CD80 / TAA fusion protein associated tumor targeting, t cell activation molecule form and use thereof

Abstract

The present invention relates to CD80 / TAA fusion protein associated tumor targeting, a T cell activation molecule form and a use thereof. Specifically, a multispecific antibody fusion protein of the present invention targets a tumor-associated antigen (TAA), targets activated T cells, and optionally targets PD1 / PD-L1, further inhibits a T cell suppression function, and can provide a co-stimulatory signal for T cells, thereby stimulating stronger and more effective killing activity.

Description

CD80 / TAA fusion protein-related tumor targeting, T cell activation molecular forms and their applications Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to CD80 / TAA fusion protein-related tumor targeting, T cell activation molecular forms and applications thereof. Background Art

[0002] T cell activation requires the participation of a dual signal system: (1) After antigen presenting cells (APCs) recognize and process antigen molecules, MHC-antigen peptide complexes are formed on their surface and the antigen information is presented to the T cell receptor (TCR). This is the first signal of T cell activation; (2) co-stimulatory signal, which is the second signal of T cell activation. The co-stimulatory signal is amplified by the interaction between co-stimulatory molecules on the surface of antigen presenting cells (APCs) and ligands on the surface of T cells. The co-stimulatory signal fully activates T cells. Without the co-stimulatory signal, T cells cannot be activated and lose their function.

[0003] Co-stimulation helps the immune system determine which antigenic stimuli are worthy of a response. Currently, the most studied co-stimulatory molecules are the B7 family proteins CD80 (B7-1) and CD86 (B7-2), which are expressed on the surface of antigen-presenting cells (APCs).

[0004] PD-1 / PD-L1 immune checkpoint inhibitors have made great progress in the treatment of tumors. Currently, there are many PD-1 / PD-L1 immune checkpoint inhibitors, which are mainly used to treat melanoma, non-small cell tumors, etc. However, existing clinical studies have shown that PD-1 / L1 inhibitors also have significant limitations in their effects on immune cell activation, such as a small number of responsive patients, recurrence and drug resistance. Clinical studies have shown that only about 25% of melanoma patients can ensure long-term tumor control (more than 21 months) after using PD-1 inhibitors. Based on the current limitations of PD-1 / L1 inhibitors, the combination of PD-1 / L1 inhibitors and other therapies has become another important development direction in the industry, thereby enhancing the treatment effect in a targeted manner.

[0005] Immune checkpoint inhibitors only benefit a subset of patients, with most cancer patients experiencing low response rates and developing drug resistance. This may be due to a lack of sufficient T cell co-stimulation within the tumor microenvironment, such as insufficient CD28 ligands. CD28 agonists are crucial signals for T cell activation, and their successful development would significantly expand the boundaries of cancer immunotherapy.

[0006] The research and development of CD28 agonists faces great challenges. In 2006, the Phase I clinical trial of an agonist antibody targeting CD28 (TNG1412) was terminated because all volunteers who received treatment experienced severe adverse reactions.

[0007] In addition to serving as a secondary signal for T cell activation, recent studies have also revealed that activation of the CD28 pathway is also of significant significance for immunotherapy targeting PD-1 / -L1. Compared to the TCR, the PD-1 inhibitory signal is more likely to target the T cell co-stimulatory receptor CD28, reducing T cell activity. Furthermore, after blocking the PD-1 signal, T cell recovery still relies on activation of the CD28 / B7 signaling pathway. Daisuke Sugiura et al. also revealed that CD80 enhances T cell activation not only by mediating CD28 co-stimulatory signals but also by attenuating PD-1-driven co-inhibitory signals.

[0008] Therefore, there is an urgent need in the art to develop a class of fusion proteins or multispecific antibodies that can address the clinical safety issues of CD28-targeted agonists. Summary of the Invention

[0009] The purpose of the present invention is to design, through genetic engineering, a fusion protein containing a CD80 recombinant protein, a tumor-targeting anti-TAA antibody, and optionally a PD-1 / L1 inhibitor, or a fusion protein containing a CD80 recombinant protein and a tumor-targeting anti-TAA antibody, thereby effectively addressing the clinical safety of CD28 agonists.

[0010] The CD80 / TAA-related fusion protein molecules designed in this invention can only activate the CD28 signaling pathway by binding to tumor TAAs, ensuring the clinical safety of CD28 agonists. The molecular mechanism of tumor killing is to enrich the candidate drug in the tumor through anti-TAA antibodies. The CD80 recombinant protein can bind to CD28 / CTLA4 respectively, activating the CD28 signaling pathway or de-inhibiting the CTLA4 signaling pathway through multiple mechanisms to activate T cells and kill and eliminate tumor cells through various immune effector mechanisms.

[0011] In a first aspect of the present invention, a fusion protein is provided, comprising the following elements:

[0012] (a) a first targeting domain D1, wherein D1 binds to the first target protein CD28;

[0013] (b) a second targeting domain D2, said D2 binding to a second target protein, said second target protein being a tumor-associated antigen (TAA); and

[0014] Optional (c) a third targeting domain D3, wherein D3 binds to a third target protein, wherein the third target protein is PD1 and / or PD-L1.

[0015] In another preferred embodiment, the first targeting domain D1 is the CD80 extracellular domain (ECD).

[0016] In another preferred embodiment, the first targeting domain D1 also binds to PD-L1 and / or CTLA-4.

[0017] In another preferred example, the second targeting domain D2 is an antibody or an active fragment thereof.

[0018] In another preferred embodiment, the third targeting domain D3 is an antibody or an active fragment thereof.

[0019] In another preferred embodiment, the first targeting domain D1, the second targeting domain D2 and the third targeting domain D3 are fused together by a connection method selected from the following group: direct connection by peptide bonds, connection via connecting fragments, non-covalent fusion through intermolecular interactions, covalent fusion through disulfide bonds or chemical cross-linking, or a combination thereof.

[0020] In another preferred embodiment, the first targeting domain D1 and the second targeting domain D2 are fused together by a connection method selected from the group consisting of direct connection via a peptide bond, connection via a connecting fragment, or a combination thereof.

[0021] In another preferred embodiment, the second targeting domain D2 and the third targeting domain D3 are fused together by a connection method selected from the group consisting of direct connection via a peptide bond, connection via a connecting fragment, or a combination thereof.

[0022] In another preferred embodiment, the first targeting domain D1 and the third targeting domain D3 are fused together by a connection method selected from the group consisting of direct connection via a peptide bond, connection via a connecting fragment, or a combination thereof.

[0023] In another preferred embodiment, the antibody or active fragment thereof is selected from the following group: complete antibody, nanobody (VHH), single chain antibody (scFv), Fab antibody, or a combination thereof.

[0024] In another preferred embodiment, the tumor-associated antigen TAA is selected from the following group: CD33, CD30, HER2, CD22, CD79b, Nectin-4, BCMA, EGFR, CD19, tissue factor, folr1 (folate receptor α), CLDN18.2, TROP2, c-Met, PSMA, Muc1, PDL1, ROR1, MSLN, TNF-α, CD25, ENPP3, Axl, CD20, ROR2, GPNMB, CEACAM6, CD13 8. CA6, FUT3, CD56, CD37, HER3, GPRC5D, STING, CEA, CD205, B7H4, CTLA4, RNF43, CDH3, DPEP3, 5T4, ITGB6, EFNA4, B7H3, CD228, Notch-3, CD46, CAIX, SLAMF6, ADAM9, GD3, TDGF1, SLAMF2, CLL-1, CD123, IGF-1R, FCRL5, TIM1, Globo H, CDH6, CD38, Ly6E, SLITRK6, GPR20, FGFR2, Muc16, CD51, SLAMF7, LAMP-1, CD74, CCR7, PTK7, SEZ6, CLDN 9. CLDN6, c-kit, LYPD3, TAA, PRL receptor, FGFR3, KAAG1, STEAP1, Flt3, LRRC15, CD44, CD70, EphA2, PDL2, p53, DLK1, ENB-FN, FOLR, CD45, DSG2, ALK, TRAIL, DDR1, EpCAM, VEGFR2, CD47, CD99, VEGF2, SSEA-4, DCLK1, OAcGD2, IL1RAP, ADAM17, CD7, CD73, ENO1, BSG, CD24, GLUT1, CXCR4, CD40, CD52, CD133, CD239, TAG72, EGFR VIII, PSCA, EphA2, NKG2D ligand, MCSP, LGR5, SSEA3, SLC34A2, Glypican-3, or a combination thereof.

[0025] In another preferred example, the second targeting domain D2 is an anti-TAA antibody element.

[0026] In another preferred embodiment, the anti-TAA antibody element specifically binds to TAA.

[0027] In another preferred embodiment, the multi-specific fusion protein includes a homodimer or a heterodimer.

[0028] In another preferred embodiment, two or three monomers of the homodimer or heterodimer form a dimer through a disulfide bond.

[0029] In another preferred embodiment, the multi-specific fusion protein is a homodimer and comprises the following parts:

[0030] (a) CD80 extracellular domain (ECD);

[0031] (b) anti-TAA antibody element;

[0032] (c) FC segment; and

[0033] (d) Anti-PD1 / PD-L1 antibody fragments.

[0034] In another preferred embodiment, the multi-specific fusion protein is a homodimer and comprises the following parts:

[0035] (a) CD80 extracellular domain (ECD);

[0036] (b) anti-PD1 / PD-L1 antibody fragments;

[0037] (c) FC segment; and

[0038] (d) Anti-TAA antibody element.

[0039] In another preferred embodiment, the multi-specific fusion protein is a homodimer and comprises the following parts:

[0040] (a) CD80 extracellular domain (ECD);

[0041] (b) an anti-TAA antibody element; and

[0042] (c) FC segment.

[0043] In another preferred embodiment, the multi-specific fusion protein is a homodimer and comprises the following parts:

[0044] (a) CD80 extracellular domain (ECD);

[0045] (b) FC segment; and

[0046] (c) Anti-TAA antibody element.

[0047] In another preferred embodiment, the multi-specific fusion protein is a heterodimer and includes a first monomer, a second monomer and a third monomer;

[0048] Wherein, the first monomer comprises: (a) CD80 extracellular domain (ECD); (b) first FC segment; (c) anti-PD1 / PD-L1 antibody fragment;

[0049] The second monomer includes: (a) an anti-TAA antibody heavy chain fragment (VH-CH1); (b) a second FC segment; (c) an anti-PD1 / PD-L1 antibody fragment;

[0050] The third monomer includes: an anti-TAA antibody light chain (VL-CL) fragment.

[0051] In another preferred embodiment, the multi-specific fusion protein is a heterodimer and includes a first monomer and a second monomer;

[0052] Wherein, the first monomer comprises: (a) CD80 extracellular domain (ECD); (b) first FC segment; (c) anti-PD1 / PD-L1 antibody fragment;

[0053] The second monomer includes: (a) an anti-TAA nanobody (VHH) or scFv antibody fragment; (b) a second FC segment; and (c) an anti-PD1 / PD-L1 antibody fragment.

[0054] In another preferred embodiment, the multi-specific fusion protein is a heterodimer and includes a first monomer and a second monomer;

[0055] Wherein, the first monomer comprises: (a) CD80 extracellular domain; (b) anti-TAA antibody element; (c) first FC segment;

[0056] The second monomer includes: (a) an anti-PD1 / PD-L1 antibody fragment; (b) an anti-TAA antibody element; and (c) a second FC segment.

[0057] In another preferred embodiment, the multi-specific fusion protein is a heterodimer and includes a first monomer and a second monomer;

[0058] Wherein, the first monomer comprises: (a) CD80 extracellular domain; (b) first FC segment; (c) anti-TAA antibody element;

[0059] The second monomer includes: (a) an anti-PD1 / PD-L1 antibody fragment; (b) a second FC segment; and (c) an anti-TAA antibody element.

[0060] In another preferred example, the anti-TAA antibody element includes an antibody selected from the following group: anti-TAA antibody heavy chain (VH-CH1), anti-TAA antibody light chain (VL-CL), anti-TAA nanobody (VHH), anti-TAA single-chain antibody (scFv), and anti-TAA Fab antibody.

[0061] In another preferred embodiment, the multi-specific fusion protein is a heterodimer and includes a first monomer and a second monomer;

[0062] Wherein, the first monomer comprises: (a) CD80 extracellular domain; (b) anti-TAA antibody element; (c) first FC segment;

[0063] The second monomer includes: (a) an anti-TAA antibody element; and (b) a second FC segment.

[0064] In another preferred embodiment, the multi-specific fusion protein is a heterodimer and includes a first monomer and a second monomer;

[0065] Wherein, the first monomer comprises: (a) CD80 extracellular domain; (b) first FC segment; (c) anti-TAA antibody element;

[0066] The second monomer includes: (a) a second FC segment; and (b) an anti-TAA antibody element.

[0067] In another preferred embodiment, the multi-specific fusion protein is a heterodimer and includes a first monomer and a second monomer;

[0068] Wherein, the first monomer comprises: (a) CD80 extracellular domain; (b) anti-TAA antibody element; (c) first FC segment;

[0069] The second monomer includes: (a) a second FC segment.

[0070] In another preferred embodiment, the CD80 extracellular domain includes a structure or fragment selected from the following groups: an IgV domain or a specific binding fragment thereof, an IgC domain or a specific binding fragment thereof, or a combination thereof.

[0071] In another preferred example, the CD80 extracellular domain specifically binds to the extracellular domain of a protein selected from the group consisting of CD28, PD-L1, CTLA-4, or a combination thereof.

[0072] In another preferred embodiment, the amino acid sequence of the CD80 extracellular domain is as shown in SEQ ID NO: 44, or has at least 90% sequence identity thereto.

[0073] In another preferred embodiment, the anti-PD1 / PD-L1 antibody fragment specifically binds to PD1 or PD-L1.

[0074] In another preferred embodiment, the PD1 / PD-L1 antibody fragment includes an antibody selected from the following group: anti-PD1 / PD-L1 nanobody (VHH), anti-PD1 / PD-L1 single-chain antibody (scFv), and anti-PD1 / PD-L1 Fab antibody.

[0075] In another preferred embodiment, the anti-PD1 / PD-L1 antibodies include: anti-PD1 antibodies, anti-PD-L1 antibodies, and PD-1 fusion molecules.

[0076] In another preferred embodiment, the anti-PD1 antibodies include: nivolumab, pilizumab, peliguzumab, and CS1003.

[0077] In another preferred embodiment, the anti-PD-L1 antibodies include: BMS-936559, MPDL3280A, MEDI4736, and MSB0010718C.

[0078] In another preferred embodiment, the PD-1 fusion molecule includes AMP224 (eg, polypeptide (AUR-012)).

[0079] In another preferred example, the amino acid sequence of the anti-PD1 / PD-L1 antibody is as shown in a sequence selected from the following group or a sequence having at least 90% sequence identity therewith: SEQ ID NO: 2, SEQ ID NO: 3.

[0080] In another preferred embodiment, the FC segment is selected from the group consisting of human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, human IgG4 Fc, or variants thereof.

[0081] In another preferred embodiment, the FC segment is selected from the following group: human IgG1 Fc, human IgG4 Fc, or variants thereof.

[0082] In another preferred example, the first FC segment and the second FC segment are aggregated to form a FC segment.

[0083] In another preferred embodiment, the FC segment is an FC heterodimer.

[0084] In another preferred embodiment, the FC segment may be selected as a mutant to eliminate the affinity for Fcγ receptors and C1q complement proteins, thereby weakening or eliminating the immune effector function.

[0085] In another preferred embodiment, the multi-specific fusion protein is a bispecific or trispecific fusion protein.

[0086] In another preferred embodiment, the multi-specific fusion protein is a bi-specific fusion protein.

[0087] In another preferred embodiment, the tri-specific fusion protein comprises:

[0088] A first targeting domain, wherein the first targeting domain is a CD28 antigen binding domain;

[0089] a second targeting domain, which is a TAA antigen binding domain; and

[0090] The third targeting domain is a PD1 / PD-L1 antigen binding domain.

[0091] In another preferred embodiment, the trispecific fusion protein is a trispecific antibody.

[0092] In another preferred embodiment, the structure of the trispecific antibody is symmetrical or asymmetrical.

[0093] In another preferred embodiment, the trispecific antibody is a bivalent, trivalent, tetravalent or multivalent molecule.

[0094] In another preferred embodiment, the trispecific antibody is a trivalent trispecific antibody with an asymmetric structure.

[0095] In another preferred embodiment, the multi-specific fusion protein is obtained by fusion of amino acid fragments selected from the following group:

[0096] (1) SEQ ID NO: 1; SEQ ID NO: 77;

[0097] (2) SEQ ID NO:4, SEQ ID NO:5; SEQ ID NO:78, SEQ ID NO:79;

[0098] (3) SEQ ID NO:4, SEQ ID NO:6; SEQ ID NO:78, SEQ ID NO:80;

[0099] (4) SEQ ID NO:7; SEQ ID NO:81;

[0100] (5) SEQ ID NO:8, SEQ ID NO:9; SEQ ID NO:82, SEQ ID NO:83;

[0101] (6) SEQ ID NO:8, SEQ ID NO:10; SEQ ID NO:82, SEQ ID NO:84;

[0102] (7) SEQ ID NO: 11, SEQ ID NO: 12;

[0103] (8) SEQ ID NO:11, SEQ ID NO:15;

[0104] (9) SEQ ID NO:4, SEQ ID NO:16;

[0105] (10) SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20; EQ ID NO: 18, SEQ ID NO: 85, SEQ ID NO: 86;

[0106] (11) SEQ ID NO: 21;

[0107] (12) SEQ ID NO: 22;

[0108] (13) SEQ ID NO: 4, SEQ ID NO: 26, SEQ ID NO: 27; SEQ ID NO: 78, SEQ ID NO: 26, SEQ ID NO: 27;

[0109] (14) SEQ ID NO: 4, SEQ ID NO: 29, SEQ ID NO: 30; SEQ ID NO: 78, SEQ ID NO: 29, SEQ ID NO: 30;

[0110] (15) SEQ ID NO: 4, SEQ ID NO: 31, SEQ ID NO: 32; SEQ ID NO: 78, SEQ ID NO: 31, SEQ ID NO: 32;

[0111] (16) SEQ ID NO:38, SEQ ID NO:20; SEQ ID NO:38, SEQ ID NO:86;

[0112] (17)SEQ ID NO:39.

[0113] In the second aspect of the present invention, a polynucleotide is provided, which encodes the fusion protein as described in the first aspect of the present invention.

[0114] In the third aspect of the present invention, a vector is provided, wherein the vector contains the polynucleotide according to the second aspect of the present invention.

[0115] In another preferred embodiment, the vector includes plasmid, phage, yeast plasmid, plant cell virus, mammalian cell virus such as adenovirus, retrovirus, or other vectors.

[0116] In a fourth aspect of the present invention, a host cell is provided, wherein the host cell comprises the vector according to the third aspect of the present invention or the polynucleotide according to the second aspect of the present invention is integrated into its genome. In another preferred embodiment, the host cell is a eukaryotic cell, preferably a mammalian cell.

[0117] In a fifth aspect of the present invention, a method for preparing the multi-specific fusion protein according to the first aspect of the present invention is provided, comprising the steps of:

[0118] (i) culturing the host cell according to the fourth aspect of the present invention under appropriate conditions to obtain a mixture containing the multi-specific fusion protein according to the first aspect of the present invention;

[0119] (ii) purifying and / or separating the mixture obtained in step (i) to obtain the multi-specific fusion protein according to the first aspect of the present invention.

[0120] In a sixth aspect of the present invention, a pharmaceutical composition is provided, comprising:

[0121] (I) the multi-specific fusion protein according to the first aspect of the present invention; and

[0122] (II) a pharmaceutically acceptable carrier.

[0123] In a seventh aspect of the present invention, an immunoconjugate is provided, comprising:

[0124] (a) the multi-specific fusion protein according to the first aspect of the present invention; and

[0125] (b) a conjugated moiety selected from the group consisting of a detectable label, a drug, a toxin, a cytokine, a radionuclide, an enzyme, or a combination thereof.

