WO2025162198A1 - Bispecific antibody-cytokine fusion protein and use thereof - Google Patents

Abstract

Provided is a bispecific antibody-cytokine fusion protein. Specifically provided is a bispecific antibody-cytokine fusion protein targeting to TIGIT and PD-1 and having immune effector cell activation effect. The bispecific antibody-cytokine fusion protein has good medication safety and anti-tumor effect. Also provided is the use of the bispecific antibody-cytokine fusion protein in disease treatment.

Description

Dual anti-cytokine fusion protein and its use

This application claims the benefit of Chinese patent application No. 2024101349441, filed on January 31, 2024. This application incorporates the entirety of the aforementioned Chinese patent application.

Technical Field

The present disclosure relates to dual anti-cytokine fusion proteins, and specifically provides dual anti-cytokine fusion proteins that target TIGIT, target PD-1, and have immune effector cell activation, and their use in disease treatment.

Background Art

T cell immunoglobulin and ITIM domain protein (TIGIT) consists of an extracellular immunoglobulin variable region (IgV) domain, a type 1 transmembrane domain, and an intracellular domain with a canonical immunoreceptor tyrosine-based inhibitory motif (ITIM) and an immunoglobulin tyrosine-based tail (ITT) motif. TIGIT signaling has been primarily studied in natural killer (NK) cells, where the ITT and ITIM motifs have been shown to be essential for TIGIT's function. These motifs recruit GRB2 and, subsequently, SHIP1, thereby inhibiting downstream PI3K and MAPK signaling pathways. TIGIT also binds to β-arrestin to recruit SHIP1, blocking TRAF6 ubiquitination and NF-κB activation. TIGIT can inhibit lymphocytes through three distinct mechanisms: 1) After binding to the poliovirus receptor (PVR), TIGIT can signal via the ITIM and/or ITT motifs in its intracellular tail. 2) TIGIT can bind to PVR and induce PVR signaling in adjacent dendritic cells or tumor cells. 3) TIGIT can inhibit CD226 signaling by binding to PVR with higher affinity or disrupting CD226 homodimerization. All three mechanisms of action of this target are immunosuppressive. Preclinical and clinical studies on the TIGIT target have shown that the anti-tumor effect of TIGIT alone cannot exceed the currently widely used mature strategies such as targeting PD-1 or PD-1 + CTLA-4; it is necessary to select appropriate targets to pair with TIGIT.

Current studies on CD8+ T cells have shown that the more inhibitory receptors expressed, the higher the degree of CD8+ T cell exhaustion. How to solve the problem of immune cell exhaustion is the key to the development of immunotherapy drugs. Current clinical studies have shown that TIGIT antibodies have little effect on patients with resistance to anti-PD1 therapy, suggesting that TIGIT antibodies alone cannot solve the problem of resistance to anti-PD1 therapy. However, multiple studies have shown that TIGIT and its ligand CD155 are upregulated in tumor tissues after PD-1 treatment, suggesting its correlation with resistance to anti-PD1 therapy.

In addition to the correlation with immune exhaustion, how to continuously provide active effector T cells and NK cells is another difficult problem that affects how long patients can continue to respond to treatment. Many studies have shown that IL-15 has the ability to activate NK cells to exert anti-tumor effects and regulate TIGIT and CD226 expression. Studies on soft tissue sarcoma (STS) have shown that intratumoral NK and T cells have significantly high activation and exhaustion markers, and IL-15 has the effect of promoting the expression of NK and T cell activation signals and exhaustion markers. In vitro experiments show that the combination of IL-15 and TIGIT significantly enhances the killing ability of STS. Clinical studies have shown that the combination of IL-15 recombinant protein N-803 and PD-1 can restore the response of immune-tolerant tumor patients and overcome the patient's resistance to immunosuppressants.

Therefore, the present disclosure provides a TIGIT/PD-1/IL-15 dual anti-cytokine fusion protein with a specific structural design, which is expected to enhance the synergistic effect of immune cells, expand the immune regulation function from multiple angles, overcome the drug resistance caused by the immune escape of tumor cells, and thus improve the effect of tumor immunotherapy.

Summary of the Invention

To address the above issues, the present disclosure provides a dual anti-cytokine fusion protein targeting TIGIT, PD-1, and an IL-15/IL-15Rα complex that activates immune effector cells. The dual anti-cytokine fusion protein has good drug safety and anti-tumor effects. The following aspects are provided.

Fusion protein

In a first aspect, the present disclosure relates to a fusion protein comprising:

Targeting the first antigen-binding domain of TIGIT,

Targeting the second antigen-binding domain of PD-1,

IL-15 polypeptide or its functional fragment, and

An IL-15Rα polypeptide or a functional fragment thereof, wherein:

(i) The first antigen-binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL) selected from any one of the following groups, wherein:

(i-a) VH comprising the following three CDRs: CDR-H1 comprising the sequence shown in SEQ ID NO: 3, CDR-H2 comprising the sequence shown in SEQ ID NO: 4, and CDR-H3 comprising the sequence shown in SEQ ID NO: 5; and

A VL comprising the following three CDRs: a CDR-L1 comprising the sequence set forth in SEQ ID NO:6, a CDR-L2 comprising the sequence set forth in SEQ ID NO:7, and a CDR-L3 comprising the sequence set forth in SEQ ID NO:8;

or

(i-b) VH comprising the following three CDRs: CDR-H1 comprising the sequence shown in SEQ ID NO: 11, CDR-H2 comprising the sequence shown in SEQ ID NO: 12, and CDR-H3 comprising the sequence shown in SEQ ID NO: 13; and

A VL comprising the following three CDRs: a CDR-L1 comprising the sequence set forth in SEQ ID NO: 14, a CDR-L2 comprising the sequence set forth in SEQ ID NO: 15, and a CDR-L3 comprising the sequence set forth in SEQ ID NO: 16;

or

(i-c) VH comprising the following three CDRs: CDR-H1 comprising the sequence shown in SEQ ID NO: 19, CDR-H2 comprising the sequence shown in SEQ ID NO: 20, and CDR-H3 comprising the sequence shown in SEQ ID NO: 21; and

A VL comprising the following three CDRs: a CDR-L1 comprising the sequence set forth in SEQ ID NO: 22, a CDR-L2 comprising the sequence set forth in SEQ ID NO: 23, and a CDR-L3 comprising the sequence set forth in SEQ ID NO: 24;

and/or, (ii) the second antigen-binding domain comprises a VH and a VL selected from any one of the following groups, wherein,

(ii-a) a VH comprising the following three CDRs: a CDR-H1 comprising the sequence set forth in SEQ ID NO: 27, a CDR-H2 comprising the sequence set forth in SEQ ID NO: 28, and a CDR-H3 comprising the sequence set forth in SEQ ID NO: 29; and

A VL comprising the following three CDRs: CDR-L1 comprising the sequence set forth in SEQ ID NO: 30, CDR-L2 comprising the sequence set forth in SEQ ID NO: 31, and CDR-L3 comprising the sequence set forth in SEQ ID NO: 32;

or

(ii-b) a VH comprising the following three CDRs: a CDR-H1 comprising the sequence shown in SEQ ID NO: 35, a CDR-H2 comprising the sequence shown in SEQ ID NO: 36, and a CDR-H3 comprising the sequence shown in SEQ ID NO: 37; and

VL comprising the following three CDRs: CDR-L1 comprising the sequence shown in SEQ ID NO:38, CDR-L2 comprising the sequence shown in SEQ ID NO:39, and CDR-L3 comprising the sequence shown in SEQ ID NO:40.

In certain embodiments, the fusion protein, wherein

(i) The first antigen-binding domain comprises a VH and a VL selected from any one of the following groups, wherein:

(i-a) VH comprising the sequence shown in SEQ ID NO: 1 and VL comprising the sequence shown in SEQ ID NO: 2;

(i-b) VH comprising the sequence shown in SEQ ID NO:9 and VL comprising the sequence shown in SEQ ID NO:10;

(i-c) VH comprising the sequence shown in SEQ ID NO:17 and VL comprising the sequence shown in SEQ ID NO:18;

(i-d) VH comprising the sequence shown in SEQ ID NO:41 and VL comprising the sequence shown in SEQ ID NO:42;

(i-e) VH comprising the sequence shown in SEQ ID NO:43 and VL comprising the sequence shown in SEQ ID NO:44;

(i-f) VH comprising the sequence shown in SEQ ID NO:45 and VL comprising the sequence shown in SEQ ID NO:46;

(i-g) VH comprising the sequence shown in SEQ ID NO:45 and VL comprising the sequence shown in SEQ ID NO:47;

(i-h) VH comprising the sequence shown in SEQ ID NO:48 and VL comprising the sequence shown in SEQ ID NO:49;

(i-i) VH comprising the sequence shown in SEQ ID NO:48 and VL comprising the sequence shown in SEQ ID NO:50;

and/or,

(ii) the second antigen-binding domain comprises a VH and a VL selected from any one of the following groups, wherein:

(ii-a) a VH comprising the sequence shown in SEQ ID NO: 25 and a VL comprising the sequence shown in SEQ ID NO: 26;

(ii-b) a VH comprising the sequence shown in SEQ ID NO: 33 and a VL comprising the sequence shown in SEQ ID NO: 34;

(ii-c) a VH comprising the sequence shown in SEQ ID NO:54 and a VL comprising the sequence shown in SEQ ID NO:55;

(ii-d) VH comprising the sequence shown in SEQ ID NO:56 and VL comprising the sequence shown in SEQ ID NO:57;

(ii-e) a VH comprising the sequence shown in SEQ ID NO:58 and a VL comprising the sequence shown in SEQ ID NO:59;

(ii-f) VH comprising the sequence shown in SEQ ID NO:56 and VL comprising the sequence shown in SEQ ID NO:59;

(ii-g) VH comprising the sequence shown in SEQ ID NO:60 and VL comprising the sequence shown in SEQ ID NO:57;

(ii-h) VH comprising the sequence shown in SEQ ID NO:60 and VL comprising the sequence shown in SEQ ID NO:61;

(ii-i) VH comprising the sequence shown in SEQ ID NO:62 and VL comprising the sequence shown in SEQ ID NO:61;

(ii-j) VH comprising the sequence shown in SEQ ID NO:51 and VL comprising the sequence shown in SEQ ID NO:52;

(ii-k) VH comprising the sequence shown in SEQ ID NO:53 and VL comprising the sequence shown in SEQ ID NO:52.

In certain optional embodiments, the VH and VL are compared to the VH and VL in any of groups (i-a)-(i-i), (ii-a)-(ii-k), VH has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; and, VL has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity;

and/or,

(iii) the IL-15 polypeptide comprises the native amino acid sequence as shown in SEQ ID NO: 63, or comprises an IL-15 variant that is different from the native amino acid sequence as shown in SEQ ID NO: 63, wherein the IL-15 variant still maintains the function of promoting effector cell activation; in certain preferred embodiments, the amino acid sequence of the IL-15 variant has at least one amino acid substitution, deletion or addition (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the native amino acid sequence as shown in SEQ ID NO: 63;

And/or, (iiii) the IL-15Rα polypeptide comprises the natural amino acid sequence as shown in SEQ ID NO:64, or comprises an IL-15Rα variant different from the natural amino acid sequence as shown in SEQ ID NO:64, wherein the IL-15Rα variant can still maintain the function of promoting effector cell activation; in certain preferred embodiments, the amino acid sequence of the IL-15Rα variant has at least one amino acid substitution, deletion or addition (for example, 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared with the natural amino acid sequence as shown in SEQ ID NO:64, and in certain preferred embodiments, the IL-15Rα variant comprises the sequence as shown in SEQ ID NO:75.

In a second aspect, the present disclosure relates to a fusion protein comprising: a first antigen-binding domain targeting TIGIT or PD-1, a second antigen-binding domain targeting TIGIT or PD-1, an IL-15 polypeptide or a functional fragment thereof, and an IL-15Rα polypeptide or a functional fragment thereof, wherein the first antigen-binding domain and the second antigen-binding domain bind to different targets respectively, the first antigen-binding domain is a Fab fragment, the second antigen-binding domain is a single-chain antibody, and the IL-15 polypeptide and IL-15Rα polypeptide are located on different peptide chains, respectively.

In certain preferred embodiments, the first antigen-binding domain is an antigen-binding domain targeting TIGIT; and the second antigen-binding domain is an antigen-binding domain targeting PD-1.

In certain preferred embodiments, the single-chain antibody is a scFv.

In certain embodiments, the fusion protein further comprises an Fc domain, wherein the Fc domain comprises a first monomer and a second monomer; wherein:

The N-terminus of the first monomer is optionally connected to one domain of the Fab fragment (e.g., the heavy chain CH1 domain thereof) via a linker, and the C-terminus thereof is optionally connected to an IL-15 polypeptide or a functional fragment thereof, or an IL-15Rα polypeptide or a functional fragment thereof via a linker; preferably, the N-terminus of the first monomer is optionally connected to the heavy chain CH1 domain of the Fab fragment via a linker, and the C-terminus thereof is optionally connected to an IL-15 polypeptide or a functional fragment thereof via a linker; and

The N-terminus of the second monomer is optionally connected to another domain of the Fab fragment (e.g., its light chain CL domain) through a linker, and the C-terminus thereof is optionally connected to an IL-15Rα polypeptide or a functional fragment thereof, or an IL-15 polypeptide or a functional fragment thereof through a linker; preferably, the N-terminus of the second monomer is optionally connected to the light chain CL domain of the Fab fragment through a linker, and the C-terminus thereof is optionally connected to an IL-15Rα polypeptide or a functional fragment thereof through a linker.

In certain preferred embodiments, the Fc domain comprises modifications to promote dimerization of the first monomer and the second monomer.

In certain preferred embodiments, the modification comprises a "knob" modification in one of the first monomer and the second monomer and a "hole" modification in the other of the first monomer and the second monomer to form a "knob-into-hole" modification.

In certain embodiments, the fusion protein, wherein

The IL-15 polypeptide comprises the amino acid sequence of a natural IL-15 polypeptide as shown in SEQ ID NO: 63, or comprises an IL-15 variant having an amino acid sequence different from that of the natural IL-15 polypeptide, wherein the IL-15 variant can still maintain the function of the original natural IL-15 polypeptide in promoting effector cell activation; in certain preferred embodiments, the amino acid sequence of the IL-15 variant has at least one amino acid substitution, deletion or addition (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared with the amino acid sequence of the natural IL-15 polypeptide; and/or,

The IL-15Rα polypeptide comprises the amino acid sequence of a natural IL-15Rα polypeptide as shown in SEQ ID NO:64, or comprises an IL-15Rα variant having an amino acid sequence different from that of the natural IL-15Rα polypeptide, wherein the IL-15Rα variant can still maintain the function of the original natural IL-15Rα polypeptide in promoting effector cell activation; in certain preferred embodiments, the amino acid sequence of the IL-15Rα variant has at least one amino acid substitution, deletion or addition (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared with the amino acid sequence of the natural IL-15Rα polypeptide. In certain preferred embodiments, the IL-15Rα variant comprises the sequence shown in SEQ ID NO:75.

In certain embodiments, the fusion protein comprises:

(i) a first peptide chain comprising the VH of the first antigen-binding domain, a heavy chain constant region 1 (CH1), an Fc domain monomer, an IL-15 polypeptide or an IL-15Rα polypeptide, and a single-chain antibody of the second antigen-binding domain; preferably, the CH1 is the human IgG1 heavy chain constant region CH1; preferably, the N-terminus of the Fc domain monomer is linked to the C-terminus of the CH1 via a first hinge region (e.g., a hinge region comprising a PPCP peptide), and the C-terminus of the Fc domain monomer is linked to the N-terminus of the IL-15 polypeptide or IL-15Rα polypeptide via a linker (e.g., a flexible peptide comprising (G4S)n); preferably, the C-terminus of the IL-15 polypeptide or IL-15Rα polypeptide is linked to the N-terminus of the single-chain antibody via a linker (e.g., a flexible peptide comprising (G4S)n); preferably, the single-chain antibody is a scFv;

and

(ii) a second peptide chain, which comprises the VL of the first antigen-binding domain, a light chain constant region (CL), an Fc domain monomer, an IL-15 polypeptide or an IL-15Rα polypeptide, and a single-chain antibody of the second antigen-binding domain; preferably, the CL is a human kappa light chain constant region; preferably, the N-terminus of the Fc domain monomer is connected to the C-terminus of the CL via a second hinge region (e.g., a hinge region comprising a PPCP peptide), and the C-terminus of the Fc domain monomer is connected to the N-terminus of the IL-15 polypeptide or IL-15Rα polypeptide via a linker (e.g., a flexible peptide comprising (G4S)n); preferably, the C-terminus of the IL-15 polypeptide or IL-15Rα polypeptide is connected to the N-terminus of the single-chain antibody via a linker (e.g., a flexible peptide comprising (G4S)n); preferably, the single-chain antibody is a scFv.

In certain preferred embodiments, the Fc domain monomer of the first peptide chain is capable of forming a dimer with the Fc domain monomer of the second peptide chain.

In certain preferred embodiments, the IL-15 polypeptide is located on the first peptide chain; and the IL-15Rα polypeptide is located on the second peptide chain.

In certain embodiments, the fusion protein, wherein the Fc domain monomer of the first peptide chain can contain modifications with the Fc domain monomer of the second peptide chain to promote dimerization.

In certain preferred embodiments, the modification comprises an amino acid substitution in the CH3 domain of the Fc domain.

In certain preferred embodiments, the modification comprises a "knob" modification in one of the two Fc domains and a "hole" modification in the other of the two Fc domains to form a "knob-into-hole" modification.

In certain preferred embodiments, the Fc domain monomer of the first peptide chain comprises the amino acid sequence shown in SEQ ID NO:71.

In certain preferred embodiments, the Fc domain monomer of the second peptide chain comprises the amino acid sequence shown in SEQ ID NO:72.

In certain preferred embodiments, the two Fc domain monomers comprise the amino acid sequences shown in SEQ ID NO: 71 and 72, respectively.

In certain preferred embodiments, CH1 in the first peptide chain contains the amino acid sequence shown in SEQ ID NO:74, and/or CL in the second peptide chain contains the amino acid sequence shown in SEQ ID NO:73.

In certain preferred embodiments, the first hinge region comprises the amino acid sequence shown in SEQ ID NO:65; the second hinge region comprises the amino acid sequence shown in SEQ ID NO:66.

In certain preferred embodiments, the C-terminus of the first peptide chain and/or the second peptide chain connected to the Fc domain monomer via a linker to the N-terminus of the IL-15 polypeptide or IL-15Rα polypeptide comprises the sequence SEQ ID NO: 67.

In certain preferred embodiments, the linker in the first peptide chain and/or the second peptide chain connecting the C-terminus of the IL-15 polypeptide or IL-15Rα polypeptide and the N-terminus of the single-chain antibody comprises the sequence SEQ ID NO: 68.

In certain embodiments, the fusion protein, wherein the first antigen binding domain targeting TIGIT comprises a VH and a VL selected from any one of the following groups, wherein:

(a) a VH comprising the following three CDRs: a CDR-H1 comprising the sequence set forth in SEQ ID NO: 3, a CDR-H2 comprising the sequence set forth in SEQ ID NO: 4, and a CDR-H3 comprising the sequence set forth in SEQ ID NO: 5; and

A VL comprising the following three CDRs: a CDR-L1 comprising the sequence set forth in SEQ ID NO:6, a CDR-L2 comprising the sequence set forth in SEQ ID NO:7, and a CDR-L3 comprising the sequence set forth in SEQ ID NO:8;

or

(b) a VH comprising the following three CDRs: a CDR-H1 comprising the sequence set forth in SEQ ID NO: 11, a CDR-H2 comprising the sequence set forth in SEQ ID NO: 12, and a CDR-H3 comprising the sequence set forth in SEQ ID NO: 13; and

A VL comprising the following three CDRs: a CDR-L1 comprising the sequence set forth in SEQ ID NO: 14, a CDR-L2 comprising the sequence set forth in SEQ ID NO: 15, and a CDR-L3 comprising the sequence set forth in SEQ ID NO: 16;

or

(c) a VH comprising the following three CDRs: a CDR-H1 comprising the sequence set forth in SEQ ID NO: 19, a CDR-H2 comprising the sequence set forth in SEQ ID NO: 20, and a CDR-H3 comprising the sequence set forth in SEQ ID NO: 21; and

A VL comprising the following three CDRs: a CDR-L1 comprising the sequence set forth in SEQ ID NO: 22, a CDR-L2 comprising the sequence set forth in SEQ ID NO: 23, and a CDR-L3 comprising the sequence set forth in SEQ ID NO: 24;

Preferably, the first antigen binding domain targeting TIGIT comprises a VH and a VL selected from any one of the following groups, wherein:

(i-a) VH comprising the sequence shown in SEQ ID NO: 1 and VL comprising the sequence shown in SEQ ID NO: 2;

(i-b) VH comprising the sequence shown in SEQ ID NO:9 and VL comprising the sequence shown in SEQ ID NO:10;

(i-c) VH comprising the sequence shown in SEQ ID NO:17 and VL comprising the sequence shown in SEQ ID NO:18;

(i-d) VH comprising the sequence shown in SEQ ID NO:41 and VL comprising the sequence shown in SEQ ID NO:42;

(i-e) VH comprising the sequence shown in SEQ ID NO:43 and VL comprising the sequence shown in SEQ ID NO:44;

(i-f) VH comprising the sequence shown in SEQ ID NO:45 and VL comprising the sequence shown in SEQ ID NO:46;

(i-g) VH comprising the sequence shown in SEQ ID NO:45 and VL comprising the sequence shown in SEQ ID NO:47;

(i-h) VH comprising the sequence shown in SEQ ID NO:48 and VL comprising the sequence shown in SEQ ID NO:49;

(i-i) VH comprising the sequence shown in SEQ ID NO:48 and VL comprising the sequence shown in SEQ ID NO:50;

In certain optional embodiments, the VH and VL are compared with the VH and VL in any of groups (i-a) to (i-i), VH has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; and, VL has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

In certain embodiments, the fusion protein, wherein the second antigen-binding domain targeting PD-1 comprises a VH and a VL selected from any one of the following groups, wherein:

(A) VH comprising the following three CDRs: CDR-H1 comprising the sequence set forth in SEQ ID NO: 27, CDR-H2 comprising the sequence set forth in SEQ ID NO: 28, and CDR-H3 comprising the sequence set forth in SEQ ID NO: 29; and

A VL comprising the following three CDRs: CDR-L1 comprising the sequence set forth in SEQ ID NO: 30, CDR-L2 comprising the sequence set forth in SEQ ID NO: 31, and CDR-L3 comprising the sequence set forth in SEQ ID NO: 32;

or

(B) VH comprising the following three CDRs: CDR-H1 comprising the sequence set forth in SEQ ID NO: 35, CDR-H2 comprising the sequence set forth in SEQ ID NO: 36, and CDR-H3 comprising the sequence set forth in SEQ ID NO: 37; and

A VL comprising the following three CDRs: a CDR-L1 comprising the sequence set forth in SEQ ID NO:38, a CDR-L2 comprising the sequence set forth in SEQ ID NO:39, and a CDR-L3 comprising the sequence set forth in SEQ ID NO:40;

Preferably, the second antigen-binding domain targeting PD-1 comprises a VH and a VL selected from any one of the following groups, wherein:

(ii-a) a VH comprising the sequence shown in SEQ ID NO: 25 and a VL comprising the sequence shown in SEQ ID NO: 26;

(ii-b) a VH comprising the sequence shown in SEQ ID NO: 33 and a VL comprising the sequence shown in SEQ ID NO: 34;

(ii-c) a VH comprising the sequence shown in SEQ ID NO:54 and a VL comprising the sequence shown in SEQ ID NO:55;

(ii-d) VH comprising the sequence shown in SEQ ID NO:56 and VL comprising the sequence shown in SEQ ID NO:57;

(ii-e) a VH comprising the sequence shown in SEQ ID NO:58 and a VL comprising the sequence shown in SEQ ID NO:59;

(ii-f) VH comprising the sequence shown in SEQ ID NO:56 and VL comprising the sequence shown in SEQ ID NO:59;

(ii-g) VH comprising the sequence shown in SEQ ID NO:60 and VL comprising the sequence shown in SEQ ID NO:57;

(ii-h) VH comprising the sequence shown in SEQ ID NO:60 and VL comprising the sequence shown in SEQ ID NO:61;

(ii-i) VH comprising the sequence shown in SEQ ID NO:62 and VL comprising the sequence shown in SEQ ID NO:61;

(ii-j) VH comprising the sequence shown in SEQ ID NO:51 and VL comprising the sequence shown in SEQ ID NO:52;

(ii-k) a VH comprising the sequence shown in SEQ ID NO:53 and a VL comprising the sequence shown in SEQ ID NO:52;

In certain optional embodiments, the VH and VL are compared with the VH and VL in any of groups (ii-a) to (ii-k), VH having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; and, VL having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity.

