD278), CD30, CD40, PD-1 (CD279), CD2, CD7, NKG2C (CD94), Intracellular domain of B7-H3 (CD276); and/or, the signaling domain comprises the signaling domains of CD3ζ, FcRγ, FcRβ, FcRε, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b and CD66d; And/or, the chimeric antigen receptor comprises the anti-CLD18A2 single domain antibody, hinge region, transmembrane domain and intracellular domain from N-terminal to C-terminal in sequence, and the intracellular domain is from N-terminal to C-terminal Including costimulatory domain and signaling domain in turn; And/or, the amino acid sequence of the chimeric antigen receptor includes the sequence shown in one of SEQ ID NO. 137-140. The cell of claim 18, wherein when the chimeric antigen receptor binds to the CLD18A2 antigen, the cell can be activated and/or stimulated to proliferate; And/or, the cell surface expresses the anti-CLD18A2 single domain antibody.

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