[0126] In another preferred embodiment, the conjugate portion is selected from: fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computer tomography) contrast agents, or enzymes capable of producing detectable products, radionuclides, biotoxins, cytokines (such as IL-2, etc.), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorods, viral particles, liposomes, and nanomagnetic particles.

[0127] In the eighth aspect of the present invention, there is provided a use of the multi-specific fusion protein as described in the first aspect of the present invention, the pharmaceutical composition as described in the sixth aspect of the present invention, or the immunoconjugate as described in the seventh aspect of the present invention, characterized in that it is used to prepare (a) a detection reagent or kit; and / or (b) a drug for preventing and / or treating cancer / tumors.

[0128] In another preferred embodiment, the cancer / tumor includes solid tumors and blood tumors.

[0129] In another preferred embodiment, the cancer / tumor is a solid tumor.

[0130] In another preferred embodiment, the cancer / tumor is selected from the following group: colorectal, breast, ovarian, pancreatic, gastric, prostate, kidney, cervical, bone marrow cancer, lymphoma, leukemia, thyroid, endometrial, uterine, bladder, neuroendocrine, head and neck, liver, nasopharyngeal, testicular, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, dermatofibrosarcoma protuberans, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, and myelodysplastic syndrome.

[0131] In another preferred embodiment, the cancer is selected from cancer that is recurrent or progressive after treatment with surgery, chemotherapy, radiotherapy, or a combination thereof.

[0132] In the ninth aspect of the present invention, a kit is provided, comprising the multi-specific fusion protein as described in the first aspect of the present invention, the polynucleotide as described in the second aspect of the present invention, the vector as described in the third aspect of the present invention, the host cell as described in the fourth aspect of the present invention, the pharmaceutical composition as described in the sixth aspect of the present invention, or the immunoconjugate as described in the seventh aspect of the present invention.

[0133] In the tenth aspect of the present invention, provided is the use of the multi-specific fusion protein as described in the first aspect of the present invention, the polynucleotide as described in the second aspect of the present invention, the vector as described in the third aspect of the present invention, the host cell as described in the fourth aspect of the present invention, the pharmaceutical composition as described in the sixth aspect of the present invention, or the immunoconjugate as described in the seventh aspect of the present invention in the preparation of a medicament for preventing, treating and / or detecting cancer / tumors.

[0134] In the eleventh aspect of the present invention, a method for preventing, treating and / or detecting cancer / tumors is provided, wherein a safe and effective amount of the multi-specific fusion protein, pharmaceutical composition or immunoconjugate thereof as described in the first aspect of the present invention is administered to a subject in need.

[0135] It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features described in detail below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be listed here one by one. BRIEF DESCRIPTION OF THE DRAWINGS

[0136] Figures 1 to 17 show schematic structural diagrams of fusion proteins used in the examples of the present invention;

[0137] Figure 18 shows ELISA detection of Trop2 nanobody Fc fusion protein binding to human Trop2 protein;

[0138] Figure 19A shows a cell-based ELISA assay detecting the binding of Trop2 nanobody Fc fusion protein to A431 cells naturally expressing human Trop2;

[0139] Figure 19B shows a cell-based ELISA assay detecting the binding of Trop2 nanobody Fc fusion protein to MCF-7 cells naturally expressing human Trop2;

[0140] Figure 20 shows the binding of Trop2 humanized Nanobody Fc fusion protein to human Trop2 protein (QP1174) detected by ELISA;

[0141] Figure 21A shows a cell-based ELISA assay detecting the binding of Trop2 humanized Nanobody Fc fusion protein to naturally expressing human Trop2 cells A431;

[0142] Figure 21B shows a cell-based ELISA assay detecting the binding of Trop2 humanized Nanobody Fc fusion protein to naturally expressing human Trop2 cells MCF-7;

[0143] Figures 22A to 22D show FACS detection of the binding of Trop2-containing nanobody fusion protein to naturally expressing human Trop2 cells HCC827-CLDN18.2;

[0144] Figure 22E shows FACS detection of the binding of Trop2 nanobody or PD-L1 antibody fusion protein to naturally expressing human Trop2 / PD-L1 cells HCC827-CLDN18.2;

[0145] FIG22F shows FACS detection of the binding of EGFR antibody-containing fusion protein to HCC827 cells naturally expressing human EGFR;

[0146] Figure 22G shows FACS detection of the binding of EGFR antibody-containing fusion protein to naturally expressing human EGFR cells HCC827-CLDN18.2;

[0147] Figure 22H shows FACS detection of the binding of the PDL1 antibody-containing fusion protein to HCC827-CLDN18.2 cells naturally expressing human PD-L1;

[0148] Figures 23A to 23D show ELISA detection of CD80 fusion protein binding to human CTLA4;

[0149] Figures 24A to 24C show the detection of binding of fusion proteins containing PD-1 antibodies to human PD-1 by ELISA;

[0150] Figures 25A and 25B show ELISA detection of fusion proteins containing PD-1 antibodies blocking the binding of human PD-1 to PD-L1;

[0151] Figures 26A and 26B show the ELISA detection of fusion proteins containing PD-L1 antibodies binding to human PD-L1;

[0152] Figures 27A and 27B show that fusion proteins containing PD-L1 antibodies blocked the binding of human PD-L1 to PD-1 by ELISA;

[0153] Figures 28A to 28D show that CD80 / TAA fusion protein combined with CD3 dual antibody enhances tumor cell killing; Figure 28A shows PBMC-mediated killing activity of HCC827-CLDN18.2; donor P121112105C; E:T=10:1; 72 hours; Figure 28B shows PBMC-mediated killing activity of HCC827-CLDN18.2; donor P121 112105C; E:T = 10:1; 48 hours; FIG28C shows the PBMC-mediated killing activity of HCC827-CLDN18.2; donor P121112105C; E:T = 10:1; 48 hours; FIG28D shows the PBMC-mediated killing activity of HCC827-CLDN18.2; donor P121112105C; E:T = 5:1; 48 hours;

[0154] Figures 29A to 29E show that CD80 / PD(L)1 / TAA fusion protein combined with CD3 dual antibody enhances tumor cell killing, wherein Figure 29A shows PBMC-mediated killing activity of HCC827-CLDN18.2; donor P121112105C; E:T=10:1; 48 hours; Figure 29B shows PBMC-mediated killing activity of HCC827-CLDN18.2; donor P121112105C; E:T=10:1; 48 hours; Figure 29C shows Figure 29D shows the PBMC-mediated killing activity of HCC827-CLDN18.2; donor P121112105C; E:T = 5:1; 48 hours; Figure 29E shows the PBMC-mediated killing activity of HCC827-CLDN18.2; donor P123050308C; E:T = 5:1; 72 hours;

[0155] Figures 30A to 30C show that CD80 / PD(L)1 / TAA fusion proteins combined with CD3 dual antibodies enhance tumor cell killing; Figure 30A shows PBMC-mediated killing activity of HCC827-CLDN18.2; donor P121061004C; E:T = 5:1; 48 hours; Figure 30B shows PBMC-mediated killing activity of HCC827-CLDN18.2; donor P121070501C; E:T = 6:1; 48 hours; Figure 30C shows PBMC-mediated killing activity of HCC827-CLDN18.2; donor P121061004C; E:T = 5:1; 48 hours;

[0156] Figures 31A and 31B show that CD80 / PD(L)1 / TAA fusion proteins combined with CD3 dual antibodies enhance tumor cell killing. Figure 31A shows PBMC-mediated killing activity of HCC827-CLDN18.2; donor P121061004C; E:T = 5:1; 48 hours; Figure 31B shows PBMC-mediated killing activity of HCC827-CLDN18.2; donor P121070501C; E:T = 10:1; 48 hours;

[0157] Figures 32A and 32B show that CD80 / PD(L)1 / TAA fusion proteins combined with CD3 dual antibodies enhance tumor cell killing; Figure 32A shows PBMC-mediated killing activity of HCC827-CLDN18.2; donor P122030710C; E:T = 5:1; 48 hours; Figure 32B shows PBMC-mediated killing activity of HCC827-CLDN18.2; donor P123050308C; E:T = 5:1; 48 hours;

[0158] FIG33 shows the release of TNF-α induced by the CD80 fusion protein of the present invention, 48 hours;

[0159] FIG34A shows the in vivo efficacy curve of CD80 fusion protein in mice;

[0160] FIG34B shows that the CD80 fusion protein inhibits mouse tumor volume in in vivo animal efficacy analysis; Two-factor ANOVA followed by Dunnett's multiple comparison test was used, G2 / 3 vs G1: ****<0.0001;

[0161] FIG34C shows that the CD80 fusion protein inhibits mouse tumor weight in in vivo animal efficacy analysis; using Two-factor ANOVA followed by Dunnett's multiple comparison test, G2 / 3 vs. G1: *p<0.05; **p<0.01; ****p<0.0001;

[0162] FIG35A shows the in vivo efficacy curve of CD80 fusion protein in mice;

[0163] Figure 35B shows that the CD80 fusion protein inhibited the tumor volume in mice in vivo; statistical analysis was performed using Two-factor ANOVA followed by Dunnett's multiple comparison test, G2 / 3 / 4 vs G1, G2 vs G4: *<0.05, ***<0.001, ****<0.0001. DETAILED DESCRIPTION

[0164] After extensive and in-depth research, the inventors unexpectedly developed a multi-specific fusion protein for the first time. The multi-specific fusion protein of the present invention comprises a CD28 targeting domain, a TAA targeting domain, and an optional PD1 / PD-L1 targeting domain. The multi-specific fusion protein of the present invention binds to CD28 on T cells with weak affinity, and can only activate the CD28 signaling pathway when bound to tumor TAAs, thereby ensuring the clinical safety of CD28 agonists. The fusion protein of the present invention enriches candidate drugs in tumors through anti-TAA antibodies, utilizes the natural weak affinity of CD80 recombinant protein to CD28 / CTLA4 / PD-L1, further targets tumors through TAAs, activates the CD28 pathway to activate T cells, and kills and eliminates tumor cells through multiple immune effector mechanisms. The present invention was completed on this basis.

[0165] In one embodiment, the present invention provides a fusion protein, wherein the fusion protein comprises the following elements:

[0166] a) a first targeting domain D1, wherein the first targeting domain D1 is a CD80 extracellular domain (ECD);

[0167] and

[0168] b) a second targeting domain D2, wherein said D2 binds to a second target protein, wherein said second target protein is a tumor-associated antigen (TAA).

[0169] According to the fusion protein of the present invention, the CD80 extracellular domain (ECD) comprises a CD80 IgV domain or a CD80 IgC domain, or a combination thereof.

[0170] In a specific embodiment, the CD80 extracellular domain (ECD) of the present invention has the amino acid sequence shown in SEQ ID NO:41, or an amino acid sequence having 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 thereto, as long as it is capable of binding to one or a combination of the following target proteins: CD28, CTLA-4 and PD-L1.

[0171] In a specific embodiment, the second targeting domain D2 of the present invention is an anti-TAA antibody or a functional fragment thereof, and the functional fragment of the antibody is selected from Fab, scFv or VHH.

[0172] In a specific embodiment, the TAA of the present invention is selected from:

[0173] CD33, CD30, HER2, CD22, CD79b, Nectin-4, BCMA, EGFR, CD19, tissue factor, folr1 (folate receptor α), CLDN18.2, TROP2, c-Met, PSMA, Muc1, PDL1, ROR1, MSLN, TNF-α, CD25, ENPP3, Axl, CD20, ROR2, GPNMB, CEACAM6, CD138, CA6, FUT3, C D56, CD37, HER3, GPRC5D, STING, CEA, CD205, B7H4, CTLA4, RNF43, CDH3, DPEP3, 5T4, ITGB6, EFNA4, B7H3, C D228, Notch-3, CD46, CAIX, SLAMF6, ADAM9, GD3, TDGF1, SLAMF2, CLL-1, CD123, IGF-1R, FCRL5, TIM1, Globo H, CDH6, CD38, Ly6E, SLITRK6, GPR20, FGFR2, Muc16, CD51, SLAMF7, LAMP-1, CD74, CCR7, PTK7, SEZ6, CLDN 9. CLDN6, c-kit, LYPD3, TAA, PRL receptor, FGFR3, KAAG1, STEAP1, Flt3, LRRC15, CD44, CD70, EphA2, PDL2, p53, DLK1, ENB-FN, FOLR, CD45, DSG2, ALK, TRAIL, DDR1, EpCAM, VEGFR2, CD47, CD99, VEGF2, SSEA-4, DCLK1, OAcGD2, IL1RAP, ADAM17, CD7, CD73, ENO1, BSG, CD24, GLUT1, CXCR4, CD40, CD52, CD133, CD239, TAG72, EGFR VIII, PSCA, EphA2, NKG2D ligand, MCSP, LGR5, SSEA3, SLC34A2, Glypican-3, or a combination thereof.

[0174] In another embodiment, the present invention provides a fusion protein, wherein the fusion protein comprises the following elements:

[0175] a) a first targeting domain D1, wherein the first targeting domain D1 is a CD80 extracellular domain (ECD);

[0176] and

[0177] b) a second targeting domain D2, wherein the second targeting domain D2 is an anti-TROP2 antibody.

[0178] In a specific embodiment, the CD80 extracellular domain (ECD) has the amino acid sequence shown in SEQ ID NO:41, or an amino acid sequence having 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 thereto, as long as it is capable of binding to one or a combination of the following target proteins: CD28, CTLA-4 and PD-L1.

[0179] In an embodiment of the present invention, the anti-TROP2 antibody is a single-domain antibody (VHH), which comprises CDR1 shown in SEQ ID NO: 47, CDR2 shown in SEQ ID NO: 48, and CDR3 shown in SEQ ID NO: 49.

[0180] Specifically, the anti-TROP2 antibody described in the present invention is a single-domain antibody (VHH), which comprises the amino acid sequence described in SEQ ID NO: 46, or an amino acid sequence having 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 thereto.

[0181] In yet another embodiment, the present invention provides a fusion protein, wherein the fusion protein comprises the following elements:

[0182] a) a first targeting domain D1, wherein the first targeting domain D1 is a CD80 extracellular domain (ECD);

[0183] and

[0184] b) a second targeting domain D2, wherein the second targeting domain D2 is an anti-EGFR antibody or a functional fragment thereof.

[0185] In a specific embodiment, the CD80 extracellular domain (ECD) has the amino acid sequence shown in SEQ ID NO:41, or an amino acid sequence having 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 thereto, as long as it is capable of binding to one or a combination of the following target proteins: CD28, CTLA-4 and PD-L1.

[0186] In an embodiment of the present invention, the anti-EGFR antibody or a functional fragment thereof comprises VH shown in SEQ ID NO: 59 and VL shown in SEQ ID NO: 60; or

[0187] The anti-EGFR antibody or a functional fragment thereof comprises VH shown in SEQ ID NO: 70 and VL shown in SEQ ID NO: 71.

[0188] In another embodiment, the present invention provides a fusion protein, wherein the fusion protein comprises the following elements:

[0189] a) a first targeting domain D1, wherein the first targeting domain D1 is a CD80 extracellular domain (ECD);

[0190] b) a second targeting domain D2, said D2 binding to a second target protein, said second target protein being a tumor associated antigen (TAA); and

[0191] c) a third targeting domain D3, wherein the D3 binds to a third target protein, and the third target protein is PD1 and / or PD-L1.

[0192] According to the fusion protein of an embodiment of the present invention, the CD80 extracellular domain (ECD) comprises a CD80 IgV domain or a CD80 IgC domain, or a combination thereof.

[0193] In a specific embodiment, the CD80 extracellular domain (ECD) of the present invention has the amino acid sequence shown in SEQ ID NO:41, or an amino acid sequence having 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 thereto, as long as it is capable of binding to one or a combination of the following target proteins: CD28, CTLA-4 and PD-L1.

[0194] According to the fusion protein of the embodiment of the present invention, the second targeting domain D2 is an anti-TAA antibody or a functional fragment thereof, and the functional fragment of the antibody is selected from Fab, scFv or VHH.

[0195] In a specific embodiment, the TAA of the present invention is selected from:

[0196] CD33, CD30, HER2, CD22, CD79b, Nectin-4, BCMA, EGFR, CD19, tissue factor, folr1 (folate receptor α), CLDN18.2, TROP2, c-Met, PSMA, Muc1, PDL1, ROR1, MSLN, TNF-α, CD25, ENPP3, Axl, CD20, ROR2, GPNMB, CEACAM6, CD138, CA6, FUT3, C D56, CD37, HER3, GPRC5D, STING, CEA, CD205, B7H4, CTLA4, RNF43, CDH3, DPEP3, 5T4, ITGB6, EFNA4, B7H3, C D228, Notch-3, CD46, CAIX, SLAMF6, ADAM9, GD3, TDGF1, SLAMF2, CLL-1, CD123, IGF-1R, FCRL5, TIM1, Globo H, CDH6, CD38, Ly6E, SLITRK6, GPR20, FGFR2, Muc16, CD51, SLAMF7, LAMP-1, CD74, CCR7, PTK7, SEZ6, CLDN 9. CLDN6, c-kit, LYPD3, TAA, PRL receptor, FGFR3, KAAG1, STEAP1, Flt3, LRRC15, CD44, CD70, EphA2, PDL2, p53, DLK1, ENB-FN, FOLR, CD45, DSG2, ALK, TRAIL, DDR1, EpCAM, VEGFR2, CD47, CD99, VEGF2, SSEA-4, DCLK1, OAcGD2, IL1RAP, ADAM17, CD7, CD73, ENO1, BSG, CD24, GLUT1, CXCR4, CD40, CD52, CD133, CD239, TAG72, EGFR VIII, PSCA, EphA2, NKG2D ligand, MCSP, LGR5, SSEA3, SLC34A2, Glypican-3, or a combination thereof.

[0197] According to the fusion protein of the embodiment of the present invention, the third targeting domain D3 is an anti-PD-1 antibody or a functional fragment thereof, or an anti-PD-L1 antibody or a functional fragment thereof, and the functional fragment of the antibody is selected from Fab, scFv or VHH.

[0198] In another embodiment, the present invention provides a fusion protein, wherein the fusion protein comprises the following elements:

[0199] a) a first targeting domain D1, wherein the first targeting domain D1 is a CD80 extracellular domain (ECD);

[0200] b) a second targeting domain D2, wherein the second targeting domain D2 is an anti-TROP2 antibody, and

[0201] c) a third targeting domain D3, wherein the third targeting domain D3 is an anti-PD-1 antibody or a functional fragment thereof, and the functional fragment of the antibody is selected from Fab, scFv or VHH.

[0202] In a specific embodiment, the CD80 extracellular domain (ECD) has the amino acid sequence shown in SEQ ID NO:41, or an amino acid sequence having 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 thereto, as long as it is capable of binding to one or a combination of the following target proteins: CD28, CTLA-4 and PD-L1.

[0203] In an embodiment of the present invention, the anti-TROP2 antibody is a single-domain antibody (VHH), which comprises CDR1 shown in SEQ ID NO: 47, CDR2 shown in SEQ ID NO: 48, and CDR3 shown in SEQ ID NO: 49.

[0204] Specifically, the anti-TROP2 antibody described in the present invention is a single-domain antibody (VHH), which comprises the amino acid sequence described in SEQ ID NO: 46, or an amino acid sequence having 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 thereto.