In certain embodiments, the fusion protein comprises:

(i) a first peptide chain having the structure [VH1]-[CH1]-[hinge region 1]-[Fc monomer 1]-[L1]-[IL-15]-[L1]-[VL2]-[L2]-[VH2], and

(ii) a second peptide chain having the structure [VL1]-[CL]-[hinge region 2]-[Fc monomer 2]-[L1]-[IL-15Rα]-[L1]-[VL2]-[L2]-[VH2];

One of the following:

(1) The VH1 comprises the sequence shown in SEQ ID NO: 9, the VL1 comprises the sequence shown in SEQ ID NO: 10, the VH2 comprises the sequence shown in SEQ ID NO: 25, the VL2 comprises the sequence shown in SEQ ID NO: 26, the CL comprises the sequence shown in SEQ ID NO: 73, the CH1 comprises the sequence shown in SEQ ID NO: 74, the Fc monomer 1 comprises the sequence shown in SEQ ID NO: 71, the Fc monomer 2 comprises the sequence shown in SEQ ID NO: 72, and the IL-15 comprises the sequence shown in SEQ ID NO: 63. The IL-15Rα comprises the sequence shown in SEQ ID NO: 64; L1 and L2 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and/or one or more serine (e.g., a peptide linker represented by (G4S)n), preferably, L1 is the sequence shown in SEQ ID NO: 67, and L2 is the sequence shown in SEQ ID NO: 68; the hinge region 1 and hinge region 2 are peptide hinge regions, preferably each independently selected from a peptide hinge region comprising PPCP, preferably, hinge region 1 is the sequence shown in SEQ ID NO: 65, and hinge region 2 is the sequence shown in SEQ ID NO: 66;

(2) The VH1 comprises the sequence shown in SEQ ID NO: 1, the VL1 comprises the sequence shown in SEQ ID NO: 2, the VH2 comprises the sequence shown in SEQ ID NO: 25, the VL2 comprises the sequence shown in SEQ ID NO: 26, the CL comprises the sequence shown in SEQ ID NO: 73, the CH1 comprises the sequence shown in SEQ ID NO: 74, the Fc monomer 1 comprises the sequence shown in SEQ ID NO: 71, the Fc monomer 2 comprises the sequence shown in SEQ ID NO: 72, and the IL-15 comprises the sequence shown in SEQ ID NO: 63. The IL-15Rα comprises the sequence shown in SEQ ID NO: 64; L1 and L2 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and/or one or more serine (e.g., a peptide linker represented by (G4S)n), preferably, L1 is the sequence shown in SEQ ID NO: 67, and L2 is the sequence shown in SEQ ID NO: 68; the hinge region 1 and hinge region 2 are peptide hinge regions, preferably each independently selected from a peptide hinge region comprising PPCP, preferably, hinge region 1 is the sequence shown in SEQ ID NO: 65, and hinge region 2 is the sequence shown in SEQ ID NO: 66;

(3) The VH1 comprises the sequence shown in SEQ ID NO:41, the VL1 comprises the sequence shown in SEQ ID NO:42, the VH2 comprises the sequence shown in SEQ ID NO:54, the VL2 comprises the sequence shown in SEQ ID NO:55, the CL comprises the sequence shown in SEQ ID NO:73, the CH1 comprises the sequence shown in SEQ ID NO:74, the Fc monomer 1 comprises the sequence shown in SEQ ID NO:71, the Fc monomer 2 comprises the sequence shown in SEQ ID NO:72, and the IL-15 comprises the sequence shown in SEQ ID NO:63 The IL-15Rα comprises the sequence shown in SEQ ID NO: 75; L1 and L2 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and/or one or more serine (e.g., a peptide linker represented by (G4S)n), preferably, L1 is the sequence shown in SEQ ID NO: 67, and L2 is the sequence shown in SEQ ID NO: 68; the hinge region 1 and hinge region 2 are peptide hinge regions, preferably each independently selected from a peptide hinge region comprising PPCP, preferably, hinge region 1 is the sequence shown in SEQ ID NO: 65, and hinge region 2 is the sequence shown in SEQ ID NO: 66;

(4) The VH1 comprises the sequence shown in SEQ ID NO:41, the VL1 comprises the sequence shown in SEQ ID NO:42, the VH2 comprises the sequence shown in SEQ ID NO:56, the VL2 comprises the sequence shown in SEQ ID NO:57, the CL comprises the sequence shown in SEQ ID NO:73, the CH1 comprises the sequence shown in SEQ ID NO:74, the Fc monomer 1 comprises the sequence shown in SEQ ID NO:71, the Fc monomer 2 comprises the sequence shown in SEQ ID NO:72, and the IL-15 comprises the sequence shown in SEQ ID NO:63 The IL-15Rα comprises the sequence shown in SEQ ID NO: 75; L1 and L2 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and/or one or more serine (e.g., a peptide linker represented by (G4S)n), preferably, L1 is the sequence shown in SEQ ID NO: 67, and L2 is the sequence shown in SEQ ID NO: 68; the hinge region 1 and hinge region 2 are peptide hinge regions, preferably each independently selected from a peptide hinge region comprising PPCP, preferably, hinge region 1 is the sequence shown in SEQ ID NO: 65, and hinge region 2 is the sequence shown in SEQ ID NO: 66;

(5) The VH1 comprises the sequence shown in SEQ ID NO:41, the VL1 comprises the sequence shown in SEQ ID NO:42, the VH2 comprises the sequence shown in SEQ ID NO:62, the VL2 comprises the sequence shown in SEQ ID NO:61, the CL comprises the sequence shown in SEQ ID NO:73, the CH1 comprises the sequence shown in SEQ ID NO:74, the Fc monomer 1 comprises the sequence shown in SEQ ID NO:71, the Fc monomer 2 comprises the sequence shown in SEQ ID NO:72, and the IL-15 comprises the sequence shown in SEQ ID NO:63. The IL-15Rα comprises the sequence shown in SEQ ID NO: 75; L1 and L2 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and/or one or more serine (e.g., a peptide linker represented by (G4S)n), preferably, L1 is the sequence shown in SEQ ID NO: 67, and L2 is the sequence shown in SEQ ID NO: 68; the hinge region 1 and hinge region 2 are peptide hinge regions, preferably each independently selected from a peptide hinge region comprising PPCP, preferably, hinge region 1 is the sequence shown in SEQ ID NO: 65, and hinge region 2 is the sequence shown in SEQ ID NO: 66;

(6) The VH1 comprises the sequence shown in SEQ ID NO:45, the VL1 comprises the sequence shown in SEQ ID NO:47, the VH2 comprises the sequence shown in SEQ ID NO:51, the VL2 comprises the sequence shown in SEQ ID NO:52, the CL comprises the sequence shown in SEQ ID NO:73, the CH1 comprises the sequence shown in SEQ ID NO:74, the Fc monomer 1 comprises the sequence shown in SEQ ID NO:71, the Fc monomer 2 comprises the sequence shown in SEQ ID NO:72, and the IL-15 comprises the sequence shown in SEQ ID NO:63 The IL-15Rα comprises the sequence shown in SEQ ID NO: 75; L1 and L2 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and/or one or more serine (e.g., a peptide linker represented by (G4S)n), preferably, L1 is the sequence shown in SEQ ID NO: 67, and L2 is the sequence shown in SEQ ID NO: 68; the hinge region 1 and hinge region 2 are peptide hinge regions, preferably each independently selected from a peptide hinge region comprising PPCP, preferably, hinge region 1 is the sequence shown in SEQ ID NO: 65, and hinge region 2 is the sequence shown in SEQ ID NO: 66;

(7) The VH1 comprises the sequence shown in SEQ ID NO:45, the VL1 comprises the sequence shown in SEQ ID NO:47, the VH2 comprises the sequence shown in SEQ ID NO:58, the VL2 comprises the sequence shown in SEQ ID NO:59, the CL comprises the sequence shown in SEQ ID NO:73, the CH1 comprises the sequence shown in SEQ ID NO:74, the Fc monomer 1 comprises the sequence shown in SEQ ID NO:71, the Fc monomer 2 comprises the sequence shown in SEQ ID NO:72, and the IL-15 comprises the sequence shown in SEQ ID NO:63. The IL-15Rα comprises the sequence shown in SEQ ID NO: 75; L1 and L2 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and/or one or more serine (e.g., a peptide linker represented by (G4S)n), preferably, L1 is the sequence shown in SEQ ID NO: 67, and L2 is the sequence shown in SEQ ID NO: 68; the hinge region 1 and hinge region 2 are peptide hinge regions, preferably each independently selected from a peptide hinge region comprising PPCP, preferably, hinge region 1 is the sequence shown in SEQ ID NO: 65, and hinge region 2 is the sequence shown in SEQ ID NO: 66;

(8) The VH1 comprises the sequence shown in SEQ ID NO:45, the VL1 comprises the sequence shown in SEQ ID NO:47, the VH2 comprises the sequence shown in SEQ ID NO:56, the VL2 comprises the sequence shown in SEQ ID NO:57, the CL comprises the sequence shown in SEQ ID NO:73, the CH1 comprises the sequence shown in SEQ ID NO:74, the Fc monomer 1 comprises the sequence shown in SEQ ID NO:71, the Fc monomer 2 comprises the sequence shown in SEQ ID NO:72, and the IL-15 comprises the sequence shown in SEQ ID NO:63. The IL-15Rα comprises the sequence shown in SEQ ID NO: 75; L1 and L2 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and/or one or more serine (e.g., a peptide linker represented by (G4S)n), preferably, L1 is the sequence shown in SEQ ID NO: 67, and L2 is the sequence shown in SEQ ID NO: 68; the hinge region 1 and hinge region 2 are peptide hinge regions, preferably each independently selected from a peptide hinge region comprising PPCP, preferably, hinge region 1 is the sequence shown in SEQ ID NO: 65, and hinge region 2 is the sequence shown in SEQ ID NO: 66;

(9) The VH1 comprises the sequence shown in SEQ ID NO:45, the VL1 comprises the sequence shown in SEQ ID NO:47, the VH2 comprises the sequence shown in SEQ ID NO:56, the VL2 comprises the sequence shown in SEQ ID NO:59, the CL comprises the sequence shown in SEQ ID NO:73, the CH1 comprises the sequence shown in SEQ ID NO:74, the Fc monomer 1 comprises the sequence shown in SEQ ID NO:71, the Fc monomer 2 comprises the sequence shown in SEQ ID NO:72, and the IL-15 comprises the sequence shown in SEQ ID NO:63. The IL-15Rα comprises the sequence shown in SEQ ID NO: 75; L1 and L2 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and/or one or more serine (e.g., a peptide linker represented by (G4S)n), preferably, L1 is the sequence shown in SEQ ID NO: 67, and L2 is the sequence shown in SEQ ID NO: 68; the hinge region 1 and hinge region 2 are peptide hinge regions, preferably each independently selected from a peptide hinge region comprising PPCP, preferably, hinge region 1 is the sequence shown in SEQ ID NO: 65, and hinge region 2 is the sequence shown in SEQ ID NO: 66;

(10) The VH1 comprises the sequence shown in SEQ ID NO:45, the VL1 comprises the sequence shown in SEQ ID NO:46, the VH2 comprises the sequence shown in SEQ ID NO:60, the VL2 comprises the sequence shown in SEQ ID NO:57, the CL comprises the sequence shown in SEQ ID NO:73, the CH1 comprises the sequence shown in SEQ ID NO:74, the Fc monomer 1 comprises the sequence shown in SEQ ID NO:71, the Fc monomer 2 comprises the sequence shown in SEQ ID NO:72, and the IL-15 comprises the sequence shown in SEQ ID NO:63. The IL-15Rα comprises the sequence shown in SEQ ID NO: 75; L1 and L2 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and/or one or more serine (e.g., a peptide linker represented by (G4S)n), preferably, L1 is the sequence shown in SEQ ID NO: 67, and L2 is the sequence shown in SEQ ID NO: 68; the hinge region 1 and hinge region 2 are peptide hinge regions, preferably each independently selected from a peptide hinge region comprising PPCP, preferably, hinge region 1 is the sequence shown in SEQ ID NO: 65, and hinge region 2 is the sequence shown in SEQ ID NO: 66;

(11) The VH1 comprises the sequence shown in SEQ ID NO:45, the VL1 comprises the sequence shown in SEQ ID NO:47, the VH2 comprises the sequence shown in SEQ ID NO:62, the VL2 comprises the sequence shown in SEQ ID NO:61, the CL comprises the sequence shown in SEQ ID NO:73, the CH1 comprises the sequence shown in SEQ ID NO:74, the Fc monomer 1 comprises the sequence shown in SEQ ID NO:71, the Fc monomer 2 comprises the sequence shown in SEQ ID NO:72, and the IL-15 comprises the sequence shown in SEQ ID NO:63. The IL-15Rα comprises the sequence shown in SEQ ID NO: 75; L1 and L2 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and/or one or more serine (e.g., a peptide linker represented by (G4S)n), preferably, L1 is the sequence shown in SEQ ID NO: 67, and L2 is the sequence shown in SEQ ID NO: 68; the hinge region 1 and hinge region 2 are peptide hinge regions, preferably each independently selected from a peptide hinge region comprising PPCP, preferably, hinge region 1 is the sequence shown in SEQ ID NO: 65, and hinge region 2 is the sequence shown in SEQ ID NO: 66;

(12) The VH1 comprises the sequence shown in SEQ ID NO:48, the VL1 comprises the sequence shown in SEQ ID NO:50, the VH2 comprises the sequence shown in SEQ ID NO:60, the VL2 comprises the sequence shown in SEQ ID NO:57, the CL comprises the sequence shown in SEQ ID NO:73, the CH1 comprises the sequence shown in SEQ ID NO:74, the Fc monomer 1 comprises the sequence shown in SEQ ID NO:71, the Fc monomer 2 comprises the sequence shown in SEQ ID NO:72, and the IL-15 comprises the sequence shown in SEQ ID NO:63. The IL-15Rα comprises the sequence shown in SEQ ID NO: 75; L1 and L2 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and/or one or more serine (e.g., a peptide linker represented by (G4S)n), preferably, L1 is the sequence shown in SEQ ID NO: 67, and L2 is the sequence shown in SEQ ID NO: 68; the hinge region 1 and hinge region 2 are peptide hinge regions, preferably each independently selected from a peptide hinge region comprising PPCP, preferably, hinge region 1 is the sequence shown in SEQ ID NO: 65, and hinge region 2 is the sequence shown in SEQ ID NO: 66;

(13) The VH1 comprises the sequence shown in SEQ ID NO:48, the VL1 comprises the sequence shown in SEQ ID NO:50, the VH2 comprises the sequence shown in SEQ ID NO:51, the VL2 comprises the sequence shown in SEQ ID NO:52, the CL comprises the sequence shown in SEQ ID NO:73, the CH1 comprises the sequence shown in SEQ ID NO:74, the Fc monomer 1 comprises the sequence shown in SEQ ID NO:71, the Fc monomer 2 comprises the sequence shown in SEQ ID NO:72, and the IL-15 comprises the sequence shown in SEQ ID NO:63 The IL-15Rα comprises the sequence shown in SEQ ID NO: 75; L1 and L2 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and/or one or more serine (e.g., a peptide linker represented by (G4S)n), preferably, L1 is the sequence shown in SEQ ID NO: 67, and L2 is the sequence shown in SEQ ID NO: 68; the hinge region 1 and hinge region 2 are peptide hinge regions, preferably each independently selected from a peptide hinge region comprising PPCP, preferably, hinge region 1 is the sequence shown in SEQ ID NO: 65, and hinge region 2 is the sequence shown in SEQ ID NO: 66;

(14) The VH1 comprises the sequence shown in SEQ ID NO:48, the VL1 comprises the sequence shown in SEQ ID NO:49, the VH2 comprises the sequence shown in SEQ ID NO:60, the VL2 comprises the sequence shown in SEQ ID NO:61, the CL comprises the sequence shown in SEQ ID NO:73, the CH1 comprises the sequence shown in SEQ ID NO:74, the Fc monomer 1 comprises the sequence shown in SEQ ID NO:71, the Fc monomer 2 comprises the sequence shown in SEQ ID NO:72, and the IL-15 comprises the sequence shown in SEQ ID NO:63. The IL-15Rα comprises the sequence shown in SEQ ID NO: 75; L1 and L2 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and/or one or more serine (e.g., a peptide linker represented by (G4S)n), preferably, L1 is the sequence shown in SEQ ID NO: 67, and L2 is the sequence shown in SEQ ID NO: 68; the hinge region 1 and hinge region 2 are peptide hinge regions, preferably each independently selected from a peptide hinge region comprising PPCP, preferably, hinge region 1 is the sequence shown in SEQ ID NO: 65, and hinge region 2 is the sequence shown in SEQ ID NO: 66;

(15) The VH1 comprises the sequence shown in SEQ ID NO:48, the VL1 comprises the sequence shown in SEQ ID NO:49, the VH2 comprises the sequence shown in SEQ ID NO:53, the VL2 comprises the sequence shown in SEQ ID NO:52, the CL comprises the sequence shown in SEQ ID NO:73, the CH1 comprises the sequence shown in SEQ ID NO:74, the Fc monomer 1 comprises the sequence shown in SEQ ID NO:71, the Fc monomer 2 comprises the sequence shown in SEQ ID NO:72, and the IL-15 comprises the sequence shown in SEQ ID NO:63. The IL-15Rα comprises the sequence shown in SEQ ID NO: 75; L1 and L2 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and/or one or more serine (e.g., a peptide linker represented by (G4S)n), preferably, L1 is the sequence shown in SEQ ID NO: 67, and L2 is the sequence shown in SEQ ID NO: 68; the hinge region 1 and hinge region 2 are peptide hinge regions, preferably each independently selected from a peptide hinge region comprising PPCP, preferably, hinge region 1 is the sequence shown in SEQ ID NO: 65, and hinge region 2 is the sequence shown in SEQ ID NO: 66;

(16) The VH1 comprises the sequence shown in SEQ ID NO:43, the VL1 comprises the sequence shown in SEQ ID NO:44, the VH2 comprises the sequence shown in SEQ ID NO:60, the VL2 comprises the sequence shown in SEQ ID NO:57, the CL comprises the sequence shown in SEQ ID NO:73, the CH1 comprises the sequence shown in SEQ ID NO:74, the Fc monomer 1 comprises the sequence shown in SEQ ID NO:71, the Fc monomer 2 comprises the sequence shown in SEQ ID NO:72, and the IL-15 comprises the sequence shown in SEQ ID NO:63. The IL-15Rα comprises the sequence shown in SEQ ID NO: 75; L1 and L2 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and/or one or more serine (e.g., a peptide linker represented by (G4S)n), preferably, L1 is the sequence shown in SEQ ID NO: 67, and L2 is the sequence shown in SEQ ID NO: 68; the hinge region 1 and hinge region 2 are peptide hinge regions, preferably each independently selected from a peptide hinge region comprising PPCP, preferably, hinge region 1 is the sequence shown in SEQ ID NO: 65, and hinge region 2 is the sequence shown in SEQ ID NO: 66;

(17) The VH1 comprises the sequence shown in SEQ ID NO:43, the VL1 comprises the sequence shown in SEQ ID NO:44, the VH2 comprises the sequence shown in SEQ ID NO:60, the VL2 comprises the sequence shown in SEQ ID NO:61, the CL comprises the sequence shown in SEQ ID NO:73, the CH1 comprises the sequence shown in SEQ ID NO:74, the Fc monomer 1 comprises the sequence shown in SEQ ID NO:71, the Fc monomer 2 comprises the sequence shown in SEQ ID NO:72, and the IL-15 comprises the sequence shown in SEQ ID NO:63. The IL-15Rα comprises the sequence shown in SEQ ID NO: 75; L1 and L2 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and/or one or more serine (e.g., a peptide linker represented by (G4S)n), preferably, L1 is the sequence shown in SEQ ID NO: 67, and L2 is the sequence shown in SEQ ID NO: 68; the hinge region 1 and hinge region 2 are peptide hinge regions, preferably each independently selected from a peptide hinge region comprising PPCP, preferably, hinge region 1 is the sequence shown in SEQ ID NO: 65, and hinge region 2 is the sequence shown in SEQ ID NO: 66;

(18) The VH1 comprises the sequence shown in SEQ ID NO:45, the VL1 comprises the sequence shown in SEQ ID NO:46, the VH2 comprises the sequence shown in SEQ ID NO:53, the VL2 comprises the sequence shown in SEQ ID NO:52, the CL comprises the sequence shown in SEQ ID NO:73, the CH1 comprises the sequence shown in SEQ ID NO:74, the Fc monomer 1 comprises the sequence shown in SEQ ID NO:71, the Fc monomer 2 comprises the sequence shown in SEQ ID NO:72, and the IL-15 comprises the sequence shown in SEQ ID NO:63. The IL-15Rα comprises the sequence shown in SEQ ID NO:75; the L1 and L2 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycines and/or one or more serines (for example, a peptide linker shown in (G4S)n), preferably, L1 is the sequence shown in SEQ ID NO:67, and L2 is the sequence shown in SEQ ID NO:68; the hinge region 1 and hinge region 2 are peptide hinge regions, preferably each independently selected from a peptide hinge region comprising PPCP, preferably, hinge region 1 is the sequence shown in SEQ ID NO:65, and hinge region 2 is the sequence shown in SEQ ID NO:66.

