WO2023134657A1

WO2023134657A1 - Anti-cldn18.2 and 4-1bb bispecific antigen binding protein and use thereof

Info

Publication number: WO2023134657A1
Application number: PCT/CN2023/071504
Authority: WO
Inventors: 杨沙沙; 陈晓锐; 王华菁; 杨焕凤; 何晓文
Original assignee: 原启生物科技(上海)有限责任公司
Priority date: 2022-01-11
Filing date: 2023-01-10
Publication date: 2023-07-20

Abstract

An isolated antigen binding protein, a preparation method therefor, and a use thereof. The antigen binding protein comprises: a first antigen binding domain and a second antigen binding domain, wherein the first antigen binding domain can specifically bind to claudin 18.2 (CLDN18.2), and the first antigen binding domain contains at least one CDR in an amino acid sequence as shown in SEQ ID NO: 100.

Description

Bispecific antigen-binding protein against CLDN18.2 and 4-1BB and use thereof

technical field

This application relates to the field of biomedicine, in particular to an anti-CLDN18.2 bispecific antigen-binding protein and its application.

Background technique

CLDN18.2 (Claudin 18.2) is a family of proteins that form an important part of tight cellular junctions. CLDN18.2 is only expressed in differentiated gastric parietal cells, but not in normal tissues. The latest research shows that CLDN18.2 is overexpressed in more than 77% of gastric cancer patients and more than 80% of pancreatic cancer patients. In addition, it is overexpressed in solid tumors such as lung cancer, esophageal cancer, and ovarian cancer.

4-1BB (CD137, tumor cell necrosis factor receptor superfamily 9), a member of the TNF receptor superfamily (TNFRSF), is a co-stimulatory molecule that activates in immune cells (innate and adaptive immune cells) post expression. 4-1BB plays an important role in regulating the activity of a variety of immune cells. 4-1BB agonists enhance immune cell proliferation, survival, cytokine secretion, and cytolytic activity of CD8+ T cells. Many other studies have shown that activation of 4-1BB enhances the immune response and eliminates tumors in mice. Therefore, 4-1BB may be a promising tumor immune target molecule.

There are currently CLDN18.2 antibodies. However, there is still a lot of room for improvement in the therapeutic effect and immune persistence of existing antibodies. Therefore, it is urgent to develop more effective antibodies against CLDN18.2.

Contents of the invention

The present application provides an isolated antigen-binding protein, the antigen-binding protein comprises a first antigen-binding domain and a second antigen-binding domain, and the first antigen-binding domain can specifically bind CLDN18.2. In the present application, the isolated antigen-binding protein has one of the following properties: 1) can specifically bind CLDN18.2; 2) can specifically bind 4-1BB; 3) can specifically bind human, mouse and CLDN18.2 protein of cynomolgus monkey; 4) can stimulate T cell activation and promote the secretion of IL-2 factor; 5) can inhibit the growth and/or proliferation of tumor cells; 6) have tumor immune memory function.

In one aspect, the application provides an isolated antigen-binding protein, which includes: a first antigen-binding domain and a second antigen-binding domain, the first antigen-binding domain can specifically bind tight junction protein 18.2 (CLDN18.2) , and the first antigen-binding domain comprises at least one CDR in the variable region VH of the antibody heavy chain, and the VH comprises the amino acid sequence shown in SEQ ID NO:100.

In certain embodiments, in the isolated antigen binding protein, the first antigen binding domain comprises HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:97.

In certain embodiments, in the isolated antigen binding protein, the first antigen binding domain comprises HCDR3, and the HCDR3 comprises SEQ ID NO:3, SEQ ID NO:57 and SEQ ID NO:71 The amino acid sequence shown in any one.

In certain embodiments, in the isolated antigen binding protein, the first antigen binding domain comprises HCDR2, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:98.

In certain embodiments, in said isolated antigen binding protein, said first antigen binding domain comprises HCDR2, and said HCDR2 comprises SEQ ID NO:2, SEQ ID NO:56 and SEQ ID NO:70 The amino acid sequence shown in any one.

In certain embodiments, in said isolated antigen binding protein, said first antigen binding domain comprises HCDR1, and said HCDR1 comprises SEQ ID NO:99 (X ₁ YX ₂ X ₃ X ₄ , wherein X ₁ is N or R, X ₂ is G, I or V, X ₃ is I or M, X ₄ is the amino acid sequence shown in H, N or S).

In certain embodiments, in the isolated antigen binding protein, the first antigen binding domain comprises HCDR1, and the HCDR1 comprises SEQ ID NO:1, SEQ ID NO:55 and SEQ ID NO:69 The amino acid sequence shown in any one.

In certain embodiments, in said isolated antigen binding protein, said first antigen binding domain comprises HCDR1, HCDR2 and HCDR3, said HCDR1 comprising SEQ ID NO: 99 (X ₁ YX ₂ X ₃ X ₄ , Wherein, _X1 is N or R, _X2 is G, I or V, _X3 is I or M, _X4 is the amino acid sequence shown in H, N or S), and the HCDR2 comprises SEQ ID NO:98 The amino acid sequence shown, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:97.

In certain embodiments, in the isolated antigen binding protein, the first antigen binding domain comprises HCDR1, HCDR2 and HCDR3, and the HCDR1, HCDR2 and HCDR3 comprise an amino acid sequence selected from any one of the following groups:

(1) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:3;

(2) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:55, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:56, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:57; and

(3) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO:69, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:70, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:71.

In certain embodiments, in the isolated antigen-binding protein, the first antigen-binding domain comprises H-FR1, the C-terminus of the H-FR1 is directly or indirectly linked to the N-terminus of the HCDR1, and The H-FR1 comprises the amino acid sequence shown in any one of SEQ ID NO:4, SEQ ID NO:43, SEQ ID NO:58 and SEQ ID NO:75.

In certain embodiments, in said isolated antigen binding protein, said first antigen binding domain comprises H-FR2, said H-FR2 is located between said HCDR1 and said HCDR2, and said H- FR2 comprises the amino acid sequence shown in any one of SEQ ID NO:5, SEQ ID NO:44 and SEQ ID NO:59.

In certain embodiments, in said isolated antigen binding protein, said first antigen binding domain comprises H-FR3, said H-FR3 is located between said HCDR2 and said HCDR3, and said H-FR3 is located between said HCDR2 and said HCDR3, and said H- FR3 comprises the amino acid sequence shown in any one of SEQ ID NO:6, SEQ ID NO:45, SEQ ID NO:60 and SEQ ID NO:72.

In certain embodiments, in the isolated antigen-binding protein, the first antigen-binding domain comprises H-FR4, the N-terminus of the H-FR4 is directly or indirectly linked to the C-terminus of the HCDR3, and The H-FR4 comprises the amino acid sequence shown in any one of SEQ ID NO:7, SEQ ID NO:46 and SEQ ID NO:61.

In certain embodiments, in the isolated antigen-binding protein, the first antigen-binding domain comprises a heavy chain variable region VH, and the VH comprises the amino acid sequence shown in SEQ ID NO:100.

In certain embodiments, in said isolated antigen binding protein, said first antigen binding domain comprises a heavy chain variable region VH comprising SEQ ID NO:8, SEQ ID NO:47, SEQ ID NO: The amino acid sequence shown in any one of NO:62, SEQ ID NO:73 and SEQ ID NO:76.

In certain embodiments, in the isolated antigen binding protein, the first antigen binding domain comprises LCDR3, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:101.

In certain embodiments, in said isolated antigen binding protein, said first antigen binding domain comprises LCDR3, and said LCDR3 comprises SEQ ID NO: 11, SEQ ID NO: 63, SEQ ID NO: 79 and The amino acid sequence shown in any one of SEQ ID NO:86.

In certain embodiments, in the isolated antigen binding protein, the first antigen binding domain comprises LCDR2, and the LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 102.

In certain embodiments, in the isolated antigen binding protein, the first antigen binding domain comprises LCDR2, and the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:10 or SEQ ID NO:78.

In certain embodiments, in the isolated antigen binding protein, the first antigen binding domain comprises LCDR1, and the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 103.

In certain embodiments, in said isolated antigen binding protein, said first antigen binding domain comprises LCDR1, and said LCDR1 comprises SEQ ID NO:9, SEQ ID NO:48 and SEQ ID NO:77 The amino acid sequence shown in any one.

In certain embodiments, in the isolated antigen binding protein, the first antigen binding domain comprises LCDR1, LCDR2 and LCDR3, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 103, and the LCDR2 comprises The amino acid sequence shown in SEQ ID NO:102, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:101.

In certain embodiments, in the isolated antigen-binding protein, the first antigen-binding domain comprises LCDR1, LCDR2 and LCDR3, and the LCDR1, LCDR2 and LCDR3 comprise an amino acid sequence selected from any one of the following groups:

(1) the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:9, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:10, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:11;

(2) the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:48, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:10, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:11;

(3) the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:48, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:10, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:63;

(4) the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:48, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:10, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:86; and

(5) The LCDR1 includes the amino acid sequence shown in SEQ ID NO:77, the LCDR2 includes the amino acid sequence shown in SEQ ID NO:78, and the LCDR3 includes the amino acid sequence shown in SEQ ID NO:79.

In certain embodiments, in the isolated antigen-binding protein, the first antigen-binding domain comprises L-FR1, the C-terminus of the L-FR1 is directly or indirectly connected to the N-terminus of the LCDR1, and The L-FR1 comprises the amino acid sequence shown in any one of SEQ ID NO:12, SEQ ID NO:49, SEQ ID NO:64, SEQ ID NO:80 and SEQ ID NO:87.

In certain embodiments, in said isolated antigen binding protein, said first antigen binding domain comprises L-FR2, said L-FR2 is located between said LCDR1 and said LCDR2, and said L- FR2 comprises the amino acid sequence shown in SEQ ID NO: 13.

In certain embodiments, in said isolated antigen binding protein, said first antigen binding domain comprises L-FR3, said L-FR3 is located between said LCDR2 and said LCDR3, and said L- FR3 comprises the amino acid sequence shown in any one of SEQ ID NO:14, SEQ ID NO:50, SEQ ID NO:65 and SEQ ID NO:81.

In certain embodiments, in the isolated antigen-binding protein, the first antigen-binding domain comprises L-FR4, the N-terminus of the L-FR4 is directly or indirectly connected to the C-terminus of the LCDR3, and The L-FR4 comprises the amino acid sequence shown in any one of SEQ ID NO:15, SEQ ID NO:51, SEQ ID NO:82 and SEQ ID NO:88.

In certain embodiments, in the isolated antigen binding protein, the first antigen binding domain comprises a light chain variable region VL, and the VL comprises the amino acid sequence shown in SEQ ID NO:104.

In certain embodiments, in said isolated antigen binding protein, said first antigen binding domain comprises a light chain variable region VL comprising SEQ ID NO: 16, SEQ ID NO: 52, SEQ ID NO: The amino acid sequence shown in any one of NO:66, SEQ ID NO:83 and SEQ ID NO:89.

In certain embodiments, in said isolated antigen binding protein, said first antigen binding domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 Comprising an amino acid sequence selected from any one of the following groups:

(1) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:3, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:9, shown LCDR2 comprises the amino acid sequence shown in SEQ ID NO:10, and said LCDR3 comprises the amino acid sequence shown in SEQ ID NO:11;

(2) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:3, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:48, shown LCDR2 comprises the amino acid sequence shown in SEQ ID NO:10, and said LCDR3 comprises the amino acid sequence shown in SEQ ID NO:11;

(3) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:55, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:56, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:57, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:48, shown LCDR2 comprises the amino acid sequence shown in SEQ ID NO:10, and said LCDR3 comprises the amino acid sequence shown in SEQ ID NO:63;

(4) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:69, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:70, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:71, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:48, shown LCDR2 comprises the amino acid sequence shown in SEQ ID NO:10, and said LCDR3 comprises the amino acid sequence shown in SEQ ID NO:11;

(5) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:69, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:70, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:71, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:77, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:78, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:79; and

(6) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO:69, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:70, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:71, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:48, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:10, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:86.

In certain embodiments, in the isolated antigen binding protein, the first antigen binding domain comprises VH and VL, the VH comprises the amino acid sequence shown in SEQ ID NO: 100, and the VL comprises SEQ ID NO: 100 Amino acid sequence shown in ID NO:104.

In certain embodiments, in the isolated antigen-binding protein, the first antigen-binding domain comprises VH and VL, and the VH and VL are selected from any one of the following amino acid sequences:

(1) the VH comprises the amino acid sequence shown in SEQ ID NO:8, and the VL shown comprises the amino acid sequence shown in SEQ ID NO:16;

(2) the VH comprises the amino acid sequence shown in SEQ ID NO:47, and the VL shown comprises the amino acid sequence shown in SEQ ID NO:52;

(3) the VH comprises the amino acid sequence shown in SEQ ID NO:62, and the VL shown comprises the amino acid sequence shown in SEQ ID NO:66;

(4) the VH comprises the amino acid sequence shown in SEQ ID NO:73, and the VL shown comprises the amino acid sequence shown in SEQ ID NO:52;

(5) the VH comprises the amino acid sequence shown in SEQ ID NO:76, and the VL comprises the amino acid sequence shown in SEQ ID NO:83; and

(6) The VH comprises the amino acid sequence shown in SEQ ID NO:76, and the VL comprises the amino acid sequence shown in SEQ ID NO:89.

In certain embodiments, in said isolated antigen binding protein, said first antigen binding domain comprises a heavy chain constant region.

In certain embodiments, in said isolated antigen binding protein, said heavy chain constant region comprises an IgG-derived heavy chain constant region.

In certain embodiments, in said isolated antigen binding protein, said heavy chain constant region comprises a heavy chain constant region derived from human IgG.

In certain embodiments, in said isolated antigen binding protein, said heavy chain constant region comprises a heavy chain constant region derived from human IgG1.

In certain embodiments, in said isolated antigen binding protein, said heavy chain constant region comprises an Fc fragment.

In certain embodiments, in said isolated antigen binding protein, said Fc fragment comprises one or more mutations.

In certain embodiments, in said isolated antigen binding protein, said Fc fragment comprises one or more mutations of N180A, D239E and L241M.

In certain embodiments, in the isolated antigen binding protein, the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO: 17.

In certain embodiments, in said isolated antigen binding protein, said first antigen binding domain comprises a light chain constant region.

In certain embodiments, in said isolated antigen binding protein, said light chain constant region comprises a human Igκ constant region.

In certain embodiments, in the isolated antigen binding protein, the light chain constant region comprises the amino acid sequence shown in SEQ ID NO: 18.

In certain embodiments, in said isolated antigen binding protein, said first antigen binding domain comprises an antibody or antigen binding fragment thereof.

In certain embodiments, in said isolated antigen binding protein, said antigen binding fragment comprises Fab, Fab', Fv fragment, F(ab)' ₂ , scFv, di-scFv and/or dAb.

In certain embodiments, in said isolated antigen binding protein, said second antigen binding domain is capable of specifically binding 4-1BB.

In certain embodiments, in the isolated antigen binding protein, the second antigen binding domain comprises at least one CDR in the variable region VH of an antibody heavy chain, the VH comprising SEQ ID NO: 105 amino acid sequence.

In certain embodiments, in the isolated antigen binding protein, the second antigen binding domain comprises HCDR3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:24.

In certain embodiments, in the isolated antigen binding protein, the second antigen binding domain comprises HCDR2, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:23.

In certain embodiments, in the isolated antigen binding protein, the second antigen binding domain comprises HCDR1, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:22.

In certain embodiments, in the isolated antigen binding protein, the second antigen binding domain comprises HCDR1, HCDR2 and HCDR3, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 22, and the HCDR2 comprises The amino acid sequence shown in SEQ ID NO:23, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:24.

In certain embodiments, in the isolated antigen binding protein, the second antigen binding domain comprises H-FR1, the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1, and The H-FR1 comprises the amino acid sequence shown in SEQ ID NO:25 or SEQ ID NO:91.

In certain embodiments, in said isolated antigen binding protein, said second antigen binding domain comprises H-FR2, said H-FR2 is located between said HCDR1 and said HCDR2, and said H- FR2 comprises the amino acid sequence shown in SEQ ID NO:26.