[0205] In an embodiment of the present invention, the anti-PD-1 antibody or a functional fragment thereof is selected from any one of i) to iv):

[0206] i) a VHH comprising the CDR1 set forth in SEQ ID NO: 53, the CDR2 set forth in SEQ ID NO: 54, and the CDR3 set forth in SEQ ID NO: 55;

[0207] Preferably, the VHH has the amino acid sequence shown in SEQ ID NO: 52; or a sequence identity thereof of 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%;

[0208] ii) a VHH comprising the CDR1 set forth in SEQ ID NO: 53, the CDR2 set forth in SEQ ID NO: 54, and the CDR3 set forth in SEQ ID NO: 57;

[0209] Preferably, the VHH has the amino acid sequence shown in SEQ ID NO: 56; or a sequence identity thereof of 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%;

[0210] iii) an anti-PD-1 antibody or a functional fragment thereof, comprising VH and VL,

[0211] The VH has the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence having 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 thereto; and

[0212] The VL has the amino acid sequence of SEQ ID NO: 64, or an amino acid sequence having 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 thereto; and

[0213] iv) an anti-PD-1 antibody or a functional fragment thereof, comprising VH and VL,

[0214] The VH has the amino acid sequence of SEQ ID NO: 65, or an amino acid sequence having 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 thereto; and

[0215] The VL has the amino acid sequence shown in SEQ ID NO:66, or an amino acid sequence having 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 thereto.

[0216] In another embodiment, the present invention provides a fusion protein, wherein the fusion protein comprises the following elements:

[0217] a) a first targeting domain D1, wherein the first targeting domain D1 is a CD80 extracellular domain (ECD);

[0218] b) a second targeting domain D2, wherein the second targeting domain D2 is an anti-TROP2 antibody, and

[0219] c) a third targeting domain D3, wherein the third targeting domain D3 is an anti-PD-L1 antibody or a functional fragment thereof, and the functional fragment of the antibody is selected from Fab, scFv or VHH.

[0220] In a specific embodiment, the CD80 extracellular domain (ECD) has the amino acid sequence shown in SEQ ID NO:41, or an amino acid sequence having 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 thereto, as long as it is capable of binding to one or a combination of the following target proteins: CD28, CTLA-4 and PD-L1.

[0221] In an embodiment of the present invention, the anti-TROP2 antibody is a single-domain antibody (VHH), which comprises CDR1 shown in SEQ ID NO: 47, CDR2 shown in SEQ ID NO: 48, and CDR3 shown in SEQ ID NO: 49.

[0222] Specifically, the anti-TROP2 antibody described in the present invention is a single-domain antibody (VHH), which comprises the amino acid sequence described in SEQ ID NO: 46, or an amino acid sequence having 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 thereto.

[0223] In an embodiment of the present invention, the anti-PD-L1 antibody or a functional fragment thereof is selected from any one of v) to vi):

[0224] v) an anti-PD-L1 antibody or a functional fragment thereof, which comprises VH and VL,

[0225] The VH has the amino acid sequence of SEQ ID NO: 61, or an amino acid sequence having 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 thereto; and

[0226] The VL has the amino acid sequence of SEQ ID NO: 62, or an amino acid sequence having 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 thereto; and

[0227] vi) a VHH having an amino acid sequence as shown in SEQ ID NO: 58, or a sequence identity thereof of 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%.

[0228] In another embodiment, the present invention provides a fusion protein, wherein the fusion protein comprises the following elements:

[0229] a) a first targeting domain D1, wherein the first targeting domain D1 is a CD80 extracellular domain (ECD);

[0230] b) a second targeting domain D2, wherein the second targeting domain D2 is an anti-EGFP antibody, and

[0231] c) a third targeting domain D3, wherein the third targeting domain D3 is an anti-PD-1 antibody or a functional fragment thereof, and the functional fragment of the antibody is selected from Fab, scFv or VHH.

[0232] In a specific embodiment, the CD80 extracellular domain (ECD) has the amino acid sequence shown in SEQ ID NO:41, or an amino acid sequence having 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 thereto, as long as it is capable of binding to one or a combination of the following target proteins: CD28, CTLA-4 and PD-L1.

[0233] In an embodiment of the present invention, the anti-EGFR antibody or a functional fragment thereof comprises VH shown in SEQ ID NO: 59 and VL shown in SEQ ID NO: 60; or

[0234] The anti-EGFR antibody or a functional fragment thereof comprises VH shown in SEQ ID NO: 70 and VL shown in SEQ ID NO: 71.

[0235] In an embodiment of the present invention, the anti-PD-1 antibody or a functional fragment thereof is selected from any one of i) to iv):

[0236] i) a VHH comprising the CDR1 set forth in SEQ ID NO: 53, the CDR2 set forth in SEQ ID NO: 54, and the CDR3 set forth in SEQ ID NO: 55;

[0237] Preferably, the VHH has the amino acid sequence shown in SEQ ID NO: 52; or a sequence identity thereof of 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%;

[0238] ii) a VHH comprising the CDR1 set forth in SEQ ID NO: 53, the CDR2 set forth in SEQ ID NO: 54, and the CDR3 set forth in SEQ ID NO: 57;

[0239] Preferably, the VHH has the amino acid sequence shown in SEQ ID NO: 56; or a sequence identity thereof of 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%;

[0240] iii) an anti-PD-1 antibody or a functional fragment thereof, comprising VH and VL,

[0241] The VH has the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence having 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 thereto; and

[0242] The VL has the amino acid sequence of SEQ ID NO: 64, or an amino acid sequence having 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 thereto; and

[0243] iv) an anti-PD-1 antibody or a functional fragment thereof, comprising VH and VL,

[0244] The VH has the amino acid sequence of SEQ ID NO: 65, or an amino acid sequence having 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 thereto; and

[0245] The VL has the amino acid sequence shown in SEQ ID NO:66, or an amino acid sequence having 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 thereto.

[0246] In another embodiment, the present invention provides a fusion protein, wherein the fusion protein comprises the following elements:

[0247] a) a first targeting domain D1, wherein the first targeting domain D1 is a CD80 extracellular domain (ECD);

[0248] b) a second targeting domain D2, wherein the second targeting domain D2 is an anti-EGFP antibody, and

[0249] c) a third targeting domain D3, wherein the third targeting domain D3 is an anti-PD-L1 antibody or a functional fragment thereof, and the functional fragment of the antibody is selected from Fab, scFv or VHH.

[0250] In a specific embodiment, the CD80 extracellular domain (ECD) has the amino acid sequence shown in SEQ ID NO:41, or an amino acid sequence having 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 thereto, as long as it is capable of binding to one or a combination of the following target proteins: CD28, CTLA-4 and PD-L1.

[0251] In an embodiment of the present invention, the anti-EGFR antibody or a functional fragment thereof comprises VH shown in SEQ ID NO: 59 and VL shown in SEQ ID NO: 60; or

[0252] The anti-EGFR antibody or a functional fragment thereof comprises VH shown in SEQ ID NO: 70 and VL shown in SEQ ID NO: 71.

[0253] In an embodiment of the present invention, the anti-PD-L1 antibody or a functional fragment thereof is selected from any one of v) to vi):

[0254] v) an anti-PD-L1 antibody or a functional fragment thereof, which comprises VH and VL,

[0255] The VH has the amino acid sequence of SEQ ID NO: 61, or an amino acid sequence having 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 thereto; and

[0256] The VL has the amino acid sequence of SEQ ID NO: 62, or an amino acid sequence having 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 thereto; and

[0257] vi) a VHH having an amino acid sequence as shown in SEQ ID NO: 58, or a sequence identity thereof of 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%.

[0258] In a specific embodiment, the fusion protein of the present invention is a homodimer comprising two identical monomers, and from the N-terminus to the C-terminus, each monomer has a structure selected from the group consisting of:

[0259] i) CD80 extracellular domain (ECD) - anti-TAA antibody or its functional fragment - Fc region - anti-PD-1 or anti-PD-L1 antibody or its functional fragment;

[0260] In particular, the monomer has the amino acid sequence shown in SEQ ID NO: 21 or SEQ ID NO: 22;

[0261] ii) CD80 extracellular domain (ECD)-anti-PD-1 or anti-PD-L1 antibody or its functional fragment-Fc region-anti-TAA antibody or its functional fragment;

[0262] iii) CD80 extracellular domain (ECD)-anti-TAA antibody or its functional fragment-Fc region;

[0263] In particular, the monomer has the amino acid sequence shown in SEQ ID NO: 7 or SEQ ID NO: 81; or

[0264] iv) CD80 extracellular domain (ECD)-Fc region-anti-TAA antibody or a functional fragment thereof;

[0265] In particular, the monomer has the amino acid sequence shown in SEQ ID NO: 1, SEQ ID NO: 39 or SEQ ID NO: 77.

[0266] In a specific embodiment, the fusion protein of the present invention is a heterodimer comprising a first monomer and a second monomer, and from the N-terminus to the C-terminus, the fusion protein has a structure selected from any one of the following i)-viii):

[0267] i) the first monomer comprises: an anti-PD1 / PD-L1 antibody or a functional fragment thereof - a first Fc region - an anti-TAA antibody or a functional fragment thereof; and

[0268] The second monomer comprises: CD80 extracellular domain (ECD)-second Fc region-anti-TAA antibody or a functional fragment thereof;

[0269] In particular, the first monomer has the amino acid sequence shown in SEQ ID NO: 6 or SEQ ID NO: 80, and the second monomer has the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 78; or

[0270] The first monomer has the amino acid sequence shown in SEQ ID NO:16, and the second monomer has the amino acid sequence shown in SEQ ID NO:4 or SEQ ID NO:78;

[0271] ii) the first monomer comprises: an anti-PD1 / PD-L1 antibody or a functional fragment thereof - an anti-TAA antibody or a functional fragment thereof - a first Fc region; and

[0272] The second monomer comprises: CD80 extracellular domain (ECD)-anti-TAA antibody or a functional fragment thereof-second Fc region;

[0273] In particular, the first monomer has the amino acid sequence shown in SEQ ID NO: 9 or SEQ ID NO: 83, and the second monomer has the amino acid sequence shown in SEQ ID NO: 8 or SEQ ID NO: 82;

[0274] iii) the first monomer comprises: CD80 extracellular domain (ECD)-first Fc region-anti-PD1 / PD-L1 antibody or a functional fragment thereof; and

[0275] The second monomer comprises: an anti-TAA antibody or a functional fragment thereof-a second Fc region-an anti-PD1 / PD-L1 antibody or a functional fragment thereof;

[0276] In particular, the first monomer has the amino acid sequence shown in SEQ ID NO:20 or SEQ ID NO:86, and the second monomer has the amino acid sequence shown in SEQ ID NO:18 and SEQ ID NO:19 or SEQ ID NO:85;

[0277] iv) the first monomer comprises: an anti-TAA antibody or a functional fragment thereof - a first Fc region; and

[0278] The second monomer comprises: CD80 extracellular domain (ECD)-anti-TAA antibody or a functional fragment thereof-second Fc region;

[0279] v) the first monomer comprises: a first Fc region - an anti-TAA antibody or a functional fragment thereof; and

[0280] The second monomer comprises: CD80 extracellular domain (ECD)-second Fc region-anti-TAA antibody or a functional fragment thereof;

[0281] In particular, the first monomer has the amino acid sequence shown in SEQ ID NO: 5 or SEQ ID NO: 79, and the second monomer has the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 78;

[0282] The first monomer has the amino acid sequence shown in SEQ ID NO:38, and the second monomer has the amino acid sequence shown in SEQ ID NO:20 or SEQ ID NO:86;

[0283] vi) the first monomer comprises: a first Fc region; and

[0284] The second monomer comprises: CD80 extracellular domain (ECD)-anti-TAA antibody or a functional fragment thereof-second Fc region;

[0285] In particular, the first monomer has the amino acid sequence shown in SEQ ID NO: 10 or SEQ ID NO: 84, and the second monomer has the amino acid sequence shown in SEQ ID NO: 8 or SEQ ID NO: 82;

[0286] vii) the first monomer comprises: CD80 extracellular domain (ECD) - first Fc region - anti-TAA antibody or a functional fragment thereof; and

[0287] The second monomer comprises: an anti-PD1 / PD-L1 antibody or a functional fragment thereof - a second Fc region - an anti-TAA antibody or a functional fragment thereof;

[0288] In particular, the first monomer has the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 78, and the second monomer has the amino acid sequence shown in SEQ ID NO: 26 and 27; or

[0289] The first monomer has the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 78, and the second monomer has the amino acid sequence shown in SEQ ID NO: 28; or

[0290] The first monomer has the amino acid sequence shown in SEQ ID NO: 4 or SEQ ID NO: 78, and the second monomer has the amino acid sequence shown in SEQ ID NO: 29 and 30; or

[0291] The first monomer has the amino acid sequence shown in SEQ ID NO:4 or SEQ ID NO:78, and the second monomer has the amino acid sequence shown in SEQ ID NO:31 and 32;

[0292] or

[0293] viii) the first monomer comprises: a CD80 extracellular domain (ECD)-a first Fc region; and

[0294] The second monomer comprises: an anti-TAA antibody or a functional fragment thereof - a second Fc region;

[0295] Wherein, - indicates direct connection or connection through a peptide linker;

[0296] wherein the first Fc region is the same as or different from the second Fc region;

[0297] Optionally, the first Fc region and the second Fc region form a knob-into-hole structure.

[0298] Specifically, the fusion polypeptide has an amino acid sequence combination selected from any one of the following 1)-11):

[0299] In another aspect, the present invention provides a polynucleotide encoding a polypeptide having an amino acid sequence selected from any one of the following:

[0300] SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:39 and SEQ ID NO:40, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, SEQ ID NO:86.

[0301] In another aspect, the present invention provides an anti-TROP2 antibody capable of forming a fusion protein with the CD80 extracellular domain (ECD) and / or anti-PD1 and / or PD-L1 antibodies.

[0302] In a specific embodiment, the TROP2 antibody provided by the present invention is a VHH, and the anti-TROP2 antibody comprises CDR1 shown in SEQ ID NO:47, CDR2 shown in SEQ ID NO:48, and CDR3 shown in SEQ ID NO:49.

[0303] In a specific embodiment, the anti-TROP2 antibody comprises the amino acid sequence of SEQ ID NO:46, or an amino acid sequence having 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 thereto.

[0304] In another aspect, the present invention provides the fusion protein of the present invention or a pharmaceutical composition comprising the same, for use in preventing and / or treating tumors.

[0305] In another aspect, the present invention provides a method for treating and / or curing a tumor, comprising administering a therapeutically effective amount of the fusion protein of the present invention or a pharmaceutical composition comprising the same to a subject in need thereof.

[0306] In a specific embodiment, the present invention provides a method for treating and / or curing a tumor, comprising administering a therapeutically effective amount of the fusion protein of the present invention or a pharmaceutical composition comprising the same, or one or more CD3 bispecific antibodies to a subject in need thereof.

[0307] In a specific embodiment, the tumor is selected from a solid tumor or a hematological tumor, such as colorectal cancer, gastric cancer, breast cancer, non-small cell lung cancer, small cell lung cancer, melanoma, head and neck cancer, ovarian cancer, pancreatic cancer, liver cancer, bladder cancer, kidney cancer, endometrial cancer, cervical cancer, glioma, leukemia, lymphoma, multiple myeloma, myelodysplastic syndrome and myeloproliferative neoplasms.

[0308] the term

[0309] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0310] The term "about" can refer to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined.

[0311] As used herein, the terms "comprising" or "including" may be open, semi-closed, or closed. In other words, the terms also include "consisting essentially of" or "consisting of."

[0312] The term "fusion" refers to the connection of components directly by peptide bonds or with the help of connecting fragments, or non-covalent fusion through intermolecular interactions or covalent fusion through one or more disulfide bonds or chemical cross-linking. In a single peptide chain, fusion refers to the connection directly by peptide bonds or with the help of connecting fragments. "Multifunctional fusion protein" refers to a protein comprising two or more targeting domains (such as domains that bind to antigens and / or domains that bind to receptors) that can bind to two or more different epitopes (for example, two, three or more different epitopes). "Fusion position" refers to the position of a functional region or domain in a peptide chain, indicating the order in which the functional fragments on the peptide chain are connected.

[0313] The term "polypeptide" refers to an amino acid chain of any length, including proteins and fragments thereof. The present invention discloses polypeptides as sequences of amino acid residues. Those sequences are written from left to right in the direction from amino terminus to carboxyl terminus. According to standard nomenclature, amino acid residue sequences are named by three-letter or single-letter codes as follows: alanine (Ala, A), arginine (Arg, R), asparagine (Asn, N), aspartic acid (Asp, D), cysteine ​​(Cys, C), glutamine (Gln, Q), glutamic acid (Glu, E), glycine (Gly, G), histidine (His, H), isoleucine (Ile, I), leucine (Leu, L), lysine (Lys, K), methionine (Met, M), phenylalanine (Phe, F), proline (Pro, P), serine (Ser, S), threonine (Thr, T), tryptophan (Trp, W), tyrosine (Tyr, Y), and valine (Val, V).

[0314] The term "peptide chain" refers to a molecule of amino acids linearly linked by peptide bonds.

[0315] The term "variant" or "mutant" refers to a polypeptide or polynucleotide that differs in the amino acids or nucleotides it contains but retains essential properties. Typically, the differences between variants or between a variant and the parent antibody are limited, with the amino acid sequences generally being very similar. In this specification, the antibody or antibody fragment before mutation is referred to as the parent antibody, and the antibody or antibody fragment after mutation is referred to as the variant. The variant still possesses antigen-binding activity.

[0316] The term "antibody" (Ab) refers to an immunoglobulin (Ig) molecule that contains at least one antigen-binding site and can specifically bind to an antigen.

[0317] The term "antigen" refers to a substance in the body that can induce an immune response and specifically bind to an antibody. The binding between the antibody and antigen is mediated by interactions between the two, including hydrogen bonds, van der Waals forces, ionic bonds, and hydrophobic bonds. The region on the antigen surface where the antibody binds is called an "antigenic determinant" or "epitope." Generally, each antigen has multiple determinants.

[0318] The term "antibody" as used herein is understood in its broadest sense and includes monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, antibody fragments, and multispecific antibodies (e.g., bispecific antibodies) comprising at least two different antigen-binding domains. Antibodies also include murine antibodies, humanized antibodies, chimeric antibodies, human antibodies, and antibodies from other sources. The antibodies of the present invention can be derived from any animal, including but not limited to immunoglobulin molecules of humans, non-human primates, mice, rats, cattle, horses, chickens, camels, and alpacas. Antibodies may contain additional changes, such as unnatural amino acids, Fc effector function mutations, and glycosylation site mutations. Antibodies also include post-translationally modified antibodies, fusion proteins comprising antigenic determinants of antibodies, and immunoglobulin molecules comprising any other modifications to antigen recognition sites, as long as these antibodies exhibit the desired biological activity. In other words, antibodies include immunoglobulin molecules and immunoactive fragments of immunoglobulin molecules, i.e., molecules containing at least one antigen-binding domain.

[0319] The basic structure of an antibody consists of two identical heavy chains (H) and two identical light chains (L) connected by disulfide bonds, forming a Y-shaped monomer. Each chain is composed of two to five structural domains (also known as functional regions) containing approximately 110 amino acids, with similar sequences but distinct functions. The amino acid sequences near the N-termini of the light and heavy chains in an antibody molecule vary significantly, forming a domain called the variable region (V region). The region near the C-terminus, where the amino acid sequence remains relatively constant, is called the constant region (C region).