Preparation of fusion proteins

The fusion proteins described in any of the above aspects can be prepared by various methods known in the art, such as by genetic engineering and recombinant techniques. For example, DNA molecules encoding them can be obtained by chemical synthesis or PCR amplification. The resulting DNA molecules are inserted into expression vectors and then transfected into host cells. The transfected host cells are then cultured under specific conditions to express the fusion proteins disclosed herein.

In another aspect, the present disclosure provides isolated nucleic acid molecules encoding:

The fusion protein or polypeptide chain thereof described in the first or second aspect.

In certain embodiments, the vector comprises a nucleotide sequence encoding each peptide chain of the present disclosure, and the nucleotide sequence encoding each peptide chain is present on the same or different vectors.

On the other hand, the invention provides host cells comprising nucleic acid molecules or vectors as described above. Such host cells include, but are not limited to, prokaryotic cells such as bacterial cells (such as Escherichia coli cells), and eukaryotic cells such as fungal cells (such as yeast cells), insect cells, plant cells and animal cells (such as mammalian cells, such as mouse cells, human cells etc.).

On the other hand, the present invention provides a method for preparing the fusion protein described in any of the above aspects, which comprises culturing the host cell described above under conditions allowing protein expression, and collecting the fusion protein from the culture of the cultured host cell.

Conjugate

The present disclosure provides a conjugate comprising the fusion protein according to any one of the above aspects, an isolated nucleic acid molecule, and a coupling moiety connected thereto.

In certain embodiments, the conjugated moiety is selected from a detectable label, such as a radioisotope, a fluorescent substance, a luminescent substance, a colored substance, or an enzyme.

In certain embodiments, the conjugated moiety is selected from therapeutic agents such as cytotoxic agents, cytokines, toxins, radionuclides, immune agonists, immunosuppressants, and other active substances that inhibit tumor cell growth, promote tumor cell apoptosis or necrosis.

Pharmaceutical composition

The fusion proteins or conjugates disclosed herein (also referred to as active ingredients) can be incorporated into pharmaceutical compositions suitable for administration.

In another aspect, the present disclosure relates to a pharmaceutical composition comprising the fusion protein, isolated nucleic acid molecule, vector, host cell, or conjugate according to any one of the above aspects, and a pharmaceutically acceptable carrier and/or excipient.

Reagent test kit

The present disclosure provides a kit comprising the fusion protein, isolated nucleic acid molecule, vector, host cell, conjugate, or pharmaceutical composition described in any one aspect.

Therapeutic uses

In another aspect, the present disclosure relates to a method for preventing and/or treating and/or neoadjuvant treatment and/or adjuvant treatment of a disease in a subject, comprising administering to a subject in need thereof the fusion protein, isolated nucleic acid molecule, vector, host cell, conjugate, or pharmaceutical composition of any of the above aspects. The present disclosure also relates to the use of the fusion protein, isolated nucleic acid molecule, vector, host cell, conjugate, or pharmaceutical composition of any of the above aspects for preventing and/or treating and/or neoadjuvant treatment and/or adjuvant treatment of a disease, or for the preparation of a medicament for preventing and/or treating and/or neoadjuvant treatment and/or adjuvant treatment of a disease.

In certain embodiments, the disease is a tumor. In certain embodiments, the tumor is selected from a solid tumor or a hematological tumor. In certain embodiments, the solid tumor is selected from melanoma, lung cancer, kidney cancer, bladder cancer, head and neck cancer, stomach cancer, esophageal cancer, liver cancer, cervical cancer, breast cancer, or skin cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1A: Anti-TIGIT chimeric antibody 7TI-027 enhances IFN-γ secretion by SEA-activated PBMCs.

Figure 1B: Detection results of anti-TIGIT chimeric antibodies 7TI-042 and 7TI-046 enhancing the secretion of IFN-γ by SEA-activated PBMCs.

Figure 2A: Anti-TIGIT chimeric antibody 7TI-027 enhances IL-2 secretion by SEA-activated PBMCs.

Figure 2B: Anti-TIGIT chimeric antibodies 7TI-042 and 7TI-046 enhance the secretion of IL-2 by SEA-activated PBMCs.

Figure 3A: Anti-PD-1 chimeric antibody 1KP1-1E10-F6-hz enhances IFN-γ secretion by SEA-activated PBMCs.

Figure 3B: Anti-PD-1 chimeric antibody 1KP1-3A6-3C6-hz enhances IFN-γ secretion by SEA-activated PBMCs.

Figure 4: Anti-PD-1 chimeric antibody 1KP1-1E10-F6-hz enhances IL-2 secretion by SEA-activated PBMCs.

Figure 5A: Schematic diagram of structure I of TIGIT/PD-1/IL-15 dual anti-cytokine fusion protein.

Figure 5B: Schematic diagram of structure II of TIGIT/PD-1/IL-15 dual anti-cytokine fusion protein; Figure 5B-a shows that the N-terminus of chain 1 is the anti-PD-1 antibody heavy chain variable region; the N-terminus of chain 2 is the anti-PD-1 antibody light chain variable region, and the C-terminus is composed of the anti-TIGIT antibody light chain variable region connected to the anti-TIGIT heavy chain variable region via a linker; Figure 5B-b shows that the N-terminus of chain 1 is the anti-TIGIT antibody heavy chain variable region, the N-terminus of chain 2 is the anti-TIGIT antibody light chain variable region, and the C-terminus is composed of the anti-PD-1 antibody light chain variable region connected to the anti-PD-1 heavy chain variable region via a linker.

Figure 5C: Schematic diagram of structure III of the TIGIT/PD-1/IL-15 dual anti-cytokine fusion protein; Figure 5C-a shows a schematic diagram of the IL-15/IL-15Ra complex connected to the N-terminus of the anti-PD-1 heavy chain variable region via a linker, and Figure 5C-b shows a schematic diagram of the IL-15/IL-15Ra complex connected to the C-terminus of the anti-PD-1 heavy chain variable region via a linker.

Figure 5D: Schematic diagram of structure IV of TIGIT/PD-1/IL-15 dual anti-cytokine fusion protein.

Figure 6A: Detection of the activation effect of chimeric dual anti-cytokine fusion protein on jurkat/NFAT-luc/PD1 Reporter cells.

Figure 6B: Detection of the activation effect of chimeric dual anti-cytokine fusion protein on Jurkat-TIGIT-NFAT-Luc Reporter cells.

FIG7A : Detection of the effect of chimeric dual anti-cytokine fusion protein in promoting the proliferation of CTLL-2 cells.

FIG7B : Detection of the proliferation effect of the chimeric dual anti-cytokine fusion protein on Mo7e cells.

FIG8A : Detection of the effect of chimeric dual anti-cytokine fusion protein 7Y2-104 in promoting IL-2 secretion.

FIG8B : Detection of the effect of chimeric dual anti-cytokine fusion protein 7Y2-102 in promoting IL-2 secretion.

FIG9A : Detection of the effect of the chimeric dual anti-cytokine fusion protein 7Y2-104 in promoting IFN-γ secretion.

FIG9B : Detection of the effect of the chimeric dual anti-cytokine fusion protein 7Y2-102 in promoting IFN-γ secretion.

Figure 10: Long-term toxicity survival curve of mice induced by chimeric dual anti-cytokine fusion protein.

Figure 11: Detection of the activation effect of humanized bispecific anti-cytokine fusion protein on TIGIT/PD-1 dual-target effector cells.

Figure 12A: Detection of the proliferation-promoting effect of humanized dual-anti-cytokine fusion proteins 7Y2-120, 7Y2-123, 7Y2-126, and 7Y2-127 on Mo7e cells.

Figure 12B: Detection of the proliferation-promoting effect of humanized dual anti-cytokine fusion proteins 7Y2-129, 7Y2-130, 7Y2-132, 7Y2-134, and 7Y2-136 on Mo7e cells.

Figure 12C: Detection of the proliferation-promoting effect of humanized dual-anti-cytokine fusion proteins 7Y2-128, 7Y2-135, and 7Y2-137 on Mo7e cells.

Figure 13: Results of tumor inhibitory effects of different doses of humanized dual anti-cytokine fusion protein. The significance level was set at p < 0.05; *, P < 0.05, **, P < 0.01, ***, P < 0.001 indicate statistically significant differences.

Figure 14: Detection results of the effects of different doses of humanized dual-anti-cytokine fusion protein on mouse body weight.

DETAILED DESCRIPTION

Definition of terms

In this disclosure, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. At the same time, in order to better understand the present disclosure, the definitions and explanations of relevant terms are provided below.

When the terms "for example," "such as," "including," "including," "comprising," or variations thereof are used herein, these terms will not be considered as limiting terms, but will be interpreted to mean "but not limited to" or "not limited to."

The terms "a" and "an" and "the" and similar referents in the context of describing the disclosure (especially in the context of the following claims) should be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.

As used herein, the terms "fusion protein" and "dual anti-cytokine fusion protein" are used interchangeably and refer to the dual anti-cytokine fusion protein provided by the present disclosure that targets TIGIT, targets PD-1, and has an IL-15/IL-15Rα complex that activates immune effector cells.

As used herein, the term "antigen-binding domain" refers to a domain that is capable of specifically binding to a target antigen. In certain embodiments, the antigen-binding domain may comprise an antigen-binding site derived from an antibody molecule that specifically binds to the target antigen; preferably, the antigen-binding site in the antibody molecule comprises the heavy chain variable region (VH) and light chain variable region (VL) of the antibody.

The term "antibody," as used herein, refers to an immunoglobulin-derived molecule that is capable of specifically binding to a target antigen through at least one antigen-binding site located in its variable region. When referring to the term "antibody," unless the context clearly indicates otherwise, it includes not only intact antibodies but also antigen-binding fragments that are capable of specifically binding to a target antigen. "Intact antibodies" are typically composed of two pairs of polypeptide chains, each pair having one light chain (LC) and one heavy chain (HC). Antibody light chains can be classified as kappa (κ) and lambda (λ). Heavy chains can be classified as μ, δ, γ, α, or ε, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Within light and heavy chains, the variable and constant regions are connected by a "J" region of approximately 12 or more amino acids, with heavy chains also containing a "D" region of approximately 3 or more amino acids. Each heavy chain consists of a heavy chain variable region (VH) and a heavy chain constant region (CH). The heavy chain constant region is composed of three domains (CH1, CH2, and CH3). Each light chain is composed of a light chain variable region (VL) and a light chain constant region (CL). The light chain constant region is composed of one domain, CL. The constant domain does not directly participate in the binding of the antibody to the antigen, but exhibits various effector functions, such as mediating the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The VH and VL regions can be further subdivided into highly variable regions called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each _VH and _VL consists of three CDRs and four FRs arranged from amino terminus to carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions (VH and VL) of each heavy chain/light chain pair form the antigen-binding site.

As used herein, the term "complementarity determining region" or "CDR" refers to the amino acid residues in the variable region of an antibody that are responsible for antigen binding. The variable regions of the heavy and light chains each contain three CDRs, designated CDR1, CDR2, and CDR3. The precise boundaries of these CDRs can be defined according to various numbering systems known in the art, for example, the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md., 1991), the Chothia numbering system (Chothia & Lesk (1987) J. Mol. Biol. 196:901-917; Chothia et al. (1989) Nature 342:878-883), the IMGT numbering system (Lefranc et al., Dev. Comparat. Immunol. 27:55-77, 2003), the ), AbM numbering system (Martin ACR, Cheetham JC, Rees AR (1989) Modelling antibody hypervariable loops: A combined algorithm. Proc Natl Acad Sci USA 86: 9268–9272) or Contact numbering system (MacCallum, R.M., Martin, A.C.R., & Thornton, J.M. (1996). Antibody-antigen Interactions: Contact Analysis and Binding Site Topography. Journal of Molecular Biology, 262(5), 732-745.). For a given antibody, those skilled in the art will easily identify the CDRs defined by each numbering system. Moreover, the correspondence between different numbering systems is well known to those skilled in the art.

As used herein, the term "framework region" or "FR" residues refers to those amino acid residues in an antibody variable region other than the CDR residues as defined above.

The term "antibody" is not limited to any particular method of producing the antibody. For example, it includes recombinant antibodies, monoclonal antibodies, and polyclonal antibodies. The antibody can be of different isotypes, for example, IgG (e.g., IgG1, IgG2, IgG3, or IgG4 subtypes), IgA1, IgA2, IgD, IgE, or IgM antibodies.

As used herein, the term "multispecific antibody" refers to an antibody that has binding specificity for at least two (e.g., two, three, or four) different antigens (or epitopes). A multispecific antibody comprises multiple antigen-binding domains that have binding specificity for different antigens (or epitopes), thereby being able to bind to at least two different binding sites and/or target molecules. Each antigen-binding domain comprised by a multispecific antibody can be independently selected from a full-length antibody (e.g., an IgG antibody) or an antigen-binding fragment thereof (e.g., an Fv fragment, a Fab fragment, a F(ab')2 fragment, or a scFv). In some cases, each antigen-binding domain is connected by a peptide linker.

As used herein, the term "antigen-binding fragment" of an antibody refers to a polypeptide comprising a fragment of a full-length antibody that retains the ability to specifically bind to the same antigen to which the full-length antibody is bound, and/or competes with the full-length antibody for specific binding to the antigen, and is also referred to as an "antigen-binding portion thereof". Antigen-binding fragments of antibodies can be produced by recombinant DNA techniques or by enzymatic or chemical fragmentation of intact antibodies. Non-limiting examples of antigen-binding fragments include Fab fragments, Fab' fragments, F(ab') ₂ , Fd, Fv, complementary determining region (CDR) fragments, single-chain antibodies (e.g., scFv or scFab), diabodies, single-domain antibodies, chimeric antibodies, linear antibodies, nanobodies (technology from Domantis), probodies, and polypeptides comprising at least a portion of an antibody sufficient to confer specific antigen-binding ability to a polypeptide.

As used herein, the term "full-length antibody" means an antibody consisting of two "full-length heavy chains" and two "full-length light chains". Wherein, "full-length heavy chain" refers to a polypeptide chain that, in the direction from N-terminus to C-terminus, consists of a heavy chain variable region (VH), a heavy chain constant region CH1 domain, a hinge region (HR), a heavy chain constant region CH2 domain, and a heavy chain constant region CH3 domain; and, when the full-length antibody is of IgE or IgM isotype, optionally further comprises a heavy chain constant region CH4 domain. Preferably, a "full-length heavy chain" is a polypeptide chain consisting of VH, CH1, HR, CH2, and CH3 in the direction from N-terminus to C-terminus. A "full-length light chain" is a polypeptide chain consisting of a light chain variable region (VL) and a light chain constant region (CL) in the direction from N-terminus to C-terminus. The two pairs of full-length antibody chains are linked together by a disulfide bond between CL and CH1 and a disulfide bond between the HRs of the two full-length heavy chains. A full-length antibody contains two antigen-binding sites formed by a VH and VL pair, respectively, and these two antigen-binding sites specifically recognize/bind to the same antigen.

The term "chimeric antibody" refers to an antibody formed by fusing the variable region of a mouse antibody with the constant region of a human antibody, which can reduce the immune response induced by the mouse antibody. To create a chimeric antibody, one must first establish a hybridoma that secretes mouse-specific monoclonal antibodies. Then, the variable region genes are cloned from the mouse hybridoma cells. The constant region genes of the human antibody are cloned as needed. The mouse variable region genes and the human constant region genes are connected to form a chimeric gene, which is then inserted into a human vector. Finally, the chimeric antibody molecule is expressed in a eukaryotic or prokaryotic industrial system. The antibody may further comprise a light chain constant region of a human kappa or lambda chain, or variants thereof. The antibody may further comprise a heavy chain constant region of a human IgG1, IgG2, IgG3, IgG4, or variants thereof. The constant region of the human antibody can be selected from the heavy chain constant region of human IgG1, IgG2, IgG3 or IgG4 or their variants, preferably comprising a human IgG2 or IgG4 heavy chain constant region, or using IgG4 that is free of ADCC (antibody-dependent cell-mediated cytotoxicity) toxicity after amino acid mutation.

"Humanized antibody" refers to a class of engineered antibodies that have CDRs derived from non-human donor immunoglobulins, while the remaining immunoglobulin portion of the humanized antibody is derived from one (or more) human immunoglobulins. In addition, framework support residues can be changed to retain binding affinity (see, for example, Queen et al., Proc. Natl. Acad. Sci. USA, 86: 10029-10032 (1989), Hodgson et al., Bio/Technology, 9: 421 (1991)). Suitable human acceptor antibodies can be antibodies selected from conventional databases such as the Los Alamos database and the Swiss protein database by homology to the nucleotide and amino acid sequences of the donor antibody. Human antibodies characterized by homology (based on amino acids) to the framework regions of the donor antibody can be suitable for providing heavy chain constant regions and/or heavy chain variable framework regions for insertion of the donor CDRs. Suitable acceptor antibodies that can provide light chain constant or variable framework regions can be selected in a similar manner. It should be noted that the acceptor antibody heavy and light chains do not need to be derived from the same acceptor antibody.

As used herein, the term "Fab fragment" means an antibody fragment consisting of the VL, VH, CL and CH1 domains; the term "F(ab') ₂ fragment" means an antibody fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; the term "Fab'fragment" means the fragment obtained after reducing the disulfide bonds linking the two heavy chain fragments in the F(ab') ₂ fragment, consisting of one complete light chain and the Fd fragment (consisting of the VH and CH1 domains) of the heavy chain.

As used herein, the term "scFv" refers to a single polypeptide chain comprising VL and VH domains, wherein the VL and VH are connected by a linker. Such scFv molecules may have the general structure: NH2 _- VL-linker-VH-COOH or NH2 _- VH-linker-VL-COOH. Suitable prior art linkers consist of repeated GGGGS amino acid sequences or variants thereof. For example, a linker having the amino acid sequence (GGGGS) ₂ may be used, although variants thereof may also be used. In some cases, a disulfide bond may also be present between the VH and VL of the scFv.

As used herein, the term "Fc domain" or "Fc region" refers to a portion of the heavy chain constant region comprising CH2 and CH3. The Fc fragment of an antibody has a variety of different functions, and the "effector functions" mediated by the Fc region include Fc receptor binding; Clq binding and complement-dependent cytotoxicity (CDC); antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; downregulation of cell surface receptors (e.g., B cell receptors); and B cell activation, among others. In some embodiments, the Fc region comprises a hinge, CH2, and CH3. When the Fc region comprises a hinge, the hinge regulates the dimerization between two Fc-containing polypeptides. The Fc region can be of any antibody heavy chain constant region isotype, such as IgG1, IgG2, IgG3, or IgG4.

An Fc domain can include both a native Fc region and a variant Fc region. A native Fc region comprises an amino acid sequence that is consistent with the amino acid sequence of an Fc region found in nature, for example, a native sequence human Fc region includes a native sequence human IgG1 Fc region (non-A and A allotypes); a native sequence human IgG2 Fc region; a native sequence human IgG3 Fc region; and a native sequence human IgG4 Fc region, as well as naturally occurring variants thereof. A variant Fc region comprises an amino acid sequence that differs from the amino acid sequence of a native sequence Fc region due to at least one amino acid modification. In some embodiments, a variant Fc region may have altered effector functions (e.g., Fc receptor binding, antibody glycosylation, the number of cysteine residues, effector cell function, or complement function) compared to a native Fc region.

The Fc domain may also include modifications to promote dimerization of the Fc domain. In some embodiments, the modifications include introducing amino acid mutations in the Fc domain monomer (e.g., replacing a smaller amino acid with a larger amino acid) to form a "knob" modification; while simultaneously introducing amino acid mutations in another Fc domain (e.g., replacing multiple larger amino acids with smaller amino acids) to form a "hole" modification, thereby changing the local spatial structure of the Fc. In this case, the modified Fc domain is prone to heterodimerization through a "knob-into-hole" mode of action. This design facilitates the correct assembly of two heterologous antibody heavy chains and avoids mispairing of light and heavy chains.

Each of the above antibody fragments retains the ability to specifically bind to the same antigen as the full-length antibody and/or competes with the full-length antibody for specific binding to the antigen.

Antigen-binding fragments of antibodies (e.g., those described above) can be obtained from a given antibody (e.g., an antibody provided herein) using conventional techniques known to those skilled in the art (e.g., recombinant DNA techniques or enzymatic or chemical cleavage methods), and the antigen-binding fragments of antibodies can be screened for specificity in the same manner as for intact antibodies.

As used herein, the term "identity" is used to refer to the matching of sequences between two polypeptides or between two nucleic acids. In order to determine the percent identity of two amino acid sequences or two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., a gap can be introduced in the first amino acid sequence or nucleic acid sequence to optimally align with the second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position. The percent identity between two sequences is a function of the number of identical positions shared by the sequences (i.e., percent identity = number of identical overlapping positions/total number of positions × 100%). In certain embodiments, the two sequences used for comparison are the same length.

The determination of percent identity between two sequences can also be achieved using a mathematical algorithm. A non-limiting example of a mathematical algorithm for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Natl. Acad. Sci. U.S.A. 87:2264-2268, as modified in Karlin and Altschul, 1993, Proc. Natl. Acad. Sci. U.S.A. 90:5873-5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403.