In certain embodiments, in said isolated antigen binding protein, said second antigen binding domain comprises H-FR3, said H-FR3 is located between said HCDR2 and said HCDR3, and said H- FR3 comprises the amino acid sequence shown in SEQ ID NO:27.

In certain embodiments, in the isolated antigen binding protein, the second antigen binding domain comprises H-FR4, the N-terminus of the H-FR4 is directly or indirectly linked to the C-terminus of the HCDR3, and The H-FR4 comprises the amino acid sequence shown in SEQ ID NO:28.

In certain embodiments, in the isolated antigen-binding protein, the second antigen-binding domain comprises a heavy chain variable region VH, and the VH comprises SEQ ID NO:29 or SEQ ID NO:92 amino acid sequence.

In certain embodiments, in the isolated antigen binding protein, the second antigen binding domain comprises LCDR3, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:34.

In certain embodiments, in the isolated antigen binding protein, the second antigen binding domain comprises LCDR2, and the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:33.

In certain embodiments, in the isolated antigen binding protein, the second antigen binding domain comprises LCDR1, and the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:32.

In certain embodiments, in the isolated antigen binding protein, the second antigen binding domain comprises LCDR1, LCDR2 and LCDR3, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 32, and the LCDR2 comprises The amino acid sequence shown in SEQ ID NO:33, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:34.

In certain embodiments, in the isolated antigen binding protein, the second antigen binding domain comprises L-FR1, and the L-FR1 comprises the amino acid sequence shown in SEQ ID NO:35.

In certain embodiments, in the isolated antigen binding protein, the second antigen binding domain comprises L-FR2, and the L-FR2 comprises the amino acid sequence shown in SEQ ID NO:36.

In certain embodiments, in the isolated antigen binding protein, the second antigen binding domain comprises L-FR3, and the L-FR3 comprises the amino acid sequence shown in SEQ ID NO:37.

In certain embodiments, in the isolated antigen binding protein, the second antigen binding domain comprises L-FR4, and the L-FR4 comprises the amino acid sequence shown in SEQ ID NO:38.

In certain embodiments, in the isolated antigen binding protein, the second antigen binding domain comprises a light chain variable region VL, and the VL comprises the amino acid sequence shown in SEQ ID NO:39.

In certain embodiments, in the isolated antigen-binding protein, wherein the second antigen-binding domain comprises VH and VL, the VH and VL are selected from the amino acid sequences of any of the following groups:

(1) the VH comprises the amino acid sequence shown in SEQ ID NO:29, and the VL comprises the amino acid sequence shown in SEQ ID NO:39; and

(2) The VH comprises the amino acid sequence shown in SEQ ID NO:92, and the VL comprises the amino acid sequence shown in SEQ ID NO:39.

In certain embodiments, in said isolated antigen binding protein, the VH and VL of said second antigen binding domain are directly or indirectly linked.

In certain embodiments, in said isolated antigen binding protein, the VH and VL of said second antigen binding domain are linked by a linker. In certain embodiments, the linker comprises the amino acid sequence shown in SEQ ID NO:40.

In certain embodiments, in said isolated antigen binding protein, said second antigen binding domain comprises an antibody or antigen binding fragment thereof.

In certain embodiments, the antigen-binding fragment comprises a scFv.

In certain embodiments, the scFv comprises the amino acid sequence shown in SEQ ID NO:41 or SEQ ID NO:96.

In certain embodiments, in said isolated antigen binding protein, said first antigen binding domain and said second antigen binding domain are directly or indirectly linked.

In certain embodiments, in said isolated antigen binding protein, said first antigen binding domain and said second antigen binding domain are linked by a linker.

In certain embodiments, in the isolated antigen binding protein, the linker comprises the amino acid sequence shown in SEQ ID NO:21.

In certain embodiments, in the isolated antigen-binding protein, the scFv of the second antigen-binding domain is directly or indirectly linked to the C-terminus of the Fc fragment of the first antigen-binding domain.

On the other hand, the present application also provides an isolated antigen-binding protein, which comprises two first polypeptides and two second polypeptides, and the first polypeptide comprises VH, CH1 in sequence from the N-terminal to the C-terminal , CH2, CH3 and scFv capable of specifically binding to 4-1BB protein, the second polypeptide comprising VL and light chain variable region CL; wherein the VH is paired with the VL and capable of specifically binding to CLDN18.2.

In certain embodiments, in the isolated antigen binding protein, the VH comprises HCDR1, HCDR2 and HCDR3, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 1, and the HCDR2 comprises SEQ ID NO: The amino acid sequence shown in 2, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:3.

In certain embodiments, in the isolated antigen binding protein, the VH comprises HCDR1, HCDR2 and HCDR3, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:55, and the HCDR2 comprises SEQ ID NO: The amino acid sequence shown in 56, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 57.

In certain embodiments, in the isolated antigen binding protein, the VH comprises HCDR1, HCDR2 and HCDR3, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:69, and the HCDR2 comprises SEQ ID NO: The amino acid sequence shown in 70, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:71.

In certain embodiments, in the isolated antigen binding protein, the VL comprises LCDR1, LCDR2 and LCDR3, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 9, and the LCDR2 comprises SEQ ID NO: The amino acid sequence shown in 10, and shown LCDR3 comprises the amino acid sequence shown in SEQ ID NO:11.

In certain embodiments, in the isolated antigen binding protein, the VL comprises LCDR1, LCDR2 and LCDR3, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:48, and the LCDR2 comprises SEQ ID NO: The amino acid sequence shown in 10, and shown LCDR3 comprises the amino acid sequence shown in SEQ ID NO:11.

In certain embodiments, in the isolated antigen binding protein, the VL comprises LCDR1, LCDR2 and LCDR3, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 48, and the LCDR2 comprises SEQ ID NO: The amino acid sequence shown in 10, and shown LCDR3 comprises the amino acid sequence shown in SEQ ID NO:63.

In certain embodiments, in the isolated antigen binding protein, the VL comprises LCDR1, LCDR2 and LCDR3, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:77, and the LCDR2 comprises SEQ ID NO: The amino acid sequence shown in 78, and shown LCDR3 comprises the amino acid sequence shown in SEQ ID NO:79.

In certain embodiments, in the isolated antigen binding protein, the VL comprises LCDR1, LCDR2 and LCDR3, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 48, and the LCDR2 comprises SEQ ID NO: The amino acid sequence shown in 10, and shown LCDR3 comprises the amino acid sequence shown in SEQ ID NO:86.

In certain embodiments, in said isolated antigen binding protein, said CH2 and said CH3 constitute an Fc fragment.

In certain embodiments, in said isolated antigen binding protein, said Fc fragment comprises one or more amino acid mutations selected from the group consisting of N180A, D239E and L241M.

In certain embodiments, in the isolated antigen binding protein, the scFv comprises HCDR1, HCDR2 and HCDR3, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 22, and the HCDR2 comprises SEQ ID NO: The amino acid sequence shown in 23, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 24.

In certain embodiments, in the isolated antigen binding protein, the scFv comprises LCDR1, LCDR2 and LCDR3, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:32, and the LCDR2 comprises SEQ ID NO: The amino acid sequence shown in 33, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO: 34.

In certain embodiments, in the isolated antigen binding protein, the scFv comprises VH, and the VH comprises the amino acid sequence shown in SEQ ID NO:29 or SEQ ID NO:92.

In certain embodiments, in the isolated antigen binding protein, the scFv comprises a VL, and the VL comprises the amino acid sequence shown in SEQ ID NO:39.

In another aspect, the present application also provides a polypeptide comprising the antigen-binding protein described in the present application.

In another aspect, the present application also provides one or more isolated nucleic acid molecules encoding the isolated antigen binding proteins described herein.

On the other hand, the present application also provides a vector comprising the nucleic acid molecule described in the present application.

On the other hand, the present application also provides a cell comprising the nucleic acid molecule or the vector.

On the other hand, the present application also provides a pharmaceutical composition, which comprises the antigen-binding protein described in the present application, and optionally a pharmaceutically acceptable carrier.

On the other hand, the present application also provides a method for preparing the antigen-binding protein, which comprises culturing the cell described in the present application under the condition that the isolated antigen-binding protein is expressed.

On the other hand, the present application also provides the use of the isolated antigen-binding protein, the polypeptide, the nucleic acid molecule, the carrier, the cell and/or the pharmaceutical composition in the preparation of a medicament, the Medicines are used to prevent, alleviate and/or treat diseases and/or conditions.

In certain embodiments, the diseases and/or disorders include diseases and/or disorders associated with abnormal expression of CLDN18.2.

In certain embodiments, the disease and/or condition comprises a tumor.

In certain embodiments, the tumor comprises solid tumors and/or non-solid tumors.

In certain embodiments, the tumor is selected from gastric cancer, pancreatic cancer, ovarian cancer, lung cancer, gastroesophageal junction cancer and/or colon cancer.

On the other hand, the present application also provides the isolated antigen-binding protein, the polypeptide, the nucleic acid molecule, the carrier, the cell and/or the pharmaceutical composition, which are used for preventing, alleviating and/or or to treat diseases and/or conditions.

In certain embodiments, the disease and/or condition comprises a tumor.

In another aspect, the present application also provides a method for preventing, alleviating and/or treating diseases and/or conditions, the method comprising administering the isolated antigen-binding protein described in the present application to a subject in need, said Polypeptide, said nucleic acid molecule, said vector, said cell and/or said pharmaceutical composition.

In certain embodiments, the disease and/or condition comprises a tumor.

Those skilled in the art can easily perceive other aspects and advantages of the present application from the following detailed description. In the following detailed description, only exemplary embodiments of the present application are shown and described. As those skilled in the art will appreciate, the content of the present application enables those skilled in the art to make changes to the specific embodiments which are disclosed without departing from the spirit and scope of the invention to which this application relates. Correspondingly, the drawings and descriptions in the specification of the present application are only exemplary rather than restrictive.

Description of drawings

The particular features of the invention to which this application relates are set forth in the appended claims. The features and advantages of the invention to which this application relates can be better understood with reference to the exemplary embodiments described in detail hereinafter and the accompanying drawings. A brief description of the accompanying drawings is as follows:

Figure 1A shows a schematic diagram of the construction of the isolated antigen-binding protein described in this application; Figure 1B shows the SEC-HPLC purity test results of the purified antigen-binding protein.

Figure 2A shows the binding activity of the antigen-binding protein to 4-1BB-NFκB-293T cells; Figure 2B shows the binding activity of the antigen-binding protein to CHO-hCLDN18.2 cells.

Figure 3 shows the flow cytometric verification of the activity assay of the double-head simultaneous binding of the antigen-binding protein.

Figure 4A shows the flow cytometric binding activity of the antigen-binding protein and 293T-mouse CLDN18.2 cells; Figure 4B shows the flow cytometric binding activity of the antigen-binding protein and 293T-cynomolgus monkey CLDN18.2 cells.

Figure 5 shows a flow cytometric competition binding experiment of the antigen binding proteins described in this application.

Figure 6 shows the results of luciferase activity analysis experiments.

Figure 7A shows the secretion level of IL-2 under the condition of PBMC activated by SEA superantigen; Figure 7B shows the level of IL-2 secretion under the condition of activation of PBMC by anti-human CD3 antibody.

Figure 8 shows the detection of tumor inhibitory activity of antigen-binding proteins in a mouse colon cancer tumor model.

Figure 9 shows the statistics of tumor volume in mice after administration.

Figure 10 shows the detection of the flow cytometry binding activity between the targeting part of CLDN18.2 described in the present application and the cells with high expression of human CLDN18.2.

Figure 11 shows the detection of the flow cytometric binding activity between the targeting part of CLDN18.2 described in the present application and high-expressing human CLDN18.1 cells.

Figure 12 shows the detection of the flow cytometry binding activity of the targeting moiety of CLDN18.2 described in the present application to tumor cell lines.

Detailed ways

The implementation of the invention of the present application will be described by specific specific examples below, and those skilled in the art can easily understand other advantages and effects of the invention of the present application from the content disclosed in this specification.

Definition of Terms

In this application, the terms "CLDN18.2", or "Claudin18.2" are used interchangeably and generally refer to subtype 2 of the cell junction claudin Claudin18. The term encompasses "full length", unprocessed CLDN18.2 as well as any form of CLDN18.2 produced by cellular processing. CLDN18.2 may include intact CLDN18.2 and fragments thereof, functional variants, isoforms, species homologues, derivatives, analogs and analogs having at least one common epitope with CLDN18.2. The amino acid sequence of CLDN18.2 (eg, human CLDN18.2) is known in the art. For example, the human CLDN18.2 nucleotide sequence can be shown under GeneBank accession number NM_001002026.3. For example, the mouse CLDN18.2 nucleotide sequence can be shown under GeneBank Accession No. NM_001194921.1. For example, the cynomolgus monkey CLDN18.2 nucleotide sequence can be shown under GeneBank accession number XM_001114708.4.

In this application, belonging to "4-1BB", also known as 4-1BB or TNFRS9, generally refers to a transmembrane protein of the tumor necrosis factor receptor superfamily (TNFRS), which is an activation-induced co-stimulatory molecule is an immune response important regulator. Studies have shown that CD137 agonistic monoclonal antibodies increase the expression of co-stimulatory molecules in many models, and significantly enhance the response of cytolytic T lymphocytes, exerting anti-tumor effects. The anti-tumor effect of CD137-targeted therapy can be confirmed by in vivo anti-tumor efficacy studies in mice using agonistic anti-mouse CD137 monoclonal antibodies. CD137 has emerged as a potent activator of immune cells and an important candidate antigen for the treatment of various diseases. (See Vinay, Dass S., and Byoung S. Kwon."4-1BB(CD137), an inducible costimulatory receptor, as a specific target for cancer therapy."BMB reports 47.3(2014):122.)

In the present application, the term "isolated" generally means obtained from the natural state by artificial means. If an "isolated" substance or component occurs in nature, it may be that its natural environment has been altered, the substance has been isolated from its natural environment, or both. For example, an unisolated polynucleotide or polypeptide naturally exists in a living animal, and the same polynucleotide or polypeptide with high purity isolated from this natural state is called isolation. of. The term "isolated" does not exclude the admixture of artificial or synthetic substances, nor the presence of other impure substances which do not affect the activity of the substance.

In this application, the term "isolated antigen-binding protein" generally refers to a protein with antigen-binding ability obtained from a natural state through artificial means. The "isolated antigen binding protein" may comprise an antigen-binding moiety and, optionally, a framework or framework portion that permits the antigen-binding moiety to adopt a conformation that facilitates binding of said antigen-binding moiety to antigen. Antigen binding proteins may comprise, for example, antibody-derived protein framework regions (FR) or alternative protein framework regions or artificial framework regions with grafted CDRs or CDR derivatives. Such frameworks include, but are not limited to, antibody-derived framework regions comprising mutations introduced, eg, to stabilize the three-dimensional structure of the antigen binding protein, and fully synthetic framework regions comprising, eg, biocompatible polymers. See eg Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53(1):121-129 (2003); Roque et al., Biotechnol. Prog. 20:639-654 (2004). Examples of antigen binding proteins include, but are not limited to: human antibodies, humanized antibodies; chimeric antibodies; recombinant antibodies; single chain antibodies; diabodies; triabodies; tetrabodies; (ab') ₂ , F(ab) ₂ , scFv, di-scFv, dAb, IgD antibody; IgE antibody; IgM antibody; IgGl antibody; IgG2 antibody; IgG3 antibody; or IgG4 antibody and fragments thereof.

In the present application, the isolated antigen binding protein may comprise more than one antigen binding domain. In the present application, the antigen binding domains may target different antigens. In the present application, the antigen binding domains may target different epitopes of the same antigen. For example, the isolated antigen binding protein can comprise a first antigen binding domain and a second antigen binding domain. For example, the first antigen binding domain can target CLDN18.2 protein, for example, the second antigen binding domain can target 4-1BB protein.

In this application, the terms "variable domain" and "variable region" are used interchangeably and generally refer to a portion of an antibody heavy and/or light chain. The variable domains of the heavy and light chains may be referred to as " _VH " and " _VL ", respectively (or "VH" and "VL", respectively). These domains are usually the most variable parts of an antibody (relative to other antibodies of the same class) and comprise the antigen binding site.