[0320] The V regions of the heavy and light chains are called VH and VL, respectively. Each VH and VL has three regions of highly variable amino acid composition and sequence, known as hypervariable regions (HVRs). These regions form the spatial conformation complementary to the antigen epitope and are also known as complementarity determining regions (CDRs). The three CDRs of VH are designated VHCDR1, VHCDR2, and VHCDR3, respectively, while the three CDRs of VL are designated VLCDR1, VLCDR2, and VLCDR3, respectively. The six CDRs in VH and VL together form the antigen-binding site. The amino acid diversity of the CDR regions is the molecular basis for the specific binding of antibodies to a vast array of different antigens. The amino acid composition and sequence outside the CDRs within the V region remain relatively unchanged and are known as the framework regions (FRs). VH and VL each have four framework regions (or framework regions), designated FR1, FR2, FR3, and FR4, respectively. Each VH and VL consists of three CDRs and four FRs, and the order from amino terminus to carboxyl terminus is: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0321] Based on the amino acid sequence of the constant region of the antibody heavy chain, human immunoglobulins can be divided into five classes: IgM, IgG, IgA, IgD, and IgE. These can be further divided into different subclasses (isotypes). For example, human IgG can be divided into IgG1, IgG2, IgG3, and IgG4; and IgA can be divided into IgA1 and IgA2. IgM, IgD, and IgE have not yet been classified into subclasses. Based on the amino acid sequence of the light chain, light chains can be classified into kappa chains and lambda chains. The antibodies of the present invention can be of any class (e.g., IgM, IgG, IgA, IgD, IgE) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, IgA2).

[0322] The constant regions of the heavy and light chains are called CH and CL, respectively. The heavy chain constant regions of IgG, IgA, and IgD have three domains: CH1, CH2, and CH3, while those of IgM and IgE have four domains: CH1, CH2, CH3, and CH4.

[0323] The hinge region is located between CH1 and CH2 and is rich in proline, so it is easy to stretch and bend, which can change the distance between the two arms of the Y shape, facilitating the simultaneous binding of the two arms to the antigen epitope.

[0324] The term "Fab fragment" stands for antigen-binding fragment (Fab), referring to an antibody fragment consisting of the VL, VH, CL, and CH1 domains that binds to a single antigenic epitope (monovalently). Those skilled in the art are aware that, under certain conditions, certain portions of the antibody molecule chain are susceptible to proteolytic hydrolysis into various fragments. Papain hydrolyzes the antibody molecule near the N-terminus of the hinge region into two identical antigen-binding fragments (Fab) and a crystallizable fragment (Fc).

[0325] The term "Fv fragment" is a monovalent small molecule composed of the L chain and the V region of the H chain (VH+VL), and is the minimum functional fragment that binds to an antigen.

[0326] The terms "Fc," "Fc segment," "Fc fragment," or "Fc domain" refer to a crystallizable fragment without antigen-binding activity. It is the site of interaction between an antibody and effector molecules or cell-surface Fc receptors (FcRs). The Fc fragment binds to cells bearing corresponding Fc receptors on their surfaces, producing diverse biological effects. In the ADCC effect (antibody-dependent cell-mediated cytotoxicity), the Fab fragment of an antibody binds to antigenic epitopes on virus-infected or tumor cells, and its Fc segment binds to FcRs on the surface of killer cells (NK cells, macrophages, etc.), mediating direct killing of target cells by these cells. The Fc fragment comprises the constant region polypeptides of an antibody, excluding the heavy chain constant region CH1. These include the two constant region domains CH2 and CH3 at the carboxyl terminus of the heavy chain constant region of human immunoglobulins IgA, IgD, and IgG, and the three constant region domains CH2, CH3, and CH4 at the carboxyl terminus of the heavy chain constant region of human immunoglobulins IgE and IgM. The Fc fragment is often selected from human IgG1 Fc, human IgG2 Fc, human IgG3 Fc, human IgG4 Fc or variants thereof, preferably IgG1 Fc, or human IgG4 Fc or variants thereof.

[0327] The Fc region can be composed of two chains, which are described herein as the first and second Fc segments. The first and second Fc segments can be mutated independently. An Fc fragment can also refer to the single polypeptide chain within the Fc domain. Mutations can be made to the Fc segment of an antibody to eliminate immune effector functions.

[0328] Mutational design of Fc variants can generate space-filling effects, electrostatic steering, hydrogen bonding, hydrophobic interactions, and the like. Interactions between Fc variants contribute to the formation of stable heterodimers. A preferred mutational design is a "knob-in-hole" mutational design.

[0329] A "linker" is one or more amino acid residues inserted into an immunoglobulin domain that provide sufficient mobility to ensure proper protein folding and peptide stability. A "linker" is preferably (GGGGS)n, where n can be 0, 1, 2, 3, 4, 5, or more. If the linker sequence is too short, it may affect the folding of the higher-order structures of the two proteins, leading to mutual interference. If the linker sequence is too long, immunogenicity may be a concern, as the linker sequence itself becomes a new antigen.

[0330] The term "vector" means a polynucleotide molecule capable of transporting another polynucleotide connected thereto. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop, wherein an additional DNA segment can be connected. Another type of vector is a viral vector, in which an additional DNA segment can be connected to the viral genome. Some vectors can replicate autonomously (for example, bacterial vectors and additional mammalian vectors with bacterial replication origins) in the host cell introducing them. Other vectors (for example, non-additional mammalian vectors) can be integrated into the genome of the host cell after being introduced into the host cell, thereby replicating together with the host genome. In addition, some vectors can instruct the expression of the gene operably connected thereto. Usually, expression vectors useful in recombinant DNA technology are usually in the form of plasmids. The present invention can provide a vector comprising a nucleic acid fragment capable of encoding a trifunctional fusion protein.

[0331] By "effector function" is meant the biochemical events resulting from the interaction of an antibody Fc region with an Fc receptor or ligand. Effector functions include, but are not limited to, ADCC, ADCP, and CDC. "ADCC" or "antibody-dependent cell-mediated cytotoxicity" refers to a cell-mediated reaction in which nonspecific cytotoxic cells expressing FcγRs recognize bound antibody on a target cell and subsequently cause lysis of the target cell. ADCC is associated with binding to FcγRIIIa; increased binding to FcγRIIIa results in an increase in ADCC activity. As discussed herein, many embodiments of the present invention completely eliminate ADCC activity. "ADCP" or antibody-dependent cell-mediated phagocytosis refers to a cell-mediated reaction in which nonspecific cytotoxic cells expressing FcγRs recognize bound antibody on a target cell and subsequently cause phagocytosis of the target cell.

[0332] "Percent (%) amino acid sequence identity" with respect to protein sequences is defined as the percentage of amino acid residues in a candidate sequence that are identical with the amino acid residues in a specified (parent) sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. In some embodiments, two or more amino acid sequences are at least 80%, 85%, or 90% identical. In some embodiments, two or more amino acid sequences are at least 95%, 97%, 98%, 99%, or even 100% identical.

[0333] As used herein, the terms "fragment," "derivative," and "analog" refer to polypeptides that substantially retain the same biological function or activity as the antibodies of the present invention. A polypeptide fragment, derivative, or analog of the present invention may be (i) a polypeptide having one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) substituted, where such substituted amino acid residues may or may not be encoded by the genetic code, or (ii) a polypeptide having a substituent group in one or more amino acid residues, or (iii) a polypeptide formed by fusion of a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iv) a polypeptide formed by fusion of an additional amino acid sequence to the polypeptide sequence (e.g., a leader sequence or secretory sequence, or a sequence or proprotein sequence used to purify the polypeptide, or a fusion protein formed with a 6His tag). Based on the teachings herein, these fragments, derivatives, and analogs are well known to those skilled in the art.

[0334] As used herein, the terms "antibody fragment," "antigen-binding fragment," "targeting domain," or "antigen-binding domain" are used to refer to a portion of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv), fragments comprising a VL or VH domain, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies. Regardless of the structure, antibody fragments bind to the same antigen recognized by the intact antibody. Examples of targeting domains of the present invention include, for example, but are not limited to, Fab, Fab', F(ab')2, Fv fragments, single-chain Fv (scFv) fragments, and single-domain fragments.

[0335] "Fv" fragment is the smallest fragment of an antibody containing a complete target recognition and binding site. This region is composed of a dimer (VH-VL dimer) of a heavy chain and a light chain variable domain in tight non-covalent binding. In this configuration, the three CDRs of each variable domain interact to define the target binding site on the surface of the VH-VL dimer. Typically, six CDRs confer target binding specificity to antibodies. However, in some cases, even a single variable domain (or only half of an Fv comprising three CDRs specific for a target) can have the ability to recognize and bind to a target, although its affinity is lower than that of the entire binding site.

[0336] "Single-chain Fv" or "scFv" antibody binding fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Typically, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form a structure that is conducive to target binding.

[0337] A "single domain fragment" consists of a single VH or VL domain that exhibits sufficient affinity for coronavirus RBD. In a specific embodiment, the single domain fragment is camelized.

[0338] As used herein, the term "light chain constant region (CL)" includes the amino acid sequence CL derived from an antibody light chain. Preferably, the light chain constant region includes at least one of a constant kappa domain or a constant lambda domain.

[0339] As used herein, the term "heavy chain constant region (CH)" includes an amino acid sequence derived from an immunoglobulin heavy chain. A polypeptide comprising a heavy chain constant region comprises at least one of the following: a CH1 domain, a hinge region (e.g., an upper, middle, and / or lower hinge region) domain, a CH2 domain, a CH3 domain, or a variant or fragment thereof. It should be understood that the heavy chain constant region can be modified such that its amino acid sequence differs from that of a naturally occurring immunoglobulin molecule.

[0340] As used herein, the term "antigen" or "target antigen" refers to a molecule or portion of a molecule that can be bound by an antibody or antibody-like binding protein. The term further refers to a molecule or portion of a molecule that can be used in an animal to produce an antibody that can bind to an epitope of the antigen. A target antigen may have one or more epitopes. For each target antigen recognized by an antibody or by an antibody-like binding protein, the antibody-like binding protein can compete with an intact antibody that recognizes the target antigen.

[0341] As used herein, the term "linker" refers to one or more amino acid residues inserted into an immunoglobulin domain that provide sufficient mobility for the domains of the light chain and heavy chain to fold into an exchange dual variable region immunoglobulin. Suitable examples of linkers include single glycine (Gly) or serine (Ser) residues. The identity and sequence of the amino acid residues in the linker can vary depending on the type of secondary structural element to be achieved in the linker. Preferred linkers can be (GS)n, (G3S)n, (G4S)n (n is selected from 1-6).

[0342] As used herein, a "variant" of an antibody, antibody fragment, or antibody domain refers to an antibody, antibody fragment, or antibody domain that: (1) has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to the original antibody, antibody fragment, or antibody domain, and (2) specifically binds to the same target to which the original antibody, antibody fragment, or antibody domain specifically binds. It will be understood that where sequence identity is expressed in the form of "at least x% identical" or "at least x% identical," such embodiments include any and all numerical percentages equal to or above the lower limit. Furthermore, it will be understood that where an amino acid sequence is presented in this application, it should be construed as further disclosing or encompassing an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to that amino acid sequence.

[0343] TAA and its targeting domain

[0344] Tumor-associated antigens (TAAs) refer to antigenic molecules present on tumor cells or normal cells. Representative examples include (but are not limited to): embryonic proteins, glycoprotein antigens, squamous cell antigens, etc.

[0345] It should be understood that tumor-associated antigens (TAAs) can be specific to tumor cells. Some TAAs are not specific to tumor cells and can be synthesized in trace amounts by normal cells. However, these antigens are highly expressed when tumor cells proliferate, and are therefore called "tumor-associated antigens."

[0346] Representative tumor-associated antigens include (but are not limited to): CD33, CD30, HER2, CD22, CD79b, Nectin-4, BCMA, EGFR, CD19, tissue factor, folr1 (folate receptor α), CLDN18.2, TROP2, c-Met, PSMA, Muc1, PDL1, ROR1, MSLN, TNF-α, CD25, ENPP3, Axl, CD20, ROR2, GPNMB, CEACAM6, CD138, CA6, FUT3, CD56, CD37, HER3, GPRC5D, STING, CEA, CD205, B7H4, CTLA4, RNF43, CDH3, DPEP3, 5T4, ITGB6, EFNA4, B 7H3, CD228, Notch-3, CD46, CAIX, SLAMF6, ADAM9, GD3, TDGF1, SLAMF2, CLL-1, CD123, IGF-1R, FCRL5, TIM1, Globo H, CDH6, CD38, Ly6E, SLITRK6, GPR20, FGFR2, Muc16, CD51, SLAMF7, LAMP-1, CD74, CCR7, PTK7, SEZ6, CLDN 9. CLDN6, c-kit, LYPD3, TAA, PRL receptor, FGFR3, KAAG1, STEAP1, Flt3, LRRC15, CD44, CD70, EphA2, PDL2, p53, DLK1, ENB-FN, FOLR, CD45, DSG2, ALK, TRAIL, DDR1, EpCAM, VEGFR2, CD47, CD99, VEGF2, SSEA-4, DCLK1, OAcGD2, IL1RAP, ADAM17, CD7, CD73, ENO1, BSG, CD24, GLUT1, CXCR4, CD40, CD52, CD133, CD239, TAG72, EGFR VIII, PSCA, EphA2, NKG2D ligand, MCSP, LGR5, SSEA3, SLC34A2, Glypican-3, or a combination thereof.

[0347] CD28 and its targeting domain

[0348] The CD28 protein is a receptor located on the surface of T cells. When CD80 and CD86 bind to the CD28 protein receptor on the T cell surface, they provide a signal for initial T cell activation, producing a stimulatory effect: promoting T cell activation, proliferation, and differentiation; inducing upregulation of the IL-2 receptor on the T cell surface, increasing IL-2 mRNA transcription, and promoting cytokine secretion.

[0349] CD28 can regulate the Fas / FasL-mediated apoptosis pathway, among other things. After activation, T cells migrate to sites of inflammation and attack cells expressing antigens, destroying them directly or indirectly, thereby forming an immune response to defend against pathogen invasion and the development of malignant tumors. Simultaneously, after T cell activation, the immune negative regulatory factor CTLA-4 (cytotoxic T lymphocyte-associated protein 4, CD152) protein is transported from the cytoplasm to the cell membrane surface, where it competes with CD28 for binding to CD80 and CD86 (its binding ability is 20-100 times higher than that of CD28), thereby blocking CD28's co-stimulatory signal and inhibiting further activation of helper T cells (Th). It also promotes the proliferation of regulatory T cells (Tregs), keeping immune system activation within a normal range and preventing the occurrence of autoimmune reactions.

[0350] CD80, also known as B7.1, belongs to the B7 family of membrane-bound proteins. Other members of the B7 family include CD86, inducible co-stimulatory ligand (ICOS-L), programmed death-1 ligand (PD-L1), programmed death-2 ligand (PD-L2), B7-H3 and B7-H4.

[0351] CD80 is expressed on activated B lymphocytes, activated T lymphocytes, macrophages, peripheral blood monocytes, and dendritic cells. CD80 participates in immune regulation by delivering co-stimulatory or co-inhibitory responses through its ligand binding activity. It belongs to the immunoglobulin superfamily, and its receptors are CD28, CD152 (CTLA4), and PD-L1.

[0352] In the present invention, the preferred CD28 targeting domain is CD80. PD1 / PD-L1 and its targeting domain

[0353] In the present invention, the third targeting domain D3 is a structural element that targets PD1 / PD-L1. Preferably, the targeting domain is an anti-PD1 / PD-L1 antibody fragment that specifically binds to PD1 and / or PD-L1.

[0354] PD-1 (Programmed death 1) is a small protein present on the surface of immune cells that is involved in immune self-regulation. PD-L1 (Programmed cell death-Ligand 1) is the ligand for PD-1. PD-L1 binds to PD-1, activating the immune brakes and preventing the activation of immune cells (primarily T lymphocytes), thereby preventing T cell overactivation.

[0355] Inventive fusion protein

[0356] As used herein, the term "multispecific fusion protein" refers to a molecule comprising at least two targeting domains with different binding specificities, wherein at least one targeting domain specifically binds to a T cell surface antigen. In some embodiments, a multispecific inhibitor is a polypeptide comprising a scaffold and two or more immunoglobulin antigen-binding domains that target different antigens or epitopes. In certain embodiments, the multispecific fusion protein is a bispecific antibody or a trispecific antibody.

[0357] As used herein, the terms "bispecific antibody" and "bispecific fusion protein" are used interchangeably herein to refer to an antibody that can specifically bind to two different antigens (or epitopes).

[0358] As used herein, the terms "trispecific antibody of the present invention," "trispecific fusion protein of the present invention," and "3Sbody" are used interchangeably herein and relate to molecules comprising three targeting domains with three different binding specificities. Each targeting domain is capable of specifically binding to a target molecule and, when bound to the target molecule, inhibits the biological function of the target molecule. In some embodiments, the trispecific antagonist is a polymer molecule having two or more peptides. In some embodiments, the targeting domain comprises an antigen-binding domain or CDR of an antibody. In some embodiments, the targeting domain comprises a ligand or a fragment thereof that specifically binds to a target protein.

[0359] It should be understood that the trispecific fusion proteins of the present invention are essentially trispecific antibodies, antibody molecules that can simultaneously and specifically bind to three antigens. Based on symmetry, trispecific antibodies can be divided into structurally symmetrical and asymmetrical molecules. Based on the number of binding sites, bispecific antibodies can be divided into bivalent, trivalent, tetravalent, and multivalent molecules. The two trispecific antibodies of the present invention are structurally asymmetrical trivalent trispecific antibodies, and each trispecific antibody is monovalent for each specific target.

[0360] As used herein, the term "Fc fragment" or "Fc" refers to a portion of an antibody that does not have antigen binding activity but was initially observed to crystallize readily, and is therefore named an Fc fragment (for fragment crystallizability). This fragment corresponds to a paired CH2 and CH3 domain and is the portion of the antibody molecule that interacts with effector molecules and cells. The Fc fragments described herein can be derived from IgG1, IgG2, and IgG4 antibodies. For specific uses, a specific IgG subclass may be preferred. In addition, the effector function of an antibody can be increased or decreased by introducing one or more mutations into the Fc. Preferably, the Fc segment may contain L234A / L235A / G237A mutations to weaken the interaction between Fc and Fcγ receptors.

[0361] Furthermore, mutations can be introduced to form a knob-into-hole (KIH) structure. Its primary function is to promote heterodimerization of the two different heavy chains of a bispecific antibody. Its structural characteristics are as follows: of the two different heavy chains comprising the bispecific antibody, the CH3 region of one heavy chain mutates to form a protruding "knob" structure, while the CH3 region of the other heavy chain mutates to form a recessed "hole" structure. This knob-into-hole design facilitates the correct assembly of the two heterologous antibody heavy chains. Specifically, the T at position 366 of the CH3 domain of the first heavy chain is mutated to a W with a larger side chain volume, forming a protruding "knob" structure; simultaneously, the T at position 366 of the CH3 domain of the second heavy chain is mutated to an S, the L at position 368 is mutated to an A, and the Y at position 407 is mutated to a V. These three mutations all reduce the side chain volume, thereby forming a recessed "hole" structure. Furthermore, the S at position 354 of the first heavy chain was mutated to C, and the Y at position 349 of the second heavy chain was mutated to C. This allows the first and second heavy chains to add a covalent disulfide bond after forming a knob-in-hole structure, which can further stabilize the formed knob-in-hole structure.

[0362] Specifically, for the knob-hole format, in addition to introducing the T366W mutation in the first Fc to produce a "knob", the T366S, L368A and Y407V mutations can be introduced in the second Fc to produce a "hole". For the charge pair format, ionic interactions are stabilized by introducing connecting charge residues in opposite Fc domains, thereby facilitating heterodimerization. For example, D356K, E357K and D399K in the first Fc domain, and K370E, K409D and K439E mutations in the second Fc domain, or a combination thereof (residues are numbered according to the Kabat EU numbering system). In addition, cysteine ​​can be introduced to stabilize the pairing of the heterodimer, such as S234C in the first Fc and Y349C in the second Fc, or Y349C in the first Fc and S344C in the second Fc. Mutating H at position 435 of the "hole" chain to R and Y at position 436 to F can eliminate the binding of the "hole" chain to Protein A, so that the homologous dimer of the "hole" chain can be removed during the purification step.