As used herein, the term "variant" also refers to a polypeptide or peptide comprising an amino acid sequence that has been altered by introducing amino acid residue substitutions, deletions, or additions in the context of a polypeptide (including polypeptides). In some cases, the term "variant" also refers to a polypeptide or peptide that has been modified (i.e., by covalently linking any type of molecule to a polypeptide or peptide). For example, but not limited to, a polypeptide can be modified, such as by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protection/blocking groups, proteolytic cleavage, connection to a cellular ligand or other protein, etc. Derivatized polypeptides or peptides can be produced by chemical modification using techniques known to those skilled in the art, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. In addition, a variant has a function that is similar, identical, or improved to the polypeptide or peptide from which it is derived. In certain embodiments, the variant has at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity compared to the sequence from which it is derived.

As used herein, the term "specific binding" refers to a non-random binding reaction between two molecules, such as the reaction between an antibody and its antigen. The strength or affinity of a specific binding interaction can be expressed by the equilibrium dissociation constant ( _KD ) of the interaction. In the present disclosure, the term " _KD " refers to the equilibrium dissociation constant of a specific antibody-antigen interaction, which is used to describe the binding affinity between the antibody and the antigen. The smaller the equilibrium dissociation constant, the tighter the antibody-antigen binding and the higher the affinity between the antibody and the antigen.

The specific binding properties between two molecules can be measured using methods well known in the art. One method involves measuring the speed of formation and dissociation of the antigen-antibody complex. Both the "association rate constant" (kas or kon) and the "dissociation rate constant" (kdis or koff) can be calculated by concentration and the actual rate of association and dissociation (see Malmqvist M, Nature, 1993, 361: 186-187). The ratio of kdis/kon is equal to the dissociation constant _KD (see Davies et al., Annual Rev Biochem, 1990; 59: 439-473). _KD , kon and kdis values can be measured by any effective method. In certain embodiments, the dissociation constant can be measured in Biacore using surface plasmon resonance (SPR), and the dissociation constant can also be measured using bioluminescence interferometry or Kinexa. In some embodiments, the fusion proteins of the present disclosure bind to human TIGIT protein with a K value of no greater than 1E-8M, 2E-8M, 3E-8M, 4E-8M, 5E-8M, 6E-8M, 7E-8M, 8E-8M, 9E-8M, 1E-9M, 2E-9M, 3E-9M, 4E-9M, 5E-9M, 6E-9M, 7E-9M, 8E-9M, 9E-9M, 1E-10M, 2E- _10M , 3E-10M, 4E-10M or 5E-10M. In some embodiments, the fusion proteins of the present disclosure bind to human PD-1 protein with a K value of no greater than 1E-8M, 2E-8M, 3E-8M, 4E-8M, 5E-8M, 6E-8M, 7E-8M, 8E-8M, 9E-8M, 1E-9M, 2E-9M, 3E-9M, 4E-9M, 5E-9M, 6E-9M, 7E-9M, 8E-9M, 9E-9M, 1E- _10M , 2E-10M, 3E-10M, 4E-10M, or 5E-10M.

As used herein, the term "vector" refers to a nucleic acid delivery vehicle into which a polynucleotide can be inserted. When a vector is capable of expressing a protein encoded by the inserted polynucleotide, it is referred to as an expression vector. A vector can be introduced into a host cell via transformation, transduction, or transfection, allowing the genetic material it carries to be expressed in the host cell. Vectors are well known to those skilled in the art and include, but are not limited to, plasmids; phagemids; cosmids; artificial chromosomes, such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs); bacteriophages, such as lambda phage or M13 phage, and animal viruses. Animal viruses that can be used as vectors include, but are not limited to, retroviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpes viruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomas (such as SV40). A vector can contain a variety of elements that control expression, including, but not limited to, promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. In addition, a vector may also contain an origin of replication.

As used herein, the term "host cell" refers to a cell that can be used to introduce a vector, including but not limited to prokaryotic cells such as Escherichia coli or Bacillus subtilis, fungal cells such as yeast cells or Aspergillus, insect cells such as S2 Drosophila cells or Sf9, or human or animal cells such as fibroblasts, CHO cells, COS cells, NSO cells, HeLa cells, BHK cells, HEK 293 cells.

The twenty conventional amino acids referred to herein are compiled in accordance with conventional usage. See, for example, Immunology—A Synthesis (2nd Edition, E.S. Golub and D.R. Gren, Eds., Sinauer Associates, Sunderland, Mass. (1991)), which is incorporated herein by reference. In this disclosure, the terms "polypeptide" and "protein" have the same meaning and are used interchangeably. In this disclosure, amino acids are generally represented by single-letter and three-letter abbreviations known in the art. For example, alanine can be represented by A or Ala.

As used herein, the term "pharmaceutically acceptable carrier and/or excipient" refers to a carrier and/or excipient that is pharmacologically and/or physiologically compatible with the subject and the active ingredient, which is well known in the art (see, for example, Remington's Pharmaceutical Sciences. Edited by Gennaro AR, 19th ed. Pennsylvania: Mack Publishing Company, 1995), and includes, but is not limited to: pH adjusters, surfactants, adjuvants, ionic strength enhancers, diluents, agents that maintain osmotic pressure, agents that delay absorption, preservatives. Pharmaceutically acceptable carriers and/or excipients include any and all solvents, dispersion media, isotonic agents, and absorption delaying agents that are physiologically compatible. Pharmaceutical carriers suitable for use in the present invention can be sterile liquids, such as water and oils, including those of petroleum, animal, plant or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be used as liquid carriers, particularly for injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. For the use of excipients and their applications, see also "Handbook of Pharmaceutical Excipients", Fifth Edition, R.C. Rowe, P.J. Seskey and S.C. Owen, Pharmaceutical Press, London, Chicago. If desired, the composition may also contain a small amount of a wetting agent or emulsifier, or a pH buffer. These compositions can be in the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like. Oral formulations may contain standard pharmaceutical carriers and/or excipients, such as pharmaceutical grade mannitol, lactose, starch, magnesium stearate, saccharin. Pharmaceutical preparations or pharmaceutical compositions comprising the present invention may be prepared by mixing the fusion protein or expressed nucleic acid of the present invention having the desired purity with one or more optional pharmaceutical excipients (Remington’s Pharmaceutical Sciences, 16th edition, Osol, A. ed. (1980)), preferably in the form of a lyophilized preparation or aqueous solution. The pharmaceutical composition or preparation of the present invention may also contain more than one active ingredient, which is required for the specific indication being treated, preferably those with complementary activities that do not adversely affect each other. For example, it is desirable to also provide other anti-infective active ingredients, such as other antibodies, anti-infective active agents, small molecule drugs or immunomodulators, etc. The active ingredients are suitably combined in amounts effective for the intended use. Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semi-permeable matrices of solid hydrophobic polymers containing the antibodies of the present invention or their antigen-binding fragments, the matrix being in the form of shaped articles, such as films or microcapsules.

As used herein, the term "prevention" refers to a method implemented to prevent or delay the occurrence of a disease, disorder or symptom in a subject. As used herein, the term "treatment" refers to a method implemented to obtain a beneficial or desired clinical result. For the purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, alleviating symptoms, reducing the scope of the disease, stabilizing (i.e., no longer worsening) the state of the disease, delaying or slowing the progression of the disease, improving or alleviating the state of the disease, and alleviating symptoms (whether partial or complete), whether detectable or undetectable. In addition, "treatment" can also refer to prolonging survival compared to the expected survival if not receiving treatment. In this article, treatment can include neoadjuvant therapy and/or adjuvant therapy. "Neoadjuvant therapy" refers to a therapy administered to a patient before a planned surgery for the treatment of the disease. "Adjuvant therapy" refers to a therapy administered to a patient after surgery for the treatment of the disease.

As used herein, the term "subject" refers to a mammal, such as a primate mammal, such as a human. In certain embodiments, the subject (e.g., a human) suffers from a tumor, an inflammatory disease, or an autoimmune disease, or is at risk of suffering from the above diseases.

As used herein, the term "effective amount" refers to an amount sufficient to obtain or at least partially obtain the desired effect. For example, an effective amount for preventing a disease (e.g., a tumor, an inflammatory disease, or an autoimmune disease) refers to an amount sufficient to prevent, stop, or delay the occurrence of a disease (e.g., a tumor, an inflammatory disease, or an autoimmune disease); an effective amount for treating a disease refers to an amount sufficient to cure or at least partially stop the disease and its complications in a patient already suffering from the disease. Determining such an effective amount is well within the capabilities of those skilled in the art. For example, an amount effective for therapeutic use will depend on the severity of the disease to be treated, the overall state of the patient's own immune system, the patient's general condition such as age, weight, and sex, the mode of administration of the drug, and other treatments administered simultaneously, etc.

Advantageous Effects of the Invention

Compared with the limitations of the efficacy of known immune checkpoint anti-tumor drugs, the dual anti-cytokine fusion protein provided by the present disclosure, which targets TIGIT, targets PD-1, and has immune effector cell activation, has obvious advantages. The dual anti-cytokine fusion protein provided by the present disclosure can effectively relieve the immunosuppression of T cells and NK cells, and can moderately promote the activation and proliferation of immune cells that infiltrate tumors, overcoming the problem of poor efficacy of TIGIT alone. At the same time, combined with the modified IL-15/IL-15Ra complex, it can reduce the toxicity of IL-15 while widening the medication window; it has good drug safety and anti-tumor effect.

The embodiments of the present disclosure will be described in detail below with reference to the accompanying drawings and examples, but those skilled in the art will understand that the following drawings and examples are only used to illustrate the present disclosure and are not intended to limit the scope of the present disclosure. Various objects and advantages of the present disclosure will become apparent to those skilled in the art based on the following detailed description of the drawings and preferred embodiments.

The present disclosure will now be described with reference to the following examples which are intended to illustrate the present disclosure rather than to limit it.

Those skilled in the art will appreciate that the examples are provided by way of example only and are not intended to limit the scope of the invention. The experimental methods in the examples are conventional methods unless otherwise specified. Where specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. Where the manufacturer of the reagents or instruments is not specified, they are all commercially available conventional products.

Example 1: Construction of anti-TIGIT monoclonal antibodies and anti-PD-1 monoclonal antibodies

1.1 Obtaining anti-TIGIT monoclonal antibodies and their coding sequences

Anti-TIGIT antibodies are multiple anti-TIGIT monoclonal antibodies invented by the applicant. The preparation method and amino acid sequence of the antibodies can be found in the Chinese invention patent application (application number CN202310994050.5, "Anti-TIGIT antibodies and their uses", application date 2023/8/9).

Specifically, according to conventional methods, 100 μg of human TIGIT extracellular domain huTIGIT-msIgG2a-Fc-tag antigen (purchased from Arco; TIT-H5253) was fully emulsified with complete Freund's adjuvant at a ratio of 1:1 and then injected intraperitoneally to immunize mice. The antigen without Freund's adjuvant was then injected into the tail vein to shock-immunize mice, and then the spleen cells of the immunized mice were obtained. Splenocytes were fused with Sp2/0 cells (Nanjing Kebai Biotechnology, CBP60881), and after fusion, positive clones were screened by ELISA with human TIGIT-His (huTIGIT-His, Kaixia Biotechnology, TIG-HM110) and monkey TIGIT-His (cynoTIGIT-His, Acro; TIT-C5223). Jurkat-huTIGIT cells (Nanjing Kebai Biotechnology, CBP60520) were added with 10 μl of lentiviral vector of human TIGIT (huTIGIT) target gene (SEQ ID NO: 77) in 6-well plates at 60% density. Virus (customized by Hanheng Biotechnology), after culturing for 24 hours, new culture medium was replaced and puromycin was added to make the working final concentration of 10μg/ml for resistance screening. After 4 days, the positive polyclonal cells obtained by screening were expanded and cultured and preserved to obtain Jurkat-huTIGIT cells) and 293T-cynoTIGIT cells (according to the method for preparing Jurkat-huTIGIT cells, 293T cells (Nanjing Kebai Biotechnology, CBP60439) were infected with lentivirus packaged with the cynomolgus macaque TIGIT (cynoTIGIT) target gene (SEQ ID NO: 78) to prepare 293T-cynoTIGIT cells). Positive clones with strong binding effects to both cell lines were screened by FACS (flow cytometry fluorescence sorting technology). The positive clones with binding effects after dual screening by ELISA and FACS were subcloned to obtain monoclones. Positive monoclones were also screened by ELISA and flow cytometry, and their NF-κB signaling pathway activation effects were further tested. In this way, functional anti-TIGIT positive clones were further screened, and mouse antibodies named 3A03A03, 2IT1-10A3-2H6 and 41A07A03 were obtained.

Total RNA was extracted from hybridoma cells and reverse transcribed. The resulting cDNA was used as a template to amplify the VH and VL sequences of the 3A03A03, 2IT1-10A3-2H6, and 41A07A03 antibodies, respectively. The VH sequence was linked to the human IgG1 heavy chain constant region (SEQ ID NO: 70), and the VL sequence was linked to the human light chain kappa chain constant region (SEQ ID NO: 73). The chimeric antibody 7TI-042 was generated from 3A03A03, the chimeric antibody 7TI-027 was generated from 2IT1-10A3-2H6, and the chimeric antibody 7TI-046 was generated from 41A07A03.

Table 1: VH and VL amino acid sequences of anti-TIGIT murine/chimeric antibodies

SPR (surface plasmon resonance) was used to detect the ability of anti-TIGIT chimeric antibodies to bind to huTIGIT-his and cynoTIGIT-his proteins. The results showed that 7TI-042, 7TI-027, and 7TI-046 could bind to human TIGIT protein with KD of 1.5E-10M, 9.26E-12M, and 5.2E-11M, respectively, and bind to monkey TIGIT protein with KD of 2.3E-08M, 2.06E-10M, and 5.8E-10M, respectively.

Flow cytometry was used to detect the binding of anti-TIGIT chimeric antibodies to Jurkat-huTIGIT cells and 293T-cynoTIGIT cells. The results showed that 7TI-042, 7TI-027, and 7TI-046 could bind to Jurkat-huTIGIT cells with _EC50 of 0.47nM, 0.45nM, and 0.66nM, respectively, and bind to 293T-cynoTIGIT cells with _EC50 of 0.28nM, 0.14nM, and 0.21nM, respectively.

Jurkat-NFAT-LUC-huTIGIT effector cells (Nanjing Kebai Biotechnology, CBP74020) and CHO-TCR-CD155 (Nanjing Kebai Biotechnology, CBP74073) target cells were used to evaluate the ability of anti-TIGIT chimeric antibodies to compete with CD155 for binding to human TIGIT antigen and downstream reporter gene activation. The results showed that 7TI-042, 7TI-027, and 7TI-046 were able to activate reporter genes with _EC50 of 2.21nM, 3.17nM, and 3.46nM, respectively.

Human PBMC donor 007 (SC12007) purchased from Saili Bio was co-incubated with SEA (Staphylococcal enterotoxin A, purchased from Toxin Technology, AT101) and an anti-TIGIT chimeric antibody, and the concentrations of IFN-γ and IL-2 were detected. The IFN-γ results are shown in Figures 1A and 1B, and the IL-2 results are shown in Figures 2A and 2B. As can be seen from the figures, the anti-TIGIT chimeric antibody disclosed herein can effectively promote the secretion of IFN-γ and IL-2.

1.2 Humanization of anti-TIGIT mouse antibodies

The mouse antibodies 3A03A03, 2IT1-10A3-2H6, and 41A07A03 were humanized. The mouse antibody sequences were compared with the human germline antibody amino acids to identify sequences with high homology and better physicochemical properties as humanized antibody framework sequences. The mouse antibody CDR regions were transplanted onto the human antibody framework sequences to obtain anti-TIGIT humanized antibodies with the following VH and VL sequences.

Table 2: VH and VL amino acid sequences of anti-TIGIT humanized antibodies

SPR (surface plasmon resonance) was used to detect the ability of anti-TIGIT humanized antibodies to bind to huTIGIT-his protein. The results showed that 7TI-208, 7TI-202, 7TI-172, 7TI-174, 7TI-224, and 7TI-225 were able to bind to human TIGIT protein with _KD of 1.1E-10M, 1.7E-09M, 3.80E-12M, 2.70E-11M, 1.99E-10M, and 1.42E-10M, respectively.

Flow cytometry was used to detect the binding of anti-TIGIT humanized antibodies to Jurkat-huTIGIT cells. The results showed that 7TI-208, 7TI-202, 7TI-172, 7TI-174, 7TI-224, and 7TI-225 were able to bind to Jurkat-huTIGIT cells with _EC50 of 0.19nM, 1.82nM, 7.51nM, 1.13nM, 0.18nM, and 0.18nM, respectively.

Jurkat-NFAT-LUC-huTIGIT effector cells and CHO-TCR-CD155 target cells were used to evaluate the competitive binding of anti-TIGIT humanized antibodies to CD155 and the reporter gene activation ability. The results showed that 7TI-208, 7TI-172, and 7TI-174 were able to activate the reporter gene with _EC50 of 3.96nM, 7.25nM, and 4.22nM, respectively.

1.3 Obtaining anti-PD-1 monoclonal antibodies and their coding sequences

The anti-PD-1 antibodies are multiple anti-PD-1 monoclonal antibodies invented by the applicant. The preparation method and amino acid sequence of the antibodies can be found in the Chinese invention patent application (application number CN202311267267.2, "Anti-PD-1 antibodies and their uses", application date 2023/9/27).

Specifically, according to conventional methods, recombinant human PD1-hFc protein (KACTUS, PD1-HM101) was mixed with equal volumes of Freund's complete adjuvant and emulsified, then injected subcutaneously into the left and right groins of 6-8 week-old BALb/c mice. Subsequently, PD1-hFc protein-containing Freund's incomplete adjuvant emulsion (protein and adjuvant were emulsified in equal volumes at a 1:1 ratio) was injected subcutaneously every two weeks for a total of four immunizations. One week after the fourth immunization, mice whose antibody titers met the fusion conditions were stimulated by intravenous injection of recombinant human PD1-hFc protein without adjuvant, and spleen cells from the stimulated mice were obtained. Splenocytes were fused with Sp2/0 cells (Nanjing Kebai Biotechnology, CBP60881). After fusion, positive clones were screened by ELISA using human PD-1 extracellular domain protein (ACROBiosystems Group, PD1-H5221) or cynomolgus monkey PD-1 extracellular domain protein (ACROBiosystems Group, PD1-C5223). CHO-K1-hPD-1 cells (CHO-K1 cells that overexpress human PD-1, which is a cell line that overexpresses human PD-1) were further used to screen positive clones. Stable cell lines were obtained by infecting CHO-K1 cells with PD-1 (gene number NM_005018.3) lentiviral vectors (the lentiviral vectors were customized by Hanbio). Positive clones with strong binding effects to CHO-K1 cells expressing human PD-1 were screened by FACS (flow cytometry fluorescence sorting technology). The positive clones were subcloned to obtain monoclonal clones. Positive monoclonal clones were also screened by ELISA and flow cytometry to obtain mouse antibodies named 1KP-3A6-3C6 and 1KP1-1E10-F6.

Total RNA was extracted from hybridoma cells and reverse transcribed. The resulting cDNA was used as a template to amplify the VH and VL sequences of the 1KP-3A6-3C6 and 1KP1-1E10-F6 antibodies, respectively. The VH sequence was linked to the human IgG1 heavy chain constant region, and the VL sequence was linked to the human light chain κ chain constant region to generate the chimeric antibodies 1KP1-3A6-3C6-hz and 1KP1-1E10-F6-hz.

Table 3: VH and VL amino acid sequences of anti-PD-1 murine/chimeric antibodies

SPR (surface plasmon resonance) was used to detect the ability of anti-PD-1 chimeric antibodies to bind to human PD-1 protein and cynomolgus macaque PD-1 protein. The results showed that 1KP1-3A6-3C6-hz and 1KP1-1E10-F6-hz could bind to human PD-1 protein with KD of 3.20E-10M and 3.93E-10M, respectively, and bind to cynomolgus macaque PD-1 protein with _KD of 3.87E-09M and 2.6E-09M, respectively.

Flow cytometry was used to detect the antigen binding ability of anti-PD-1 chimeric antibodies to CHO-K1-hPD-1 cells and CHO-K1-cynoPD-1 cells (CHO-K1 cells overexpressing cynomolgus monkey PD-1, which is a stably transfected cell line obtained by infecting CHO-K1 cells with a cynomolgus monkey PD-1 (gene number NM_001114358.1) lentiviral vector). The results showed that 1KP1-3A6-3C6-hz and 1KP1-1E10-F6-hz were able to bind to CHO-K1 cells expressing human PD-1 with _EC50 of 2.558nM and 9.13nM, respectively, and to CHO-K1 cells expressing cynomolgus monkey PD-1 with _EC50 of 2.075nM and 1.208nM, respectively.

CHO-hPDL1-TCR cells (GenScript, M00613) overexpressing human PD-L1 were used as target cells, and Jurkat/NFAT-luc/PD1 cells (GenScript, M00612) were used as effector cells to evaluate the ability of anti-PD-1 chimeric antibodies to activate effector cells. The results showed that 1KP1-3A6-3C6-hz and 1KP1-1E10-F6-hz were able to activate effector cells with _EC50s of 0.192 nM and 0.43 nM, respectively.

CHO-K1 cells overexpressing human PD-1, biotin-labeled PD-L1 (329703, Biolegend), mouse Fc-labeled PD-L2 (329604, Biolegend), AF647-labeled goat anti-mouse IgG Fc secondary antibody (Jackson Immunoresearch, 115-605-008), and APC-labeled SA (streptavidin) were used to evaluate the competitive binding effect of anti-PD-1 chimeric antibodies in blocking the binding of PD-L1/PD-L2 to PD-1. The results showed that 1KP1-3A6-3C6-hz and 1KP1-1E10-F6-hz were able to block the binding of human PD-L1 to human PD-1 on the surface of CHO-K1 cells with _EC50 of 1.868 nM and 1.27 nM, respectively; 1KP1-1E10-F6-hz had an EC50 of 1.209 nM. ₅₀ blocks the binding of human PD-L2 to human PD-1 on the surface of CHO-K1 cells.

Human PBMCs (Donor: 007) purchased from Saili Biotechnology were co-incubated with SEA (Staphylococcal enterotoxin A, purchased from Toxin Technology, AT101) and anti-PD-1 chimeric antibodies to detect IFN-γ and IL-2 concentrations. The IFN-γ results are shown in Figures 3A and 3B, and the IL-2 results are shown in Figure 4. As can be seen from the figures, the anti-PD-1 chimeric antibodies disclosed herein can effectively promote the secretion of IFN-γ and IL-2.