In the present application, the term "variable" generally means that some segments of the variable domain may have large differences in sequence between antibodies. The variable domains mediate antigen binding and determine the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains. It is usually concentrated in three segments called hypervariable regions (CDRs or HVRs) in the light and heavy chain variable domains. The more highly conserved portions of variable domains are called the framework regions (FR). The variable domains of native heavy and light chains each comprise four FR regions, most adopting a β-sheet configuration, connected by three CDRs, which form a circular connection and in some cases form part of the β-sheet structure . The CDRs in each chain are held in close proximity by the FR regions, and the CDRs from the other chain together contribute to the formation of the antibody's antigen-binding site (see Kabat et al, Sequences of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)).

In this application, the term "antibody" generally refers to an immunoglobulin or fragment or derivative thereof, encompassing any polypeptide that includes an antigen combining site, whether produced in vitro or in vivo. The term includes, but is not limited to, polyclonal, monoclonal, monospecific, multispecific, nonspecific, humanized, single-stranded, chimeric, synthetic, recombinant, hybrid , mutated and transplanted antibodies. Unless otherwise modified by the term "intact", as in "intact antibody", for the purposes of the present invention, the term "antibody" also includes antibody fragments, such as Fab, F(ab') ₂ , Fv, scFv, Fd, dAbs and other antibody fragments that retain antigen binding function (eg, specifically bind CLDN18.2). Typically, such fragments will include the antigen binding domain. The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies consist of 5 basic heterotetrameric units and another polypeptide called the J chain, and contain 10 antigen-binding sites, while IgA antibodies include 2-5 antigen-binding sites that can be combined with the J chain to form a multivalent A basic 4-chain unit for combinations. For IgG, the 4-chain unit is typically about 150,000 Daltons. Each L chain is linked to an H chain by a covalent disulfide bond, while the two H chains are linked to each other by one or more disulfide bonds depending on the H chain isotype. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has a variable domain (VH) at the N-terminus followed by three constant domains (CH) for the alpha and gamma chains each, and four CH domains for the mu and epsilon isoforms. Each L chain has a variable domain (VL) at its N-terminus and a constant domain at its other end. VL corresponds to VH, and CL corresponds to the first constant domain (CH1) of the heavy chain. Certain amino acid residues are believed to form the interface between the light and heavy chain variable domains. VH and VL pair together to form a single antigen-binding site. For the structure and properties of different classes of antibodies see, e.g., Basic and Clinical Immunology, 8th Edition, Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw (eds), Appleton & Lange, Norwalk, Conn., 1994, p. 71 and Chapter 6. L chains from any vertebrate species can be classified into one of two distinct types, called kappa and lambda, based on the amino acid sequence of their constant domains. Depending on the amino acid sequence of the heavy chain (CH) constant domain, immunoglobulins can be assigned to different classes, or isotypes. There are currently five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains designated alpha, delta, epsilon, gamma, and mu, respectively. The gamma and alpha classes are further divided into subclasses based on relatively minor differences in CH sequence and function, eg, humans express the following subclasses: IgGl, IgG2A, IgG2B, IgG3, IgG4, IgAl and IgKl.

In this application, the term "CDR", also referred to as "complementarity determining region", generally refers to the region in the variable domain of an antibody, the sequence of which is highly variable and/or forms a structurally defined loop. Typically, antibodies comprise six CDRs; three in the VH (HCDR1, HCDR2, HCDR3), and three in the VL (LCDR1, LCDR2, LCDR3). In certain embodiments, naturally occurring camelid antibodies consisting only of heavy chains are capable of functioning and stabilizing in the absence of light chains. See, eg, Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al, Nature Struct. Biol. 3:733-736 (1996).

In this application, the term "FR" generally refers to the more highly conserved portions of antibody variable domains, known as the framework regions. Typically, the variable domains of native heavy and light chains each comprise four FR regions, four in VH (H-FR1, H-FR2, H-FR3, and H-FR4), and four in VL. (L-FR1, L-FR2, L-FR3, and L-FR4). For example, the VL of an isolated antigen binding protein described herein can include the framework regions L-FR1, L-FR2, L-FR3, and L-FR4. The VH of the isolated antigen binding proteins described herein can include framework regions H-FR1, H-FR2, H-FR3, and H-FR4.

In this application, the term "antigen-binding fragment" generally refers to one or more fragments that have the ability to specifically bind an antigen (eg, CLDN18.2). In the present application, the antigen-binding fragment may include Fab, Fab', F(ab) ₂ , Fv fragment, F(ab') ₂ , scFv, di-scFv and/or dAb.

In this application, the term "Fab" generally refers to an antigen-binding fragment of an antibody. Intact antibodies can be digested using papain as described above. Papain digestion of antibodies yields two identical antigen-binding fragments, the "Fab" fragment, and a residual "Fc" fragment (ie, the Fc region, supra). Fab fragments may consist of a complete L chain with the variable region of a heavy chain and the first constant region ( _CH 1 ) of the H chain ( _VH ).

In this application, the term "Fab' fragment" generally refers to a monovalent antigen-binding fragment of a human monoclonal antibody, which fragment is slightly larger than a Fab fragment. For example, a Fab' fragment may include all of the light chain, all of the variable domains of the heavy chain, and all or part of the first and second constant domains of the heavy chain. For example, a Fab' fragment may also include part or all of the 220-330 amino acid residues of the heavy chain.

In this application, the term "F(ab')2" generally refers to antibody fragments produced by pepsin digestion of intact antibodies. The F(ab')2 fragment contains two Fab fragments and part of the hinge region held together by disulfide bonds. F(ab')2 fragments have bivalent antigen binding activity and are capable of cross-linking antigen.

In this application, the term "Fv fragment" generally refers to a monovalent antigen-binding fragment of a human monoclonal antibody comprising all or part of the heavy and light chain variable regions and lacking the heavy and light chain constant regions. The heavy and light chain variable regions include, for example, CDRs. For example, an Fv fragment includes all or part of the approximately 110 amino acid amino-terminal variable regions of the heavy and light chains.

In this application, the term "scFv" generally refers to a fusion protein comprising at least one antibody fragment comprising a variable region of a light chain and at least one antibody fragment comprising a variable region of a heavy chain, wherein the light and heavy chains are variable The regions are contiguous (eg via a synthetic linker such as a short flexible polypeptide linker) and can be expressed as a single chain polypeptide wherein the scFv retains the specificity of the intact antibody from which it was derived. Unless otherwise specified, as used in this application, a scFv can have the VL and VH variable regions described in any order (eg, relative to the N-terminal and C-terminal of the polypeptide), and the scFv can include a VL-linker-VH Or VH-linker-VL can be included.

In the present application, the term "dAb" generally refers to an antigen-binding fragment having a VH domain, a VL domain, or having a VH domain or a VL domain, see e.g. Ward et al. (Nature, 1989 Oct 12; 341(6242): 544-6) , with reference to Holt et al., Trends Biotechnol., 2003, 21(11):484-490; and to other published patent applications such as WO 06/030220, WO 06/003388 and Domantis Ltd.

In this application, the term "monoclonal antibody" generally refers to a preparation of antibody molecules of single molecular composition. Monoclonal antibodies are usually highly specific against a single antigenic site. Furthermore, each monoclonal antibody is directed against a single determinant on the antigen, unlike conventional polyclonal antibody preparations, which typically have different antibodies directed against different determinants. In addition to their specificity, monoclonal antibodies have the advantage that they can be synthesized by hybridoma cultures without contamination from other immunoglobulins. The modifier "monoclonal" denote the characteristics of an antibody obtained from a substantially homogeneous population of antibodies and is not to be construed as requiring that the antibody be produced by any particular method. For example, monoclonal antibodies used herein can be produced in hybridoma cells, or can be produced by recombinant DNA methods.

In this application, the term "chimeric antibody" generally refers to an antibody in which the variable regions are derived from one species and the constant regions are derived from another species. Typically, the variable regions are derived from an antibody of a laboratory animal, such as a rodent ("parent antibody"), and the constant regions are derived from a human antibody, such that the resulting chimeric antibody is more effective in a human individual than the parental (e.g., mouse-derived) antibody. Less likely to trigger an adverse immune response.

In this application, the term "humanized antibody" generally refers to an antibody in which some or all of the amino acids other than the CDR region of a non-human antibody (such as a mouse antibody) are replaced with corresponding amino acids derived from human immunoglobulins. In the CDR regions, small additions, deletions, insertions, substitutions or modifications of amino acids may also be permissible so long as they still retain the ability of the antibody to bind a particular antigen. A humanized antibody optionally will comprise at least a portion of a human immunoglobulin constant region. A "humanized antibody" retains antigen specificity similar to the original antibody. "Humanized" forms of non-human (eg, murine) antibodies may contain, at a minimum, chimeric antibodies of sequence derived from non-human immunoglobulin. In some cases, CDR region residues in a human immunoglobulin (recipient antibody) can be replaced with a non-human species (donor antibody) (such as mouse, rat) having the desired properties, affinity and/or capabilities. , rabbit or non-human primate) residue substitution in the CDR region. In certain instances, FR region residues of the human immunoglobulin may be replaced with corresponding non-human residues. In addition, humanized antibodies can comprise amino acid modifications that are absent in the recipient antibody or in the donor antibody. These modifications may be made to further refine antibody properties, such as binding affinity.

The term "fully human antibody" generally refers to an antibody that comprises only human immunoglobulin protein sequences. Fully human antibodies may contain murine sugar chains if they are produced in mice, in mouse cells, or in hybridomas derived from mouse cells. Similarly, a "mouse antibody" or "rat antibody" refers to an antibody comprising only mouse or rat immunoglobulin sequences, respectively. Fully human antibodies can be produced in humans, in transgenic animals with human immunoglobulin germline sequences, by phage display or other molecular biology methods. Exemplary techniques that can be used to make antibodies are described in US Patents: 6,150,584, 6,458,592, 6,420,140. Other techniques, such as using libraries, are known in the art.

In this application, the term "directly connected" is opposite to the term "indirectly connected", and the term "directly connected" generally refers to a direct connection. For example, the direct connection may be a case where substances are directly connected without a spacer. The spacer may be a linker. For example, the linker can be a peptide linker. The term "indirectly linked" generally refers to the situation where substances are not directly linked. For example, the indirect connection may be through a spacer. For example, in the isolated antigen-binding protein described herein, the C-terminus of L-FR1 may be directly or indirectly linked to the N-terminus of LCDR1.

In this application, the term "isolated nucleic acid molecule" generally refers to an isolated form of nucleotides of any length, deoxyribonucleotides or ribonucleotides, or analogs isolated from their natural environment or artificially synthesized.

In this application, the term "vector" generally refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein can be inserted and the protein can be expressed. The vector can be expressed by transforming, transducing or transfecting the host cell, so that the genetic material elements carried by it can be expressed in the host cell. For example, vectors may include: plasmids; phagemids; cosmids; artificial chromosomes such as yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC) or P1-derived artificial chromosome (PAC); phage such as lambda phage or M13 Phages and animal viruses, etc. Animal virus species used as vectors may include retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, papillomaviruses, and papillomaviruses. Vascular viruses (such as SV40). A vector may contain a variety of elements that control expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, the vector may also contain an origin of replication. Vectors may also include components that facilitate their entry into cells, such as viral particles, liposomes or protein coats, but not only.

In this application, the term "cell" generally refers to a single cell, cell line or cell culture that can be or has been the recipient of a subject's plasmid or vector, which includes a nucleic acid molecule as described herein or a nucleic acid molecule as described herein. Carrier. Cells can include progeny of a single cell. Due to natural, accidental or deliberate mutations, the progeny may not necessarily be completely identical (either in the morphology of the total DNA complement or in the genome) to the original parent cell. Cells may include cells transfected in vitro with the vectors described herein. The cells can be bacterial cells (e.g., E. coli), yeast cells, or other eukaryotic cells, such as COS cells, Chinese hamster ovary (CHO) cells, CHO-K1 cells, LNCAP cells, HeLa cells, HEK293 cells, COS-1 cells , NS0 cells. In certain embodiments, the cells are mammalian cells. In certain embodiments, the mammalian cells are HEK293 cells.

In this application, the term "pharmaceutical composition" generally refers to a composition for preventing/treating a disease or condition. The pharmaceutical composition may comprise the isolated antigen binding protein described herein, the nucleic acid molecule described herein, the carrier described herein and/or the cell described herein, and optionally a pharmaceutically acceptable adjuvant. In addition, the pharmaceutical composition may also comprise one or more suitable (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and/or preservatives. preparations. The acceptable ingredients of the compositions are preferably nontoxic to recipients at the dosages and concentrations employed. Pharmaceutical compositions of the present invention include, but are not limited to, liquid, frozen and lyophilized compositions.

As used herein, the term "pharmaceutically acceptable carrier" generally includes a pharmaceutically acceptable carrier, excipient or stabilizer that is inert to the cells or mammals to which it is exposed at the dosage and concentration employed. poisonous. Physiologically acceptable carriers can include, for example, buffers, antioxidants, low molecular weight (less than about 10 residues) polypeptides, proteins, hydrophilic polymers, amino acids, monosaccharides, disaccharides and other carbohydrates, chelating agents, sugar alcohols, salt-forming counterions such as sodium; and/or nonionic surfactants.

In this application, the term "specifically binds" or "specific" generally refers to a measurable and reproducible interaction, such as the binding between a target and an antibody, that can occur in a heterogeneous population of molecules, including biomolecules. Presence determines the presence of a target. For example, an antibody that specifically binds a target (which may be an epitope) can be an antibody that binds that target with greater affinity, avidity, greater ease, and/or for a greater duration than it binds other targets . In certain embodiments, an antibody specifically binds an epitope on a protein that is conserved among proteins of different species. In certain embodiments, specific binding can include, but does not require exclusive binding.

In this application, the term "subject" generally refers to human or non-human animals, including but not limited to cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats or monkeys.

In this application, the term "tumor" generally refers to a neoplasm or solid lesion formed by abnormal cell growth. In this application, a tumor may be a solid tumor or a non-solid tumor. For example, in the present application, the tumor may be a CLDN18.2 positive tumor. For example, in the present application, a tumor may be a disease and/or disorder related to abnormal expression of CLDN18.2.

The term "cancer" generally refers to a disease characterized by the rapid and uncontrolled growth of abnormal cells. Cancer cells can spread to other parts of the body locally or through the bloodstream and lymphatic system. Cancer in this application includes but not limited to gastric cancer, colon cancer and the like. The terms "tumor" and "cancer" are used interchangeably herein, eg, both terms encompass solid tumors and liquid tumors, eg, diffuse or circulating tumors. As used herein, the term "cancer" or "tumor" can include premalignant as well as malignant cancers and tumors.

In this application, the protein, polypeptide and/or amino acid sequence involved should also be understood to include at least the following scope: variants or homologues having the same or similar functions as the protein or polypeptide.

In the present application, the variant may be, for example, passed through in the amino acid sequence of the protein and/or the polypeptide (for example, an antibody or fragment thereof that specifically binds to CLDN18.2 and/or 4-1BB protein). A protein or polypeptide that has one or more amino acids substituted, deleted, or added. For example, the functional variant may comprise at least 1, such as 1-30, 1-20 or 1-10, further such as 1, 2, 3, 4 or 5 amino acid substitutions , proteins or polypeptides with amino acid changes by deletion and/or insertion. Said functional variant may substantially retain the biological properties of said protein or said polypeptide prior to alteration (eg, substitution, deletion or addition). For example, the functional variant may retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (eg, antigen binding ability) of the protein or polypeptide prior to the alteration. For example, the substitutions may be conservative substitutions.

In the present application, the homologue may have at least about 85% (e.g., having at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or higher) sequence homology of proteins or polypeptides.