[0363] The multispecific molecules of the present invention can include various structures selected from the group consisting of: asymmetric IgG-like antibodies (e.g., triomab / quadroma); knobs-into-holes antibodies; cross monoclonal antibodies (Cross MAb); electrostatic matching antibodies; LUZ-Y; chain exchange engineered domain (SEED) bodies; Fab exchange antibodies; symmetric IgG-like antibodies; two-in-one antibodies; cross-linked monoclonal antibodies, mAb2; Cov X-body; dual variable domain (DVD)-Ig fusion protein; IgG-like bispecific antibody; Ts2Ab; BsAb; scFv / Fc fusion; double (scFv)2-Fabs; F(ab)2 fusion protein; dual-action or Bis-Fab; Dock-and-Lock (DNL); Fab-Fv; scFv antibodies and diabodies (e.g., bispecific fusion proteins (BiTEs); tandem diabodies (Tandab); DARTs; single-chain diabodies; TCR-like antibodies; human serum albumin scFv fusion proteins, COMBODIES and IgG / non-IgG fusion proteins.

[0364] Encoding nucleic acid and expression vector

[0365] The present invention also provides polynucleotide molecules encoding the above-mentioned antibodies or fragments thereof. The polynucleotides of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or artificially synthesized DNA. DNA may be single-stranded or double-stranded. DNA may be a coding strand or a non-coding strand. The coding region sequence encoding the mature polypeptide may be identical to the coding region sequence of the antibody of the present invention or a degenerate variant. As used herein, "degenerate variant" in the present invention refers to a nucleic acid sequence encoding an amino acid sequence identical to that of the polypeptide of the present invention, but having a different coding region sequence.

[0366] The polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence encoding only the mature polypeptide; a coding sequence of the mature polypeptide and various additional coding sequences; a coding sequence of the mature polypeptide (and optional additional coding sequences) and non-coding sequences.

[0367] The term "polynucleotide encoding a polypeptide" may include a polynucleotide encoding the polypeptide, or may also include additional coding and / or non-coding sequences.

[0368] The present invention also relates to polynucleotides that hybridize to the above-mentioned sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that hybridize to the polynucleotides of the present invention under stringent conditions. In the present invention, "stringent conditions" refer to: (1) hybridization and elution at relatively low ionic strength and relatively high temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) the addition of a denaturing agent during hybridization, such as 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C; or (3) hybridization occurs only when the identity between the two sequences is at least 90%, preferably at least 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptides shown in SEQ ID NO.4 and SEQ ID NO.9.

[0369] The full-length nucleotide sequence of the antibody of the present invention or its fragments can generally be obtained by PCR amplification, recombinant methods, or artificial synthesis methods. One feasible method is to synthesize the relevant sequence by artificial synthesis, especially when the fragment length is relatively short. Generally, by first synthesizing multiple small fragments and then ligating them, very long fragments of sequence can be obtained. In addition, the coding sequence of the heavy chain can be fused with an expression tag (such as 6His) to form a fusion protein.

[0370] Once the relevant sequence is obtained, recombinant methods can be used to obtain it in large quantities. This is typically accomplished by cloning it into a vector, transferring it into cells, and then isolating the relevant sequence from the proliferated host cells using conventional methods. The biomolecules (nucleic acids, proteins, etc.) referred to in the present invention include biomolecules in isolated form.

[0371] Currently, DNA sequences encoding proteins of the present invention (or fragments thereof, or derivatives thereof) can be obtained entirely by chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. In addition, mutations can also be introduced into protein sequences of the present invention by chemical synthesis.

[0372] The present invention also relates to vectors comprising the above-mentioned appropriate DNA sequence and appropriate promoter or control sequence. These vectors can be used to transform appropriate host cells to enable them to express proteins.

[0373] Host cells can be prokaryotic cells, such as bacterial cells; lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells, such as yeast; insect cells such as Drosophila S2 or Sf9; and animal cells such as CHO, COS7, and 293 cells.

[0374] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryotic organism such as Escherichia coli, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated using the CaCl2 method, using procedures well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryotic organism, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0375] The obtained transformants can be cultured using conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cell used, the culture medium used can be selected from various conventional culture media. Culture is carried out under conditions suitable for the growth of the host cells. After the host cells grow to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature conversion or chemical induction), and the cells are cultured for a period of time.

[0376] The recombinant polypeptide in the above method can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein can be isolated and purified by various separation methods utilizing its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to, conventional renaturation treatment, treatment with a protein precipitant (salting out method), centrifugation, osmotic sterilization, ultrafiltration, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations of these methods.

[0377] The antibodies of the present invention may be used alone or in combination with a detectable label (for diagnostic purposes), a therapeutic agent, a PK (protein kinase) modifying moiety, or any combination of these.

[0378] Detectable labels for diagnostic purposes include, but are not limited to, fluorescent or luminescent labels, radioactive labels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes capable of producing a detectable product.

[0379] Therapeutic agents that can be conjugated include, but are not limited to, insulin, IL-2, interferon, calcitonin, GHRH peptide, intestinal peptide analogs, albumin, antibody fragments, cytokines, and hormones.

[0380] Composition

[0381] The present invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition comprising the above-mentioned antibody or active fragment thereof or fusion protein thereof, and a pharmaceutically acceptable carrier. Generally, these substances can be formulated in a non-toxic, inert and pharmaceutically acceptable aqueous carrier medium, wherein the pH is generally about 5-8, preferably about 6-8, although the pH value may vary depending on the properties of the formulated substance and the condition to be treated. The formulated pharmaceutical composition can be administered by conventional routes, including (but not limited to): oral, respiratory, intratumoral, intraperitoneal, intravenous, or topical administration.

[0382] The pharmaceutical composition of the present invention can be used to treat cancer / tumor, especially solid tumor, especially solid tumor with high expression of MUC17.

[0383] The pharmaceutical composition of the present invention contains a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-mentioned monoclonal antibody of the present invention (or its conjugate) and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical preparation should match the mode of administration. The pharmaceutical composition of the present invention can be prepared in the form of an injection, for example, using physiological saline or an aqueous solution containing glucose and other adjuvants by conventional methods. Pharmaceutical compositions such as injections and solutions are preferably manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 1 μg / kg body weight to about 10 mg / kg body weight per day. In addition, the pharmaceutical composition of the present invention can also be used in conjunction with other therapeutic agents.

[0384] When using a pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to a mammal, wherein the safe and effective amount is generally at least about 10 μg / kg body weight, and in most cases does not exceed about 8 mg / kg body weight. Preferably, the dose is about 10 μg / kg body weight to about 1 mg / kg body weight. Of course, the specific dose should also take into account factors such as the route of administration and the patient's health status, which are all within the skill of a skilled physician.

[0385] application

[0386] The present invention relates to a method for preventing, treating and / or detecting cancer / tumor or autoimmune disease. The method comprises administering an effective amount of a multi-specific fusion protein or specific antibody of the present invention to a subject in need thereof. On the other hand, the method for preventing, treating and / or detecting cancer / tumor comprises administering an effective amount of one or more expression vectors expressing the multi-specific antibody of the present invention to a subject in need thereof. In another preferred embodiment, the cancer / tumor is a TAA-associated cancer / tumor, a disease / disorder characterized by an increased number / proportion / activity of cells expressing the TAA, compared to the number / proportion / activity of cells expressing the TAA in the absence of the disease / disorder.

[0387] Among them, tumor-associated antigens (TAA) can be tumor-associated antigens such as CD33, CD30, HER2, CD22, CD79b, Nectin-4, BCMA, EGFR, CD19, tissue factor, folr1 (folate receptor α), CLDN18.2, TROP2, c-Met, PSMA, Muc1, PDL1, ROR1, MSLN, TNF-α, CD25, ENPP3, Axl, CD20, ROR2, GPNMB, CEACAM6, CD 138, CA6, FUT3, CD56, CD37, HER3, GPRC5D, STING, CEA, CD205, B7H4, CTLA4, RNF43, CDH3, DPEP3, 5T4, ITGB6, EFNA4 , B7H3, CD228, Notch-3, CD46, CAIX, SLAMF6, ADAM9, GD3, TDGF1, SLAMF2, CLL-1, CD123, IGF-1R, FCRL5, TIM1, Globo H, CDH6, CD38, Ly6E, SLITRK6, GPR20, FGFR2, Muc16, CD51, SLAMF7, LAMP-1, CD74, CCR7, PTK7, SEZ6, CLDN 9. CLDN6, c-kit, LYPD3, TAA, PRL receptor, FGFR3, KAAG1, STEAP1, Flt3, LRRC15, CD44, CD70, EphA2, PDL2, p53, DLK1, ENB-FN, FOLR, CD45, DSG2, ALK, TRAIL, DDR1, EpCAM, VEGFR2, CD47, CD99, VEGF2, SSEA-4, DCLK1, OAcGD2, IL1RAP, ADAM17, CD7, CD73, ENO1, BSG, CD24, GLUT1, CXCR4, CD40, CD52, CD133, CD239, TAG72, EGFR VIII, PSCA, EphA2, NKG2D ligand, MCSP, LGR5, SSEA3, SLC34A2, Glypican-3, or a combination thereof.

[0388] The cancer / tumor includes solid tumors and hematological tumors. Preferably, it is selected from the group consisting of colorectal, breast, ovarian, pancreatic, gastric, prostate, kidney, cervical, bone marrow cancer, lymphoma, leukemia, thyroid, endometrial, uterine, bladder, neuroendocrine, head and neck, liver, nasopharyngeal, testicular, small cell lung cancer, non-small cell lung cancer, melanoma, basal cell skin cancer, squamous cell skin cancer, dermatofibrosarcoma protuberans, Merkel cell carcinoma, glioblastoma, glioma, sarcoma, mesothelioma, and myelodysplastic syndrome.

[0389] The main advantages of the present invention include:

[0390] (a) The CD80 multifunctional fusion protein of the present invention can simultaneously target the tumor-associated antigen TAA and the co-stimulatory molecule CD28 expressed by T cells. Only by binding to the tumor TAA can it activate the CD28 signaling pathway, thereby ensuring the clinical safety of the CD28 agonist.

[0391] (b) The CD80 multifunctional fusion protein of the present invention enriches candidate drugs in tumors through anti-TAA antibodies, and utilizes the natural weak affinity of CD80 recombinant protein with CD28 / CTLA4 / PD-L1 to further target tumors through TAA; binds to the T cell co-stimulatory molecule CD28, activates the CD28 pathway to activate T cells; binds to the T cell inhibitory molecule CTLA4, eliminates the inhibition of CTLA4 on T cells, and further activates T cells; kills and eliminates tumor cells through multiple immune effector mechanisms, can stimulate stronger and more effective killing activity on tumors; and has good in vitro safety.

[0392] (c) The invented CD80 multifunctional fusion protein has high purity and good physical and chemical properties, avoiding problems such as aggregates, degradation fragments and incomplete assembly that are prone to occur during the production and storage of antibodies, and is conducive to industrial production.

[0393] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the scope of the present invention. The experimental methods in the following examples, for which detailed conditions are not specified, were generally performed under conventional conditions such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer. Unless otherwise stated, percentages and parts are calculated by weight.

[0394] Material

[0395] The schematic structural diagrams of the multi-specific fusion proteins of the present invention used in the examples of the present invention are shown in Figures 1 to 17.

[0396] Part 1: Construction of Trop2 nanobody.

[0397] Example 1: Preparation of Trop2 recombinant protein and control antibody

[0398] Trop2-hFc recombinant protein (protein ID QP1173), produced in-house, consists of a Trop2 extracellular domain sequence (selected from amino acids 27-274 of UNIPROT, SEQ ID NO: P09758) fused to Fc (see the following sequence). This protein was inserted into the eukaryotic expression vector pQD to construct an expression clone. The protein was transiently transfected into 293E cells and purified.

[0399] The sequence of QP1173:Trop2-FC is as follows:

[0400] The Trop2-flag-his recombinant protein (protein ID QP1174) is produced in-house and consists of a Trop2 extracellular domain sequence (selected from amino acids 27-274 of UNIPROT, SEQ ID NO: P09758) fused to the flag-his region. The sequence is shown below. This protein was inserted into the eukaryotic expression vector pQD to construct an expression clone. The protein was transiently transfected into 293E cells and purified.

[0401] The sequence of QP1174:Trop2-flag-his is as follows:

[0402] Example 2: Alpaca immunization and construction of phage immune library

[0403] Alpaca immunization: QP1174:Trop2-flag-his recombinant protein was used as the antigen to immunize two healthy alpacas (vicugna pacos, alpaca, lama pacos). The first immunization was with Freund's complete adjuvant (CFA) and 0.3 mg of antigen protein. The second to fifth immunizations were with Freund's incomplete adjuvant (IFA) emulsified and mixed with the antigen and then injected subcutaneously at multiple points, with 0.2 mg of antigen protein.

[0404] Library construction: 50 ml of peripheral blood was collected to separate peripheral blood mononuclear cells (PBMCs), and total RNA from PBMCs was extracted. RT-PCR was used to reverse transcribe the cells into cDNA. Primers were designed to PCR amplify the VHH gene and construct an immune library. Peripheral blood lymphocyte isolation: 50 ml of peripheral blood was collected, and PBMCs were isolated according to the instructions for lymphocyte separation solution. Total RNA from PBMCs was extracted using TRIzol reagent. Reverse transcription, see III First-Strand Synthesis System for RT-PCR Instructions: 8 μg of RNA was transcribed. After the first round of nested PCR, a VHH fragment of approximately 750 bp was recovered by gel tapping for the second round of nested PCR. The phage library was constructed using pComb3XSS. The pComb3XSS phagemid vector was digested with SfiI to form two large fragments: 1672 bp (SS stuffer) and 3301 bp (vector target fragment). This vector carries a His tag and an HA tag for easy purification and detection. Ligation of the vector and target fragment: The vector and target fragment were each digested with SfiI overnight at 50°C, followed by gel tapping to recover the target fragment. The molar ratio of vector to VHH was 1:3. A total of 10 electroporation transformations were performed. Immediately after electroporation, 1 mL of 2YT medium (preheated at 37°C) was added to the electroporation cup for recovery. The electroporation product was aspirated and the electroporation cup was washed with 2YT medium. A total of 100 mL of recovery product was obtained. The product was recovered at 37°C, 180 rpm for 45 minutes. 100 μL was taken for gradient dilution to 10-3 and 10-4 to determine the library capacity. The product was spread on a 90 mm plate. The rest was centrifuged, 8 mL of 2YT was added for resuspending, and the product was spread on 8 200 mm plates. The next day, there were 128 clones in the NB088 project 10-4 on the plate for determining the library capacity, with a library capacity of 1.28×10 9 (128*1000*10 4 ), NB089 project 10-4 has a total of 175 clones, and the library capacity is 1.75×10 9 (175*1000*10 4 ).

[0405] Example 3: Screening and identification of nanobody immune library

[0406] Two rounds of screening were performed using the Trop2 recombinant proteins QP1173 and QP1174.

[0407] First round (1st) panning: Coat the immunotube with antigen at 5 ng / ul in 1 ml at 4°C overnight. Blocking: Block the immunotube with 2% MPBS at 37°C for 1 hour. Subtraction: Add 1800ul of 2% MPBS, then 200ul of input phage, and the subtraction antigen at a final concentration of 50 ng / ul, and rotate at room temperature for 1 hour. Binding: Transfer the supernatant to the antigen-coated immunotube and rotate at room temperature for 1 hour. Wash: Wash the immunotube 8 times with 1xPBST and 4 times with 1xPBS. Elution: Elute 800ul of 100mM TEA at room temperature for 10 minutes. Neutralization: Transfer the eluate to a 1.5ml EP tube and add 400ul of 1M Tris, pH 7.4. Infection: Add the neutralized eluted phage to 10ml of TG1 at OD600 = 0.5. Incubate at 37°C for 4 minutes. Titer the supernatant and plate the remaining bacterial solution on a large plate. Incubate at 37°C overnight.

[0408] Packaging the second input phage: Scrape the cells from 2xTY medium and inoculate them into 50ml of 2xTY medium with amp and 1% glucose to an OD of ~0.1. Grow at 37°C at 200 rpm for ~1 hour 20 minutes, to an OD of ~0.4-0.6. Add 500µl of M13KO7, infect at 37°C for 40 minutes, centrifuge, and remove the supernatant. Resuspend the pellet in 100ml of 2xTY medium with amp and kana. Incubate at 30°C at 200 rpm overnight. Second input phage precipitation: Centrifuge the overnight bacterial culture at 4200 rpm for 15 minutes. Remove 40ml of the supernatant and add 10ml of PEG / NaCl. Mix well and place on ice for 20 minutes. Centrifuge at 4200 rpm for 15 minutes. Discard the supernatant. Centrifuge briefly and aspirate the supernatant. Resuspend the pellet in 1ml of 1xPBS. Centrifuge at 13,000 rpm for 10 minutes. Transfer the supernatant to a new 1.5EP tube. Use for subsequent washing, or add 0.5 ml of 50% glycerol and freeze at -80°C.

[0409] Second round (2 nd) Panning: Coat the antigen in an immunotube at 2 ng / ul in 1 ml at 4°C overnight. Blocking: Block the immunotube with 2% MPBS at 37°C for 1 hour. Subtraction: Add 1800 μl of 2% MPBS, then 200 μl of input phage, and the subtraction antigen at a final concentration of 50 ng / ul. Rotate at room temperature for 1 hour. Binding: Transfer the supernatant to the antigen-coated immunotube and rotate at room temperature for 1 hour. Wash: Wash the immunotube 8 times with 1xPBST and 4 times with 1xPBS. Elution: Elute 800 μl of 100 mM TEA at room temperature for 10 minutes. Neutralization: Transfer the eluate to a 1.5 ml EP tube and add 400 μl of 1 M Tris, pH 7.4. Infection: Add the neutralized eluted phage to 10 ml of TG1 at an OD600 of 0.5. Incubate at 37°C for 4 minutes. Titer the cells and plate the remaining bacterial solution on a large plate. Incubate at 37°C overnight.

[0410] Packaging the 2nd output phage: Scrape the cells from 2xTY medium and inoculate them into 50ml of 2xTY medium with amp and 1% glucose to an OD of ~0.1. Grow at 37°C at 200 rpm for ~1 hour 20 minutes, to an OD of ~0.4-0.6. Add 500µl of M13KO7, infect at 37°C for 40 minutes, centrifuge, and remove the supernatant. Resuspend the pellet in 100ml of 2xTY medium with amp and kana. Incubate at 30°C at 200 rpm overnight. 2nd output phage precipitation: Centrifuge the overnight culture at 4200 rpm for 15 minutes. Remove 40ml of the supernatant and add 10ml of PEG / NaCl. Mix well and place on ice for 20 minutes. Centrifuge at 4200 rpm for 15 minutes. Discard the supernatant. Centrifuge briefly and aspirate the supernatant. Resuspend the pellet in 1ml of 1xPBS. Centrifuge at 13,000 rpm for 10 minutes. Transfer the supernatant to a new 1.5EP tube. Use for subsequent washing, or add 0.5 ml of 50% glycerol and freeze at -80°C.

[0411] The panning strategy and results are shown below:

[0412] Table 1 Panning strategy

[0413] Table 2 Panning results

[0414] Immune library screening and identification: After two rounds of panning, single clones were selected for phage ELISA to screen for positive clones that bind to Trop2. Positive clones were sequenced to obtain the VHH sequence of the Trop2 antibody. The ELISA screening method is as follows:

[0415] Phage packaging and soluble expression: Select clones (P1-P4) in four 96-well plates. Inoculate a single clone in 180 μl of 2YT+amp and incubate at 37°C for 2 hours. For phage packaging: Add 100 μl of 2YT+M13K07 to 30 μl of culture medium, incubate at 37°C for 1 hour, then add 50 μl of 2YT+amp+kan and shake overnight at 30°C. Centrifuge and collect the supernatant for ELISA. For soluble expression: After incubating 120 μl of culture medium with shaking for 3 hours, add 60 μl of 2YT and 1 M IPTG to a final concentration of 1 mM. Shake overnight at 30°C. Centrifuge and collect the supernatant for ELISA.