1.4 Humanization of anti-PD-1 mouse antibodies

The mouse antibodies 1KP1-3A6-3C6 and 1KP1-1E10-F6 were humanized. The mouse antibody sequences were compared with the human germline antibody amino acids to identify sequences with high homology and better physicochemical properties as the humanized antibody framework sequences. The mouse antibody CDR regions were transplanted onto the human antibody framework sequences to obtain anti-PD-1 humanized antibodies with the following VH and VL sequences.

Table 4: VH and VL amino acid sequences of anti-PD-1 humanized antibodies

SPR (surface plasmon resonance) was used to detect the ability of anti-PD-1 humanized antibodies to bind to human PD-1 protein. The results showed that 7PD-079, 7PD-082, and 7PD-118 could bind to human PD-1 protein with KD of 3.0E-09M, 4.6E-09M, and 6.47E-10M, respectively, and 7PD-118 bound to cynomolgus monkey PD-1 protein with a _KD of 1.03E-09M.

Flow cytometry was used to detect the binding ability of anti-PD-1 chimeric antibodies to CHO-K1 cells expressing human PD-1 and cynomolgus monkey PD-1. The results showed that 7PD-079, 7PD-082, 7PD-098, 7PD-099, 7PD-104, 7PD-111, 7PD-115, 7PD-118, and 7PD-120 could bind to human PD-1 and cynomolgus monkey PD-1 with EC values of 1.363 nM, 1.597 nM, 1.131 nM, 1.248 nM, 1.029 nM, 1.207 nM, 1.189 nM, 1.133 nM, and 0.726 nM, respectively. ₅₀ binds to CHO-K1 cells expressing human PD-1 and binds to CHO-K1 cells expressing cynomolgus monkey PD-1 with _EC50 of 1.658nM, 1.686nM, 1.747nM, 1.522nM, 0.148nM, 2.550nM, 2.957nM, 4.029nM and 1.694nM, respectively.

Example 2: Construction of TIGIT/PD-1/IL-15 dual anti-cytokine fusion protein expression vector

The TIGIT/PD-1/IL-15 dual anti-cytokine fusion protein of this embodiment has four structures: Structure I, Structure II, Structure III, and Structure IV.

Structure I is shown in Figure 5A and consists of two chains: Chain 1, from N-terminus to C-terminus, consists of the anti-TIGIT antibody heavy chain variable region, human IgG1 heavy chain constant region CH1 (SEQ ID NO: 74), hinge region 1 (SEQ ID NO: 65), human IgG1 heavy chain constant region CH2 and CH3 containing Knob mutation (SEQ ID NO: 71), Linker 1 (SEQ ID NO: 67), IL-15 polypeptide (SEQ ID NO: 63), Linker 1, anti-PD-1 antibody light chain variable region, Linker 2 (SEQ ID NO: 74), and IL-15 polypeptide (SEQ ID NO: 73). O:68), anti-PD-1 antibody heavy chain variable region; chain 2, from N-terminus to C-terminus, is composed of anti-TIGIT antibody light chain variable region, human IgG1 light chain constant region CL (SEQ ID NO:73), hinge region 2 (SEQ ID NO:66), human IgG1 heavy chain constant region CH2 and CH3 containing Hole mutation (SEQ ID NO:72), Linker 1, IL-15Ra polypeptide (SEQ ID NO:64 or 75), Linker 1, anti-PD-1 antibody light chain variable region, Linker 2, and anti-PD-1 antibody heavy chain variable region.

Structure II is shown in Figure 5B-a and Figure 5B-b, where Structure II shown in Figure 5B-a consists of two chains: Chain 1, which is composed of, from N-terminus to C-terminus, the anti-PD-1 antibody heavy chain variable region, human IgG1 heavy chain constant region CH1, hinge region 1, human IgG1 heavy chain constant region CH2 and CH3 containing Knob mutation, Linker 1, IL-15Ra polypeptide, Linker 3 (SEQ ID NO: 69), and IL-15 polypeptide; 2. Chain 2, which is composed of, from N-terminus to C-terminus, the anti-PD-1 antibody light chain variable region, human IgG1 light chain constant region CL, hinge region 2, human IgG1 heavy chain constant region CH2 and CH3 containing Hole mutation, Linker 1, anti-TIGIT antibody light chain variable region, Linker 2, and anti-TIGIT heavy chain variable region. Among them, structure II shown in Figure 5B-b is composed of two chains: Chain 1, from N-terminus to C-terminus, consists of the anti-TIGIT antibody heavy chain variable region, human IgG1 heavy chain constant region CH1, hinge region 1, human IgG1 heavy chain constant region CH2 and CH3 containing Knob mutation, Linker 1, IL-15Ra polypeptide, Linker 3, and IL-15 polypeptide; 2. Chain 2, from N-terminus to C-terminus, consists of: anti-TIGIT antibody light chain variable region, human IgG1 light chain constant region CL, hinge region 2, human IgG1 heavy chain constant region CH2 and CH3 containing Hole mutation, Linker 1, anti-PD-1 antibody light chain variable region, Linker 2, and anti-PD-1 heavy chain variable region.

Structure III is shown in Figure 5C-a and Figure 5C-b, where Structure III shown in Figure 5C-a is composed of four chains: chain 1 and chain 4, which are composed of: anti-PD-1 antibody light chain variable region and human IgG1 light chain constant region CL from N-terminus to C-terminus; chain 2 and chain 3 are composed of the TIGIT/PD-1 antibody structure part and the IL-15/IL-15Ra complex, and the TIGIT/PD-1 antibody structure part is composed of anti-PD-1 heavy chain variable region, human IgG1-WT heavy chain constant region (SEQ ID NO: 70), Linker 1, anti-TIGIT antibody light chain variable region, Linker 2, and anti-TIGIT antibody heavy chain variable region from N-terminus to C-terminus; the IL-15/IL-15Ra complex is composed of IL-15Ra, Linker 3, and IL-15 from N-terminus to C-terminus, and is connected to the N-terminus of the anti-PD-1 heavy chain variable region through Linker 1. Among them, structure III shown in Figure 5C-b is composed of four chains: chain 1 and chain 4, which are composed of: anti-TIGIT antibody light chain variable region, human IgG1 light chain constant region CL from N-terminus to C-terminus; the TIGIT/PD-1 antibody structure part of chain 2 and chain 3 is composed of anti-TIGIT heavy chain variable region, human IgG1-WT heavy chain constant region, Linker 1, anti-PD-1 antibody light chain variable region, Linker 2, and anti-PD-1 antibody heavy chain variable region from N-terminus to C-terminus; the IL-15/IL-15Ra complex is connected to the C-terminus of the anti-PD-1 heavy chain variable region.

Structure IV is shown in Figure 5D and consists of two chains. Chain 1, from N-terminus to C-terminus, consists of: anti-TIGIT antibody heavy chain variable region, human IgG1 heavy chain constant region CH1, hinge region 1, human IgG1 heavy chain constant region CH2 and CH3 containing Knob mutation, Linker 1, IL-15Ra, Linker 3, IL-15, Linker 1, anti-PD-1 antibody light chain variable region, Linker 2, and anti-PD-1 antibody heavy chain variable region; Chain 2, from N-terminus to C-terminus, consists of: anti-TIGIT antibody light chain variable region, human IgG1 light chain constant region CL, hinge region 2, human IgG1 heavy chain constant region CH2 and CH3 containing Hole mutation, Linker 1, IL-15Ra polypeptide, Linker 3, IL-15, Linker 1, anti-PD-1 antibody light chain variable region, Linker 2, and anti-PD-1 antibody heavy chain variable region.

Specifically, a total of 7 anti-TIGIT/PD-1/IL-15 chimeric dual anti-cytokine fusion proteins were obtained, 7Y2-102 and 7Y2-104 belong to structure type I, 7Y2-072 (structure II shown in Figure 5B-a) and 7Y2-079 (structure II shown in Figure 5B-b) belong to structure type II, 7Y2-094 (structure III shown in Figure 5C-a) and 7Y2-078 (structure III shown in Figure 5C-b) belong to structure type III, and 7Y2-070 belongs to structure type IV. Their specific structures are shown in Table 6.

A total of 16 humanized anti-TIGIT/PD-1/IL-15 bispecific anti-cytokine fusion proteins belonging to structure type I were obtained: 7Y2-120, 7Y2-123, 7Y2-133, 7Y2-136, 7Y2-137, 7Y2-126, 7Y2-127, 7Y2-132, 7Y2-134, 7Y2-135, 7Y2-128, 7Y2-129, 7Y2-124, 7Y2-125, 7Y2-130, and 7Y2-131. Their specific structures are shown in Table 7.

Table 5: Amino acid sequences of IL-15, IL-15Ra, constant region, hinge region, and linker

Table 6: TIGIT/PD-1/IL-15 chimeric dual anti-cytokine fusion proteins

Note: The 7Y2-072 fusion protein belongs to Structure II shown in Figure 5B-a; the 7Y2-079 fusion protein belongs to Structure II shown in Figure 5B-b; the 7Y2-094 belongs to Structure III shown in Figure 5C-a; and the 7Y2-078 fusion protein belongs to Structure III shown in Figure 5C-b.

Table 7: TIGIT/PD-1/IL-15 humanized dual anti-cytokine fusion protein

Note: The 16 TIGIT/PD-1/IL-15 humanized bispecific anti-cytokine fusion proteins in Table 7 all belong to structure type I. The IL-15 sequence in this structure is shown in SEQ ID NO: 63, the IL-15Ra_2 sequence is shown in SEQ ID NO: 75, the human IgG1 heavy chain constant region CH1 sequence is shown in SEQ ID NO: 74, the human IgG1 heavy chain constant region CH2 and CH3 sequences containing Hole mutations are shown in SEQ ID NO: 72, the human IgG1 heavy chain constant region CH2 and CH3 sequences containing Knob mutations are shown in SEQ ID NO: 71, the human IgG1 light chain constant region CL sequence is shown in SEQ ID NO: 73, the hinge region 1 sequence is shown in SEQ ID NO: 65, the hinge region 2 sequence is shown in SEQ ID NO: 66, the linker 1 sequence is shown in SEQ ID NO: 67, and the linker 2 sequence is shown in SEQ ID NO: 68.

Example 3: Expression and purification of TIGIT/PD-1/IL-15 dual anti-cytokine fusion protein

3.1 Vector Construction

(1) Based on the pTT5 (Hunan Youbao Biological, VT2202) vector, which contains a signal peptide (SEQ ID NO: 76) that directs protein synthesis and secretion, the enzyme cleavage site was introduced by primer mutation, and the synthetic human IGKC sequence (SEQ ID NO: 79) was inserted into the pTT5 vector through the common biological enzyme cleavage and enzyme ligation methods to obtain the pTT5-IgG1-CL vector; similarly, the synthetic human IgG1 heavy chain constant region sequence was inserted into the pTT5 vector to obtain the pTT5-IgG1-CH vector.

(2) Mutations were introduced into the pTT5-IgG1-CH vector using a forward primer, and homologous fragments were introduced using a reverse primer. The pTT5-IgG1-Knob vector was obtained by PCR and homologous recombination. Similarly, the pTT5-IgG1-Hole vector was constructed using this method.

3.2 Expression of fusion protein

The gene sequences of the TIGIT/PD-1/IL-15 chimeric dual anti-cytokine fusion protein in Table 6 and the TIGIT/PD-1/IL-15 humanized dual anti-cytokine fusion protein in Table 7 were cloned into pTT5-IgG1-CL, pTT5-IgG1-CH, pTT5-IgG1-Knobe, or pTT5-IgG1-Hole vectors containing human IgG1 signal peptides for protein synthesis and secretion. The expression vectors were transfected into ExpiCHO-S cells (Gibco, A29127) for expression using transient transfection technology. Polyplus transfection reagent (Polyplus, REF#116-010/10mL) was used for transfection. One day before transfection (D0-1), the cell density was diluted to 2.0×10 ⁶ cells/mL with culture medium. On the day of transfection (D0), the cells were counted (cell viability should be ≥95%) and the cell density was adjusted to 4.0×10 ⁶ cells/mL. 1 μg of plasmid and 1 μl of Polyplus were used for every 4.0×10 ⁶ cells. Transfection Reagent, 1ul Dosage for transfection: Take a centrifuge tube, add 1/10 of the expression volume of culture medium to dilute the plasmid containing the dual anti-cytokine fusion protein fragment, take another centrifuge tube and add the required Polyplus DNA Transfection Reagent, slowly add the diluted plasmid dropwise to the centrifuge tube containing DNA Transfection Reagent, invert to mix, incubate at room temperature for 10 minutes, slowly add the incubated transfection reagent-DNA complex to ExpiCHO-S cells under shaking, and add within 0-4 hours. After transfection, the cells were cultured in a shaking incubator at 37°C, 120 rpm, and 8% _CO2. On the first day after transfection (D1), preheated CHOgro complete medium was added at 1/5 of the expression volume, and the culture was continued at 32°C. Advanced CHO Feed 1 was added at 8%, 5%, 5%, and 5% on D2, D4, D6, and D8, respectively, and the cells were harvested on D10.

3.3 Expression statistics of TIGIT/PD-1/IL-15 chimeric dual anti-cytokine fusion protein

The cells harvested in Example 3.2 were centrifuged to collect the supernatant. The dual-anti-cytokine fusion protein was purified using a protein purifier (AKTA pure 150) based on Protein-A affinity purification (Affinity Chromatography, AC). The steps are as follows:

(1) Sterilize the chromatography system by soaking in 0.5 M NaOH for at least 30 minutes and the chromatography column by soaking in 0.1 M NaOH for 30 minutes;

(2) Use 1× PBS buffer with pH = 7.4 to stabilize the system pH and conductivity baseline;

(3) Load the sample at a flow rate that provides a retention time of 3 to 5 minutes;

(4) After loading, wash with 0.5 M arginine hydrochloride + 1 M NaCl + 0.5% Triton X-100, pH = 5.0 ± 0.1 to remove non-specifically bound proteins and reduce the binding level of endotoxins and antibodies;

(5) Elute the target protein with 40 mM citric acid-sodium citrate + 0.15 M NaCI, pH = 3.0 ± 0.1, and adjust the pH of the target protein to 6.0 ± 0.1 with 1 M Tris, pH = 8.6 neutralization solution;

(6) Take 2-5ul of protein solution and use Nanodrop to detect the protein concentration. Then use size-exclusion chromatography (SEC-HPLC) to detect the purity: prepare 10μg, 1mg/ml sample, use liquid chromatograph (Shimadzu, Japan, LC-2030C-3D Plus), select the loading program of 0.5ml/min flow rate and 35 minutes injection, and after the test, use the corresponding software provided by the instrument for analysis (Lab solution), calculate the main peak area ratio to obtain the purity of the dual anti-cytokine fusion protein. Calculate the expression amount of fusion protein = concentration × volume × purity ÷ expression volume.

The expression data of the obtained TIGIT/PD-1/IL-15 chimeric dual anti-cytokine fusion protein were statistically analyzed, and the results are shown in Table 8.

Table 8: Expression levels of TIGIT/PD-1/IL-15 chimeric dual anti-cytokine fusion protein

The results in the table show that: (1) the expression level of the chimeric dual anti-cytokine fusion protein 7Y2-102 (Structure I) prepared by combining 1KP-3A6-3C6 with 2IT1-10A3-2H6 was significantly higher than that of 7Y2-072 (Structure II), 7Y2-094 (Structure III) and 7Y2-070 (Structure IV); (2) the expression level of the chimeric dual anti-cytokine fusion protein 7Y2-104 (Structure I) prepared by combining 1KP-3A6-3C6 with 3A03A03 was significantly higher than that of 7Y2-079 (Structure II) and 7Y2-078 (Structure III). Therefore, the TIGIT/PD-1/IL-15 dual anti-cytokine fusion protein prepared using Structure I has a better expression level than the other three structures.

3.4 Gel filtration chromatography purification

After affinity purification, the dual-anti-cytokine fusion protein was further purified by gel filtration chromatography (GFC) to obtain a highly purified recombinant fusion protein. The chromatography system and column were soaked in 0.5 M NaOH for at least 2 hours. The chromatography system was equilibrated with 1× PBS (pH 6.2) to a pH and conductivity baseline. The sample was then loaded using a sample loop, and the intact dual-anti-cytokine fusion protein monomer was collected using the molecular sieving principle.

Example 4: Physical and chemical properties detection of TIGIT/PD-1/IL-15 chimeric dual anti-cytokine fusion protein

The denaturation temperature (Melting Point, Tm), aggregation temperature (Temperature of Aggregation, Tagg), aggregation resistance (Aggregation-Resistant) and thermal stability (Thermostable) of the TIGIT/PD-1/IL-15 chimeric dual anti-cytokine fusion protein purified in Example 3.4 were detected.

Tm assay: Using differential scanning fluorescence (DSF), prepare 17.5 μl of 1 mg/ml chimeric anti-cytokine fusion protein to be tested. Add 2.5 μl of 1000× protein thermal shift dye (Thermo, 4461146) and mix in an EP tube. The sample mixture is then added to a Q-PCR system (Quant Studio) for reaction. The Q-PCR parameters are: Target (ROX), program (25°C, 3 min, 0.05°C/s temperature ramp to 99°C; 99°C, 2 min). The results are imported into Graph Prism software to calculate Tm values.

Tagg test: Prepare 9 μl (1 mg/ml) of each chimeric anti-cytokine fusion protein to be tested, load it into the detection instrument (Uncle-0734), and analyze it with the corresponding software provided with the instrument after the test.

High temperature stability at 40°C: The chimeric anti-cytokine fusion protein to be tested was placed in a 40°C constant temperature incubator for 1 day, 3 days, 7 days, and 14 days. The protein purity after 1 day, 3 days, 7 days, and 14 days of high temperature treatment was determined by referring to the SEC-HPLC method in Example 3.3.

Repeated freeze-thaw: The chimeric bispecific anti-cytokine fusion protein to be tested was placed in a -20°C freezer and then thawed at room temperature. This process was repeated 1, 3, and 5 times, respectively. The protein purity after 1, 3, and 5 freeze-thaw cycles was determined by referring to the SEC-HPLC method in Example 3.3.

The Tm value, Tagg value, 40°C high temperature stability and repeated freeze-thaw test results of the chimeric bispecific anti-cytokine fusion protein are shown in Table 9.

Table 9. Physicochemical properties of chimeric dual anti-cytokine fusion proteins

Note: “N/A” indicates None or not applicable; “/” indicates that the antibody structure is stable and no melting occurred, so no data value was obtained.

From the results in Table 9, it can be seen that in the TIGIT/PD-1/IL-15 chimeric dual anti-cytokine fusion protein prepared by combining 1KP-3A6-3C6 and 2IT1-10A3-2H6: 7Y2-102 (Structure I) has better effects than 7Y2-072 (Structure II) in terms of Tm value, Tagg value, repeated freeze-thaw and high temperature stability, indicating that the TIGIT/PD-1/IL-15 dual anti-cytokine fusion protein prepared using Structure I has better physical and chemical properties.

Example 5: Affinity determination of TIGIT/PD-1/IL-15 chimeric dual anti-cytokine fusion protein and human TIGIT antigen, cynomolgus monkey TIGIT antigen, human PD-1 antigen or cynomolgus monkey PD-1 antigen

The biomolecular interaction analyzer (Biacore, Cytiva) was used to detect the interaction of chimeric bispecific anti-cytokine fusion protein with human TIGIT (i.e. Hu-TIGIT, purchased from Acro Biosystem, TIT-H52H5), human PD-1 (i.e. Hu-PD-1, purchased from Acro Biosystem, PD1-H5221), cynomolgus monkey TIGIT (i.e. Cyno-TIGIT, purchased from Acro Biosystem, CAT: TIT-C5223), cynomolgus monkey PD-1 (i.e. Cyno-PD-1, purchased from Acro Biosystem, CAT: TIT-C5224), and human PD-1 (i.e. Hu-PD-1, purchased from Acro Biosystem, CAT: TIT-H5225). o Biosystem, PD1-C5223) protein association rate constant (ka), dissociation rate constant (kd), and equilibrium dissociation constant (KD). The specific implementation steps were as follows: the anti-human IgG antibody secondary antibody was amino-coupled and fixed on the activated CM5 biosensor chip, and the chimeric dual-antibody cytokine fusion protein to be detected was added at a final concentration of 5 μg/ml. Then, gradient dilutions of Hu-TIGIT, Cyno-TIGIT, Hu-PD-1, and Cyno-PD-1 antigens (starting at 90 nM and diluted 3 times) were added from low to high, according to the following Kinetics program: Start up (60 s)--Capture (60 s)--Association (200 s)--Dissociation (600 s)--Regeneration (40 s). After the program was completed, the result file was read using Insight Evaluation Software, and then analyzed using the kinetic fitting analysis method to obtain the affinity binding results of the chimeric dual anti-cytokine fusion protein with the corresponding antigen, as shown in Table 10.

Table 10. Affinity results of chimeric dual anti-cytokine fusion proteins

Note: N/A means None or not applicable.

As can be seen from Table 10, the TIGIT/PD-1/IL-15 chimeric dual anti-cytokine fusion protein in the table has good binding activity with human TIGIT, human PD-1, cynomolgus monkey TIGIT, and cynomolgus monkey PD-1 protein.