In this application, the homology generally refers to the similarity, similarity or association between two or more sequences. "Percentage of sequence homology" can be calculated in the following manner: compare the two sequences to be aligned in the comparison window, and determine that there are identical nucleic acid bases (for example, A, T, C, G, I) in the two sequences ) or the same amino acid residue (for example, Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) Number of positions To obtain the number of matching positions, the number of matching positions was divided by the total number of positions in the comparison window (ie, window size), and the result was multiplied by 100 to yield the percent sequence identity. Alignment for purposes of determining percent sequence homology can be accomplished in various ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared or over a region of sequence of interest. The homology can also be determined by the following methods: FASTA and BLAST. A description of the FASTA algorithm can be found in "An Improved Tool for Biological Sequence Comparison" by W.R.Pearson and D.J. Lipman, Proc. Natl. Acad. Sci., 85:2444-2448, 1988; and D.J. Lipman and W.R. Pearson "Fast and sensitive protein similarity search", Science, 227:1435-1441, 1989. A description of the BLAST algorithm can be found in S. Altschul, W. Gish, W. Miller, E. W. Myers, and D. Lipman, "A Basic Local Alignment Search Tool", Journal of Molecular Biology, 215: 403-410 , 1990.

In this application, the term "comprises" generally refers to the meanings comprising, encompassing, comprising or encompassing. In some cases, it also means "for" and "consisting of".

In this application, the term "about" generally refers to a range of 0.5%-10% above or below the specified value, such as 0.5%, 1%, 1.5%, 2%, 2.5%, above or below the specified value. 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10%.

Detailed description of the invention

The CDR of an antibody, also known as the complementarity determining region, is part of the variable region. The amino acid residues in this region may make contacts with the antigen or antigenic epitope. Antibody CDRs can be determined by various coding systems, such as CCG, Kabat, Chothia, IMGT, Kabat/Chothia, etc. These coding systems are known in the art, see http://www.bioinf.org.uk/abs/index.html#kabatnum for details. Those skilled in the art can use different coding systems to determine the CDR region according to the sequence and structure of the antibody. There may be differences in the CDR regions using different coding systems. In this application, the CDR covers the CDR sequence divided according to any CDR division method; also covers its variants, the variants include the amino acid sequence of the CDR through substitution, deletion and/or addition of one or more amino acids . For example 1-30, 1-20 or 1-10, and for example 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acid substitutions, deletions and/or or insertions; homologues thereof, which may be at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, Amino acid sequences having about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more) sequence homology.

In this application, for example, the CDR may be divided according to the Kabat method.

first antigen binding domain

In the present application, the isolated antigen-binding protein may comprise a first antigen-binding domain capable of specifically binding to CLDN18.2 or a functionally active fragment thereof.

In the present application, the first antigen-binding domain may comprise an antibody or an antigen-binding fragment thereof or a variant thereof.

In the present application, the antibody may be selected from the following groups: monoclonal antibody, single chain antibody, chimeric antibody, humanized antibody and fully human antibody.

In the present application, the antigen-binding fragment can be selected from the following group: Fab, Fab', Fv fragment, F(ab)' ₂ , scFv, di-scFv and/or dAb.

In the present application, the first antigen-binding domain may comprise at least one CDR in the variable region VH of the antibody heavy chain, and the VH comprises the amino acid sequence shown in SEQ ID NO:100.

In the present application, in the isolated antigen-binding protein, the first antigen-binding domain may include HCDR3, and the HCDR3 may include the amino acid sequence shown in SEQ ID NO:97.

In the present application, in the isolated antigen binding protein, the first antigen binding domain may comprise HCDR3, and the HCDR3 may comprise SEQ ID NO:3, SEQ ID NO:57 and SEQ ID NO:71 The amino acid sequence shown in any one.

In the present application, in the isolated antigen-binding protein, the first antigen-binding domain may comprise HCDR2, and the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:98.

In the present application, in the isolated antigen binding protein, the first antigen binding domain may comprise HCDR2, and the HCDR2 may comprise SEQ ID NO:2, SEQ ID NO:56 and SEQ ID NO:70 The amino acid sequence shown in any one.

In the present application, in the isolated antigen binding protein, the first antigen binding domain may comprise HCDR1, and the HCDR1 may comprise SEQ ID NO:99 (X ₁ YX ₂ X ₃ X ₄ , wherein, X ₁ is N or R, X ₂ is G, I or V, X ₃ is I or M, X ₄ is the amino acid sequence shown in H, N or S).

In the present application, in the isolated antigen binding protein, the first antigen binding domain may comprise HCDR1, and the HCDR1 may comprise SEQ ID NO:1, SEQ ID NO:55 and SEQ ID NO:69 The amino acid sequence shown in any one.

In the present application, in the isolated antigen binding protein, the first antigen binding domain may comprise HCDR1, HCDR2 and HCDR3, and the HCDR1 may comprise SEQ ID NO: 99 (X ₁ YX ₂ X ₃ X ₄ , Wherein, _X1 is N or R, _X2 is G, I or V, _X3 is I or M, _X4 is the amino acid sequence shown in H, N or S), and the HCDR2 may comprise SEQ ID NO:98 The amino acid sequence shown in SEQ ID NO:97, and the HCDR3 may include the amino acid sequence shown in SEQ ID NO:97.

In the present application, in the isolated antigen-binding protein, the first antigen-binding domain may comprise HCDR1, HCDR2 and HCDR3, and the HCDR1, HCDR2 and HCDR3 may comprise any amino acid sequence selected from the following group:

In the present application, in the isolated antigen-binding protein, the first antigen-binding domain may comprise H-FR1, the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1, and the The H-FR1 may comprise the amino acid sequence shown in any one of SEQ ID NO:4, SEQ ID NO:43, SEQ ID NO:58 and SEQ ID NO:75.

In the present application, in the isolated antigen binding protein, the first antigen binding domain may comprise H-FR2, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 The amino acid sequence shown in any one of SEQ ID NO:5, SEQ ID NO:44 and SEQ ID NO:59 may be included.

In the present application, in the isolated antigen binding protein, the first antigen binding domain may comprise H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 The amino acid sequence shown in any one of SEQ ID NO:6, SEQ ID NO:45, SEQ ID NO:60 and SEQ ID NO:72 may be included.

In the present application, in the isolated antigen-binding protein, the first antigen-binding domain may comprise H-FR4, the N-terminus of the H-FR4 is directly or indirectly connected to the C-terminus of the HCDR3, and the The H-FR4 may comprise the amino acid sequence shown in any one of SEQ ID NO:7, SEQ ID NO:46 and SEQ ID NO:61.

In the present application, in the isolated antigen-binding protein, the first antigen-binding domain may comprise a heavy chain variable region VH, and the VH may comprise the amino acid sequence shown in SEQ ID NO:100.

In the present application, in the isolated antigen-binding protein, the first antigen-binding domain may comprise a heavy chain variable region VH, and the VH may comprise SEQ ID NO:8, SEQ ID NO:47, SEQ ID The amino acid sequence shown in any one of NO:62, SEQ ID NO:73 and SEQ ID NO:76.

In the present application, in the isolated antigen-binding protein, the first antigen-binding domain may comprise LCDR3, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:101.

In the present application, in the isolated antigen binding protein, the first antigen binding domain may comprise LCDR3, and the LCDR3 may comprise SEQ ID NO: 11, SEQ ID NO: 63, SEQ ID NO: 79 and The amino acid sequence shown in any one of SEQ ID NO:86.

In the present application, in the isolated antigen-binding protein, the first antigen-binding domain may comprise LCDR2, and the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO: 102.

In the present application, in the isolated antigen-binding protein, the first antigen-binding domain may comprise LCDR2, and the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:10 or SEQ ID NO:78.

In the present application, in the isolated antigen-binding protein, the first antigen-binding domain may comprise LCDR1, and the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO:103.

In the present application, in the isolated antigen binding protein, the first antigen binding domain may comprise LCDR1, and the LCDR1 may comprise SEQ ID NO:9, SEQ ID NO:48 and SEQ ID NO:77. The amino acid sequence shown in any one.

In the present application, in the isolated antigen-binding protein, the first antigen-binding domain may comprise LCDR1, LCDR2 and LCDR3, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 103, and the LCDR2 may comprise Comprising the amino acid sequence shown in SEQ ID NO: 102, and the LCDR3 may include the amino acid sequence shown in SEQ ID NO: 101.

In the present application, in the isolated antigen-binding protein, the first antigen-binding domain may comprise LCDR1, LCDR2 and LCDR3, and the LCDR1, LCDR2 and LCDR3 comprise any group of amino acid sequences selected from the following group:

In the present application, in the isolated antigen-binding protein, the first antigen-binding domain may comprise L-FR1, the C-terminus of the L-FR1 is directly or indirectly connected to the N-terminus of the LCDR1, and the The L-FR1 may comprise the amino acid sequence shown in any one of SEQ ID NO:12, SEQ ID NO:49, SEQ ID NO:64, SEQ ID NO:80 and SEQ ID NO:87.

In the present application, in the isolated antigen binding protein, the first antigen binding domain may comprise L-FR2, the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 Can comprise the aminoacid sequence shown in SEQ ID NO:13.

In the present application, in the isolated antigen binding protein, the first antigen binding domain may comprise L-FR3, the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 The amino acid sequence shown in any one of SEQ ID NO:14, SEQ ID NO:50, SEQ ID NO:65 and SEQ ID NO:81 may be included.

In the present application, in the isolated antigen-binding protein, the first antigen-binding domain may comprise L-FR4, the N-terminus of the L-FR4 is directly or indirectly connected to the C-terminus of the LCDR3, and the The L-FR4 may comprise the amino acid sequence shown in any one of SEQ ID NO:15, SEQ ID NO:51, SEQ ID NO:82 and SEQ ID NO:88.

In the present application, in the isolated antigen-binding protein, the first antigen-binding domain may comprise a light chain variable region VL, and the VL may comprise the amino acid sequence shown in SEQ ID NO:104.

In the present application, in the isolated antigen binding protein, the first antigen binding domain may comprise a light chain variable region VL, and the VL may comprise SEQ ID NO: 16, SEQ ID NO: 52, SEQ ID The amino acid sequence shown in any one of NO:66, SEQ ID NO:83 and SEQ ID NO:89.

In the present application, in the isolated antigen binding protein, the first antigen binding domain may comprise HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, and the HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 may be Comprising an amino acid sequence selected from any one of the following groups:

In the present application, in the isolated antigen-binding protein, the first antigen-binding domain may comprise VH and VL, the VH may comprise the amino acid sequence shown in SEQ ID NO: 100, and the VL may comprise Amino acid sequence shown in SEQ ID NO: 104.

In the present application, in the isolated antigen-binding protein, the first antigen-binding domain may comprise VH and VL, and the VH and VL may be selected from any one of the following amino acid sequences:

In the present application, in the isolated antigen-binding protein, the first antigen-binding domain may comprise a heavy chain constant region.

In the present application, in the isolated antigen-binding protein, the heavy chain constant region may comprise an IgG-derived heavy chain constant region.

In the present application, in the isolated antigen-binding protein, the heavy chain constant region may comprise a heavy chain constant region derived from human IgG.

In the present application, in the isolated antigen-binding protein, the heavy chain constant region may comprise a heavy chain constant region derived from human IgG1.

In the present application, in the isolated antigen-binding protein, the heavy chain constant region may comprise an Fc fragment.

In the present application, in the isolated antigen-binding protein, the Fc fragment may contain one or more mutations.

In the present application, in the isolated antigen-binding protein, the Fc fragment may contain one or more mutations among N180A, D239E and L241M.

In the present application, in the isolated antigen-binding protein, the heavy chain constant region may comprise the amino acid sequence shown in SEQ ID NO:17.

In the present application, in the isolated antigen binding protein, the first antigen binding domain may comprise a light chain constant region.

In the present application, in the isolated antigen-binding protein, the light chain constant region may comprise a human Igκ constant region.

In the present application, in the isolated antigen-binding protein, the light chain constant region may comprise the amino acid sequence shown in SEQ ID NO: 18.

In the present application, the first antigen-binding domain may comprise a monoclonal antibody capable of binding CLDN18.2.

second antigen binding domain

In the present application, the isolated antigen-binding protein may comprise a second antigen-binding domain capable of specifically binding to the 4-1BB protein or a functionally active fragment thereof.

In the present application, the second antigen binding domain of the isolated antigen binding protein may comprise scFv.

In the present application, in the isolated antigen-binding protein, the second antigen-binding domain may comprise at least one CDR in the variable region VH of the heavy chain of an antibody, and the VH may comprise SEQ ID NO: 105. amino acid sequence.

In the present application, in the isolated antigen-binding protein, the second antigen-binding domain comprises HCDR3, and the HCDR3 may comprise the amino acid sequence shown in SEQ ID NO:24.

In the present application, in the isolated antigen-binding protein, the second antigen-binding domain comprises HCDR2, and the HCDR2 may comprise the amino acid sequence shown in SEQ ID NO:23.

In the present application, in the isolated antigen-binding protein, the second antigen-binding domain comprises HCDR1, and the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO:22.

In the present application, in the isolated antigen-binding protein, the second antigen-binding domain may comprise HCDR1, HCDR2 and HCDR3, the HCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 22, and the HCDR2 may comprise Comprising the amino acid sequence shown in SEQ ID NO:23, and the HCDR3 may include the amino acid sequence shown in SEQ ID NO:24.

In the present application, in the isolated antigen-binding protein, the second antigen-binding domain may comprise H-FR1, the C-terminus of the H-FR1 is directly or indirectly linked to the N-terminus of the HCDR1, and the The H-FR1 may comprise the amino acid sequence shown in SEQ ID NO:25 or SEQ ID NO:91.

In the present application, in the isolated antigen binding protein, the second antigen binding domain may comprise H-FR2, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 Can comprise the aminoacid sequence shown in SEQ ID NO:26.

In the present application, in the isolated antigen binding protein, the second antigen binding domain may comprise H-FR3, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 Can comprise the aminoacid sequence shown in SEQ ID NO:27.

In the present application, in the isolated antigen-binding protein, the second antigen-binding domain may comprise H-FR4, the N-terminus of the H-FR4 is directly or indirectly connected to the C-terminus of the HCDR3, and the The H-FR4 may comprise the amino acid sequence shown in SEQ ID NO:28.

In the present application, in the isolated antigen-binding protein, the second antigen-binding domain may comprise a heavy chain variable region VH, and the VH may comprise SEQ ID NO:29 or SEQ ID NO:92 amino acid sequence.

In the present application, in the isolated antigen-binding protein, the second antigen-binding domain may comprise LCDR3, and the LCDR3 may comprise the amino acid sequence shown in SEQ ID NO:34.

In the present application, in the isolated antigen-binding protein, the second antigen-binding domain may comprise LCDR2, and the LCDR2 may comprise the amino acid sequence shown in SEQ ID NO:33.

In the present application, in the isolated antigen-binding protein, the second antigen-binding domain may comprise LCDR1, and the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO:32.

In the present application, in the isolated antigen-binding protein, the second antigen-binding domain may comprise LCDR1, LCDR2 and LCDR3, the LCDR1 may comprise the amino acid sequence shown in SEQ ID NO: 32, and the LCDR2 may comprise Comprising the amino acid sequence shown in SEQ ID NO:33, and the LCDR3 may include the amino acid sequence shown in SEQ ID NO:34.

In the present application, in the isolated antigen-binding protein, the second antigen-binding domain may comprise L-FR1, and the L-FR1 may comprise the amino acid sequence shown in SEQ ID NO:35.

In the present application, in the isolated antigen-binding protein, the second antigen-binding domain may comprise L-FR2, and the L-FR2 may comprise the amino acid sequence shown in SEQ ID NO:36.

In the present application, in the isolated antigen-binding protein, the second antigen-binding domain may comprise L-FR3, and the L-FR3 may comprise the amino acid sequence shown in SEQ ID NO:37.

In the present application, in the isolated antigen-binding protein, the second antigen-binding domain may comprise L-FR4, and the L-FR4 may comprise the amino acid sequence shown in SEQ ID NO:38.

In the present application, in the isolated antigen-binding protein, the second antigen-binding domain may comprise a light chain variable region VL, and the VL may comprise the amino acid sequence shown in SEQ ID NO:39.

In the present application, in the isolated antigen-binding protein, wherein the second antigen-binding domain may comprise VH and VL, the VH and VL may be selected from the amino acid sequences of any of the following groups:

In the present application, in the isolated antigen-binding protein, the VH and VL of the second antigen-binding domain may be directly or indirectly linked.

In the present application, in the isolated antigen-binding protein, the VH and VL of the second antigen-binding domain may be linked by a linker. In the present application, the linker may comprise the amino acid sequence shown in SEQ ID NO:40.