[0416] Phage ELISA: Coat with QP1174 (2 ng / μl, 60 μl / well) overnight at 4°C, then wash three times with PBS. Block with 200 μl / well of 5% milk for 1 hour at room temperature. Add 20 μl of phage supernatant and 40 μl of 2% MPBS, mix, and incubate at room temperature for 1 hour. Wash the plate three times with PBST, add 60 μl of anti-M13 HRP (Sino Biological, 11973-MM05T-200), and incubate at room temperature for 1 hour. Wash the plate five times with PBST, develop with 100 μl / well of TMB for 10 minutes at room temperature, and terminate the reaction with 100 μl / well of 2 M H₂SO₄. QP1174-binding positive clones were sent for sequencing.

[0417] Positive clones that bind to Trop2 were selected and sequenced to obtain the VHH sequence of the anti-Trop2 antibody.

[0418] Example 4: Nanobody construction of FC fusion protein, cloning, expression, and purification of protein

[0419] Clone Design and Construction: The C-terminus of the anti-Trop2 nanobody VHH was fused to human IgG1 Fc to create the nanobody Fc fusion protein anti-Trop2 VHH-Fc. A eukaryotic expression plasmid was constructed for transient expression in HEK293 cells and the protein was purified by protein A affinity chromatography and other methods. The protein is designated QP3198.

[0420] Protein expression: 293E cell culture density was maintained at 0.2-3×10 6 / ml, and culture in the maintenance medium (GIBCO Freestyle 293 expression medium). The day before transfection, the cells to be transfected were centrifuged and the medium was changed, and the cell density was adjusted to 0.5-0.8×10 6 / ml. On the day of transfection, the density of 293E cells was 1-1.5×10 6Prepare the plasmid and transfection reagent PEI at a ratio of 100 μg / 100 ml of cells and a 2:1 PEI to plasmid mass ratio. Mix the plasmid and PEI well and let it sit for 15 minutes (no more than 20 minutes). Slowly add the plasmid and PEI mixture to the 293E cells and incubate in a shaker at 8% CO2, 120 rpm, and 37°C. On the fifth day of transfection, centrifuge at 4700 rpm for 20 minutes to collect the supernatant.

[0421] Protein A affinity chromatography purification: Pass the column with the equilibration solution for at least 3 CVs, using an actual volume of 20 ml. Ensure that the pH and conductivity of the final solution flowing out of the instrument are consistent with those of the equilibration solution. Flow rate: 1 ml / min. Pass the supernatant of the centrifuged culture medium through the column, loading 40 ml of the sample at a flow rate of 0.33 ml / min. Pass the column with the equilibration solution for at least 3 CVs, using an actual volume of 20 ml. Ensure that the pH and conductivity of the final solution flowing out of the instrument are consistent with those of the equilibration solution. Flow rate: 0.33 ml / min. Pass the column with the eluent. Collect the elution peak (PAC-EP) when the UV280 value rises to 15 mAU and stop collecting when the UV280 value drops to 15 mAU. Flow rate: 1 ml / min. After sample collection, adjust the PAC-EP to neutral with pH adjustment solution.

[0422] Example 5: ELISA detection of the binding of Trop2 nanobody Fc fusion protein to human Trop2 protein

[0423] Experimental steps: Plate coating: QP1174 (Trop2–FC) 1 μg / ml, 60 μl / well, overnight at 4°C, PBS*3; Blocking: 5% milk / PBS, 200 μl / well, incubated at 25°C for 1 hour; Antigen: PD-1 nanoantibodies PD-1 VHH-FC QP3120 and QP3126, respectively, were incubated at 25 μg / ml in 8 5-fold dilutions, 60 μl / well, 25°C for 1 hour, PBST*5; Secondary antibody: Anti-Fab-HRP, 1:8000 dilution, 60 μl / well, 25°C for 1 hour, PBST*5; Color development: TMB 100 μl / well, 5-10 minutes, 2M H2SO4 to terminate the reaction, and read at 450 nm. The results are shown in Figure 18 , showing that the Trop2 nanoantibody Fc fusion protein binds to the human Trop2 protein QP1174.

[0424] Example 6: Cell-based ELISA detection of the binding of Trop2 nanobody Fc fusion protein to cell lines naturally expressing Trop2

[0425] The native Trop2-expressing cell lines A431 and MCF7 were plated at 1.5E5 cells / well for A431 and 1E5 cells / well for MCF7. Blocking was performed with 250 μl / well of 3% BSA at room temperature for 1 hour. Antibody was added at a starting concentration of 20 μg / ml, diluted 1:5, incubated at 4°C for 1 hour, and washed three times with PBS. Secondary antibody, anti-hFc HRP, was added at a 1:5000 dilution, 60 μl / well, incubated at 4°C for 1 hour, and washed five times with PBS. TMB was used for color development and H2SO4 was used to terminate the reaction. A microplate reader was set to read at 450 nm. As shown in the figure, the Trop2 VHH-Fc fusion protein QP3198 bound to both the A431 and MCF7 cell lines, which naturally express Trop2.

[0426] The results are shown in FIG19A and FIG19B .

[0427] Example 7: Humanized Nanobody Design, Cloning, Expression, and Protein Purification

[0428] Humanized Nanobody Design: By comparing the IMGT human antibody heavy and light chain variable region germline gene database and MOE software, the highly homologous heavy and light chain variable region germline genes of QP3198 were used as templates. The CDRs of the alpaca nanobody were transplanted into the corresponding human templates, forming a variable region sequence with the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. Some important amino acid residues were then selected for back mutation combinations. The amino acid residues were determined and annotated using the Kabat numbering system. Primers were designed for PCR to construct the VH gene segments of each humanized antibody, which were then homologously recombined with the expression vector pQD containing a signal peptide and constant region gene (FC) segment to construct the full-length antibody expression vector VH-FC-pQD. Using the online software DNAWorks (v3.2.4) (http: / / helixweb.nih.gov / dnaworks / ), multiple primers were designed to synthesize the VH / VK gene fragments required for recombination: 5'-30 bp signal peptide + VH + 30 bp FC-3'. Following the instructions for TaKaRa's Primer STAR GXL DNA polymerase, these primers were used for two-step PCR amplification to obtain the VH / VK gene fragments required for recombination. The expression vector pQD containing the signal peptide and constant region gene (FC) fragments was constructed and digested using restriction enzymes, such as BsmBI, which have different recognition sequences and cleavage sites. The vector was digested with BsmBI and recovered from the gel for later use. The expression vector VH-FC-pQD was reconstructed. The VH gene fragment containing the required recombination and the expression vector pQD (with signal peptide and constant region gene (FC) fragment) recovered by BsmBI digestion were added to DH5a competent cells at a ratio of 3:1, ice-bathed at 0°C for 30 minutes, heat-shocked at 42°C for 90 seconds, and added with 5 times the volume of LB medium. The cells were incubated at 37°C for 45 minutes, coated on LB-Amp plates, and cultured at 37°C overnight. Single clones were picked and sent for sequencing to obtain the target clones.

[0429] The sequences of the light and heavy chain variable regions and protein expression numbers of the humanized clones are shown in Table 3 below, where all antibodies were fused with human IgG1-FC constant regions at their C-termini.

[0430] Alpaca Trop2 antibody QP3198 sequence:

[0431] Humanized Trop2 antibody QP645, QP646, QP647, QP648 sequences:

[0432] The CDR region sequence of the Trop2 VHH is:

[0433] As shown in Table 3, the humanized Trop2 VHH sequences were fused with human IgG1 FC to construct clones. The constructed plasmids were expressed in HEK293 cells and purified by protein A affinity chromatography to obtain a total of four humanized Trop2 VHH-FC fusion proteins QP645-QP648, among which the Trop2 VHH sequences are shown in SEQ ID NOs:43-46, respectively, and the human IgG1 FC segment sequence is shown in SEQ ID NO:23.

[0434] Table 3 QP3198 humanized clones

[0435] Protein expression: 293E cell culture density was maintained at 0.2-3×10 6 / ml, and culture in the maintenance medium (GIBCO Freestyle 293 expression medium). The day before transfection, the cells to be transfected were centrifuged and the medium was changed, and the cell density was adjusted to 0.5-0.8×10 6 / ml. On the day of transfection, the density of 293E cells was 1-1.5×10 6 Prepare the plasmid and transfection reagent PEI at a ratio of 100 μg / 100 ml of cells, using a 2:1 PEI to plasmid mass ratio. Mix the plasmid and PEI well and let it sit for 15 minutes (no more than 20 minutes). Slowly add the plasmid and PEI mixture to the 293E cells and incubate in a shaker at 8% CO2, 120 rpm, and 37°C. On the fifth day of transfection, centrifuge at 4700 rpm for 20 minutes to collect the supernatant.

[0436] Protein A affinity chromatography purification: Pass the column with the equilibration solution for at least 3 CVs, using an actual volume of 20 ml. Ensure that the pH and conductivity of the final solution flowing out of the instrument are consistent with those of the equilibration solution. Use a flow rate of 1 ml / min. Pass the supernatant of the centrifuged culture medium through the column, loading 40 ml of the sample at a flow rate of 0.33 ml / min. Pass the column with the equilibration solution for at least 3 CVs, using an actual volume of 20 ml. Ensure that the pH and conductivity of the final solution flowing out of the instrument are consistent with those of the equilibration solution. Use a flow rate of 0.33 ml / min. Pass the column with the eluent, starting collection of the elution peak (PAC-EP) when the UV280 rises to 15 mAU and stopping collection when the UV280 drops to 15 mAU. Use a flow rate of 1 ml / min. After sample collection, adjust the PAC-EP to neutral with a pH adjustment solution.

[0437] Example 8: ELISA detection of the binding of Trop2 nanobody Fc fusion protein to human Trop2 protein

[0438] Experimental procedures: Plate coating: QP1174 (Trop2–FC) 1 μg / ml, 60 μl / well, overnight at 4°C, PBS*3; Blocking: 5% milk / PBS, 200 μl / well, incubated at 25°C for 1 hour; Antigen: PD-1 nanobody PD-1 VHH-FC QP3120 and QP3126, respectively, incubated at 25 μg / ml in 8 5-fold dilutions, 60 μl / well, 25°C for 1 hour, PBST*5; Secondary antibody: Anti-Fab-HRP, 1:8000 dilution, 60 μl / well, 25°C for 1 hour, PBST*5; Color development: TMB 100 μl / well, 5-10 minutes, 2 M H2SO4 to terminate the reaction, and read at 450 nm. The results, as shown in Figure 20 and Table 4, show that the Trop2 nanobody Fc fusion protein binds to human Trop2 protein.

[0439] Table 4: EC50 values ​​of humanized Trop2 nanobody Fc fusion protein binding to human Trop2 protein detected by ELISA

[0440] Example 9: Cell-based ELISA detection of the binding of Trop2 nanobody Fc fusion protein to cell lines naturally expressing Trop2

[0441] The native Trop2-expressing cell lines A431 and MCF7 were plated at 1.5E5 cells / well for A431 and 1E5 cells / well for MCF7. Blocking was performed with 250 μl / well of 3% BSA at room temperature for 1 hour. Antibody was added at a starting concentration of 20 μg / ml, diluted 1:5, incubated at 4°C for 1 hour, and washed three times with PBS. Secondary antibody, anti-hFc HRP, was added at a 1:5000 dilution in 60 μl / well for 1 hour at 4°C, followed by five washes with PBS. TMB was used for color development and H₂SO₄ was used to terminate the reaction. A microplate reader was set to read at 450 nm. As shown in Figures 21A and 21B and Tables 5 and 6, the Trop2 VHH-Fc fusion protein QP3198 bound to both the A431 and MCF7 cell lines, which naturally express Trop2.

[0442] Table 5: Cell-based ELISA detection of the binding of Trop2 humanized nanobody Fc fusion protein to naturally expressing human Trop2 cells A431

[0443] Table 6: Cell-based ELISA detection of the binding of Trop2 humanized nanobody Fc fusion protein to naturally expressing human Trop2 cells MCF-7

[0444] Part II: CD80 fusion protein

[0445] Example 10: Fusion protein molecular cloning design

[0446] 1.1 Cloning construction method: Design clones as shown in Table 7 to construct full-length expression vectors.

[0447] Table 7

[0448] The plasmid names indicate the connection of the domains from N-terminus to C-terminus, which can be directly connected or connected through a peptide linker.

[0449] The specific amino acid sequence of the above plasmid is as follows:

[0450] As described in Table 7 above

[0451] CD80 is the extracellular domain of CD80 and has the following amino acid sequence:

[0452] Trop2 VHH (QD648) has the amino acid sequence shown below:

[0453] Among them, the CDR sequence of Trop2 VHH (QD648) is:

[0454] PD1 VHH (QD959) refers to patent application CN115991771A, in which the sequence of the FR region is slightly different and has the following amino acid sequence:

[0455] Among them, the CDR sequence of PD1 VHH (QD959) is:

[0456] PD1 VHH (QD960) refers to patent application CN115991771A, in which the sequence of the FR region is slightly different and has the following amino acid sequence:

[0457] Among them, the CDR sequence of PD1 VHH (QD960) is:

[0458] PD-L1 VHH (QD322) refers to patent application CN112142842B, which has the following amino acid sequence:

[0459] Necitumumab is a monoclonal antibody targeting EGFR. Referring to patent application WO2005090407A1, it has the following VH and VL:

[0460] Anti-PD-L1 (3280A) is a monoclonal antibody named atezolizumab, with the trade name Tecentriq, and is disclosed in patent application US8217149B2, which has the following VH and VL:

[0461] Nivolumab is an anti-PD-1 monoclonal antibody. Referring to patent application CN103059138B, it has the following VH and VL:

[0462] Anti-PD-1 (CS1003), referring to patent application US11414487B2, has VH and VL as shown below:

[0463] The linker sequences used between the domains are as follows:

[0464] FC has the following amino acid sequence:

[0465] Compared with human IgG Fc, it has amino acid mutations C223S, L235A, D265A, and P331S, where the amino acid positions are determined according to the EU numbering system.

[0466] The EGFR (AFM24) antibody refers to patent application WO2020043670A1, which has the following VH and VL:

[0467] Primer design: Multiple primers were designed using the online software DNAWorks (v3.2.4) (http: / / helixweb.nih.gov / dnaworks / ) to synthesize the gene fragments required for recombination.

[0468] 1.2 Fragment splicing:

[0469] According to the operating instructions of Primer STAR GXL DNA polymerase of TaKaRa Company, the gene fragment required for recombination was amplified by PCR using the multiple primers designed above.

[0470] Step 1 PCR: PCR reaction system 50 μL including 10 μL PrimerSTAR GXL Buffer (5×); 4 μL dNTP Mixture (2.5 mmol·L -1 ), 1 μL of each primer, and 1 μL of PrimeSTAR GXL DNA Polymerase. PCR reaction conditions were 98°C for 2 min, 98°C for 20 s, 55°C for 15 s, and 68°C for 30 s, for 30 cycles; and 68°C for 5 min.

[0471] Second PCR: Using the first PCR product as a template, perform PCR amplification with the first and last primers under the same conditions as the first step. PCR is then constructed to amplify the target fragment.

[0472] 1.3 Construction and enzyme digestion of expression vector pQD:

[0473] Using some special restriction endonucleases, such as BsmBI, which have different characteristics of recognition sequence and enzyme cleavage site, the expression vector pQD (with signal peptide) was designed and constructed, and the vector was digested by BsmBI and the gel was cut and recovered for later use.

[0474] 1.4 Recombinant construction of expression vector:

[0475] The recombinant target gene fragment and the expression vector pQD (with signal peptide fragment) recovered by BsmBI digestion were added to DH5α competent cells at a ratio of 3:1, ice-bathed at 0°C for 30 min, heat-shocked at 42°C for 90 s, added with 5 times the volume of LB medium, incubated at 37°C for 45 min, coated on LB-Amp plates, cultured at 37°C overnight, and single clones were picked for sequencing to obtain the target clones.

[0476] Example 11: Fusion protein expression and purification

[0477] 2.1 Protein expression

[0478] Adjust the cell density to 1 × 10 cells per well using 293E cells. 6 Prepare the plasmid and transfection reagent PEI at a concentration of 100 μg / 100 ml of cells, using a 2:1 PEI to plasmid mass ratio. Mix the plasmid and PEI well and let it sit for 15 minutes. Slowly add the plasmid and PEI mixture to the 293E cells and incubate in a shaker at 8% CO2, 120 rpm, and 37°C. On the fifth or sixth day of transfection, centrifuge the cells at 4700 rpm for 20 minutes to collect the supernatant for purification.

[0479] 2.2 Protein affinity chromatography:

[0480] After high-speed centrifugation, the cell culture supernatant was collected and affinity chromatography was performed using a GE Protein A column. The chromatography used an equilibrium buffer of 1× PBS (pH 7.4). After the cell supernatant was loaded and bound, it was washed with PBS until the UV light returned to baseline. The target protein was then eluted with 0.1M glycine (pH 3.0) elution buffer and the pH was adjusted to neutral with Tris for storage.

[0481] 2.3 Protein ion exchange chromatography:

[0482] Adjust the pH of the affinity chromatography product to 1-2 pH units below or above the pI and dilute appropriately to control the sample conductivity below 5ms / cm. Using an appropriate pH buffer (e.g., phosphate buffer, acetate buffer, etc.), perform a NaCl gradient elution under the corresponding pH conditions using conventional ion exchange chromatography methods such as anion exchange or cation exchange. Select the collection tube containing the target protein based on SDS-PAGE and store it in a single tube.

[0483] 2.4 Protein size exclusion chromatography:

[0484] The ion exchange product is concentrated by ultrafiltration and then subjected to size exclusion chromatography, such as using GE's Superdex 200 gel, to remove possible aggregates and other components, yielding a highly pure target product. The resulting protein can be analyzed for purity by SDS-PAGE and SEC-HPLC. Protein concentration is determined by UV spectrophotometry. Endotoxin levels are strictly controlled throughout the purification process, with the purified protein containing less than 1 EU / mg of endotoxin.

[0485] The results showed that the fusion proteins were produced in high yield in 293E cells, with HPLC-SEC purity greater than 95%, indicating that the proteins were manufacturable.

[0486] The present invention prepared a series of fusion proteins comprising the plasmid described in Example 1, as shown in Table 8 below.

[0487] Table 8

[0488] Example 12: FACS detection of fusion protein binding activity

[0489] Experimental Methods: 1E5 cells / well of the HCC827 cell line, which naturally expresses Trop2, were seeded in a U-shaped 96-well plate. The plates were washed once with ice-cold PBS and centrifuged at 1200 rpm for 3 minutes. After washing, 200 μL / well of 3% FBS / PBS blocking buffer was added and the plates were incubated on ice for 1 hour. After blocking, the plates were centrifuged at 1200 rpm for 3 minutes, the supernatant was discarded, and the plates were incubated with samples of varying concentrations, incubated on ice for 2 hours, and washed three times with ice-cold PBS. The plates were then incubated with 50 μL / well of PE-anti-human Fc antibody at a 1:200 dilution. After thorough mixing, the plates were incubated on ice for 1 hour and washed three times with ice-cold PBS. The cells were resuspended in 200 μL / well of PBS, and the mean fluorescence values ​​were read on a FACS instrument. The results were analyzed using GraphPad Prism software.

[0490] Experimental results: As shown in Figures 22A to 22D, the results showed that the fusion proteins containing Torp2 antibodies all bound to the cell line HCC827 that naturally expresses trop2.