Example 6: Detection of the binding ability of TIGIT/PD-1/IL-15 chimeric dual anti-cytokine fusion protein to cells expressing human TIGIT antigen, cynomolgus monkey TIGIT antigen, human PD-1 antigen or cynomolgus monkey PD-1 antigen

Flow cytometry fluorescence sorting (FACS) was used to detect the binding ability of the chimeric dual anti-cytokine fusion protein to CHO-K1-hPD-1 and CHO-K1-cynoPD-1 cells, respectively. The specific experimental steps are as follows: CHO-K1-hPD-1 cells overexpressing human PD-1 and CHO-K1-cynoPD-1 overexpressing cynomolgus monkey PD-1 in the logarithmic growth phase were taken, washed once with Ham's F-12K medium (abbreviated as H2 medium, BasalMedia, L450KJ) containing 2% FBS (ExCell Bio), and then the cell density was adjusted to 1×10 ⁶ cells/mL with H2 medium. Fc blocker (BD, 564220, please provide the manufacturer and product number) was added at a volume ratio of 1:100 and incubated at room temperature for 30 minutes. Add 100 μL of CHO-K1-hPD-1 or CHO-K1-cynoPD-1 cell suspensions (1× ¹⁰⁵ cells) to each well of a 96-well U-bottom plate. Centrifuge at 300 g for 3 minutes, and discard the supernatant. Dilute the TIGIT/PD-1/IL-15 chimeric dual-anti-cytokine fusion protein to a starting working concentration of 240 μg/mL using H2 medium. A three-fold serial dilution series was performed, and a blank control well containing H2 medium was set up. Then, 50 μL of the chimeric dual-anti-cytokine fusion protein at each dilution level was added to the plated CHO-K1-hPD-1 and CHO-K1-cynoPD-1 cells. Mix thoroughly, and incubate at 37°C for 1 hour. After incubation, cells were centrifuged at 300 g for 3 min, the supernatant discarded, and the cells were harvested and washed three times with FACS buffer (PBS containing 2% FBS and 0.002 M EDTA). Cells were resuspended in 100 μL of a fluorescently conjugated goat anti-human IgG Fc secondary antibody (diluted 1:800 in FACS buffer, Biolegend, 410712) per well and incubated at 4°C for 30 min. After incubation, cells were centrifuged at 300 g for 3 min, the supernatant discarded, and the cells were washed three times with FACS buffer. The cells were resuspended in 100 μL of FACS buffer and analyzed by flow cytometry. Flow cytometry results were analyzed and calculated using FlowJo, and the mean fluorescence intensity (MFI) value of AF647 cells in each sample well was derived. The MFI value and the corresponding antibody concentration were subjected to nonlinear regression fitting using the Log (agonist) vs. response—Variable slope (four parameters) method in GraphPad Prism software. The abscissa was Log [Ab concentration] and the ordinate was the MFI value. The binding curve of the chimeric dual anti-cytokine fusion protein and the PD-1 antigen was obtained, and the _EC50 of the binding curve of the chimeric dual anti-cytokine fusion protein and the PD-1 antigen was calculated to determine the binding ability of the fusion protein to cells overexpressing the PD-1 antigen.

Referring to the above-mentioned method for measuring CHO-K1-hPD-1 cells, the binding ability of the chimeric bispecific anti-cytokine fusion protein to Jurkat-huTIGIT cells overexpressing human TIGIT and 293T-cynoTIGIT cells overexpressing cynomolgus macaque TIGIT was determined; Jurkat cells were cultured in RPMI1640 (Shanghai Yuanpei, L210KJ) and 293T cells were cultured in DMEM high glucose (Shanghai Yuanpei, L110KJ). The affinity binding results of the chimeric bispecific anti-cytokine fusion protein to cells expressing the corresponding antigens are shown in Table 11.

Table 11. Binding results of chimeric bispecific anti-cytokine fusion proteins to cell surface targets

It can be seen from Table 11 that the TIGIT/PD-1/IL-15 chimeric dual anti-cytokine fusion proteins in the table have good binding ability with cells expressing human TIGIT antigen, cynomolgus monkey TIGIT antigen, human PD-1 antigen or cynomolgus monkey PD-1 antigen, respectively.

Example 7: Activation assay of TIGIT/PD-1/IL-15 chimeric dual anti-cytokine fusion protein with cells overexpressing human TIGIT/CD155 and overexpressing human PD-1/PD-L1 reporter

7.1 Activation Assay of TIGIT/PD-1/IL-15 Chimeric Anti-Cytokine Fusion Protein and Human PD-1/PD-L1 Reporter Cells

Logarithmically growing CHO-K1/hPDL1/TCR cells (M00613, Nanjing GenScript Biotechnology Co., Ltd.) overexpressing human PD-L1 were used as target cells, trypsinized and resuspended in fresh Ham's F-12K medium containing 10% FBS (referred to as H10 medium) to adjust the cell density to 5×10 ⁵ cells/mL. The resuspended cells were added to each well at a volume of 5×10 ⁴ cells, and 100 μL was added to each well and seeded into a white-walled, transparent-bottomed 96-well cell culture plate. The plates were cultured overnight at 37°C and 5% CO _{2 in a 5% CO 2} incubator. The supernatant was centrifuged the next day and discarded. The TIGIT/PD-1/IL-15 chimeric dual anti-cytokine fusion protein was diluted to 2 times the starting working concentration of 240 μg/mL using RPMI1640 medium containing 2% FBS (referred to as R2 medium). 11 concentrations were diluted in a 3-fold gradient. A blank R2 medium control group was also set up. Then, 50 μL of the fusion protein from each gradient was added to the plated CHO-K1/hPDL1/TCR cells. Jurkat/NFAT-luc/PD1 cells (M00612, Nanjing GenScript Biotechnology Co., Ltd.), stably transfected with human PD-1 and a fluorescent reporter gene controlled by the NFAT nuclear transcriptional response element, were used as effector cells. The cells were resuspended in R2 and adjusted to a cell density of 3 × 10 ⁶ /mL. 50 μL was added to each well of a 96-well plate and incubated at 37°C in a 5% CO ₂ incubator for 6 hours. The 96-well plate was removed and 100 μL of Bright-Lite™ (Vazyme) luciferase detection reagent was added to each well. After incubation at room temperature for 3-5 minutes, the cells were assayed in a microplate reader. The fluorescence reading corresponding to each gradient concentration well was used as the ordinate, and the logarithm of the sample concentration gradient with base 10 was used as the abscissa. The Log (agonist) vs. response—Variable slope (four parameters) method (GraphPad Prism software) was used for nonlinear regression fitting to obtain the antibody activation intensity curve on reporter cells. The _EC50 of the antibody activation curve was calculated to determine the activation ability of the antibody.

7.2 Activation Assay of TIGIT/PD-1/IL-15 Chimeric Anti-Cytokine Fusion Protein and Cells Expressing Human TIGIT/CD155 Reporter

Referring to the assay method of Example 7.1 above, the activation of the chimeric dual anti-cytokine fusion protein and CHO-K1/CD155/TCR cells overexpressing human CD155 (CBP74073, Nanjing Kebai Biotechnology Co., Ltd.)/Jurkat-TIGIT-NFAT-Luc cells stably transfected with human TIGIT and a fluorescent reporter gene controlled by the NFAT nuclear transcription response element (CBP74020, Nanjing Kebai Biotechnology Co., Ltd.) was determined; the adjusted density of the Jurkat-TIGIT-NFAT-Luc cells was 1×10 ⁶ cells/mL.

The results are shown in Table 12, Figure 6A, and Figure 6B. The TIGIT/PD-1/IL-15 chimeric dual anti-cytokine fusion protein in the table can recognize and activate Jurkat-TIGIT-NFAT-Luc or jurkat/NFAT-luc/PD1 Reporter cells; and compared with 7Y2-072 (Structure II), 7Y2-102 (Structure I) can better activate both cells, indicating that the TIGIT/PD-1/IL-15 dual anti-cytokine fusion protein prepared using Structure I has a better ability to activate reporter cells.

Table 12. Activation results of chimeric dual anti-cytokine fusion protein on reporter cells

Example 8: Functional detection of TIGIT/PD-1/IL-15 chimeric dual anti-cytokine fusion protein in promoting immune cell proliferation

CTLL-2 cells (China Food and Drug Inspection Institute, 1102MOU-NIFDC00051) and Mo7e cells (from the National Biomedical Experimental Cell Resource Bank) in the logarithmic growth phase were harvested, washed twice with PBS, and resuspended in assay medium (RPMI1640 (ATCC modified) (Shanghai Yuanpei, L240KJ) + 10% FBS). The cell density was adjusted to 8 × 10 ⁵ cells/ml. Mo7e cells and CTLL-2 cells were seeded into 96-well flat-bottom plates at a volume of 50 μl per well, and the plates were cultured in a 37°C, 5% CO ₂ incubator for 4 hours to allow the experimental cells to reach a cytokine starvation state.

The chimeric dual-anti-cytokine fusion protein was diluted to a final concentration of 1080 nM using assay medium. A two-fold serial dilution was performed followed by nine three-fold serial dilutions. 50 μl of the chimeric dual-anti-cytokine fusion protein dilutions were added to Mo7e and CTLL-2 cell plates, respectively. The CTLL-2 or Mo7e cells supplemented with the chimeric dual-anti-cytokine fusion protein were cultured for an additional 72 hours. Finally, the number of viable cells was determined using CCK-8 reagent (Biyuntian, C0040). Proliferation curves were plotted, and the _EC50 for the chimeric dual-anti-cytokine fusion protein in promoting the proliferation of Mo7e and CTLL-2 cells was calculated.

The results are shown in Table 13, Figures 7A, and 7B. Both chimeric dual-anti-cytokine fusion proteins stimulated the proliferation of CTLL-2 and Mo7e cells. Among the TIGIT/PD-1/IL-15 chimeric dual-anti-cytokine fusion proteins prepared with 1KP-3A6-3C6 and 3A03A03, the fusion protein 7Y2-078 (Structure III) exhibited higher proliferation activity than 7Y2-104 (Structure I). Among the TIGIT/PD-1/IL-15 chimeric dual-anti-cytokine fusion proteins prepared with 1KP-3A6-3C6 and 2IT1-10A3-2H6, the proliferation activity of 7Y2-072 (Structure II) and 7Y2-070 (Structure IV) was higher than that of 7Y2-102 (Structure I). The results indicate that Structure I fusion proteins can moderately activate immune cells and have a better safety profile than Structures II, III, and IV.

Table 13. Cytokine activity of IL-15 in chimeric dual anti-cytokine fusion proteins

Example 9: Functional detection of TIGIT/PD-1/IL-15 chimeric dual anti-cytokine fusion protein activating human PBMCs

Peripheral blood mononuclear cells (PBMCs) from healthy volunteers (Shanghai Aoneng Biotechnology Co., Ltd., Donor ID: Z0395) were taken out of liquid nitrogen and revived. They were resuspended to 1×10 ⁶ cells/mL in complete medium (RPMI1640 + 10% FBS). After adding SEA to the resuspended cell suspension at 10 ng/mL, 100 μl was added to each well of a 96-well flat-bottom plate. The chimeric dual anti-cytokine fusion protein was diluted to 30 μg/mL with complete medium. At the same time, the combined drug control group (Combo (P+T)) was diluted to 20 μg/ml of sample P (Keytruda) and 10 μg/ml of sample T (Tiragolumab). Then, both were diluted 5-fold and added to the corresponding PBMCs cell wells at 100 μL/well. The cells were cultured in a 37°C, 5% CO ₂ incubator for 3-4 days. The cell culture supernatant was collected on the 3rd and 4th days, respectively.

Homogeneous time-resolved fluorescence (HTRF) was used to measure human IFN-γ and IL-2 in the cell culture supernatant. Experimental procedures were performed according to the kit instructions. The IL-2 detection kit was purchased from Cisbio Bioassays SAS, catalog number 62HIL02PEH; the IFN-γ detection kit was purchased from Cisbio Bioassays SAS, catalog number 62HIFNGPEH. The test results were calculated according to the kit instructions. The results are shown in Figures 8A, 8B, 9A, and 9B. With increasing concentrations of the chimeric dual-antibody fusion protein, IFN-γ showed no significant change, but IL-2 secretion increased significantly, indicating that the chimeric dual-antibody fusion protein activates human PBMCs.

Compare the mean concentration of the sample test with that of the control group (Combo P+T):

The results are shown in Table 14. The activation of human PBMC by the chimeric dual anti-cytokine fusion proteins 7Y2-102 and 7Y2-104 was comparable to that of the control group.

Table 14 PBMCs activation results of chimeric dual anti-cytokine fusion protein

Example 10: Long-term toxicity evaluation of TIGIT/PD-1/IL-15 chimeric dual anti-cytokine fusion protein in mice

4-6 week-old humanized Balb/c mice targeting PD-1 and TIGIT were purchased (Jiangsu Jicui Pharmaceutical Kang Biotechnology Co., Ltd.) and placed in SPF mice housing for one week of recovery. At the start of the experiment, mice weighing 19-25 g were randomly divided into four groups (7Y2-102, 7Y2-104, 7Y2-070, and 7Y2-078), with six mice per group. The chimeric dual-anti-cytokine fusion protein (7Y2-102, 1.5 mg/kg, 2.7 mg/kg, and 2.0 mg/kg, respectively) was injected intraperitoneally twice weekly. The mice were weighed and observed twice weekly. After two weeks of injection, the mice were observed for one week after the drug was discontinued.

The dose, time to initial death, and mortality rate of each group of mice at the end of the study were statistically analyzed. The results are shown in Table 15, and the corresponding survival curves are shown in Figure 10. At higher doses of the structural class I fusion proteins (7Y2-102 and 7Y2-104), no mice died until the end of the study. However, the mortality rate of mice in the structural class IV fusion protein (7Y2-070) group was 66.67%, and the mortality rate of mice in the structural class III fusion protein (7Y2-078) group was 50%.

Table 15 Toxicity evaluation of chimeric dual anti-cytokine fusion protein

Example 11: Physical and chemical properties detection of TIGIT/PD-1/IL-15 humanized dual anti-cytokine fusion protein

The physical and chemical properties of the TIGIT/PD-1/IL-15 humanized antibody were tested using the physicochemical property testing method of the chimeric bispecific anti-cytokine fusion protein in Example 4. The test results are shown in Tables 16 to 18. As can be seen from the results, the humanized bispecific anti-cytokine fusion protein in the table has good properties in terms of Tm value, repeated freeze-thaw stability, and high temperature stability.

Table 16. Tm values of humanized dual anti-cytokine fusion proteins

Note: “/” indicates that the antibody structure is stable and no melting occurs, so no data value is obtained.

Table 17. Repeated freeze-thaw stability test of humanized bispecific anti-cytokine fusion proteins

Table 18. High temperature stability test of humanized bispecific anti-cytokine fusion protein

Note: ND means No Data or Not Done.

Example 12: Affinity Determination of TIGIT/PD-1/IL-15 Humanized Dual Anti-Cytokine Fusion Protein and Human TIGIT Antigen, Cynomolgus Monkey TIGIT Antigen, Human PD-1 Antigen, or Cynomolgus Monkey PD-1 Antigen

Reference Example 5 Detection of the affinity of chimeric bispecific anti-cytokine fusion proteins to human and cynomolgus monkey TIGIT antigens and human and cynomolgus monkey PD-1 antigens The affinity of the humanized bispecific anti-cytokine fusion proteins to the corresponding target antigens was detected, and the results are shown in Tables 19 and 20. As can be seen from the results, the humanized bispecific anti-cytokine fusion proteins in Tables 19 and 20 have good affinity with human and cynomolgus monkey TIGIT antigens and human and cynomolgus monkey PD-1 antigens, respectively.

Table 19 Affinity test results of humanized bispecific anti-cytokine fusion protein and TIGIT antigen

Note: N/A means None or Not Applicable

Table 20 Affinity test results of humanized bispecific anti-cytokine fusion protein and PD-1 antigen

Example 13: Detection of the binding ability of TIGIT/PD-1/IL-15 humanized dual anti-cytokine fusion protein to cells expressing human TIGIT antigen, cynomolgus monkey TIGIT antigen, human PD-1 antigen or cynomolgus monkey PD-1 antigen

The binding ability of the TIGIT/PD-1/IL-15 humanized antibody to cells expressing the corresponding target antigens was detected by the method of detecting the binding ability of the fusion protein to cells overexpressing human TIGIT antigen, cynomolgus monkey TIGIT antigen, human PD-1 antigen, or cynomolgus monkey PD-1 antigen in Example 6. The results are shown in Tables 21 and 22. As can be seen from the results, the TIGIT/PD-1/IL-15 humanized antibodies in Tables 21 and 22 have good binding ability to cells expressing human TIGIT antigen, cynomolgus monkey TIGIT antigen, human PD-1 antigen, or cynomolgus monkey PD-1 antigen, respectively.

Table 21. Binding results of humanized bispecific anti-cytokine fusion proteins to human or cynomolgus monkey TIGIT antigens on cell surfaces

Note: N/A means None or Not Applicable

Table 22. Binding results of humanized bispecific anti-cytokine fusion proteins to human or cynomolgus monkey PD-1 antigens on cell surfaces

Example 14: Activation assay of TIGIT/PD-1/IL-15 humanized dual anti-cytokine fusion protein with overexpressed human TIGIT/CD155 and overexpressed human PD-1/PD-L1 reporter cells

Referring to Example 7, the activation assay method for detecting the fusion protein with overexpressed human TIGIT/CD155 and overexpressed human PD-1/PD-L1 reporter cell pairs was used to detect the activation ability of the TIGIT/PD-1/IL-15 humanized dual anti-cytokine fusion protein with the corresponding reporter cell pairs. The results are shown in Table 23. The humanized dual anti-cytokine fusion protein molecules in Table 23 have a good activation effect on the activation of the Jurkat cell TCR pathway response mediated by the TIGIT or PD-1 signaling pathway. Reporter cells.

Table 23. Activation results of humanized dual anti-cytokine fusion protein on reporter cells

Note: N/A means None or Not Applicable

Example 15: Activation assay of TIGIT/PD-1/IL-15 humanized dual anti-cytokine fusion protein and reporter cell pairs simultaneously overexpressing human TIGIT/CD155 and human PD-1/PD-L1

Logarithmically growing PDL1/CD155/TCR Activator/CHO cells (Nanjing Kebai, CBP74127) were trypsinized and resuspended in fresh F12K medium (Shanghai Yuanpei, L450KJ) containing 10% FBS. The cell density was adjusted to 4 × 10 ⁵ /mL. The resuspended cells were seeded into a 96-well cell culture plate with a white wall and a clear bottom. 100 μL of cell suspension was added to each well and cultured in a 37°C incubator overnight. The next day, the F12K medium in the 96-well plate inoculated with PDL1/CD155/TCR Activator/CHO cells was aspirated, and then the samples were serially diluted with RPMI1640 medium containing 10% serum. Samples 7Y2-123 and 7Y2-127 started from a maximum concentration of 600 μg/mL (2-fold concentration sample), sample P (Keytruda) started from a maximum concentration of 512.8 μg/mL (2-fold concentration sample), and in sample P+T, sample P started from a maximum concentration of 684.4 μg/mL (2-fold concentration sample), and sample T (Tiragolumab) started from a maximum concentration of 342.2 μg/mL (double concentration sample). They were serially diluted 4-fold in sequence, and the serially diluted 2-fold concentration samples (50 μL/well) were added to the 96-well plate inoculated with cells. A blank culture medium control well was also set up.

Human PD1/TIGIT Dual Effector Reporter Cells (Nanjing Kebai, CBP74126) growing in the logarithmic phase were centrifuged and the supernatant was discarded. The cells were resuspended in fresh RPMI1640 medium containing 10% FBS and the cell density was adjusted to 4×10 ⁵ cells/mL. The cells were then added to the 96-well plate containing the fusion protein and PDL1/CD155/TCR Activator/CHO cells prepared above. 50 μL was added to each well and the cells were placed in a 37°C incubator for further incubation for 6 hours. The 96-well plate was removed from the incubator and 100 μL of Bright-Glo ^™ Luciferase Assay Reagent was added to each well. The plate was left for 3 to 5 minutes and then placed in a microplate reader for reading. Based on the readings corresponding to each gradient concentration well, the gradient curve of the sample on cell activation was fitted using Prism Graphpad software, and the half-maximal effect concentration (EC ₅₀ ) of the sample was calculated. The results are shown in Table 24 and Figure 11. The activation effect of 7Y2-123, 7Y2-127 and the control antibody Keytruda and Tiragolumab combined is comparable, and stronger than the activation effect of Keytruda alone.

Table 24. Reporter cell activation results of humanized dual anti-cytokine fusion proteins

Example 16: Functional detection of TIGIT/PD-1/IL-15 humanized dual anti-cytokine fusion protein in promoting immune cell proliferation

Refer to Example 8 for the method for detecting the function of the fusion protein in promoting the proliferation of immune cells to detect the function of the TIGIT/PD-1/IL-15 humanized dual anti-cytokine fusion protein in promoting the proliferation of immune cells.

The results are shown in Table 25, Figures 12A, 12B, and 12C. The TIGIT/PD-1/IL-15 humanized dual anti-cytokine fusion protein in the figures can stimulate the proliferation of the Mo7e cell line.

Table 25 Proliferation results of humanized dual anti-cytokine fusion protein

Example 17: Functional detection of human PBMC activation by TIGIT/PD-1/IL-15 humanized dual anti-cytokine fusion protein

Reference Example 9 Detection of the Function of Fusion Protein in Activating Human PBMCs The in vitro activation function of TIGIT/PD-1/IL-15 humanized dual anti-cytokine fusion protein on peripheral lymphocytes was detected. The secretion of IL-2 and IFN-γ in the sample group and the ratio of the combination drug (Keytruda and Tiragolumab) control group were calculated as shown in Table 26. A value greater than 1 indicates that the activation effect of the sample is stronger than that of the control group. It can be seen from the results that the humanized dual anti-cytokine fusion protein in the table can effectively activate PBMC cells.

Table 26. PBMCs activation results of humanized dual anti-cytokine fusion proteins

Example 18: In vivo anti-tumor efficacy evaluation of TIGIT/PD-1/IL-15 humanized dual anti-cytokine fusion protein

Fifty-six NOGdko mice (purchased from Weitonglihua) were acclimated in a SPF facility for 7 days. During this period, human bladder cancer cells 5637 (Nanjing Kebai, CBP60309) were revived and cultured. After the acclimation period, human PBMCs (Aoneng, ID: Z0406) were injected into the tail vein at a density of 5× ¹⁰⁶ cells/mouse. After 4 days of normal feeding, 5637 cells were inoculated into the left axilla at a density of 5× ¹⁰⁶ cells/mouse. After 3 days of normal feeding, tumor size was measured and the mice were randomly divided into five groups: Vehicle group, P+T (i.e., Keytruda 5mg/kg + Tiragolumab 10mg/kg) group, 7Y2-123_0.75mg/kg group, 7Y2-123_1.5mg/kg group, and 7Y2-123_3mg/kg group. The first three groups consisted of 10 mice each, and the latter two groups consisted of 13 mice each. Dosing began on day 6 after tumor cell inoculation: Each group received the corresponding dose via intraperitoneal injection twice weekly for a total of 6 doses. Mouse body weight and tumor volume were monitored twice weekly for 27 days, starting from the first dose. As shown in Figure 13, 7Y2-123 exhibited a dose-dependent tumor-suppressive effect across the different dose groups, demonstrating a good tumor-suppressing effect. The weight change rates of mice in each group are shown in Figure 14. There were no significant differences in the weight change rates among the groups during the dosing period, demonstrating a good safety profile for 7Y2-123.