In the present application, the antigen-binding fragment may comprise scFv.

In the present application, the scFv may comprise the amino acid sequence shown in SEQ ID NO:41 or SEQ ID NO:96.

In the present application, in the isolated antigen-binding protein, the second antigen-binding domain may comprise an antibody or an antigen-binding fragment thereof.

In the present application, the framework region may be selected from the group consisting of human consensus framework sequences and human germline sequences.

In the present application, the second antigen-binding domain may comprise an antibody or an antigen-binding fragment thereof or a variant thereof.

For example, the second antigen binding domain of the isolated antigen binding protein can be a scFv.

isolated antigen binding protein

The present application provides an antigen-binding protein, which includes a first antigen-binding domain and a second antigen-binding domain. For example, the first antigen binding domain can specifically bind CLDN18.2 protein. For example, the second antigen binding domain can specifically bind a 4-1BB protein.

For example, the first antigen binding domain can be a monoclonal antibody. For example, the second antigen binding domain may be a scFv.

In the present application, the first antigen-binding domain and the second antigen-binding domain may be directly or indirectly linked.

In the present application, the first antigen-binding domain may be connected to the second antigen-binding domain through a linker. For example, the linker can be a peptide linker. For example, the linker can include the amino acid sequence shown in SEQ ID NO: 21.

In the present application, the scFv of the second antigen-binding domain can be connected to the C-terminus of the Fc fragment of the first antigen-binding domain through a linker. For example, the VH of the second antigen-binding domain can be connected to the C-terminus of the Fc fragment of the first antigen-binding domain through a linker. For example, the VL of the second antigen-binding domain can be connected to the C-terminus of the Fc fragment of the first antigen-binding domain through a linker.

In another aspect, the application provides an isolated antigen binding protein, which may comprise two first polypeptides and two second polypeptides. For example, the first polypeptide may comprise a heavy chain of an antigen-binding protein capable of specifically binding CLDN18.2 protein and a scFv capable of specifically binding 4-1BB protein. For example, the second polypeptide may comprise a light chain of an antigen binding protein capable of specifically binding a CLDN18.2 protein.

For example, the first polypeptide sequentially comprises VH, CH1, CH2, CH3 and scFv capable of specifically binding to 4-1BB protein from N-terminus to C-terminus, and the second polypeptide comprises VL and light chain variable region CL ; wherein said VH is paired with said VL and is capable of specifically binding to CLDN18.2. For example, the VH may comprise HCDR1, HCDR2 and HCDR3. For example, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:3. For example, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:55, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:56, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:57. For example, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:69, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:70, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:71.

For example, the VL may comprise LCDR1, LCDR2 and LCDR3. For example, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:9, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:10, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:11. For example, the VL comprises LCDR1, LCDR2 and LCDR3, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:48, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:10, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO : the amino acid sequence shown in 11. For example, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:48, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:10, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:63. For example, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:77, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:78, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:79. For example, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:48, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:10, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:86.

For example, said CH2 and said CH3 may constitute an Fc fragment. For example, the Fc fragment may comprise one or more amino acid mutations selected from the group consisting of N180A, D239E and L241M.

For example, the scFv capable of specifically binding to 4-1BB protein may comprise HCDR1, HCDR2 and HCDR3. For example, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:22, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:23, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:24.

For example, the scFv capable of specifically binding to 4-1BB protein may comprise LCDR1, LCDR2 and LCDR3. For example, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:32, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:33, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:34.

For example, the scFv capable of specifically binding 4-1BB protein may comprise a VH. For example, the VH comprises the amino acid sequence shown in SEQ ID NO:29 or SEQ ID NO:92. For example, the scFv capable of specifically binding 4-1BB protein may comprise a VL. For example, the VL comprises the amino acid sequence shown in SEQ ID NO:39.

For example, the scFv capable of specifically binding to 4-1BB protein may comprise VH and VL. For example, the VH may comprise the amino acid sequence set forth in SEQ ID NO:29, and the VL may comprise the amino acid sequence set forth in SEQ ID NO:39. For example, the VH may comprise the amino acid sequence set forth in SEQ ID NO:92, and the VL may comprise the amino acid sequence set forth in SEQ ID NO:39.

For example, the first polypeptide of the isolated antigen binding protein may comprise the amino acid sequence shown in SEQ ID NO:42. For example, the second polypeptide of the isolated antigen binding protein may comprise any one of SEQ ID NO:20, SEQ ID NO:54, SEQ ID NO:68, SEQ ID NO:85 and SEQ ID NO:90. The amino acid sequence shown.

In the present application, the two first polypeptides of the isolated antigen binding protein may comprise the same amino acid sequence. In the present application, the two second polypeptides of the isolated antigen binding protein may comprise the same amino acid sequence.

Polypeptide molecules, nucleic acid molecules, vectors, cells and pharmaceutical compositions

In another aspect, the application provides polypeptide molecules, which may comprise an isolated antigen binding protein described herein.

In another aspect, the application provides one or more nucleic acid molecules that encode the isolated antigen binding proteins described herein. For example, it may be produced or synthesized by (i) amplified in vitro, such as by polymerase chain reaction (PCR) amplification, (ii) recombinantly produced by cloning, (iii) purified (iv) synthetic, for example by chemical synthesis.

In another aspect, the present application provides a vector, which may comprise the nucleic acid molecule described in the present application. In addition, other genes may be included in the vector, such as marker genes that allow selection of the vector in appropriate host cells and under appropriate conditions. In addition, the vector may also contain expression control elements that permit proper expression of the coding region in an appropriate host. Such control elements are well known to those skilled in the art, and may include, for example, promoters, ribosome binding sites, enhancers, and other control elements that regulate gene transcription or mRNA translation, and the like. The vector can be expressed by transforming, transducing or transfecting the host cell so that the genetic material elements it carries can be expressed in the host cell. Such vectors may include, for example, plasmids, cosmids, viruses, phages, or other vectors commonly used in, for example, genetic engineering. For example, the vector is an expression vector. In addition, the vector may also include components that facilitate its entry into cells, such as, but not exclusively, viral particles, liposomes or protein coats.

In another aspect, the present application provides a cell, which may comprise the nucleic acid molecule or the vector described in the present application. In certain embodiments, each or each host cell may comprise one or more of the nucleic acid molecules or vectors described herein. In certain embodiments, each or each host cell may comprise a plurality (eg, 2 or more) or a plurality (eg, 2 or more) of the nucleic acid molecules or vectors described herein. For example, the vectors described herein can be introduced into the host cells, such as eukaryotic cells, such as cells from plants, fungal or yeast cells, and the like. In certain embodiments, the cells can be bacterial cells (eg, E. coli), yeast cells, or other eukaryotic cells. The vectors described herein can be introduced into the host cells by methods known in the art.

On the other hand, the present application also provides a pharmaceutical composition, which may comprise the isolated antigen-binding protein described in the present application, the polypeptide molecule described in the present application, the nucleic acid molecule described in the present application, the carrier described in the present application and /or the cells described herein, and optionally a pharmaceutically acceptable carrier.

In certain embodiments, the pharmaceutical composition may further comprise one or more (pharmaceutically effective) adjuvants, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers and/or or a suitable formulation of preservatives. The acceptable ingredients of the compositions are preferably nontoxic to recipients at the dosages and concentrations employed. Pharmaceutical compositions of the present invention include, but are not limited to, liquid, frozen and lyophilized compositions.

In certain embodiments, the pharmaceutical compositions may also contain more than one active compound, generally those with complementary activities that do not adversely affect each other. The type and effective amount of such drug may depend, for example, on the amount and type of antagonist present in the formulation, as well as the clinical parameters of the subject.

In certain embodiments, the pharmaceutically acceptable carrier may include any and all solvents, dispersion media, coatings, isotonic agents and absorption delaying agents compatible with pharmaceutical administration, generally safe, nontoxic .

In certain embodiments, the pharmaceutical composition may comprise parenteral, transdermal, intracavity, intraarterial, intrathecal and/or intranasal administration or direct injection into tissue. For example, the pharmaceutical composition can be administered to a patient or subject by infusion or injection. In certain embodiments, the administration of the pharmaceutical composition can be performed by different means, such as intravenous, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration. In certain embodiments, the pharmaceutical composition can be administered without interruption. Such uninterrupted (or continuous) administration can be achieved by a small pump system worn by the patient to measure the influx of the therapeutic agent into the patient, as described in WO2015/036583.

Preparation

In another aspect, the present application provides a method for preparing the antigen-binding protein. The method may comprise culturing the host cell described herein under conditions such that the antigen binding protein is expressed. For example, by using appropriate medium, appropriate temperature and incubation time, etc., these methods are understood by those of ordinary skill in the art.

method and use

On the other hand, the present application also provides the described isolated antigen-binding protein, the described polypeptide molecule, the described nucleic acid molecule, the described carrier, the described cell and/or the described pharmaceutical composition The purposes in, described medicine is used for preventing, alleviating and/or treating disease and/or disease.

On the other hand, the present application also provides a method for preventing, alleviating or treating diseases and/or conditions, the method may include administering the isolated antigen-binding protein, the polypeptide molecule described in the present application to a subject in need , the nucleic acid molecule, the carrier, the cell and/or the pharmaceutical composition. In this application, the administration can be carried out in different ways, such as intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, topical or intradermal administration.

On the other hand, the isolated antigen-binding protein, the polypeptide molecule, the nucleic acid molecule, the carrier, the cell and/or the pharmaceutical composition described in the present application can be used to prevent , Alleviate or treat diseases and/or conditions.

In certain embodiments, the diseases and/or disorders may include diseases and/or disorders associated with abnormal expression of CLDN18.2.

In certain embodiments, the disease and/or condition may include a tumor.

In certain embodiments, the tumor may comprise solid tumors and/or non-solid tumors.

In certain embodiments, the tumor may comprise a CLDN18.2 positive tumor.

In certain embodiments, the tumor may comprise gastric cancer, pancreatic cancer, ovarian cancer, lung cancer, gastroesophageal junction cancer, and/or colon cancer.

On the other hand, the present application also provides a method for detecting CLDN18.2 in a sample. The method comprises administering the isolated antigen binding protein, the polypeptide molecule, the nucleic acid molecule, the vector, the cell and/or the pharmaceutical composition described herein. The method may be an ex vivo and/or in vitro method.

On the other hand, the present application also provides a reagent or kit for detecting CLDN18.2 in a sample, which may comprise the isolated antigen-binding protein, the polypeptide molecule, the nucleic acid molecule, the carrier, the cell And/or the pharmaceutical composition.

On the other hand, the present application also provides that the isolated antigen-binding protein, the polypeptide molecule, the nucleic acid molecule, the carrier, the cell and/or the pharmaceutical composition are used in the preparation of the detection CLDN18.2 Uses in reagents or kits.

In this application, the reagent or kit can be used to detect the presence and/or content of CLDN18.2 in a sample.

Not intending to be limited by any theory, the following examples are only for explaining various technical solutions of the invention of the present application, and are not intended to limit the scope of the invention of the present application.

Example

Example 1 Generation of anti-CLDN18.2/4-1BB bispecific antibody

The humanized Claudin18.2 monoclonal antibody 5E6 independently developed by the company (VH amino acid sequence shown in SEQ ID NO: 8, VL amino acid sequence shown in SEQ ID NO: 16) and fully human 4-1BB monoclonal antibody YN006 ( The VH amino acid sequence is shown in SEQ ID NO:29, and the VL amino acid sequence is shown in SEQ ID NO:39), and the combined construction forms an anti-CLDN18.2/4-1BB bispecific antibody. The double antibody adopts the human IgG1-Fc backbone with amino acid mutations, the anti-CLDN18.2 part of the complete IgG form is linked to the N-terminal of the Fc segment, and the anti-4-1BB part of the single-chain scFv form is linked to the C-terminal of the Fc segment ; Among them, the amino acid mutation in the human IgG1-Fc framework region can silence the ADCC and other effector functions of the double antibody. The amino acid mutation is specifically: N180A/D239E/L241M, and the molecular structure is shown in Figure 1A. The double antibody was purified by Protein A one-step affinity chromatography in the 293F transient expression system, the expression level of the antibody could reach about 130 mg/L, and the SEC-HPLC purity was 96%. The double antibody was named OriAb-362. (Figure 1B)

Example 2 Identification of Antigen Binding Activity of OriAb-362 Double Antibody

The antigen-binding activity of the OriAb-362 double antibody was verified by flow cytometry, and the specific method was as follows: target cells overexpressing human 4-1BB (41BB-NFkB-293T) or overexpressing human CLDN18. 2 target cells (hCLDN18.2-CHO); add serially diluted primary antibodies (ie OriAb-362 double antibody and 4-1BB parent monoclonal antibody YN006; OriAb-362 double antibody and CLDN18.2 parent monoclonal antibody 5E6), a The initial working concentration of the antibody was 100nM, and it was serially diluted 3 times to form a total of 11 concentration gradients; after incubation at 4°C for 1 hour, the plate was washed by centrifugation, and then the corresponding secondary antibody (Goat pAb to human IgG-Dylight 650, abcam, Cat#ab98593), incubated at 4°C for 30 min, centrifuged to wash the plate, and then used an iQue Screener flow cytometer (purchased from IntelliCyt Company) to detect the fluorescence signal on the machine. The results are shown in Figure 2A, the binding activity of OriAb-362 double antibody to 4-1BB cell antigen is slightly lower than that of the parent monoclonal antibody YN006, and the EC50 value is about 2 times; the result is shown in Figure 2B, OriAb-362 double antibody and CLDN18. The binding activity of the 2-cell antigen is basically the same as that of the parental mAb 5E6, and the antigen-binding activity of the parental mAb of CLDN18.2 is retained.

Example 3 Flow cytometric identification of the simultaneous binding activity of both ends of the OriAb-362 double antibody

The method of flow cytometry was used to verify the simultaneous binding activity of both ends of the OriAb-362 double antibody. The method was as follows: select CHO-hCLDN18.2 cells overexpressing human CLDN18.2 to plate, and then add serially diluted OriAb-362 double antibody, antibody from 25nM At the beginning of the working concentration, two-fold gradient dilution was performed to form a total of 9 concentration gradients. At the same time, monoclonal antibody 5E6 was set as a negative control, and the antibody concentration was the same as that of Ori-Ab362 double antibody; an equal volume of fixed concentration (1.5ug/ml working concentration) of biotinylated 4-1BB protein (h4-1BB-Biotin), incubated at 4°C for 1 hour, centrifuged to wash the plate, added secondary antibody (BD phamingen APC Streptavidin, Cat#554067), incubated at 4°C for 30 minutes The plate was washed by centrifugation, and then an iQue Screener flow cytometer (purchased from IntelliCyt) was used to detect the fluorescent signal on the machine. The results are shown in Figure 3. The OriAb-362 double antibody can simultaneously bind to CHO-CLDN18.2 cells and biotinylated 4-1BB protein, that is, the OriAb-362 double antibody has two simultaneous binding activities.

Example 4 Identification of species cross-binding activity of OriAb-362 double antibody

Using our company's lentivirus packaging, infection and stable cell line construction system, we screened and obtained the cell 293T-mouseCLDN18.2 overexpressing mouse CLDN18.2 and the cell 293T-macacaCLDN18.2 overexpressing cynomolgus monkey CLDN18.2, The binding activity of OriAb-362 double antibody to mouse/cynomolgus monkey CLDN18.2 was verified by flow cytometry: the primary antibody was serially diluted OriAb-362 double antibody and CLDN18.2 parent monoclonal antibody 5E6, and the antibody was from 33.3nM The starting working concentration was 3 times serially diluted to form a total of 10 concentration gradients; the secondary antibody was Goat pAb to human IgG (Dylight 650) (abcam, Cat#ab98593), incubated at 4°C for 30min and then centrifuged, then centrifuged with 30ul The flow buffer per well was resuspended, and finally the fluorescent signal was detected on the machine using an iQue Screener flow meter (purchased from IntelliCyt Company). The results are shown in Figure 4A. In terms of the binding activity to 293T-mouseCLDN18.2 cells, the OriAb-362 double antibody is basically the same as the parental monoclonal antibody 5E6, and the EC50 value is basically the same; the results are shown in Figure 4B. The binding activity to 293T-macacaCLDN18.2 cells In terms of binding activity, the OriAb-362 double antibody is slightly weaker than the parental monoclonal antibody 5E6, showing a relationship of 1.5 times the EC50 value. Therefore, the OriAb-362 double antibody basically retains the binding activity of the parent monoclonal antibody 5E6 to the mouse/cynomolgus monkey CLDN18.2 antigen, and can simultaneously bind to human, mouse, and cynomolgus monkey CLDN18.2, and has cross-species binding activity.