[0491] Example 13: ELISA detection of binding between CD80 fusion protein and CTLA-4 protein

[0492] The CD80 fusion protein has an affinity for binding to CTLA4 of 0.78 nM, an affinity for binding to CD28 fusion protein of greater than 2600 nM, and an affinity for binding to PD-L1 of greater than 37,000 nM (reference: Mark F. Maurer, NATURE COMMUNICATIONS | (2022) 13:1790). Based on the high affinity of CD80 for CTLA4, this example identified the in vitro activity of the CD80 fusion protein by detecting the binding of the CD80 fusion protein to a recombinant protein containing CTLA4. The experimental method is as follows:

[0493] His-tag antibody (GenScript, A00174-40) was added to the ELISA plate at 1 μg / ml, 100 μl per well, and incubated at 4°C overnight. After blocking with 5% non-fat milk at room temperature for 1 hour, 0.1 μg / ml CTLA-4 protein (Sino Biological, 11159-H08H-B) was added to the plate at 100 μl per well and incubated at room temperature for 1 hour. CD80-Fc fusion protein was serially diluted with blocking buffer and added to the plate at 100 μl per well and incubated at room temperature for 1 hour. HRP-goat anti-human Fc (Abcam, ab97225) was diluted 1:5000 and added to the ELISA plate at 100 μl per well and incubated at room temperature for 1 hour. TMB was added at 100 μl per well and the plate was developed in the dark at room temperature for 5 minutes. 1 M H2SO4 (100 μl per well) was added to stop the color development and the absorbance at 450 nm was measured on a microplate reader.

[0494] The experimental results are shown in Figures 23A to 23D. All the CD80-containing fusion proteins bind to CTLA4, indicating that all the CD80-containing fusion proteins have biological activity.

[0495] Example 14: ELISA detection of PD-1 binding activity of fusion protein containing PD-1 antibody

[0496] Coating: Dilute the anti-His protein to a final concentration of 1 μg / mL in 1X PBS, add 60 μL / well to the ELISA plate, and incubate at 4°C for 18 hours.

[0497] Blocking: Wash the coated ELISA plate twice with PBST. After washing, add 200 μL / well of blocking solution and block at room temperature for 1 hour.

[0498] Incubate with PD1 antibody: dilute hPD1-His protein to a final concentration of 1 μg / mL, add 60 μL / well to the ELISA plate, and incubate at room temperature for 1 hour.

[0499] Incubate the fusion protein to be tested: Wash three times with PBST. Dilute all samples to an initial concentration of 66.7 nM and perform a 5-fold dilution series, with the final dilution being 100-fold, for a total of eight series.

[0500] Incubate with enzyme-labeled antibody: dilute the anti-hFc enzyme-labeled antibody 5000 times, add 60 μL / well to the ELISA plate, incubate at room temperature for 1 hour, and wash 5 times with PBST.

[0501] TMB color development: After TMB has equilibrated to room temperature, add 100 μL / well of TMB and allow color development to proceed for 15-40 minutes at room temperature in the dark. After color development is complete, quickly add 100 μL / well of stop solution to terminate the reaction. Measure the OD value at 450 nm on a microplate reader.

[0502] As shown in Figures 24A-24C, the results showed that the fusion proteins containing PD-1 antibodies all bound to the PD-1 protein.

[0503] Example 15: ELISA detection of the blocking activity of the fusion protein on the binding of PD-1 and PD-L1

[0504] Coating: Dilute PD1-hFc protein with 1X PBS to a final concentration of 2 μg / mL, add 60 μL / well to the ELISA plate, and incubate at 4°C for 18 hours.

[0505] Blocking: Wash the coated ELISA plate twice with PBST. After washing, add 200 μL / well of blocking solution and block at room temperature for 1 hour.

[0506] Incubate PD-L1-mFc recombinant protein: dilute the PD-L1-mFc protein to a final concentration of 0.5 μg / mL, add 60 μL / well to the ELISA plate, and incubate at room temperature for 1 hour.

[0507] Incubate the fusion protein to be tested: Wash three times with PBST. Dilute all samples to an initial concentration of 333.3 nM and perform a 5-fold dilution series, with the final dilution being 100-fold, for a total of eight series.

[0508] Incubate with enzyme-labeled antibody: dilute anti-mlgG (H+L) enzyme-labeled antibody 5000 times, add 60 μL / well to ELISA plate, incubate at room temperature for 1 hour, and wash 5 times with PBST.

[0509] TMB color development: After TMB has equilibrated to room temperature, add 100 μL / well of TMB and allow color development to proceed for 15-40 minutes at room temperature in the dark. After color development is complete, quickly add 100 μL / well of stop solution to terminate the reaction. Measure the OD value at 450 nm on a microplate reader.

[0510] As shown in FIG. 25A to FIG. 25B , the results showed that the fusion proteins containing the PD-1 antibody blocked the binding of PD-1 to PD-L1.

[0511] Example 16: ELISA detection of fusion protein binding activity to PD-L1

[0512] Coating: Dilute the anti-His protein to a final concentration of 1 μg / mL in 1X PBS, add 60 μL / well to the ELISA plate, and incubate at 4°C for 18 hours.

[0513] Blocking: Wash the coated ELISA plate twice with PBST. After washing, add 200 μL / well of blocking solution and block at room temperature for 1 hour.

[0514] Incubate with PD-L1 antibody: dilute PD-L1-His (QP003) to a final concentration of 1 μg / mL, add 60 μL / well to the ELISA plate, and incubate at room temperature for 1 hour.

[0515] Incubate the fusion protein to be tested: Wash three times with PBST. Dilute all samples to an initial concentration of 66.7 nM and perform a 5-fold dilution series, with the final dilution being 100-fold, for a total of eight series.

[0516] Incubate with enzyme-labeled antibody: dilute the anti-hFc enzyme-labeled antibody 5000 times, add 60 μL / well to the ELISA plate, incubate at room temperature for 1 hour, and wash 5 times with PBST.

[0517] TMB color development: After TMB has equilibrated to room temperature, add 100 μL / well of TMB and allow color development to proceed for 15-40 minutes at room temperature in the dark. After color development is complete, quickly add 100 μL / well of stop solution to terminate the reaction. Measure the OD value at 450 nm on a microplate reader.

[0518] As shown in Figure 26A and Figure 26B, the results showed that the fusion proteins containing PD-L1 antibodies all bound to the PD-L1 recombinant protein.

[0519] Example 17: ELISA detection of the blocking activity of the fusion protein on the binding of PD-1 and PD-L1

[0520] PD-L1 blocking assay

[0521] Coating: Dilute PD1-hFc protein with 1X PBS to a final concentration of 2 μg / mL, add 60 μL / well to the ELISA plate, and incubate at 4°C for 18 hours.

[0522] Blocking: Wash the coated ELISA plate twice with PBST. After washing, add 200 μL / well of blocking solution and block at room temperature for 1 hour.

[0523] Incubate with PD-L1-mFc antibody: dilute the PD-L1-mFc protein to a final concentration of 0.5 μg / mL, add 60 μL / well to the ELISA plate, and incubate at room temperature for 1 hour.

[0524] Incubate the fusion protein to be tested: Wash three times with PBST. Dilute all samples to an initial concentration of 333.3 nM and perform a 5-fold dilution series, with the final dilution being 100-fold, for a total of eight series.

[0525] Incubate with enzyme-labeled antibody: dilute anti-mlgG (H+L) enzyme-labeled antibody 5000 times, add 60 μL / well to ELISA plate, incubate at room temperature for 1 hour, and wash 5 times with PBST.

[0526] TMB color development: After equilibration of TMB at room temperature, add 100 μL / well. Allow to develop for 15-40 minutes at room temperature in the dark. After color development is complete, quickly add 100 μL / well of stop solution to terminate the reaction. Measure the OD value at 450 nm on a microplate reader.

[0527] As shown in Figures 27A and 27B, the results showed that the fusion proteins containing PD-L1 antibodies were able to block the binding of PD-1 to PD-L1.

[0528] Example 18: Detection of cytotoxicity of co-stimulatory fusion proteins to enhance CD3 dual antibody-dependent PBMC-mediated killing of tumor cells

[0529] This patent contains a CD80 co-stimulatory fusion protein combined with a T cell engager containing a CLDN18.2 / CD3 dual antibody to enhance the anti-tumor activity of the CD3 dual antibody. The CLDN18.2 / CD3 dual antibody T cell engager used in this patent is shown in Table 9 below:

[0530] Table 9:

[0531] The sequence looks like this:

[0532] Experimental Methods: The target cells were HCC827-CLDN18.2, a human lung cancer cell line stably expressing CLDN18.2 and Trop2. The effector cells were human PBMCs. At an E:T ratio of 10:1, separate experimental groups were set up: different concentrations of the CLDN18.2 / CD3 dual antibody QP146134233424. Different concentrations of the CLDN18.2 / CD3 dual antibody QP146134233424 were then combined with fixed concentrations of different forms of CD80 co-stimulatory fusion proteins. The cells were incubated at 37°C, 5% CO2, and supernatants were collected after 48 or 72 hours. CytoTox was used to analyze the supernatants. The Non-Radioactive Cytotoxicity Assay (Promega, G1780-1000 assays) was used to detect LDH in the cell culture supernatant and quantify % cytotoxicity. The maximum target cell lysis rate (100%) was 1% Triton X-100 treatment, resulting in cell lysis and release of all LDH. Control wells were set up for spontaneous target cell activity, spontaneous effector cell activity, and spontaneous target cell + effector cell activity. Data were analyzed using the formula % Cytotoxicity = [(Experimental - Effector Spontaneous - Target Spontaneous) / (Target Maximum - Target Spontaneous)] × 100.

[0533] The experimental results were analyzed according to the four types of CD80 fusion protein molecules as follows:

[0534] (1) Fusion protein containing CD80 / TAA, specifically CD80 / anti-Trop2

[0535] As shown in Table 10 below, the bivalent CD80 fused Trop2 nanobody proteins QP5152 and QP5157; the monovalent CD80 fused Trop2 nanobody proteins QP51555156 and QP51585161.

[0536] Table 10:

[0537] The control molecules are shown in Table 11 below: CD80 fusion protein QP5153, Trop2 nanobody fusion protein QP5154.

[0538] Table 11:

[0539] The experimental results of synergistic CD3 dual antibody killing are shown in Figures 28A to 28D and Tables 12-1 to 12-4: bivalent CD80 fusion Trop2 nanoantibody proteins QP5152 and QP5157; and monovalent CD80 fusion Trop2 nanoantibody proteins QP51555156 and QP51585161 all significantly enhanced CD3 dual antibody killing of tumor cells, while the control CD80 fusion protein QP5153 and Trop2 nanoantibody fusion protein QP5154 did not synergize CD3 dual antibody killing, indicating that CD80 fusion protein antigen-dependently enhances CD3 dual antibody killing of tumor cells.

[0540] Table 12-1: Enhanced killing of tumor cells by CD80 / TAA fusion protein combined with CD3 dual antibody

[0541] Table 12-2: Enhanced killing of tumor cells by CD80 / TAA fusion protein combined with CD3 dual antibody

[0542] Table 12-3: Enhanced killing of tumor cells by CD80 / TAA fusion protein combined with CD3 dual antibody

[0543] Table 12-4: Enhanced killing of tumor cells by CD80 / TAA fusion protein combined with CD3 dual antibody

[0544] (2) Fusion protein containing CD80 / PD(L)1 / TAA, specifically CD80 / antiPD(L)1VHH / antiTrop2

[0545] As shown in Table 13 below, CD80 fusion PD-1 / PD-L1 nanobody and Trop2 nanobody trifunctional fusion proteins QP51555159, QP51585160, QP5278 and QP5279; monovalent CD80 fusion Trop2 nanobody proteins QP51555156 and QP51585161.

[0546] Table 13:

[0547] The control molecules are shown in Table 14 below:

[0548] CD80 fused with PD-1 nanoantibody bifunctional protein QP52045205, PD-1 nanoantibody QP959.

[0549] Table 14:

[0550] The experimental results of synergistic CD3 dual antibody killing are shown in Figures 29A to 29E and Tables 15-1 to 15-5: The CD80 fusion PD-1 nanobody and Trop2 nanobody trifunctional fusion proteins QP51555159, QP51585160, QP5278, and QP5279 significantly enhanced CD3 dual antibody killing of tumor cells at an effector-target ratio of 10:1 or 5:1, with significant increases in the maximum killing Emax and EC50 increases ranging from 3.8-fold to 10-fold. The effect was significantly superior to the control CD80 fusion PD-1 nanobody bifunctional protein QP52045205 and the CD80 fusion Trop2 nanobody bifunctional proteins QP51555156 and QP51555161.

[0551] Table 15-1: Enhanced tumor cell killing by CD80 / PD(L)1 / TAA fusion protein combined with CD3 dual antibody

[0552] Table 15-2: CD80 / PD(L)1 / TAA fusion protein combined with CD3 dual antibody enhances tumor cell killing

[0553] Table 15-3: Enhanced killing of tumor cells by CD80 / PD(L)1 / TAA fusion protein combined with CD3 dual antibody

[0554] Table 15-4: CD80 / PD(L)1 / TAA fusion protein combined with CD3 dual antibody enhances tumor cell killing

[0555] Table 15-5: Enhanced tumor cell killing by CD80 / PD(L)1 / TAA fusion protein combined with CD3 dual antibody

[0556] (3) Fusion protein containing CD80 / PD(L)1 / TAA, specifically CD80 / anti PD1 Fab / antiTrop2 or CD80 / anti PDL1 Fab / antiTrop2

[0557] As shown in Table 16 below, the trifunctional fusion proteins QP515532115380, QP51555381, QP515550955396, and QP515511815399 of CD80 fusion PD-1 or PD-L1 antibody and Trop2 nanobody are listed; and the monovalent CD80 fusion Trop2 nanobody proteins QP51555156 and QP51585161.

[0558] Table 16:

[0559] The control CD80 fusion PD-1 antibody bifunctional protein QP520450955394, CD80 fusion PD-L1 antibody bifunctional protein QP520411815398, and PD-1 antibody QP50955096 are listed in Table 17 below.

[0560] Table 17:

[0561] The experimental results of synergistic CD3 dual antibody killing are shown in Figures 30A and 30B and Tables 18-1 and 18-2: The CD80 fusion PD-1 antibody and Trop2 nanobody trifunctional fusion proteins QP515532115380, QP51555381, and QP515550955396 significantly enhanced the CD3 dual antibody killing of tumor cells at an effector-target ratio of 6:1 or 5:1, with a significant increase in the maximum killing Emax and an EC50 increase of 10-fold or more. The effect was significantly superior to the control CD80 fusion PD-1 antibody bifunctional protein QP520450955394 and the CD80 fusion Trop2 nanobody bifunctional protein QP51555156.

[0562] Table 18-1: Enhanced killing of tumor cells by CD80 / PD(L)1 / TAA fusion protein combined with CD3 dual antibody

[0563] Table 18-2: CD80 / PD(L)1 / TAA fusion protein combined with CD3 dual antibody enhances tumor cell killing

[0564] The experimental results of synergistic CD3 dual antibody killing are shown in Figure 30C and Table 18-3: The CD80 fusion PD-L1 antibody and Trop2 nanobody trifunctional fusion protein QP515511815399 significantly enhanced the CD3 dual antibody killing of tumor cells at an effector-target ratio of 5:1, with a significant increase in the maximum killing Emax and an EC50 increase of more than 10-fold. The effect was significantly superior to the control CD80 fusion PD-L1 antibody bifunctional protein QP520411815398 and the CD80 fusion Trop2 nanobody bifunctional protein QP51555156.

[0565] Table 18-3: Enhanced killing of tumor cells by CD80 / PD(L)1 / TAA fusion protein combined with CD3 dual antibody

[0566] (4) Fusion protein containing CD80 / EGFR / (PDL1), specifically CD80 / antiEGFR or CD80 / antiEGFR / antiPDL1

[0567] As shown in Tables 19 and 20 below, CD80, PD-L1 antibody, and EGFR antibody were fused into trifunctional or bifunctional fusion proteins QP527352745275, QP52775389, and QP5400, respectively.

[0568] Table 19

[0569] comparison

[0570] Table 20

[0571] The experimental results of synergistic CD3 dual antibody killing are shown in Figures 31A and 31B and Tables 21-1 and 21-2: CD80, PD-L1 antibodies, and EGFR antibodies were fused into trifunctional or bifunctional fusion proteins QP527352745275, QP52775389, and QP5400, respectively, which significantly enhanced the CD3 dual antibody killing of tumor cells at an effector-target ratio of 5:1 or 10:1, with the maximum killing Emax significantly increased and the EC50 enhanced by 5 to 50 times or more.

[0572] Table 21-1: Enhanced tumor cell killing by CD80 / PD(L)1 / TAA fusion protein combined with CD3 dual antibody

[0573] Table 21-2: CD80 / PD(L)1 / TAA fusion protein combined with CD3 dual antibody enhances tumor cell killing

[0574] Example 19: Detection of cytotoxicity of co-stimulatory fusion proteins to enhance PBMC-mediated killing of tumor cells in CD3 dual antibody-dependent manner

[0575] Experimental Methods: The target cells were HCC827-CLDN18.2, a human lung cancer cell line stably expressing CLDN18.2 and Trop2. The effector cells were human PBMCs. At an E:T ratio of 5:1, separate experimental groups were set up: different concentrations of CD3-CLDN18.2 antibodies, followed by a CD3-CLDN18.2 antibody plus a fixed concentration of CD80-Trop2 co-antibody. The cells were incubated at 37°C, 5% CO2, and supernatants were collected at 48 and 72 hours. CytoTox was used to analyze the supernatants. The Non-Radioactive Cytotoxicity Assay (Promega, G1780-1000 assays) was used to detect LDH in the cell culture supernatant and quantify % cytotoxicity. The maximum target cell lysis rate (100%) was 1% Triton X-100 treatment, resulting in cell lysis and release of all LDH. Control wells were set up for spontaneous target cell activity, spontaneous effector cell activity, and spontaneous target cell + effector cell activity. Data were analyzed using the formula % Cytotoxicity = [(Experimental - Effector Spontaneous - Target Spontaneous) / (Target Maximum - Target Spontaneous)] × 100.

[0576] Experimental results: As shown in Figures 32A to 32B, and Tables 22-1 and 22-2, the results show that TAA / CD80 or TAA / CD80 / PD-1 fusion proteins can also significantly synergistically enhance the cytotoxicity of CD3 bispecific antibodies-dependent PBMCs in killing tumor cells under the conditions of low efficiency target ratio (5:1) and high affinity CD3 bispecific antibodies.

[0577] Table 22-1: Enhanced tumor cell killing by CD80 / PD(L)1 / TAA fusion protein combined with CD3 dual antibody

[0578] Table 22-2: CD80 / PD(L)1 / TAA fusion protein combined with CD3 dual antibody enhances tumor cell killing

[0579] Example 20: Cytokine release experiment

[0580] To better predict the safety of CD28 agonists, studies have shown that TGN1412, a CD28 agonist that caused a severe cytokine storm in a Phase 1 clinical trial, can induce the release of cytokines such as TNF-α in a solid-phase assay, thereby predicting its safety [Findlay, J. Immunological Methods 352:1-12 (2010)]. To evaluate the safety of the CD80-related fusion proteins of the present invention, cytokine release after incubation of the fusion proteins with PBMCs was measured using a solid-phase assay established in existing literature.

[0581] Experimental methods: 96-well plates (Corning, 9018) were coated with 1 μg / well CD80 protein and incubated at 4°C overnight. The next day, the plates were washed twice with PBS. The cell concentration of revived PBMCs was adjusted to 2E6 / mL, and 100 μL of cells were added to each well. A CD80 protein group alone and a CD80 protein + 1 ng / mL OKT3 antibody group were also set up. The plates were incubated at 37°C, 5% CO2. Supernatants were collected at 24, 48, and 72 hours, and the release of TNF-α, IFN-γ, and IL-2 in the supernatants was measured using Invitrogen ELISA kits.

[0582] Experimental Results: Some of the results are shown in Figure 33. TGN1412 can activate PBMC to secrete the cytokine TNF-α. Under the same conditions, all CD80 bifunctional or trifunctional fusion proteins of the present invention did not cause significant release of any cytokine. Therefore, compared with TGN1412, all forms of the CD80 fusion protein of the present invention have excellent safety and significantly lower risk of inducing cytokine storm.