Although the specific embodiments of the present disclosure have been described in detail, it will be understood by those skilled in the art that various modifications and variations may be made to the details based on all the teachings published, and that these variations are within the scope of protection of the present disclosure. The entire disclosure is given by the appended claims and any equivalents thereof.

Claims (21)

A fusion protein comprising: Targeting the first antigen-binding domain of TIGIT, Targeting the second antigen-binding domain of PD-1, IL-15 polypeptide or a functional fragment thereof, and An IL-15Rα polypeptide or a functional fragment thereof, wherein: (i) The first antigen-binding domain comprises a heavy chain variable region (VH) and a light chain variable region (VL) selected from any one of the following groups, wherein: (i-a) VH comprising the following three CDRs: CDR-H1 comprising the sequence shown in SEQ ID NO: 3, CDR-H2 comprising the sequence shown in SEQ ID NO: 4, and CDR-H3 comprising the sequence shown in SEQ ID NO: 5; and A VL comprising the following three CDRs: a CDR-L1 comprising the sequence set forth in SEQ ID NO:6, a CDR-L2 comprising the sequence set forth in SEQ ID NO:7, and a CDR-L3 comprising the sequence set forth in SEQ ID NO:8; or (i-b) VH comprising the following three CDRs: CDR-H1 comprising the sequence shown in SEQ ID NO: 11, CDR-H2 comprising the sequence shown in SEQ ID NO: 12, and CDR-H3 comprising the sequence shown in SEQ ID NO: 13; and A VL comprising the following three CDRs: a CDR-L1 comprising the sequence set forth in SEQ ID NO: 14, a CDR-L2 comprising the sequence set forth in SEQ ID NO: 15, and a CDR-L3 comprising the sequence set forth in SEQ ID NO: 16; or (i-c) VH comprising the following three CDRs: CDR-H1 comprising the sequence shown in SEQ ID NO: 19, CDR-H2 comprising the sequence shown in SEQ ID NO: 20, and CDR-H3 comprising the sequence shown in SEQ ID NO: 21; and A VL comprising the following three CDRs: a CDR-L1 comprising the sequence set forth in SEQ ID NO: 22, a CDR-L2 comprising the sequence set forth in SEQ ID NO: 23, and a CDR-L3 comprising the sequence set forth in SEQ ID NO: 24; and/or, (ii) the second antigen-binding domain comprises a VH and a VL selected from any one of the following groups, wherein, (ii-a) a VH comprising the following three CDRs: a CDR-H1 comprising the sequence set forth in SEQ ID NO: 27, a CDR-H2 comprising the sequence set forth in SEQ ID NO: 28, and a CDR-H3 comprising the sequence set forth in SEQ ID NO: 29; and A VL comprising the following three CDRs: CDR-L1 comprising the sequence set forth in SEQ ID NO: 30, CDR-L2 comprising the sequence set forth in SEQ ID NO: 31, and CDR-L3 comprising the sequence set forth in SEQ ID NO: 32; or (ii-b) a VH comprising the following three CDRs: a CDR-H1 comprising the sequence shown in SEQ ID NO: 35, a CDR-H2 comprising the sequence shown in SEQ ID NO: 36, and a CDR-H3 comprising the sequence shown in SEQ ID NO: 37; and VL comprising the following three CDRs: CDR-L1 comprising the sequence shown in SEQ ID NO:38, CDR-L2 comprising the sequence shown in SEQ ID NO:39, and CDR-L3 comprising the sequence shown in SEQ ID NO:40. The fusion protein according to claim 1, wherein (i) The first antigen-binding domain comprises a VH and a VL selected from any one of the following groups, wherein: (i-a) VH comprising the sequence shown in SEQ ID NO: 1 and VL comprising the sequence shown in SEQ ID NO: 2; (i-b) VH comprising the sequence shown in SEQ ID NO:9 and VL comprising the sequence shown in SEQ ID NO:10; (i-c) VH comprising the sequence shown in SEQ ID NO:17 and VL comprising the sequence shown in SEQ ID NO:18; (i-d) VH comprising the sequence shown in SEQ ID NO:41 and VL comprising the sequence shown in SEQ ID NO:42; (i-e) VH comprising the sequence shown in SEQ ID NO:43 and VL comprising the sequence shown in SEQ ID NO:44; (i-f) VH comprising the sequence shown in SEQ ID NO:45 and VL comprising the sequence shown in SEQ ID NO:46; (i-g) VH comprising the sequence shown in SEQ ID NO:45 and VL comprising the sequence shown in SEQ ID NO:47; (i-h) VH comprising the sequence shown in SEQ ID NO:48 and VL comprising the sequence shown in SEQ ID NO:49; (i-i) VH comprising the sequence shown in SEQ ID NO:48 and VL comprising the sequence shown in SEQ ID NO:50; and/or, (ii) the second antigen-binding domain comprises a VH and a VL selected from any one of the following groups, wherein: (ii-a) a VH comprising the sequence shown in SEQ ID NO: 25 and a VL comprising the sequence shown in SEQ ID NO: 26; (ii-b) a VH comprising the sequence shown in SEQ ID NO: 33 and a VL comprising the sequence shown in SEQ ID NO: 34; (ii-c) a VH comprising the sequence shown in SEQ ID NO:54 and a VL comprising the sequence shown in SEQ ID NO:55; (ii-d) VH comprising the sequence shown in SEQ ID NO:56 and VL comprising the sequence shown in SEQ ID NO:57; (ii-e) a VH comprising the sequence shown in SEQ ID NO:58 and a VL comprising the sequence shown in SEQ ID NO:59; (ii-f) VH comprising the sequence shown in SEQ ID NO:56 and VL comprising the sequence shown in SEQ ID NO:59; (ii-g) VH comprising the sequence shown in SEQ ID NO:60 and VL comprising the sequence shown in SEQ ID NO:57; (ii-h) VH comprising the sequence shown in SEQ ID NO:60 and VL comprising the sequence shown in SEQ ID NO:61; (ii-i) VH comprising the sequence shown in SEQ ID NO:62 and VL comprising the sequence shown in SEQ ID NO:61; (ii-j) VH comprising the sequence shown in SEQ ID NO:51 and VL comprising the sequence shown in SEQ ID NO:52; (ii-k) a VH comprising the sequence shown in SEQ ID NO:53 and a VL comprising the sequence shown in SEQ ID NO:52; Optionally, the VH and VL are compared to the VH and VL in any of groups (i-a) to (i-i), (ii-a) to (ii-k), VH has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; and, VL has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; and/or, (iii) the IL-15 polypeptide comprises the native amino acid sequence as shown in SEQ ID NO: 63, or comprises an IL-15 variant that is different from the native amino acid sequence as shown in SEQ ID NO: 63; preferably, the amino acid sequence of the IL-15 variant has at least one amino acid substitution, deletion or addition (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared to the native amino acid sequence as shown in SEQ ID NO: 63; And/or, (iv) the IL-15Rα polypeptide comprises the natural amino acid sequence as shown in SEQ ID NO:64, or includes an IL-15Rα variant different from the natural amino acid sequence as shown in SEQ ID NO:64; preferably, the amino acid sequence of the IL-15Rα variant has at least one amino acid substitution, deletion or addition (for example, 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared with the natural amino acid sequence as shown in SEQ ID NO:64; further preferably, the IL-15Rα variant comprises the sequence as shown in SEQ ID NO:75. A fusion protein comprising: a first antigen-binding domain targeting TIGIT or PD-1, a second antigen-binding domain targeting TIGIT or PD-1, an IL-15 polypeptide or a functional fragment thereof, and an IL-15Rα polypeptide or a functional fragment thereof, wherein the first antigen-binding domain and the second antigen-binding domain bind to different targets, respectively, the first antigen-binding domain is a Fab fragment, the second antigen-binding domain is a single-chain antibody, and the IL-15 polypeptide and IL-15Rα polypeptide are located on different peptide chains; Preferably, the first antigen-binding domain is an antigen-binding domain targeting TIGIT; the second antigen-binding domain is an antigen-binding domain targeting PD-1; Preferably, the single-chain antibody is scFv. The fusion protein according to claim 3, wherein the fusion protein further comprises an Fc domain, wherein the Fc domain comprises a first monomer and a second monomer; wherein: The N-terminus of the first monomer is optionally connected to one domain of the Fab fragment (e.g., the heavy chain CH1 domain thereof) via a linker, and the C-terminus thereof is optionally connected to an IL-15 polypeptide or a functional fragment thereof, or an IL-15Rα polypeptide or a functional fragment thereof via a linker; preferably, the N-terminus of the first monomer is optionally connected to the heavy chain CH1 domain of the Fab fragment via a linker, and the C-terminus thereof is optionally connected to an IL-15 polypeptide or a functional fragment thereof via a linker; The N-terminus of the second monomer is optionally connected to another domain of the Fab fragment (e.g., the light chain CL domain thereof) via a linker, and the C-terminus thereof is optionally connected to an IL-15Rα polypeptide or a functional fragment thereof, or an IL-15 polypeptide or a functional fragment thereof via a linker; preferably, the N-terminus of the second monomer is optionally connected to the light chain CL domain of the Fab fragment via a linker, and the C-terminus thereof is optionally connected to an IL-15Rα polypeptide or a functional fragment thereof via a linker; Preferably, the Fc domain comprises modifications to promote dimerization of the first monomer and the second monomer; Further preferably, the modification comprises a "knob" modification in one of the first monomer and the second monomer and a "hole" modification in the other of the first monomer and the second monomer to form a "knob-into-hole" modification. The fusion protein according to claim 3 or 4, wherein The IL-15 polypeptide comprises the amino acid sequence of a natural IL-15 polypeptide as shown in SEQ ID NO: 63, or comprises an IL-15 variant having an amino acid sequence different from that of the natural IL-15 polypeptide, wherein the IL-15 variant can still maintain the function of the original natural IL-15 polypeptide in promoting effector cell activation; preferably, the amino acid sequence of the IL-15 variant has at least one amino acid substitution, deletion or addition (e.g., 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared with the amino acid sequence of the natural IL-15 polypeptide; and/or, The IL-15Rα polypeptide comprises the amino acid sequence of the natural IL-15Rα polypeptide as shown in SEQ ID NO:64, or comprises an IL-15Rα variant having an amino acid sequence different from that of the natural IL-15Rα polypeptide, wherein the IL-15Rα variant can still maintain the function of the original natural IL-15Rα polypeptide in promoting effector cell activation; preferably, the amino acid sequence of the IL-15Rα variant has at least one amino acid substitution, deletion or addition (for example, 1, 2, 3, 4 or 5 amino acid substitutions, deletions or additions) compared with the amino acid sequence of the natural IL-15Rα polypeptide; further preferably, the IL-15Rα variant comprises the sequence as shown in SEQ ID NO:75. The fusion protein according to any one of claims 3 to 5, wherein the fusion protein comprises: (i) a first peptide chain comprising the VH of the first antigen-binding domain, a heavy chain constant region 1 (CH1), an Fc domain monomer, an IL-15 polypeptide or an IL-15Rα polypeptide, and a single-chain antibody of the second antigen-binding domain; preferably, the CH1 is the human IgG1 heavy chain constant region CH1; preferably, the N-terminus of the Fc domain monomer is linked to the C-terminus of the CH1 via a first hinge region (e.g., a hinge region comprising a PPCP peptide), and the C-terminus of the Fc domain monomer is linked to the N-terminus of the IL-15 polypeptide or IL-15Rα polypeptide via a linker (e.g., a flexible peptide comprising (G4S)n); preferably, the C-terminus of the IL-15 polypeptide or IL-15Rα polypeptide is linked to the N-terminus of the single-chain antibody via a linker (e.g., a flexible peptide comprising (G4S)n); preferably, the single-chain antibody is a scFv; and (ii) a second peptide chain comprising the VL of the first antigen-binding domain, a light chain constant region (CL), an Fc domain monomer, an IL-15 polypeptide or an IL-15Rα polypeptide, and a single-chain antibody of the second antigen-binding domain; preferably, the CL is a human kappa light chain constant region; preferably, the N-terminus of the Fc domain monomer is linked to the C-terminus of the CL via a second hinge region (e.g., a hinge region comprising a PPCP peptide), and the C-terminus of the Fc domain monomer is linked to the N-terminus of the IL-15 polypeptide or IL-15Rα polypeptide via a linker (e.g., a flexible peptide comprising (G4S)n); preferably, the C-terminus of the IL-15 polypeptide or IL-15Rα polypeptide is linked to the N-terminus of the single-chain antibody via a linker (e.g., a flexible peptide comprising (G4S)n); preferably, the single-chain antibody is a scFv; Preferably, the Fc domain monomer of the first peptide chain is capable of forming a dimer with the Fc domain monomer of the second peptide chain; More preferably, the IL-15 polypeptide is located on the first peptide chain; and the IL-15Rα polypeptide is located on the second peptide chain. The fusion protein according to any one of claims 3 to 6, wherein the Fc domain monomer of the first peptide chain can contain modifications with the Fc domain monomer of the second peptide chain to promote dimerization; Preferably, the modification comprises an amino acid substitution in the CH3 domain of the Fc domain; Preferably, the modification comprises a "knob" modification in one of the two Fc domains and a "hole" modification in the other of the two Fc domains to form a "knob-into-hole" modification; Preferably, the Fc domain monomer of the first peptide chain comprises the amino acid sequence shown in SEQ ID NO:71; Preferably, the Fc domain monomer of the second peptide chain comprises the amino acid sequence shown in SEQ ID NO: 72; Preferably, the two Fc domain monomers comprise the amino acid sequences shown in SEQ ID NO: 71 and 72, respectively; Preferably, CH1 in the first peptide chain comprises the amino acid sequence shown in SEQ ID NO: 74, and/or CL in the second peptide chain comprises the amino acid sequence shown in SEQ ID NO: 73; Preferably, the first hinge region comprises the amino acid sequence shown in SEQ ID NO: 65; the second hinge region comprises the amino acid sequence shown in SEQ ID NO: 66; and/or Preferably, the C-terminus of the first peptide chain and/or the second peptide chain connected to the Fc domain monomer via a linker to the N-terminus of the IL-15 polypeptide or IL-15Rα polypeptide comprises the sequence SEQ ID NO: 67; Preferably, the linker connecting the C-terminus of the IL-15 polypeptide or IL-15Rα polypeptide and the N-terminus of the single-chain antibody in the first peptide chain and/or the second peptide chain comprises the sequence SEQ ID NO: 68. The fusion protein according to any one of claims 3 to 7, wherein the first antigen-binding domain targeting TIGIT comprises a VH and a VL selected from any one of the following groups, wherein: (a) a VH comprising the following three CDRs: a CDR-H1 comprising the sequence set forth in SEQ ID NO: 3, a CDR-H2 comprising the sequence set forth in SEQ ID NO: 4, and a CDR-H3 comprising the sequence set forth in SEQ ID NO: 5; and A VL comprising the following three CDRs: a CDR-L1 comprising the sequence set forth in SEQ ID NO:6, a CDR-L2 comprising the sequence set forth in SEQ ID NO:7, and a CDR-L3 comprising the sequence set forth in SEQ ID NO:8; or (b) a VH comprising the following three CDRs: a CDR-H1 comprising the sequence set forth in SEQ ID NO: 11, a CDR-H2 comprising the sequence set forth in SEQ ID NO: 12, and a CDR-H3 comprising the sequence set forth in SEQ ID NO: 13; and A VL comprising the following three CDRs: a CDR-L1 comprising the sequence set forth in SEQ ID NO: 14, a CDR-L2 comprising the sequence set forth in SEQ ID NO: 15, and a CDR-L3 comprising the sequence set forth in SEQ ID NO: 16; or (c) a VH comprising the following three CDRs: a CDR-H1 comprising the sequence set forth in SEQ ID NO: 19, a CDR-H2 comprising the sequence set forth in SEQ ID NO: 20, and a CDR-H3 comprising the sequence set forth in SEQ ID NO: 21; and A VL comprising the following three CDRs: a CDR-L1 comprising the sequence set forth in SEQ ID NO: 22, a CDR-L2 comprising the sequence set forth in SEQ ID NO: 23, and a CDR-L3 comprising the sequence set forth in SEQ ID NO: 24; Preferably, the first antigen binding domain targeting TIGIT comprises a VH and a VL selected from any one of the following groups, wherein: (i-a) VH comprising the sequence shown in SEQ ID NO: 1 and VL comprising the sequence shown in SEQ ID NO: 2; (i-b) VH comprising the sequence shown in SEQ ID NO:9 and VL comprising the sequence shown in SEQ ID NO:10; (i-c) VH comprising the sequence shown in SEQ ID NO:17 and VL comprising the sequence shown in SEQ ID NO:18; (i-d) VH comprising the sequence shown in SEQ ID NO:41 and VL comprising the sequence shown in SEQ ID NO:42; (i-e) VH comprising the sequence shown in SEQ ID NO:43 and VL comprising the sequence shown in SEQ ID NO:44; (i-f) VH comprising the sequence shown in SEQ ID NO:45 and VL comprising the sequence shown in SEQ ID NO:46; (i-g) VH comprising the sequence shown in SEQ ID NO:45 and VL comprising the sequence shown in SEQ ID NO:47; (i-h) VH comprising the sequence shown in SEQ ID NO:48 and VL comprising the sequence shown in SEQ ID NO:49; (i-i) VH comprising the sequence shown in SEQ ID NO:48 and VL comprising the sequence shown in SEQ ID NO:50; Optionally, the VH and VL are compared with the VH and VL in any of groups (i-a) to (i-i), VH has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity; and VL has at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. The fusion protein according to any one of claims 3 to 8, wherein the second antigen-binding domain targeting PD-1 comprises a VH and a VL selected from any one of the following groups: (A) VH comprising the following three CDRs: CDR-H1 comprising the sequence set forth in SEQ ID NO: 27, CDR-H2 comprising the sequence set forth in SEQ ID NO: 28, and CDR-H3 comprising the sequence set forth in SEQ ID NO: 29; and A VL comprising the following three CDRs: CDR-L1 comprising the sequence set forth in SEQ ID NO: 30, CDR-L2 comprising the sequence set forth in SEQ ID NO: 31, and CDR-L3 comprising the sequence set forth in SEQ ID NO: 32; or (B) VH comprising the following three CDRs: CDR-H1 comprising the sequence set forth in SEQ ID NO: 35, CDR-H2 comprising the sequence set forth in SEQ ID NO: 36, and CDR-H3 comprising the sequence set forth in SEQ ID NO: 37; and A VL comprising the following three CDRs: a CDR-L1 comprising the sequence set forth in SEQ ID NO:38, a CDR-L2 comprising the sequence set forth in SEQ ID NO:39, and a CDR-L3 comprising the sequence set forth in SEQ ID NO:40; Preferably, the second antigen-binding domain targeting PD-1 comprises a VH and a VL selected from any one of the following groups, wherein: (ii-a) a VH comprising the sequence shown in SEQ ID NO: 25 and a VL comprising the sequence shown in SEQ ID NO: 26; (ii-b) a VH comprising the sequence shown in SEQ ID NO: 33 and a VL comprising the sequence shown in SEQ ID NO: 34; (ii-c) a VH comprising the sequence shown in SEQ ID NO:54 and a VL comprising the sequence shown in SEQ ID NO:55; (ii-d) VH comprising the sequence shown in SEQ ID NO:56 and VL comprising the sequence shown in SEQ ID NO:57; (ii-e) a VH comprising the sequence shown in SEQ ID NO:58 and a VL comprising the sequence shown in SEQ ID NO:59; (ii-f) VH comprising the sequence shown in SEQ ID NO:56 and VL comprising the sequence shown in SEQ ID NO:59; (ii-g) VH comprising the sequence shown in SEQ ID NO:60 and VL comprising the sequence shown in SEQ ID NO:57; (ii-h) VH comprising the sequence shown in SEQ ID NO:60 and VL comprising the sequence shown in SEQ ID NO:61; (ii-i) VH comprising the sequence shown in SEQ ID NO:62 and VL comprising the sequence shown in SEQ ID NO:61; (ii-j) VH comprising the sequence shown in SEQ ID NO:51 and VL comprising the sequence shown in SEQ ID NO:52; (ii-k) a VH comprising the sequence shown in SEQ ID NO:53 and a VL comprising the sequence shown in SEQ ID NO:52; Optionally, the VH and VL are compared to the VH and VL in any of groups (ii-a) to (ii-k), VH having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity; and VL having at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. The fusion protein according to any one of claims 3 to 9, wherein the fusion protein comprises: (i) a first peptide chain having the structure [VH1]-[CH1]-[hinge region 1]-[Fc monomer 1]-[L1]-[IL-15]-[L1]-[VL2]-[L2]-[VH2], and (ii) a second peptide chain having the structure [VL1]-[CL]-[hinge region 2]-[Fc monomer 2]-[L1]-[IL-15Rα]-[L1]-[VL2]-[L2]-[VH2]; One of the following: (1) The VH1 comprises the sequence shown in SEQ ID NO: 9, the VL1 comprises the sequence shown in SEQ ID NO: 10, the VH2 comprises the sequence shown in SEQ ID NO: 25, the VL2 comprises the sequence shown in SEQ ID NO: 26, the CL comprises the sequence shown in SEQ ID NO: 73, the CH1 comprises the sequence shown in SEQ ID NO: 74, the Fc monomer 1 comprises the sequence shown in SEQ ID NO: 71, the Fc monomer 2 comprises the sequence shown in SEQ ID NO: 72, and the IL-15 comprises the sequence shown in SEQ ID NO: 63. The IL-15Rα comprises the sequence shown in SEQ ID NO: 64; L1 and L2 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and/or one or more serine (e.g., a peptide linker represented by (G4S)n), preferably, L1 is the sequence shown in SEQ ID NO: 67, and L2 is the sequence shown in SEQ ID NO: 68; the hinge region 1 and hinge region 2 are peptide hinge regions, preferably each independently selected from a peptide hinge region comprising PPCP, preferably, hinge region 1 is the sequence shown in SEQ ID NO: 65, and hinge region 2 is the sequence shown in SEQ ID NO: 66; (2) The VH1 comprises the sequence shown in SEQ ID NO: 1, the VL1 comprises the sequence shown in SEQ ID NO: 2, the VH2 comprises the sequence shown in SEQ ID NO: 25, the VL2 comprises the sequence