Example 5 Flow cytometric competition binding activity analysis of OriAb-362 double antibody

First, label OriAb-362 double antibody, human CLDN18.2 monoclonal antibody 5E6 and human CLDN18.2 positive control monoclonal antibody IMAB362 with biotin (EZ-Link Sulfo-NHS-LC-Biotin, Thermo, A39257) (see patent CN103509114A provided 175D10 clone), each labeling 500ug, biotinylated antibody molecules use molecular interaction analyzer OctetRED384 (sartorius) to measure the labeling effect, choose SA sensor to immobilize the antibody, the antibody concentration is 100nM, labeling 180s, labeling completed biotinylation The antibodies were named Biotin-OriAb362, Biotin-5E6, Biotin-IMAB362.

By flow cytometry fluorescence sorting technique (FACS), using iQue Screener flow meter (purchased from IntelliCyt Company), using PBS containing 0.1% BSA as a buffer to detect the competitive binding activity of the above antibody and CHO-humanCLDN18.2 cells, The specific process is as follows: 1) configure the test antibody (Biotin-OriAb362, Biotin-5E6, Biotin-IMAB362), so that the antibody concentration is 1.5 μg/ml, 30ul/hole, that is, the working concentration is 0.5 μg/ml; configure the competing antibody ( OriAb-362, 5E6, IMAB362), the antibody dilution concentration starts from 6666nM, dilutes the antibody according to the 4-fold ratio gradient to form 9 concentration gradients, sets PBS as the negative control well, 30ul/well, that is, the antibody working concentration starts from 2222nM Initially; the two antibodies were pre-mixed and spread in a 96-well plate; 2) CHO-humanCLDN18.2 cells with a concentration of 1x10 ⁶ cells/ml were prepared using buffer, and added to the above-mentioned 96-well pointed bottom plate (corning 3894), each 30 μl well; 3) Centrifuge and resuspend after incubation at 4°C for 1 hour, add fluorescent secondary antibody (BD phamingen APC Streptavidin, Cat#554067), and incubate at 4°C for 30 minutes; 4) Finally add 25 μl of buffer to each well and mix well detected with a flow instrument. As shown in Figure 5, the analysis results of the flow cytometric competitive binding activity test show that the OriAb-362 double antibody and the 5E6 monoclonal antibody have a completely competitive relationship with each other, while the positive control monoclonal antibody IMAB362 has never been able to completely compete with the OriAb-362 double antibody or the 5E6 monoclonal antibody , there is an imperfect competition relationship. It shows that the OriAb362 double antibody, 5E6 monoclonal antibody and IMAB362 monoclonal antibody may have different antigen-binding epitopes, and the OriAb-362 double antibody is more competitive than IMAB362 in terms of antigen binding activity, and the OriAb-362 double antibody completely retains the parental monoclonal antibody The antigen-binding epitope of 5E6 has the same competitive binding activity.

Example 6 OriAb-362 double antibody activates 4-1BB signaling pathway in a CLDN18.2-dependent manner

In order to verify the agonist activity of the OriAb-362 double antibody to activate the 4-1BB signaling pathway, this example uses the luciferase experimental method to detect: 1) The effector cell 41BB-NFkB-293T is a stably integrated NFkB luciferase reporter For 293T cells overexpressing human 4-1BB, trypsinize the cells and resuspend them with DMEM+10% FBS medium to make the cell density 5*10 ⁵ /well, 50ul/well evenly spread on a white background and opaque in a sterile 96-well plate (Nunclon Delta Surface CAT: 136101); 2) CHO-hCLDN18.2 is a lentivirus-transfected high-expression human CLDN18.2 cell, and NUGC-4 is a natural low-expression human CLDN18.2 cell For human gastric cancer cells, CHO-hCLDN18.2 and NUGC-4 were selected as target cells with different expression levels of CLDN18.2 in this example, and CHO-hCLDN18.2 and NUGC-4 cells were respectively digested with trypsin and cultured with DMEM+10% FBS Resuspend the base so that the cell density is 5*10 ⁵ /well, and spread 50ul/well evenly in the above 96 plate; 3) Add the antibody (YN006, 5E6, OriAb-362, YN006+5E6, hIgG-Fc) of serial dilution , the initial working concentration of the antibody is 50nM, 5-fold serial dilution, a total of 6 gradients, 50ul/well spread in the above-mentioned 96-well plate, and a control well without antibody is set at the same time, and two duplicate wells are set for each treatment; 4 ) put the 96-well plate into a 37-degree cell culture incubator and incubate for 24 hours; 5) use a luciferase detection kit (promega, E6120), add a luciferase substrate to obtain luminescence, and measure the luciferase content with a Tecan microplate reader; 6) Data processing: Fold stimulation=RLU antibody treatment group/RLU antibody-free group. The results are shown in Figure 6. OriAb-362 double antibody can stimulate target cells to activate 4-1BB luciferase signaling, which is dependent on the expression of CLDN18.2, and its activation intensity is positively correlated with the expression abundance of human CLDN18.2 in target cells (Fig. 6A, the target cells with high expression of human CLDN18.2, the activation fold is about 5 times; Fig. 6B, the target cells with low expression of human CLDN18.2, the activation fold is about 2 times), and it is antibody concentration dependent, while Neither the parental mAb nor its combination could activate 4-1BB luciferase signaling.

Example 7 OriAb-362 double antibody can stimulate T cell activation and promote the secretion of IL-2 factor

Superantigens such as SEA can cross-link TCR and MHC class II molecules to activate CD4+ T cells. In this example, SEA superantigen was used to continuously stimulate human PBMC, and the factor release experiment was used to verify the activity of OriAb-362 double antibody in promoting the immune response of human PBMC. "X-VIVO+5%FBS+5ug/ml SEA" culture medium was resuspended, the cell density was 1.25*10 ⁶ /well, 100ul cell suspension/well, spread evenly in a V-bottom sterile 96-well plate; Positive target cells are CHO-hCLDN18.2 cells with high expression of human CLDN18.2 and human gastric cancer cell NUGC-4 with natural low expression of human CLDN18.2. Negative target cells are CHO and CHO-hCLDN18.1 cells. The cell density of 10 ⁵ /well was resuspended (X-VIVO+5%FBS), and 50ul/well was spread in the above-mentioned 96-well plate, and the effective target ratio PBMC:target cell=5:1; antibody (YN006, 5E6, OriAb-362, YN006+5E6, hIgG-Fc) was diluted with X-VIVO+5% FBS medium, and 50ul/well was spread in the above-mentioned 96-well plate, so that the working concentration of the antibody was 100nM, 10nM and 1nM; the antibody, PBMC and target cells were co-incubated for 72 hours at 37°C in a cell culture incubator, and the cell culture supernatant was retained after centrifugation, and the level of IL-2 was detected by ELISA (R&D, DY202).

Solid-phase binding of high-density anti-CD3 antibody can cause cross-linking of TCR-CD3 complex, directly generate activation signal, and stimulate T cell activation. In this embodiment, the solid-phase-bound anti-human CD3 antibody is used to continuously stimulate human PBMC, and the factor release experiment is used to verify the activity of the OriAb-362 double antibody in promoting the immune response of human PBMC, as follows: Coating with a sterile 96-well plate that can absorb proteins Anti-human CD3 antibody, diluted with sterile PBS to a concentration of 1μg/mL, 100mL per well, coated overnight at 4°C; washing CD3 protein: suck up the PBS in the 96-well plate with an aspirator the next day, and use X-VIVO+ Wash the plate three times with 5% FBS medium; then add PBMC, target cells and antibodies in sequence, the antibody concentration and cell amount are the same as above, and the resuspension buffer is X-VIVO+5% FBS; IL-2 kit was used to detect the secretion of factor.

The results are shown in Figure 7. In the case of continuous stimulation of human PBMC by SEA superantigen (Figure 7A) or immobilized anti-CD3 antibody (Figure 7B), OriAb-362 can effectively enhance the presence of CHO-hCLDN18.2 or NUGC-4 cells T cell function leads to the release of IL-2 factor, while the presence of negative cells CHO/CHO-hCLDN18.1 does not lead to an increase in the release level of IL-2, indicating that this activation signal is dependent on CLDN18.2; at the same time only Ori -Ab362 double antibody can effectively enhance the function of T cells and lead to the release of IL-2, the corresponding parental monoclonal antibody and drug combination can not induce the secretion of IL-2.

Example 8 Antitumor activity of OriAb-362 double antibody in mouse colon cancer tumor model

In order to study the anti-tumor effect of OriAb-362 double antibody in mice, human 4-1BB transgenic C57BL/6 mice (Biocytogen, 110004, B-h4-1BB mice) were selected in this example, and tumors were inoculated subcutaneously Mouse colon cancer cells overexpressing human CLDN18.2, that is, MC38-hCLDN18.2 cells (1.5*10 ⁶ /mouse), were grouped when the average tumor volume of the mice was 80 mm ³ , and the grouping information was shown in Table 1 below. On the same day, intraperitoneal administration (BIW*3; administered twice a week for three weeks) was recorded as D1, and the body weight and tumor volume of the mice were measured three times a week, and the mice were euthanized when the tumor volume was greater than 3000 mm ³ . The body weight and tumor volume of the mice were continuously observed until D30, and the body weight of the mice was normal throughout the observation period, as shown in Figure 8A; the tumor inhibition of the mice is shown in Figure 8B, compared to Group1: PBS group and Group2: hIgG-Fc group, Group3/Group4 /Group5/Group6 all had significant tumor inhibitory activity (p<0.0001), among which Group3: 5E6 monoclonal antibody group had no mice that completely eliminated tumors (0/6), and the tumor volume inhibition rate TGI _TV (%) was 39.22% , Group4: 4 mice in the YN006 monoclonal antibody group completely eliminated tumors (4/6), TGI _TV (%) was 102.62%, Group5: 6 mice in the OriAb-362 double antibody group completely eliminated tumors (6/6 ), TGI _TV (%) was 102.81%, and in Group6: 5E6+YN006 combined administration group, 5 mice completely eliminated tumors (5/6), and TGI _TV (%) was 98.62%. The specific data are shown in Table 2. That is, the OriAb-362 double antibody group has a very significant tumor inhibitory activity in mice, which is superior to the 5E6 parental monoclonal antibody, YN006 parental monoclonal antibody and combined administration.

Table 1 Grouping of mice

组别group	抗体Antibody	剂量(mg/kg)Dose (mg/kg)	小鼠数量number of mice	给药途径&周期Administration route & cycle
Group1Group1	PBSPBS	//	66	IP,Biw3 weeksIP, Biw3 weeks
Group2Group2	hIgG-FchIgG-Fc	1010	66	IP,Biw3 weeksIP, Biw3 weeks
Group3Group3
5E65E6	1010	66	IP,Biw3 weeksIP, Biw3 weeks
Group4Group4
YN006YN006	1010	66	IP,Biw3 weeksIP, Biw3 weeks
Group5Group5	OriAb-362OriAb-362	13.313.3	66	IP,Biw3 weeksIP, Biw3 weeks
Group6Group6	5E6+YN0065E6+YN006	10+1010+10	66	IP,Biw3 weeksIP, Biw3 weeks

Table 2 Statistics of tumor suppression results

组别 group	1st给药后30天小鼠消瘤情况30 days after 1st administration, tumor elimination in mice	1st给药后30天的TGI _TV(％)值 TGI _TV (%) value 30 days after 1st administration
G1,PBS,10ml/kgG1, PBS, 10ml/kg	0/60/6	//
G2,hIgG-Fc,10mg/kgG2, hIgG-Fc, 10mg/kg	0/60/6	13.89％13.89%
G3,5E6,10mg/kgG3, 5E6, 10mg/kg	0/60/6	39.22％39.22%
G4,YN006,10mg/kgG4, YN006, 10mg/kg	4/64/6	102.62％102.62%
G5,OriAb362,13.3mg/kgG5, OriAb362, 13.3mg/kg	6/66/6	102.81％102.81%
G6,5E6+YN006,10+10mg/kgG6, 5E6+YN006, 10+10mg/kg	5/65/6	98.62％98.62%

Example 9 Antitumor activity and tumor immune memory function of OriAb-362 double antibody at low dose

In order to further study the anti-tumor activity of the OriAb-362 double antibody at a low dosage, this example reduced the dosage of the OriAb-362 double antibody and designed the following experimental scheme: the mice were human 4-1BB transgenic C57BL/6 mice (Biocytogen, 110004, B-h4-1BB mice), subcutaneously transplanted mouse colon cancer cells overexpressing human CLDN18.2, that is, MC38-hCLDN18.2 cells (1.5*10 ⁶ /only), and treated mice Grouping was performed when the average tumor volume was 80 mm ³ , and the grouping information was shown in Table 3. On the day of grouping, intraperitoneal administration (BIW*3; twice a week for three weeks) was performed and recorded as D1, and the mice were measured three times a week Body weight and tumor volume, when the mouse tumor volume is greater than 3000mm ³ Euthanize the mouse. Analyze the tumor inhibition data of mice on Day 31 after 1st administration, the tumor elimination status of mice in each group is shown in Table 4: G2: OriAb-362, 13.3mg/kg high-dose group and G3: OriAb-362, 3.325mg/kg All the mice in the kg low-dose group eliminated tumors (6/6), and 3 mice in the G4:5E6+YN006 combined administration group eliminated tumors (3/6). Compared with the G1:hIgG-Fc control group, there were significant The anti-tumor activity (p<0.01), as shown in FIG. 9 . The mice whose tumors had been eliminated continued to be observed until Day40, and then each mouse was inoculated subcutaneously on the contralateral side. The tumor cells and doses used for inoculation were the same as above (MC38-hCLDN18.2 cells, 1.5*10 ⁶ /mouse) At the same time, 6 blank mice of the same strain without any experiment were selected as the Naive control group and subcutaneously received tumor treatment at the same time. After receiving the tumor, it was observed for 20 days, and the tumor volume of the mice was measured three times a week. The tumor growth of the mice is shown in Figure 9. Only the 6 mice in the Naive control group all developed tumors, and the mice in the G2/G3/G4 groups that had completely eliminated tumors remained in the state of tumor elimination after subcutaneous tumor grafting on the opposite side. In summary, even at a low dose of 3.325 mg/kg, the OriAb-362 double antibody can induce complete tumor elimination in all the mice enrolled (6/6), and re-inoculate tumors on the contralateral side of the tumor-eliminated mice, The mice still maintain the state of tumor elimination, no new tumor formation, showing good tumor immune memory function, and have long-term protective immune memory.

Table 3 Grouping of mice

组别group	抗体Antibody	剂量(mg/kg)Dose (mg/kg)	小鼠数量number of mice	给药途径&周期Administration route & cycle
11	hIgG-FchIgG-Fc	1010	66	IP,Biw3 weeksIP, Biw3 weeks
22	Ori-Ab362Ori-Ab362	13.313.3	66	IP,Biw3 weeksIP, Biw3 weeks
33	Ori-Ab362Ori-Ab362	3.3253.325	66	IP,Biw3 weeksIP, Biw3 weeks
44	5E6+YN0065E6+YN006	10+310+3	66	IP,Biw3 weeksIP, Biw3 weeks

Table 4 Statistics of tumor elimination in mice

组别group	1st给药后31天小鼠消瘤情况31 days after the 1st administration, tumor elimination in mice
G1：hIgG-Fc,10mg/kgG1: hIgG-Fc, 10mg/kg	0/60/6
G2：OriAb-362,13.3mg/kgG2: OriAb-362, 13.3mg/kg	6/66/6
G3：OriAb-362,3.325mg/kgG3: OriAb-362, 3.325mg/kg	6/66/6
G4：5E6+YN006,10+3mg/kgG4: 5E6+YN006, 10+3mg/kg	3/63/6

Example 10 Determination of CLDN18.2 Antigen Binding Protein and CLDN18.2 Specific Binding Activity

By flow cytometry fluorescence sorting technique (FACS), using iQue Screener flow meter (purchased from IntelliCyt Company), using PBS containing 0.1% BSA as buffer to detect the specific binding activity of the above-mentioned chimeric antibody to target cells, select Three kinds of target cells: a stably transfected cell line expressing human CLDN18.2, a stably transfected cell line expressing human CLDN18.1, and a tumor cell line were tested for their binding activity.