shown in SEQ ID NO: 26, the CL comprises the sequence shown in SEQ ID NO: 73, the CH1 comprises the sequence shown in SEQ ID NO: 74, the Fc monomer 1 comprises the sequence shown in SEQ ID NO: 71, the Fc monomer 2 comprises the sequence shown in SEQ ID NO: 72, and the IL-15 comprises the sequence shown in SEQ ID NO: 63. The IL-15Rα comprises the sequence shown in SEQ ID NO: 64; L1 and L2 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and/or one or more serine (e.g., a peptide linker represented by (G4S)n), preferably, L1 is the sequence shown in SEQ ID NO: 67, and L2 is the sequence shown in SEQ ID NO: 68; the hinge region 1 and hinge region 2 are peptide hinge regions, preferably each independently selected from a peptide hinge region comprising PPCP, preferably, hinge region 1 is the sequence shown in SEQ ID NO: 65, and hinge region 2 is the sequence shown in SEQ ID NO: 66; (3) The VH1 comprises the sequence shown in SEQ ID NO:41, the VL1 comprises the sequence shown in SEQ ID NO:42, the VH2 comprises the sequence shown in SEQ ID NO:54, the VL2 comprises the sequence shown in SEQ ID NO:55, the CL comprises the sequence shown in SEQ ID NO:73, the CH1 comprises the sequence shown in SEQ ID NO:74, the Fc monomer 1 comprises the sequence shown in SEQ ID NO:71, the Fc monomer 2 comprises the sequence shown in SEQ ID NO:72, and the IL-15 comprises the sequence shown in SEQ ID NO:63 The IL-15Rα comprises the sequence shown in SEQ ID NO: 75; L1 and L2 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and/or one or more serine (e.g., a peptide linker represented by (G4S)n), preferably, L1 is the sequence shown in SEQ ID NO: 67, and L2 is the sequence shown in SEQ ID NO: 68; the hinge region 1 and hinge region 2 are peptide hinge regions, preferably each independently selected from a peptide hinge region comprising PPCP, preferably, hinge region 1 is the sequence shown in SEQ ID NO: 65, and hinge region 2 is the sequence shown in SEQ ID NO: 66; (4) The VH1 comprises the sequence shown in SEQ ID NO:41, the VL1 comprises the sequence shown in SEQ ID NO:42, the VH2 comprises the sequence shown in SEQ ID NO:56, the VL2 comprises the sequence shown in SEQ ID NO:57, the CL comprises the sequence shown in SEQ ID NO:73, the CH1 comprises the sequence shown in SEQ ID NO:74, the Fc monomer 1 comprises the sequence shown in SEQ ID NO:71, the Fc monomer 2 comprises the sequence shown in SEQ ID NO:72, and the IL-15 comprises the sequence shown in SEQ ID NO:63 The IL-15Rα comprises the sequence shown in SEQ ID NO: 75; L1 and L2 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and/or one or more serine (e.g., a peptide linker represented by (G4S)n), preferably, L1 is the sequence shown in SEQ ID NO: 67, and L2 is the sequence shown in SEQ ID NO: 68; the hinge region 1 and hinge region 2 are peptide hinge regions, preferably each independently selected from a peptide hinge region comprising PPCP, preferably, hinge region 1 is the sequence shown in SEQ ID NO: 65, and hinge region 2 is the sequence shown in SEQ ID NO: 66; (5) The VH1 comprises the sequence shown in SEQ ID NO:41, the VL1 comprises the sequence shown in SEQ ID NO:42, the VH2 comprises the sequence shown in SEQ ID NO:62, the VL2 comprises the sequence shown in SEQ ID NO:61, the CL comprises the sequence shown in SEQ ID NO:73, the CH1 comprises the sequence shown in SEQ ID NO:74, the Fc monomer 1 comprises the sequence shown in SEQ ID NO:71, the Fc monomer 2 comprises the sequence shown in SEQ ID NO:72, and the IL-15 comprises the sequence shown in SEQ ID NO:63. The IL-15Rα comprises the sequence shown in SEQ ID NO: 75; L1 and L2 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and/or one or more serine (e.g., a peptide linker represented by (G4S)n), preferably, L1 is the sequence shown in SEQ ID NO: 67, and L2 is the sequence shown in SEQ ID NO: 68; the hinge region 1 and hinge region 2 are peptide hinge regions, preferably each independently selected from a peptide hinge region comprising PPCP, preferably, hinge region 1 is the sequence shown in SEQ ID NO: 65, and hinge region 2 is the sequence shown in SEQ ID NO: 66; (6) The VH1 comprises the sequence shown in SEQ ID NO:45, the VL1 comprises the sequence shown in SEQ ID NO:47, the VH2 comprises the sequence shown in SEQ ID NO:51, the VL2 comprises the sequence shown in SEQ ID NO:52, the CL comprises the sequence shown in SEQ ID NO:73, the CH1 comprises the sequence shown in SEQ ID NO:74, the Fc monomer 1 comprises the sequence shown in SEQ ID NO:71, the Fc monomer 2 comprises the sequence shown in SEQ ID NO:72, and the IL-15 comprises the sequence shown in SEQ ID NO:63 The IL-15Rα comprises the sequence shown in SEQ ID NO: 75; L1 and L2 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and/or one or more serine (e.g., a peptide linker represented by (G4S)n), preferably, L1 is the sequence shown in SEQ ID NO: 67, and L2 is the sequence shown in SEQ ID NO: 68; the hinge region 1 and hinge region 2 are peptide hinge regions, preferably each independently selected from a peptide hinge region comprising PPCP, preferably, hinge region 1 is the sequence shown in SEQ ID NO: 65, and hinge region 2 is the sequence shown in SEQ ID NO: 66; (7) The VH1 comprises the sequence shown in SEQ ID NO:45, the VL1 comprises the sequence shown in SEQ ID NO:47, the VH2 comprises the sequence shown in SEQ ID NO:58, the VL2 comprises the sequence shown in SEQ ID NO:59, the CL comprises the sequence shown in SEQ ID NO:73, the CH1 comprises the sequence shown in SEQ ID NO:74, the Fc monomer 1 comprises the sequence shown in SEQ ID NO:71, the Fc monomer 2 comprises the sequence shown in SEQ ID NO:72, and the IL-15 comprises the sequence shown in SEQ ID NO:63. The IL-15Rα comprises the sequence shown in SEQ ID NO: 75; L1 and L2 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and/or one or more serine (e.g., a peptide linker represented by (G4S)n), preferably, L1 is the sequence shown in SEQ ID NO: 67, and L2 is the sequence shown in SEQ ID NO: 68; the hinge region 1 and hinge region 2 are peptide hinge regions, preferably each independently selected from a peptide hinge region comprising PPCP, preferably, hinge region 1 is the sequence shown in SEQ ID NO: 65, and hinge region 2 is the sequence shown in SEQ ID NO: 66; (8) The VH1 comprises the sequence shown in SEQ ID NO:45, the VL1 comprises the sequence shown in SEQ ID NO:47, the VH2 comprises the sequence shown in SEQ ID NO:56, the VL2 comprises the sequence shown in SEQ ID NO:57, the CL comprises the sequence shown in SEQ ID NO:73, the CH1 comprises the sequence shown in SEQ ID NO:74, the Fc monomer 1 comprises the sequence shown in SEQ ID NO:71, the Fc monomer 2 comprises the sequence shown in SEQ ID NO:72, and the IL-15 comprises the sequence shown in SEQ ID NO:63. The IL-15Rα comprises the sequence shown in SEQ ID NO: 75; L1 and L2 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and/or one or more serine (e.g., a peptide linker represented by (G4S)n), preferably, L1 is the sequence shown in SEQ ID NO: 67, and L2 is the sequence shown in SEQ ID NO: 68; the hinge region 1 and hinge region 2 are peptide hinge regions, preferably each independently selected from a peptide hinge region comprising PPCP, preferably, hinge region 1 is the sequence shown in SEQ ID NO: 65, and hinge region 2 is the sequence shown in SEQ ID NO: 66; (9) The VH1 comprises the sequence shown in SEQ ID NO:45, the VL1 comprises the sequence shown in SEQ ID NO:47, the VH2 comprises the sequence shown in SEQ ID NO:56, the VL2 comprises the sequence shown in SEQ ID NO:59, the CL comprises the sequence shown in SEQ ID NO:73, the CH1 comprises the sequence shown in SEQ ID NO:74, the Fc monomer 1 comprises the sequence shown in SEQ ID NO:71, the Fc monomer 2 comprises the sequence shown in SEQ ID NO:72, and the IL-15 comprises the sequence shown in SEQ ID NO:63. The IL-15Rα comprises the sequence shown in SEQ ID NO: 75; L1 and L2 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and/or one or more serine (e.g., a peptide linker represented by (G4S)n), preferably, L1 is the sequence shown in SEQ ID NO: 67, and L2 is the sequence shown in SEQ ID NO: 68; the hinge region 1 and hinge region 2 are peptide hinge regions, preferably each independently selected from a peptide hinge region comprising PPCP, preferably, hinge region 1 is the sequence shown in SEQ ID NO: 65, and hinge region 2 is the sequence shown in SEQ ID NO: 66; (10) The VH1 comprises the sequence shown in SEQ ID NO:45, the VL1 comprises the sequence shown in SEQ ID NO:46, the VH2 comprises the sequence shown in SEQ ID NO:60, the VL2 comprises the sequence shown in SEQ ID NO:57, the CL comprises the sequence shown in SEQ ID NO:73, the CH1 comprises the sequence shown in SEQ ID NO:74, the Fc monomer 1 comprises the sequence shown in SEQ ID NO:71, the Fc monomer 2 comprises the sequence shown in SEQ ID NO:72, and the IL-15 comprises the sequence shown in SEQ ID NO:63. The IL-15Rα comprises the sequence shown in SEQ ID NO: 75; L1 and L2 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and/or one or more serine (e.g., a peptide linker represented by (G4S)n), preferably, L1 is the sequence shown in SEQ ID NO: 67, and L2 is the sequence shown in SEQ ID NO: 68; the hinge region 1 and hinge region 2 are peptide hinge regions, preferably each independently selected from a peptide hinge region comprising PPCP, preferably, hinge region 1 is the sequence shown in SEQ ID NO: 65, and hinge region 2 is the sequence shown in SEQ ID NO: 66; (11) The VH1 comprises the sequence shown in SEQ ID NO:45, the VL1 comprises the sequence shown in SEQ ID NO:47, the VH2 comprises the sequence shown in SEQ ID NO:62, the VL2 comprises the sequence shown in SEQ ID NO:61, the CL comprises the sequence shown in SEQ ID NO:73, the CH1 comprises the sequence shown in SEQ ID NO:74, the Fc monomer 1 comprises the sequence shown in SEQ ID NO:71, the Fc monomer 2 comprises the sequence shown in SEQ ID NO:72, and the IL-15 comprises the sequence shown in SEQ ID NO:63. The IL-15Rα comprises the sequence shown in SEQ ID NO: 75; L1 and L2 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and/or one or more serine (e.g., a peptide linker represented by (G4S)n), preferably, L1 is the sequence shown in SEQ ID NO: 67, and L2 is the sequence shown in SEQ ID NO: 68; the hinge region 1 and hinge region 2 are peptide hinge regions, preferably each independently selected from a peptide hinge region comprising PPCP, preferably, hinge region 1 is the sequence shown in SEQ ID NO: 65, and hinge region 2 is the sequence shown in SEQ ID NO: 66; (12) The VH1 comprises the sequence shown in SEQ ID NO:48, the VL1 comprises the sequence shown in SEQ ID NO:50, the VH2 comprises the sequence shown in SEQ ID NO:60, the VL2 comprises the sequence shown in SEQ ID NO:57, the CL comprises the sequence shown in SEQ ID NO:73, the CH1 comprises the sequence shown in SEQ ID NO:74, the Fc monomer 1 comprises the sequence shown in SEQ ID NO:71, the Fc monomer 2 comprises the sequence shown in SEQ ID NO:72, and the IL-15 comprises the sequence shown in SEQ ID NO:63. The IL-15Rα comprises the sequence shown in SEQ ID NO: 75; L1 and L2 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and/or one or more serine (e.g., a peptide linker represented by (G4S)n), preferably, L1 is the sequence shown in SEQ ID NO: 67, and L2 is the sequence shown in SEQ ID NO: 68; the hinge region 1 and hinge region 2 are peptide hinge regions, preferably each independently selected from a peptide hinge region comprising PPCP, preferably, hinge region 1 is the sequence shown in SEQ ID NO: 65, and hinge region 2 is the sequence shown in SEQ ID NO: 66; (13) The VH1 comprises the sequence shown in SEQ ID NO:48, the VL1 comprises the sequence shown in SEQ ID NO:50, the VH2 comprises the sequence shown in SEQ ID NO:51, the VL2 comprises the sequence shown in SEQ ID NO:52, the CL comprises the sequence shown in SEQ ID NO:73, the CH1 comprises the sequence shown in SEQ ID NO:74, the Fc monomer 1 comprises the sequence shown in SEQ ID NO:71, the Fc monomer 2 comprises the sequence shown in SEQ ID NO:72, and the IL-15 comprises the sequence shown in SEQ ID NO:63 The IL-15Rα comprises the sequence shown in SEQ ID NO: 75; L1 and L2 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and/or one or more serine (e.g., a peptide linker represented by (G4S)n), preferably, L1 is the sequence shown in SEQ ID NO: 67, and L2 is the sequence shown in SEQ ID NO: 68; the hinge region 1 and hinge region 2 are peptide hinge regions, preferably each independently selected from a peptide hinge region comprising PPCP, preferably, hinge region 1 is the sequence shown in SEQ ID NO: 65, and hinge region 2 is the sequence shown in SEQ ID NO: 66; (14) The VH1 comprises the sequence shown in SEQ ID NO:48, the VL1 comprises the sequence shown in SEQ ID NO:49, the VH2 comprises the sequence shown in SEQ ID NO:60, the VL2 comprises the sequence shown in SEQ ID NO:61, the CL comprises the sequence shown in SEQ ID NO:73, the CH1 comprises the sequence shown in SEQ ID NO:74, the Fc monomer 1 comprises the sequence shown in SEQ ID NO:71, the Fc monomer 2 comprises the sequence shown in SEQ ID NO:72, and the IL-15 comprises the sequence shown in SEQ ID NO:63. The IL-15Rα comprises the sequence shown in SEQ ID NO: 75; L1 and L2 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and/or one or more serine (e.g., a peptide linker represented by (G4S)n), preferably, L1 is the sequence shown in SEQ ID NO: 67, and L2 is the sequence shown in SEQ ID NO: 68; the hinge region 1 and hinge region 2 are peptide hinge regions, preferably each independently selected from a peptide hinge region comprising PPCP, preferably, hinge region 1 is the sequence shown in SEQ ID NO: 65, and hinge region 2 is the sequence shown in SEQ ID NO: 66; (15) The VH1 comprises the sequence shown in SEQ ID NO:48, the VL1 comprises the sequence shown in SEQ ID NO:49, the VH2 comprises the sequence shown in SEQ ID NO:53, the VL2 comprises the sequence shown in SEQ ID NO:52, the CL comprises the sequence shown in SEQ ID NO:73, the CH1 comprises the sequence shown in SEQ ID NO:74, the Fc monomer 1 comprises the sequence shown in SEQ ID NO:71, the Fc monomer 2 comprises the sequence shown in SEQ ID NO:72, and the IL-15 comprises the sequence shown in SEQ ID NO:63. The IL-15Rα comprises the sequence shown in SEQ ID NO: 75; L1 and L2 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and/or one or more serine (e.g., a peptide linker represented by (G4S)n), preferably, L1 is the sequence shown in SEQ ID NO: 67, and L2 is the sequence shown in SEQ ID NO: 68; the hinge region 1 and hinge region 2 are peptide hinge regions, preferably each independently selected from a peptide hinge region comprising PPCP, preferably, hinge region 1 is the sequence shown in SEQ ID NO: 65, and hinge region 2 is the sequence shown in SEQ ID NO: 66; (16) The VH1 comprises the sequence shown in SEQ ID NO:43, the VL1 comprises the sequence shown in SEQ ID NO:44, the VH2 comprises the sequence shown in SEQ ID NO:60, the VL2 comprises the sequence shown in SEQ ID NO:57, the CL comprises the sequence shown in SEQ ID NO:73, the CH1 comprises the sequence shown in SEQ ID NO:74, the Fc monomer 1 comprises the sequence shown in SEQ ID NO:71, the Fc monomer 2 comprises the sequence shown in SEQ ID NO:72, and the IL-15 comprises the sequence shown in SEQ ID NO:63. The IL-15Rα comprises the sequence shown in SEQ ID NO: 75; L1 and L2 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and/or one or more serine (e.g., a peptide linker represented by (G4S)n), preferably, L1 is the sequence shown in SEQ ID NO: 67, and L2 is the sequence shown in SEQ ID NO: 68; the hinge region 1 and hinge region 2 are peptide hinge regions, preferably each independently selected from a peptide hinge region comprising PPCP, preferably, hinge region 1 is the sequence shown in SEQ ID NO: 65, and hinge region 2 is the sequence shown in SEQ ID NO: 66; (17) The VH1 comprises the sequence shown in SEQ ID NO:43, the VL1 comprises the sequence shown in SEQ ID NO:44, the VH2 comprises the sequence shown in SEQ ID NO:60, the VL2 comprises the sequence shown in SEQ ID NO:61, the CL comprises the sequence shown in SEQ ID NO:73, the CH1 comprises the sequence shown in SEQ ID NO:74, the Fc monomer 1 comprises the sequence shown in SEQ ID NO:71, the Fc monomer 2 comprises the sequence shown in SEQ ID NO:72, and the IL-15 comprises the sequence shown in SEQ ID NO:63. The IL-15Rα comprises the sequence shown in SEQ ID NO: 75; L1 and L2 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycine and/or one or more serine (e.g., a peptide linker represented by (G4S)n), preferably, L1 is the sequence shown in SEQ ID NO: 67, and L2 is the sequence shown in SEQ ID NO: 68; the hinge region 1 and hinge region 2 are peptide hinge regions, preferably each independently selected from a peptide hinge region comprising PPCP, preferably, hinge region 1 is the sequence shown in SEQ ID NO: 65, and hinge region 2 is the sequence shown in SEQ ID NO: 66; (18) The VH1 comprises the sequence shown in SEQ ID NO:45, the VL1 comprises the sequence shown in SEQ ID NO:46, the VH2 comprises the sequence shown in SEQ ID NO:53, the VL2 comprises the sequence shown in SEQ ID NO:52, the CL comprises the sequence shown in SEQ ID NO:73, the CH1 comprises the sequence shown in SEQ ID NO:74, the Fc monomer 1 comprises the sequence shown in SEQ ID NO:71, the Fc monomer 2 comprises the sequence shown in SEQ ID NO:72, and the IL-15 comprises the sequence shown in SEQ ID NO:63. The IL-15Rα comprises the sequence shown in SEQ ID NO:75; the L1 and L2 are peptide linkers, preferably each independently selected from a peptide linker comprising one or more glycines and/or one or more serines (for example, a peptide linker shown in (G4S)n), preferably, L1 is the sequence shown in SEQ ID NO:67, and L2 is the sequence shown in SEQ ID NO:68; the hinge region 1 and hinge region 2 are peptide hinge regions, preferably each independently selected from a peptide hinge region comprising PPCP, preferably, hinge region 1 is the sequence shown in SEQ ID NO:65, and hinge region 2 is the sequence shown in SEQ ID NO:66. An isolated nucleic acid molecule encoding the fusion protein according to any one of claims 1 to 10. A vector comprising the isolated nucleic acid molecule of claim 11; Preferably, the vector comprises a nucleotide sequence encoding each peptide chain of the fusion protein, and the nucleotide sequence encoding each peptide chain exists on the same or different vectors. A host cell comprising the isolated nucleic acid molecule of claim 11 or the vector of claim 12. A method for preparing a fusion protein, comprising culturing the host cell according to claim 13 under conditions allowing protein expression, and collecting the fusion protein from a culture of the cultured host cell. A conjugate comprising the fusion protein according to any one of claims 1 to 10, or the isolated nucleic acid molecule according to claim 11, and a coupling moiety linked thereto; Preferably, the coupling portion is selected from a detectable label (such as a radioisotope, a fluorescent substance, a luminescent substance, a colored substance or an enzyme) or a therapeutic agent (such as a cytotoxic agent, a cytokine, a toxin, a radionuclide, an immune agonist, an immunosuppressant, and other active substances that inhibit tumor cell growth, promote tumor cell apoptosis or necrosis). A pharmaceutical composition comprising the fusion protein of any one of claims 1 to 10, the isolated nucleic acid molecule of claim 11, the vector of claim 12, the host cell of claim 13, or the conjugate of claim 15, and a pharmaceutically acceptable carrier and/or excipient. A kit comprising the fusion protein of any one of claims 1 to 10, the isolated nucleic acid molecule of claim 11, the vector of claim 12, the host cell of claim 13, the conjugate of claim 15, or the pharmaceutical composition of claim 16. Use of the fusion protein according to any one of claims 1 to 10, the isolated nucleic acid molecule according to claim 11, the vector according to claim 12, the host cell according to claim 13, the conjugate according to claim 15, or the pharmaceutical composition according to claim 16 in the preparation of a medicament for the prevention and/or treatment and/or neoadjuvant treatment and/or adjuvant treatment of a disease. A method for preventing and/or treating and/or neoadjuvant treating and/or adjuvant treating a disease in a subject, comprising administering to a subject in need thereof an effective amount of the fusion protein according to any one of claims 1 to 10, the isolated nucleic acid molecule according to claim 11, the vector according to claim 12, the host cell according to claim 13, the conjugate according to claim 15, or the pharmaceutical composition according to claim 16. The use according to claim 18 or the method according to claim 19, wherein the disease is a tumor; Preferably, the tumor is selected from a solid tumor or a blood tumor; More preferably, the solid tumor is selected from melanoma, lung cancer, kidney cancer, bladder cancer, head and neck cancer, gastric cancer, esophageal cancer, liver cancer, cervical cancer, breast cancer or skin cancer. The fusion protein according to any one of claims 1 to 10, the isolated nucleic acid molecule according to claim 11, the vector according to claim 12, the host cell according to claim 13, the conjugate according to claim 15, or the pharmaceutical composition according to claim 16, for use in the prevention and/or treatment and/or neoadjuvant treatment and/or adjuvant treatment of a disease in a subject; Preferably, the disease is a tumor; More preferably, the tumor is selected from solid tumors or hematological tumors; Even more preferably, the solid tumor is selected from melanoma, lung cancer, kidney cancer, bladder cancer, head and neck cancer, gastric cancer, esophageal cancer, liver cancer, cervical cancer, breast cancer or skin cancer.