1. Detection of flow cytometric binding activity between the CLDN18.2 antigen binding protein of the present application and high-expression human CLDN18.2 cells

The cell lines that obtained stable and high expression of CLDN18 were constructed and marked as 293T-human CLDN18.2, CHO-human CLDN18.2 and SP2/0-human CLDN18.2 cells respectively, the cells were digested and counted, and the cells were resuspended and adjusted in flow buffer To 1×10 ⁶ /ml, add 30ul/well into a V-bottom 96-well plate; add 30ul/well of the primary antibody, starting at a concentration of 30ug/ml, dilute with flow buffer in a two- or three-fold gradient to form a 7 Each antibody was set as a PBS negative control, and the positive control antibody was zolbetuximab purified in Example 1; incubated at 4°C for 1 hour, washed once with flow buffer, and added secondary antibody (abcam, Cat#ab98593), 30ul/ Well, incubate at 4°C for 30 minutes; wash the flow buffer twice, shake the cells loose, add 25ul/well flow buffer, and wait for the machine. Substituting the original data into GraphPad8.0 software for drawing and calculation, the results are shown in Figure 10.

2. Detection of the flow cytometric binding activity of the CLDN18.2 antigen-binding protein of the present application to high-expressing human CLDN18.1 cells

The positive control antibody is a commercially available anti-CLDN18 antibody (Anti-Claudin18 antibody) (abcam, Cat#ab203563), whose antigen-binding site is located in the intracellular part of the CLDN18.2 tetrachorotin protein, and flow cytometric intracellular staining analysis is required. Specifically, the cells are constructed 293T-human CLDN18.1 and SP2/0-human CLDN18.1 cells. After the cells are digested and counted, they are fixed and ruptured, and the treated cells are resuspended to 1×10 ⁶ in flow buffer /ml, 30ul/well was added to a V-bottom 96-well plate; 30ul/well was added to the primary antibody, the antibody was initially diluted at a concentration of 30ug/ml, and was diluted with flow buffer in a three-fold ratio to form 7 gradients. PBS negative control, positive control antibody dilution conditions are the same as above; incubate at 4°C for 1 hour, wash with flow buffer, add secondary antibody (abcam, Cat#ab98593 and Cat#ab150079), 30ul/well, incubate at 4°C for 30 minutes; Wash twice with flow buffer, shake the cells loose, add 25ul/well flow buffer, and wait for the machine. Substituting the original data into GraphPad8.0 software for drawing and calculation, the results are shown in Figure 11.

3. Detection of the flow cytometric binding activity of the CLDN18.2 antigen-binding protein of the present application to tumor cell lines

Select the tumor cells MC38-human CLDN18.2, which are stably and highly expressing human CLDN18.2, as target cells, digest and count the cells, resuspend the cells with flow cytometry buffer and adjust to 1×10 ⁶ /ml, add in 30ul/well V-bottom 96-well plate; 30ul/well of primary antibody was added, starting at 30ug/ml concentration, diluted with flow buffer in a three-fold ratio to form 7 gradients, with PBS negative control and positive control antibody for each antibody To purify zolbetuximab, the dilution conditions are the same as above; incubate at 4°C for 1 hour, wash once with flow buffer, add secondary antibody (abcam, Cat#ab98593), 30ul/well, incubate at 4°C for 30 minutes; wash twice with flow buffer Once again, shake the cells loose, add 25ul/well flow buffer, and wait for the machine. Substituting the original data into GraphPad8.0 software for drawing and calculation, the results are shown in Figure 12.

Through the above steps, expression and purification obtained 5 CLDN18.2 antigen-binding proteins (wherein, the VH sequence of 5E6 is shown in SEQ ID NO: 8, the VL sequence is shown in SEQ ID NO: 16; the VH sequence of 7E3 is shown in SEQ ID As shown in NO:73, the VL sequence is shown in SEQ ID NO:52; the VH sequence of 3A6 is shown in SEQ ID NO:62, and the VL sequence is shown in SEQ ID NO:66; the VH sequence of 14E12 is shown in SEQ ID NO: 76, the VL sequence is shown in SEQ ID NO:83, the VH sequence of 17B10 is shown in SEQ ID NO:76, and the VL sequence is shown in SEQ ID NO:89), and its antigen binding activity is verified by flow cytometry: as shown in the figure As shown in Figure 10, the five antigen-binding proteins all exhibited concentration-dependent binding activity to human CLDN18.2, and most of them were stronger than the zolbetuximab positive antibody, and the detection results of the three types of cells were consistent; as shown in Figure 11, the five antigen-binding proteins All the proteins can specifically bind to human CLDN18.2 but not to human CLDN18.1; as shown in Figure 12, the five antigen-binding proteins all strongly bind to mouse colon cancer MC38 cells stably and highly expressing human CLDN18.2, The intensity of the binding activity is concentration-dependent, and the effect is equivalent to or stronger than that of the zolbetuximab positive antibody (the sequence of the heavy chain is shown in SEQ ID NO: 106, and the sequence of the light chain is shown in SEQ ID NO: 107).

The foregoing detailed description has been offered by way of explanation and example, not to limit the scope of the appended claims. Variations on the presently recited embodiments of the present application will be apparent to those of ordinary skill in the art and remain within the scope of the appended claims and their equivalents.

Claims

An isolated antigen binding protein comprising: a first antigen binding domain and a second antigen binding domain, the first antigen binding domain is capable of specifically binding tight junction protein 18.2 (CLDN18.2), and the first antigen binding domain Comprising at least one CDR in the variable region VH of the antibody heavy chain, the VH comprising the amino acid sequence shown in SEQ ID NO:100.
The isolated antigen binding protein according to claim 1, wherein said first antigen binding domain comprises HCDR3, and said HCDR3 comprises the amino acid sequence shown in SEQ ID NO:97.
The isolated antigen binding protein according to any one of claims 1-2, wherein said first antigen binding domain comprises HCDR3, and said HCDR3 comprises SEQ ID NO:3, SEQ ID NO:57 and SEQ ID NO : the amino acid sequence shown in any one of 71.
The isolated antigen binding protein according to any one of claims 1-3, wherein said first antigen binding domain comprises HCDR2, and said HCDR2 comprises the amino acid sequence shown in SEQ ID NO:98.
The isolated antigen binding protein according to any one of claims 1-4, wherein said first antigen binding domain comprises HCDR2, and said HCDR2 comprises SEQ ID NO:2, SEQ ID NO:56 and SEQ ID NO : the amino acid sequence shown in any one of 70.
The isolated antigen binding protein according to any one of claims 1-5, wherein said first antigen binding domain comprises HCDR1, and said HCDR1 comprises SEQ ID NO:99(X 1 YX 2 X 3 X 4 , Wherein, X1 is N or R, X2 is G, I or V, X3 is I or M, X4 is the amino acid sequence shown in H, N or S).
The isolated antigen binding protein according to any one of claims 1-6, wherein said first antigen binding domain comprises HCDR1, and said HCDR1 comprises SEQ ID NO:1, SEQ ID NO:55 and SEQ ID NO : the amino acid sequence shown in any one of 69.
The isolated antigen binding protein according to any one of claims 1-7, wherein said first antigen binding domain comprises HCDR1, HCDR2 and HCDR3, said HCDR1 comprising SEQ ID NO: 99 (X 1 YX 2 X 3 X 4 , wherein X 1 is N or R, X 2 is G, I or V, X 3 is I or M, X 4 is the amino acid sequence shown in H, N or S), and the HCDR2 comprises SEQ ID NO The amino acid sequence shown in SEQ ID NO: 98, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 97.
The isolated antigen-binding protein according to any one of claims 1-8, wherein said first antigen-binding domain comprises HCDR1, HCDR2 and HCDR3, said HCDR1, HCDR2 and HCDR3 comprising any group of amino acids selected from sequence:

(1) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:3;

(2) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:55, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:56, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:57; and

(3) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO:69, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:70, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:71.
The isolated antigen-binding protein according to any one of claims 6-9, wherein the first antigen-binding domain comprises H-FR1, and the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1 Linked, and the H-FR1 comprises the amino acid sequence shown in any one of SEQ ID NO:4, SEQ ID NO:43, SEQ ID NO:58 and SEQ ID NO:75.
The isolated antigen binding protein according to any one of claims 6-10, wherein said first antigen binding domain comprises H-FR2, said H-FR2 is located between said HCDR1 and said HCDR2, and said The H-FR2 comprises the amino acid sequence shown in any one of SEQ ID NO:5, SEQ ID NO:44 and SEQ ID NO:59.
The isolated antigen binding protein according to any one of claims 4-11, wherein said first antigen binding domain comprises H-FR3, said H-FR3 is located between said HCDR2 and said HCDR3, and said The H-FR3 comprises the amino acid sequence shown in any one of SEQ ID NO:6, SEQ ID NO:45, SEQ ID NO:60 and SEQ ID NO:72.
The isolated antigen-binding protein according to any one of claims 2-12, wherein the first antigen-binding domain comprises H-FR4, and the N-terminus of the H-FR4 is directly or indirectly connected to the C-terminus of the HCDR3 Linked, and the H-FR4 comprises the amino acid sequence shown in any one of SEQ ID NO:7, SEQ ID NO:46 and SEQ ID NO:61.
The isolated antigen-binding protein according to any one of claims 1-13, wherein said first antigen-binding domain comprises a heavy chain variable region VH, said VH comprising the amino acid sequence shown in SEQ ID NO:100.
The isolated antigen binding protein according to any one of claims 1-14, wherein said first antigen binding domain comprises a heavy chain variable region VH, said VH comprising SEQ ID NO:8, SEQ ID NO:47 , the amino acid sequence shown in any one of SEQ ID NO:62, SEQ ID NO:73 and SEQ ID NO:76.
The isolated antigen binding protein according to any one of claims 1-15, wherein said first antigen binding domain comprises LCDR3, and said LCDR3 comprises the amino acid sequence shown in SEQ ID NO:101.
The isolated antigen binding protein according to any one of claims 1-16, wherein said first antigen binding domain comprises LCDR3, and said LCDR3 comprises SEQ ID NO: 11, SEQ ID NO: 63, SEQ ID NO :79 and the amino acid sequence shown in any one of SEQ ID NO:86.
The isolated antigen binding protein according to any one of claims 1-17, wherein said first antigen binding domain comprises LCDR2, and said LCDR2 comprises the amino acid sequence shown in SEQ ID NO: 102.
The isolated antigen binding protein according to any one of claims 1-18, wherein said first antigen binding domain comprises LCDR2, and said LCDR2 comprises amino acids shown in SEQ ID NO:10 or SEQ ID NO:78 sequence.
The isolated antigen binding protein according to any one of claims 1-19, wherein said first antigen binding domain comprises LCDR1, and said LCDR1 comprises the amino acid sequence shown in SEQ ID NO:103.
The isolated antigen binding protein according to any one of claims 1-20, wherein said first antigen binding domain comprises LCDR1, and said LCDR1 comprises SEQ ID NO:9, SEQ ID NO:48 and SEQ ID NO : the amino acid sequence shown in any one of 77.
The isolated antigen-binding protein according to any one of claims 1-21, wherein the first antigen-binding domain comprises LCDR1, LCDR2 and LCDR3, and the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 103, wherein Said LCDR2 comprises the amino acid sequence shown in SEQ ID NO:102, and said LCDR3 comprises the amino acid sequence shown in SEQ ID NO:101.
The isolated antigen-binding protein according to any one of claims 1-22, wherein said first antigen-binding domain comprises LCDR1, LCDR2 and LCDR3, said LCDR1, LCDR2 and LCDR3 comprising any group of amino acids selected from sequence:

(1) the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:9, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:10, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:11;

(2) the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:48, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:10, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:11;

(3) the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:48, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:10, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:63;

(4) the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:48, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:10, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:86; and

(5) The LCDR1 includes the amino acid sequence shown in SEQ ID NO:77, the LCDR2 includes the amino acid sequence shown in SEQ ID NO:78, and the LCDR3 includes the amino acid sequence shown in SEQ ID NO:79.
The isolated antigen-binding protein according to any one of claims 20-23, wherein the first antigen-binding domain comprises L-FR1, the C-terminus of the L-FR1 is directly or indirectly connected to the N-terminus of the LCDR1 Linked, and the L-FR1 comprises the amino acid sequence shown in any one of SEQ ID NO:12, SEQ ID NO:49, SEQ ID NO:64, SEQ ID NO:80 and SEQ ID NO:87.
The isolated antigen binding protein according to any one of claims 20-24, wherein said first antigen binding domain comprises L-FR2, said L-FR2 is located between said LCDR1 and said LCDR2, and said Said L-FR2 comprises the amino acid sequence shown in SEQ ID NO:13.
The isolated antigen binding protein according to any one of claims 18-25, wherein said first antigen binding domain comprises L-FR3, said L-FR3 is located between said LCDR2 and said LCDR3, and said The L-FR3 comprises the amino acid sequence shown in any one of SEQ ID NO:14, SEQ ID NO:50, SEQ ID NO:65 and SEQ ID NO:81.
The isolated antigen-binding protein according to any one of claims 16-26, wherein the first antigen-binding domain comprises L-FR4, the N-terminus of the L-FR4 is directly or indirectly connected to the C-terminus of the LCDR3 Linked, and the L-FR4 comprises the amino acid sequence shown in any one of SEQ ID NO:15, SEQ ID NO:51, SEQ ID NO:82 and SEQ ID NO:88.
The isolated antigen-binding protein according to any one of claims 1-27, wherein said first antigen-binding domain comprises a light chain variable region VL, said VL comprising the amino acid sequence shown in SEQ ID NO:104.
The isolated antigen binding protein according to any one of claims 1-28, wherein said first antigen binding domain comprises a light chain variable region VL, said VL comprising SEQ ID NO:16, SEQ ID NO:52 , the amino acid sequence shown in any one of SEQ ID NO:66, SEQ ID NO:83 and SEQ ID NO:89.
The isolated antigen binding protein according to any one of claims 1-29, wherein said first antigen binding domain comprises HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3, said HCDR1, HCDR2, HCDR3, LCDR1, LCDR2 and LCDR3 comprise amino acid sequences selected from any one of the following groups:

(1) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:3, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:9, shown LCDR2 comprises the amino acid sequence shown in SEQ ID NO:10, and said LCDR3 comprises the amino acid sequence shown in SEQ ID NO:11;

(2) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:3, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:48, shown LCDR2 comprises the amino acid sequence shown in SEQ ID NO:10, and said LCDR3 comprises the amino acid sequence shown in SEQ ID NO:11;

(3) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:55, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:56, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:57, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:48, shown LCDR2 comprises the amino acid sequence shown in SEQ ID NO:10, and said LCDR3 comprises the amino acid sequence shown in SEQ ID NO:63;

(4) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:69, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:70, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:71, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:48, shown LCDR2 comprises the amino acid sequence shown in SEQ ID NO:10, and said LCDR3 comprises the amino acid sequence shown in SEQ ID NO:11;

(5) the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:69, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:70, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:71, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:77, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:78, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:79; and

(6) The HCDR1 comprises the amino acid sequence shown in SEQ ID NO:69, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:70, the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:71, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:48, the LCDR2 comprises the amino acid sequence shown in SEQ ID NO:10, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:86.
The isolated antigen binding protein according to any one of claims 1-30, wherein said first antigen binding domain comprises VH and VL, said VH comprises the amino acid sequence shown in SEQ ID NO: 100, and said VL comprises the amino acid sequence shown in SEQ ID NO: 104.
The isolated antigen-binding protein according to any one of claims 1-31, wherein the first antigen-binding domain comprises VH and VL, and the VH and VL are selected from any one of the following amino acid sequences:

(1) the VH comprises the amino acid sequence shown in SEQ ID NO:8, and the VL shown comprises the amino acid sequence shown in SEQ ID NO:16;

(2) the VH comprises the amino acid sequence shown in SEQ ID NO:47, and the VL shown comprises the amino acid sequence shown in SEQ ID NO:52;

(3) the VH comprises the amino acid sequence shown in SEQ ID NO:62, and the VL shown comprises the amino acid sequence shown in SEQ ID NO:66;

(4) the VH comprises the amino acid sequence shown in SEQ ID NO:73, and the VL shown comprises the amino acid sequence shown in SEQ ID NO:52;

(5) the VH comprises the amino acid sequence shown in SEQ ID NO:76, and the VL comprises the amino acid sequence shown in SEQ ID NO:83; and

(6) The VH comprises the amino acid sequence shown in SEQ ID NO:76, and the VL comprises the amino acid sequence shown in SEQ ID NO:89.
The isolated antigen binding protein of any one of claims 1-32, wherein the first antigen binding domain comprises a heavy chain constant region.
The isolated antigen binding protein of claim 33, wherein the heavy chain constant region comprises an IgG-derived heavy chain constant region.
The isolated antigen binding protein of any one of claims 33-34, wherein the heavy chain constant region comprises a heavy chain constant region derived from human IgG.
The isolated antigen binding protein of any one of claims 33-35, wherein the heavy chain constant region comprises a heavy chain constant region derived from human IgG1.
The isolated antigen binding protein of any one of claims 33-36, wherein the heavy chain constant region comprises an Fc fragment.
The isolated antigen binding protein of claim 37, wherein said Fc fragment comprises one or more mutations.
The isolated antigen binding protein of any one of claims 37-38, wherein the Fc fragment comprises one or more mutations in N180A, D239E and L241M.
The isolated antigen binding protein according to any one of claims 33-39, wherein the heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:17.
The isolated antigen binding protein of any one of claims 1-40, wherein the first antigen binding domain comprises a light chain constant region.
The isolated antigen binding protein of claim 41, wherein the light chain constant region comprises a human Igκ constant region.
The isolated antigen binding protein according to any one of claims 41-42, wherein the light chain constant region comprises the amino acid sequence shown in SEQ ID NO: 18.
The isolated antigen binding protein of any one of claims 41-43, wherein said first antigen binding domain comprises an antibody or antigen binding fragment thereof.
The isolated antigen binding protein of claim 44, wherein the antibody comprises a monoclonal antibody, a single chain antibody, a chimeric antibody, a humanized antibody and/or a fully human antibody.
The isolated antigen binding protein of claim 44, wherein said antigen binding fragment comprises Fab, Fab', Fv fragment, F(ab)' 2 , scFv, di-scFv and/or dAb.
The isolated antigen binding protein of any one of claims 1-46, wherein the second antigen binding domain is capable of specifically binding 4-1BB.
The isolated antigen binding protein according to any one of claims 1-47, wherein said second antigen binding domain comprises at least one CDR in the VH of an antibody heavy chain variable region, said VH comprising SEQ ID NO: 105 Amino acid sequence shown.
The isolated antigen binding protein according to any one of claims 1-48, wherein said second antigen binding domain comprises HCDR3, and said HCDR3 comprises the amino acid sequence shown in SEQ ID NO:24.
The isolated antigen binding protein according to any one of claims 1-49, wherein said second antigen binding domain comprises HCDR2, and said HCDR2 comprises the amino acid sequence shown in SEQ ID NO:23.
The isolated antigen binding protein according to any one of claims 1-50, wherein said second antigen binding domain comprises HCDR1, and said HCDR1 comprises the amino acid sequence shown in SEQ ID NO:22.
The isolated antigen-binding protein according to any one of claims 1-51, wherein the second antigen-binding domain comprises HCDR1, HCDR2 and HCDR3, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 22, wherein Said HCDR2 comprises the amino acid sequence shown in SEQ ID NO:23, and said HCDR3 comprises the amino acid sequence shown in SEQ ID NO:24.
The isolated antigen binding protein according to any one of claims 51-52, wherein the second antigen binding domain comprises H-FR1, the C-terminus of the H-FR1 is directly or indirectly connected to the N-terminus of the HCDR1 Linked, and the H-FR1 comprises the amino acid sequence shown in SEQ ID NO:25 or SEQ ID NO:91.
The isolated antigen binding protein according to any one of claims 51-53, wherein said second antigen binding domain comprises H-FR2, said H-FR2 is located between said HCDR1 and said HCDR2, and said The H-FR2 comprises the amino acid sequence shown in SEQ ID NO:26.
The isolated antigen binding protein according to any one of claims 50-54, wherein said second antigen binding domain comprises H-FR3, said H-FR3 is located between said HCDR2 and said HCDR3, and said Said H-FR3 comprises the amino acid sequence shown in SEQ ID NO:27.
The isolated antigen-binding protein according to any one of claims 49-55, wherein the second antigen-binding domain comprises H-FR4, the N-terminus of the H-FR4 is directly or indirectly connected to the C-terminus of the HCDR3 Linked, and the H-FR4 comprises the amino acid sequence shown in SEQ ID NO:28.
The isolated antigen binding protein according to any one of claims 1-56, wherein said second antigen binding domain comprises a heavy chain variable region VH, and said VH comprises SEQ ID NO: 29 or SEQ ID NO: The amino acid sequence shown in 92.
The isolated antigen binding protein according to any one of claims 1-57, wherein said second antigen binding domain comprises LCDR3, and said LCDR3 comprises the amino acid sequence shown in SEQ ID NO:34.
The isolated antigen binding protein according to any one of claims 1-58, wherein said second antigen binding domain comprises LCDR2, and said LCDR2 comprises the amino acid sequence shown in SEQ ID NO:33.
The isolated antigen binding protein according to any one of claims 1-59, wherein said second antigen binding domain comprises LCDR1, and said LCDR1 comprises the amino acid sequence shown in SEQ ID NO:32.
The isolated antigen-binding protein according to any one of claims 1-60, wherein the second antigen-binding domain comprises LCDR1, LCDR2 and LCDR3, and the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 32, wherein Said LCDR2 comprises the amino acid sequence shown in SEQ ID NO:33, and said LCDR3 comprises the amino acid sequence shown in SEQ ID NO:34.
The isolated antigen binding protein according to any one of claims 1-61, wherein said second antigen binding domain comprises L-FR1, and said L-FR1 comprises the amino acid sequence shown in SEQ ID NO:35.
The isolated antigen binding protein according to any one of claims 1-62, wherein said second antigen binding domain comprises L-FR2, and said L-FR2 comprises the amino acid sequence shown in SEQ ID NO:36.
The isolated antigen binding protein according to any one of claims 1-63, wherein said second antigen binding domain comprises L-FR3, and said L-FR3 comprises the amino acid sequence shown in SEQ ID NO:37.
The isolated antigen binding protein according to any one of claims 1-64, wherein said second antigen binding domain comprises L-FR4, and said L-FR4 comprises the amino acid sequence shown in SEQ ID NO:38.
The isolated antigen-binding protein according to any one of claims 1-65, wherein the second antigen-binding domain comprises a light chain variable region VL, and the VL comprises the amino acid sequence shown in SEQ ID NO:39 .
The isolated antigen binding protein according to any one of claims 1-66, wherein said second antigen binding domain comprises VH and VL, said VH and VL being selected from the amino acid sequences of any of the following groups:

(1) the VH comprises the amino acid sequence shown in SEQ ID NO:29, and the VL comprises the amino acid sequence shown in SEQ ID NO:39; and

(2) The VH comprises the amino acid sequence shown in SEQ ID NO:92, and the VL comprises the amino acid sequence shown in SEQ ID NO:39.
The isolated antigen binding protein of any one of claims 1-67, wherein the VH and VL of the second antigen binding domain are directly or indirectly linked.
The isolated antigen binding protein of any one of claims 1-68, wherein the VH and VL of the second antigen binding domain are linked by a linker.
The isolated antigen-binding protein of claim 69, wherein the linker comprises the amino acid sequence shown in SEQ ID NO:40.
The isolated antigen binding protein of any one of claims 1-70, wherein the second antigen binding domain comprises an antibody or antigen binding fragment thereof.
The isolated antigen binding protein of claim 71, wherein said antigen binding fragment comprises a scFv.
The isolated antigen binding protein of claim 72, wherein the scFv comprises the amino acid sequence shown in SEQ ID NO:41 or SEQ ID NO:96.
The isolated antigen binding protein of any one of claims 1-73, wherein the first antigen binding domain and the second antigen binding domain are directly or indirectly linked.
The isolated antigen binding protein of any one of claims 1-74, wherein the first antigen binding domain and the second antigen binding domain are linked by a linker.
The isolated antigen binding protein of claim 75, wherein the linker comprises the amino acid sequence shown in SEQ ID NO:21.
The isolated antigen binding protein of any one of claims 72-76, wherein the scFv of the second antigen binding domain is directly or indirectly linked to the C-terminus of the Fc fragment of the first antigen binding domain.
An isolated antigen-binding protein comprising two first polypeptides and two second polypeptides, the first polypeptides sequentially comprising VH, CH1, CH2, CH3 and capable of specifically binding 4- scFv of 1BB protein, the second polypeptide comprises a VL and a light chain variable region CL; wherein the VH is paired with the VL and can specifically bind to CLDN18.2.
The isolated antigen-binding protein according to claim 78, wherein the VH comprises HCDR1, HCDR2 and HCDR3, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:1, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:2 The amino acid sequence of the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:3.
The isolated antigen-binding protein according to claim 78, wherein the VH comprises HCDR1, HCDR2 and HCDR3, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:55, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:56 The amino acid sequence of the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:57.
The isolated antigen-binding protein according to claim 78, wherein the VH comprises HCDR1, HCDR2 and HCDR3, the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:69, and the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:70 The amino acid sequence of the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:71.
The isolated antigen-binding protein according to any one of claims 78-81, wherein said VL comprises LCDR1, LCDR2 and LCDR3, said LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 9, and said LCDR2 comprises SEQ ID NO:9 The amino acid sequence shown in ID NO:10, and the shown LCDR3 comprises the amino acid sequence shown in SEQ ID NO:11.
The isolated antigen-binding protein according to any one of claims 78-81, wherein the VL comprises LCDR1, LCDR2 and LCDR3, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 48, and the LCDR2 comprises SEQ ID NO:48 The amino acid sequence shown in ID NO:10, and the shown LCDR3 comprises the amino acid sequence shown in SEQ ID NO:11.
The isolated antigen-binding protein according to any one of claims 78-81, wherein the VL comprises LCDR1, LCDR2 and LCDR3, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 48, and the LCDR2 comprises SEQ ID NO:48 The amino acid sequence shown in ID NO:10, and the shown LCDR3 comprises the amino acid sequence shown in SEQ ID NO:63.
The isolated antigen-binding protein according to any one of claims 78-81, wherein the VL comprises LCDR1, LCDR2 and LCDR3, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO:77, and the LCDR2 comprises SEQ ID NO:77 The amino acid sequence shown in ID NO:78, and shown LCDR3 comprises the amino acid sequence shown in SEQ ID NO:79.
The isolated antigen-binding protein according to any one of claims 78-81, wherein the VL comprises LCDR1, LCDR2 and LCDR3, the LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 48, and the LCDR2 comprises SEQ ID NO:48 The amino acid sequence shown in ID NO:10, and the shown LCDR3 comprises the amino acid sequence shown in SEQ ID NO:86.
The isolated antigen binding protein according to any one of claims 78-86, wherein said CH2 and said CH3 constitute an Fc fragment.
The isolated antigen binding protein of claim 87, wherein said Fc fragment comprises one or more amino acid mutations selected from the group consisting of N180A, D239E and L241M.
The isolated antigen-binding protein according to any one of claims 78-88, wherein said scFv comprises HCDR1, HCDR2 and HCDR3, said HCDR1 comprises the amino acid sequence shown in SEQ ID NO:22, and said HCDR2 comprises SEQ ID NO:22 The amino acid sequence shown in ID NO:23, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO:24.
The isolated antigen-binding protein according to any one of claims 78-89, wherein said scFv comprises LCDR1, LCDR2 and LCDR3, said LCDR1 comprises the amino acid sequence shown in SEQ ID NO: 32, and said LCDR2 comprises SEQ ID NO:32 The amino acid sequence shown in ID NO:33, and the LCDR3 comprises the amino acid sequence shown in SEQ ID NO:34.
The isolated antigen binding protein according to any one of claims 78-90, wherein said scFv comprises VH, and said VH comprises the amino acid sequence shown in SEQ ID NO:29 or SEQ ID NO:92.
The isolated antigen binding protein according to any one of claims 78-91, wherein the scFv comprises a VL, and the VL comprises the amino acid sequence shown in SEQ ID NO:39.
A polypeptide comprising the antigen binding protein of any one of claims 1-92.
One or more isolated nucleic acid molecules encoding the isolated antigen binding protein of any one of claims 1-92 or the polypeptide of claim 93.
A vector comprising the nucleic acid molecule of claim 94.
A cell comprising the nucleic acid molecule of claim 94 or the vector of claim 95.
A pharmaceutical composition comprising the antigen binding protein of any one of claims 1-92, and optionally a pharmaceutically acceptable carrier.
A method of making the isolated antigen binding protein of any one of claims 1-92, comprising culturing the cell of claim 96 under conditions such that the isolated antigen binding protein is expressed.
The isolated antigen binding protein of any one of claims 1-92, the polypeptide of claim 93, the nucleic acid molecule of claim 94, the carrier of claim 95, the cell of claim 96 And/or the purposes of the pharmaceutical composition described in claim 97 in the preparation of medicine, described medicine is used for preventing, alleviating and/or treating disease and/or disease.
The use according to claim 99, wherein the diseases and/or disorders include diseases and/or disorders associated with abnormal expression of CLDN18.2.
Use according to any one of claims 99-100, wherein the disease and/or condition comprises a tumor.
The use according to claim 101, wherein the tumor comprises solid tumors and/or non-solid tumors.
The use according to any one of claims 101-102, wherein the tumor is selected from gastric cancer, pancreatic cancer, ovarian cancer, lung cancer, gastroesophageal junction cancer and/or colon cancer.
The isolated antigen binding protein of any one of claims 1-92, the polypeptide of claim 93, the nucleic acid molecule of claim 94, the carrier of claim 95, the cell of claim 96 And/or the pharmaceutical composition described in claim 97, it is used for preventing, alleviating and/or treating disease and/or disease.
The isolated antigen binding protein, said polypeptide, said nucleic acid molecule, said carrier, said cell and/or said pharmaceutical composition according to claim 104, wherein said disease and/or Conditions include diseases and/or conditions associated with abnormal expression of CLDN18.2.
The isolated antigen binding protein, said polypeptide, said nucleic acid molecule, said carrier, said cell and/or said pharmaceutical composition according to any one of claims 104-105, wherein The diseases and/or conditions include tumors.
The isolated antigen binding protein, said polypeptide, said nucleic acid molecule, said carrier, said cell and/or said pharmaceutical composition according to claim 106, wherein said tumor comprises a solid tumor and/or non-solid tumors.
The isolated antigen binding protein, said polypeptide, said nucleic acid molecule, said carrier, said cell and/or said pharmaceutical composition according to any one of claims 106-107, wherein Said tumor is selected from gastric cancer, pancreatic cancer, ovarian cancer, lung cancer, gastroesophageal junction cancer and/or colon cancer.
A method for preventing, alleviating and/or treating diseases and/or disorders, said method comprising administering the isolated antigen-binding protein of any one of claims 1-92, claim 93 to a subject in need The polypeptide, the nucleic acid molecule of claim 94, the carrier of claim 95, the cell of claim 96 and/or the pharmaceutical composition of claim 97.
The method according to claim 109, wherein the disease and/or disorder comprises a disease and/or disorder associated with abnormal expression of CLDN18.2.
The method according to any one of claims 109-110, wherein the disease and/or condition comprises a tumor.
The method of claim 111, wherein the tumor comprises a solid tumor and/or a non-solid tumor.
The method according to any one of claims 111-112, wherein the tumor is selected from gastric cancer, pancreatic cancer, ovarian cancer, lung cancer, gastroesophageal junction cancer and/or colon cancer.