[0583] Example 21: CD80 fusion protein inhibits tumor growth in animal models

[0584] Experimental purpose: To verify the inhibitory effect of hCD80 fusion protein on tumor growth through a cell line xenograft model (CDX animal model)

[0585] Experimental steps: The cultured mouse colon cancer cell line CT26-hTrop2 cells stably expressing human Trop2 antibody were collected by centrifugation and dispersed with 1× PBS to prepare a cell density of 3×10 7 0.1 ml of cell suspension was subcutaneously inoculated into the right rib of Balb / c mice to establish a CT26-hTrop2 tumor-bearing mouse model. When the average tumor volume of the tumor-bearing mice reached about 91 mm 3At 4 hr, mice were randomly divided into groups of 8. All animals were weighed, and tumor volumes were measured with a vernier caliper. Groups were randomly divided based on tumor volume, ensuring that tumor volumes were similar between groups. The day of grouping was designated D0, and dosing began on the same day. Detailed dosage, route of administration, and dosing cycle are shown in the table below.

[0586] Table 23: Dosage regimen for animal efficacy studies

[0587] The administration volume was 10 μL / g.

[0588] After the start of drug administration, the body weight and tumor volume of mice were measured three times a week. Tumor volume calculation formula: Tumor volume (mm 3 )=1 / 2×(a×b 2 ) (where a represents the major diameter and b represents the minor diameter) The experiment was terminated six days after the last administration, the mice were killed, the tumors were removed, and the tumor weight was measured.

[0589] The following analysis methods were used for data analysis:

[0590] Tumor proliferation rate, T / C (%) = (T-T0) / (C-C0) × 100% (T0 and C0 are the tumor volumes of the treatment group and the control group at the time of grouping, respectively; T and C are the tumor volumes of the treatment group and the control group at a specific time point after drug administration, respectively)

[0591] Tumor inhibition rate, TGI (%), is calculated as follows: TGI% = (1-T / C) × 100%

[0592] Experimental results:

[0593] Refer to Figure 34A for changes in mouse body weight. During the treatment period, all treatment groups were well tolerated and there was no obvious weight loss.

[0594] The tumor inhibitory effects are shown in Figures 34B and 34C and Table 24. The experimental results showed that both QP5153 and QP51555156 had significant anti-tumor effects. The monovalent CD80-fused anti-TAA antibody and FC fusion protein (CD80 / TAA / FC: QP51555156) was superior to the bivalent CD80-fused FC fusion protein (CD80 / FC: QP5153).

[0595] Table 24

[0596] Example 22: hCD80 fusion protein inhibits tumor growth in animal models

[0597] Objective: To verify that hCD80 fusion protein inhibits tumor growth by conducting efficacy tests in a cell line xenograft model (CDX animal model)

[0598] Experimental steps: The cultured mouse colon cancer cell line CT26-hTrop2 cells stably expressing human Trop2 antibody were collected by centrifugation and dispersed with 1× PBS to prepare a cell density of 2×10 7 0.1 ml of cell suspension was subcutaneously inoculated into the right rib of Balb / c mice to establish a CT26-hTrop2 tumor-bearing mouse model. When the average tumor volume of the tumor-bearing mice reached about 65 mm 3 At 4 hr, mice were randomly divided into groups of 9. All animals were weighed, and tumor volumes were measured with a vernier caliper. Groups were randomly divided based on tumor volume, ensuring that tumor volumes were similar between groups. Grouping was designated D0, and dosing began on the same day. Detailed dosage, route of administration, and dosing cycle are shown in the table below.

[0599] Table 25: Dosage regimen for animal efficacy studies

[0600] The administration volume was 10 μL / g.

[0601] After the start of drug administration, the body weight and tumor volume of mice were measured three times a week. Tumor volume calculation formula: Tumor volume (mm 3 )=1 / 2×(a×b 2 ) (where a represents the major diameter and b represents the minor diameter) The experiment was terminated two days after the last administration and the mice were killed.

[0602] The following analysis methods were used for data analysis:

[0603] Tumor proliferation rate, T / C (%) = (T-T0) / (C-C0) × 100% (T0 and C0 are the tumor volumes of the treatment group and the control group at the time of grouping, respectively; T and C are the tumor volumes of the treatment group and the control group at a specific time point after drug administration, respectively)

[0604] Tumor inhibition rate, TGI (%), is calculated as follows: TGI% = (1-T / C) × 100%

[0605] Experimental results:

[0606] Please refer to Figure 35A below for changes in mouse body weight. During the treatment period, all treatment groups were well tolerated and there was no obvious weight loss.

[0607] The tumor inhibitory effects are shown in Figure 35B and Table 26. The experimental results showed that QP5153, QP5152, and QP515550955396 all had significant anti-tumor effects, with the order of efficacy being QP515550955396 > QP5152 > QP5153. Specifically, the bivalent CD80-fused anti-TAA antibody (CD80 / TAA: QP5152) was superior to the bivalent CD80-fused FC fusion protein (CD80 / FC: QP5153). Furthermore, the fusion protein containing a monovalent CD80-fused anti-PD-1 antibody and an anti-TAA antibody (CD80 / PD-1 / TAA: QP515550955366) was superior to the bivalent CD80-fused anti-TAA antibody (CD80 / TAA: QP5152) and the bivalent CD80-fused FC fusion protein (CD80 / FC: QP5153) in inhibiting tumor growth.

[0608] Table 26

[0609] All documents mentioned in this application are incorporated herein by reference, just as if each document were incorporated herein by reference individually. It should also be understood that after reading the above teachings of the present invention, those skilled in the art may make various changes or modifications to the present invention, and that such equivalents also fall within the scope of the claims appended hereto.

Claims

1. A fusion protein, wherein the fusion protein comprises the following elements: a) a first targeting domain D1, wherein the first targeting domain D1 is a CD80 extracellular domain (ECD); and b) a second targeting domain D2, wherein said D2 binds to a second target protein, wherein said second target protein is a tumor-associated antigen (TAA).

2. The fusion protein according to claim 1, wherein the fusion protein further comprises c) a third targeting domain D3, wherein the D3 binds to a third target protein, and the third target protein is PD1 and / or PD-L1.

3. The fusion protein according to claim 1 or 2, wherein the fusion protein further comprises an Fc region, Preferably, The Fc region is selected from the Fc region of human IgG1, human IgG2, human IgG3 and human IgG4, or variants thereof; The variant comprises a point mutation introduced based on the knob-into-hole technology compared to the Fc region of human IgG1, human IgG2, human IgG3 or human IgG4, or Compared to the Fc region of human IgG1, human IgG2, human IgG3 or human IgG4, the variant comprises a point mutation that eliminates affinity for Fcγ receptors and C1q complement proteins, and weakens or eliminates immune effector functions; More preferably, Compared to human IgG1 Fc, the variant has amino acid mutations of L235A, D265A, and P331S, wherein amino acid positions are determined according to the EU numbering system; More preferably, The Fc region has an amino acid sequence as shown in SEQ ID NO: 69, or an amino acid sequence having 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 thereto.

4. The fusion protein according to any one of claims 1 to 3, wherein the CD80 extracellular domain (ECD) comprises a CD80 IgV domain or a CD80 IgC domain, or a combination thereof; Preferably, The CD80 extracellular domain (ECD) has an amino acid sequence as shown in SEQ ID NO: 41, or an amino acid sequence having 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 thereto, as long as it is capable of binding to one or a combination of the following target proteins: CD28, CTLA-4 and PD-L1.

5. The fusion protein according to any one of claims 1 to 4, wherein the second targeting domain D2 is an anti-TAA antibody or a functional fragment thereof, and the functional fragment of the antibody is selected from Fab, scFv or VHH; Preferably, The second targeting domain D2 is an anti-TAA antibody or a functional fragment thereof, and the TAA is selected from: CD33, CD30, HER2, CD22, CD79b, Nectin-4, BCMA, EGFR, CD19, tissue factor, folr1 (folate receptor α), CLDN18.2, TROP2, c-Met, PSMA, Muc1, PDL1, ROR1, MSLN, TNF-α, CD25, ENPP3, Axl, CD20, ROR2, GPNMB, CEACAM6, CD138, CA6, FUT3, C D56, CD37, HER3, GPRC5D, STING, CEA, CD205, B7H4, CTLA4, RNF43, CDH3, DPEP3, 5T4, ITGB6, EFNA4, B7H3, C D228, Notch-3, CD46, CAIX, SLAMF6, ADAM9, GD3, TDGF1, SLAMF2, CLL-1, CD123, IGF-1R, FCRL5, TIM1, Globo H, CDH6, CD38, Ly6E, SLITRK6, GPR20, FGFR2, Muc16, CD51, SLAMF7, LAMP-1, CD74, CCR7, PTK7, SEZ6, CLDN 9. CLDN6, c-kit, LYPD3, TAA, PRL receptor, FGFR3, KAAG1, STEAP1, Flt3, LRRC15, CD44, CD70, EphA2, PDL2, p53, DLK1, ENB-FN, FOLR, CD45, DSG2, ALK, TRAIL, DDR1, EpCAM, VEGFR2, CD47, CD99, VEGF2, SSEA-4, DCLK1, OAcGD2, IL1RAP, ADAM17, CD7, CD73, ENO1, BSG, CD24, GLUT1, CXCR4, CD40, CD52, CD133, CD239, TAG72, EGFR VIII, PSCA, EphA2, NKG2D ligand, MCSP, LGR5, SSEA3, SLC34A2, Glypican-3, or a combination thereof; More preferably, The second targeting domain D2 is an anti-TROP2 antibody or a functional fragment thereof; or an anti-EGFR antibody or a functional fragment thereof; or an anti-PDL1 antibody or a functional fragment thereof; More preferably, The second targeting domain D2 is an anti-TROP2 VHH, which comprises CDR1 shown in SEQ ID NO: 47, CDR2 shown in SEQ ID NO: 48, and CDR3 shown in SEQ ID NO: 49; Still more preferably, the second targeting domain D2 is an anti-TROP2 VHH comprising the amino acid sequence of SEQ ID NO: 46, or an amino acid sequence having 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 thereto; or The second targeting domain D2 is an anti-EGFR antibody or a functional fragment thereof, which comprises VH shown in SEQ ID NO: 59 and VL shown in SEQ ID NO: 60; or The second targeting domain D2 is an anti-EGFR antibody or a functional fragment thereof, which comprises VH shown in SEQ ID NO: 70 and VL shown in SEQ ID NO: 71; or The second targeting domain D2 is an anti-PDL1 antibody or a functional fragment thereof, which comprises the VHH shown in SEQ ID NO:

58.

6. The fusion protein according to any one of claims 2 to 5, wherein the third targeting domain D3 is an anti-PD-1 antibody or a functional fragment thereof, or an anti-PD-L1 antibody or a functional fragment thereof, and the functional fragment of the antibody is selected from Fab, scFv or VHH; Preferably, The anti-PD-1 antibody or a functional fragment thereof is selected from any one of i) to iv): i) a VHH comprising the CDR1 set forth in SEQ ID NO: 53, the CDR2 set forth in SEQ ID NO: 54, and the CDR3 set forth in SEQ ID NO: 55; Preferably, the VHH has the amino acid sequence shown in SEQ ID NO: 52; or a sequence identity thereof of 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%; ii) a VHH comprising the CDR1 set forth in SEQ ID NO: 53, the CDR2 set forth in SEQ ID NO: 54, and the CDR3 set forth in SEQ ID NO: 57; Preferably, the VHH has the amino acid sequence shown in SEQ ID NO: 56; or a sequence identity thereof of 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%; iii) an anti-PD-1 antibody or a functional fragment thereof, comprising VH and VL, The VH has the amino acid sequence of SEQ ID NO: 63, or an amino acid sequence having 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 thereto; and The VL has the amino acid sequence of SEQ ID NO: 64, or an amino acid sequence having 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 thereto; and iv) an anti-PD-1 antibody or a functional fragment thereof, comprising VH and VL, The VH has the amino acid sequence of SEQ ID NO: 65, or an amino acid sequence having 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 thereto; and The VL has the amino acid sequence of SEQ ID NO: 66, or an amino acid sequence having 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 thereto; or The anti-PD-L1 antibody or a functional fragment thereof is selected from any one of v) to vi): v) an anti-PD-L1 antibody or a functional fragment thereof, which comprises VH and VL, The VH has the amino acid sequence of SEQ ID NO: 61, or an amino acid sequence having 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 thereto; and The VL has the amino acid sequence of SEQ ID NO: 62, or an amino acid sequence having 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 thereto; and vi) a VHH having an amino acid sequence as shown in SEQ ID NO: 58, or a sequence identity thereof of 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%.

7. The fusion protein according to any one of claims 1 to 6, wherein the fusion protein is a homodimer comprising two identical monomers, and from the N-terminus to the C-terminus, each monomer has a structure selected from the group consisting of: i) CD80 extracellular domain (ECD) - anti-TAA antibody or its functional fragment - Fc region - anti-PD-1 or anti-PD-L1 antibody or its functional fragment; ii) CD80 extracellular domain (ECD)-anti-PD-1 or anti-PD-L1 antibody or its functional fragment-Fc region-anti-TAA antibody or its functional fragment; iii) CD80 extracellular domain (ECD)-anti-TAA antibody or a functional fragment thereof-Fc region; or iv) CD80 extracellular domain (ECD)-Fc region-anti-TAA antibody or a functional fragment thereof; in, - indicates direct connection or connection through a peptide linker; Preferably, Each monomer has an amino acid sequence selected from any one of the following: SEQ ID NO:1, SEQ ID NO:77, SEQ ID NO:7, SEQ ID NO:81, SEQ ID NO:21, SEQ ID NO:22 and SEQ ID NO:

39.

8. The fusion protein according to any one of claims 1 to 6, wherein the fusion protein is a heterodimer comprising a first monomer and a second monomer, and from the N-terminus to the C-terminus, the fusion protein has a structure selected from any one of the following i) to viii): i) the first monomer comprises: an anti-PD1 / PD-L1 antibody or a functional fragment thereof - a first Fc region - an anti-TAA antibody or a functional fragment thereof; and The second monomer comprises: CD80 extracellular domain (ECD)-second Fc region-anti-TAA antibody or a functional fragment thereof; ii) the first monomer comprises: an anti-PD1 / PD-L1 antibody or a functional fragment thereof - an anti-TAA antibody or a functional fragment thereof - a first Fc region; and The second monomer comprises: CD80 extracellular domain (ECD)-anti-TAA antibody or a functional fragment thereof-second Fc region; iii) the first monomer comprises: CD80 extracellular domain (ECD)-first Fc region-anti-PD1 / PD-L1 antibody or a functional fragment thereof; and The second monomer comprises: an anti-TAA antibody or a functional fragment thereof-a second Fc region-an anti-PD1 / PD-L1 antibody or a functional fragment thereof; iv) the first monomer comprises: an anti-TAA antibody or a functional fragment thereof - a first Fc region; and The second monomer comprises: CD80 extracellular domain (ECD)-anti-TAA antibody or a functional fragment thereof-second Fc region; v) the first monomer comprises: a first Fc region - an anti-TAA antibody or a functional fragment thereof; and The second monomer comprises: CD80 extracellular domain (ECD)-second Fc region-anti-TAA antibody or a functional fragment thereof; vi) the first monomer comprises: a first Fc region; and The second monomer comprises: CD80 extracellular domain (ECD)-anti-TAA antibody or a functional fragment thereof-second Fc region; vii) the first monomer comprises: CD80 extracellular domain (ECD) - first Fc region - anti-TAA antibody or a functional fragment thereof; and The second monomer comprises: an anti-PD1 / PD-L1 antibody or a functional fragment thereof-a second Fc region-an anti-TAA antibody or a functional fragment thereof; or viii) the first monomer comprises: a CD80 extracellular domain (ECD)-a first Fc region; and The second monomer comprises: an anti-TAA antibody or a functional fragment thereof - a second Fc region; in, - indicates direct connection or connection through a peptide linker; Wherein the anti-TAA antibody or its functional fragment, anti-PD1 / PD-L1 antibody or its functional fragment may include Fab form, ScFv form, VHH form; wherein the first Fc region is the same as or different from the second Fc region; Optionally, the first Fc region forms a heterodimer structure with the second Fc region; Preferably, The fusion polypeptide has an amino acid sequence combination selected from any one of the following 1)-21): 1) SEQ ID NO: 4 and SEQ ID NO: 5; 2) SEQ ID NO: 78 and SEQ ID NO: 79; 3) SEQ ID NO: 4 and SEQ ID NO: 6; 4) SEQ ID NO: 78 and SEQ ID NO: 80; 5) SEQ ID NO: 8 and SEQ ID NO: 9; 6) SEQ ID NO: 82 and SEQ ID NO: 83; 7) SEQ ID NO: 8 and SEQ ID NO: 10; 8) SEQ ID NO: 82 and SEQ ID NO: 84; 9) SEQ ID NO: 4 and SEQ ID NO: 16; 10) SEQ ID NO:18, SEQ ID NO:19 and SEQ ID NO:20; 11) SEQ ID NO:18, SEQ ID NO:85 and SEQ ID NO:86 12) SEQ ID NO:4, SEQ ID NO:26 and SEQ ID NO:27; 13) SEQ ID NO:78, SEQ ID NO:26 and SEQ ID NO:27; 14) SEQ ID NO: 4 and SEQ ID NO: 28; 15) SEQ ID NO: 78 and SEQ ID NO: 28; 16) SEQ ID NO:4, SEQ ID NO:29 and SEQ ID NO:30; 17) SEQ ID NO:78, SEQ ID NO:29 and SEQ ID NO:30; 18) SEQ ID NO:4, SEQ ID NO:31 and SEQ ID NO:32; 19) SEQ ID NO:78, SEQ ID NO:31 and SEQ ID NO:32; 20) SEQ ID NO: 20 and SEQ ID NO: 38; and 21) SEQ ID NO:86 and SEQ ID NO:

38.

9. A polynucleotide encoding a polypeptide having an amino acid sequence selected from any one of the following: SEQ ID NO:1, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:77, SEQ ID NO:78, SEQ ID NO:79, SEQ ID NO:80, SEQ ID NO:81, SEQ ID NO:82, SEQ ID NO:83, SEQ ID NO:84, SEQ ID NO:85, and SEQ ID NO:

86.

10. A vector comprising the polynucleotide according to claim 9.

11. A host cell comprising the polynucleotide according to claim 9 or the vector according to claim 10.

12. A method for preparing the fusion protein according to any one of claims 1 to 8, comprising: i) culturing the host cell according to claim 11 under appropriate conditions to obtain the fusion protein according to any one of claims 1 to 8; Optionally, the method further comprises ii) purifying and / or isolating the fusion protein obtained in step (i).

13. A pharmaceutical composition comprising: i) the fusion protein of any one of claims 1 to 8; and ii) one or more pharmaceutically acceptable carriers.

14. Use of the fusion protein according to any one of claims 1 to 8 or the pharmaceutical composition according to claim 13 for preparing a drug for preventing and / or treating tumors. Preferably, the tumor is selected from solid tumors or hematological tumors, such as colorectal cancer, gastric cancer, breast cancer, non-small cell lung cancer, small cell lung cancer, melanoma, head and neck cancer, ovarian cancer, pancreatic cancer, liver cancer, bladder cancer, kidney cancer, endometrial cancer, cervical cancer, glioma, leukemia, lymphoma, multiple myeloma, myelodysplastic syndrome and myeloproliferative neoplasms, Preferably, the drug is administered simultaneously, separately or sequentially with the CD3 bispecific antibody for treating tumors.

15. An anti-TROP2 antibody, which is a VHH, comprising a CDR1 as set forth in SEQ ID NO: 47, a CDR2 as set forth in SEQ ID NO: 48, and a CDR3 as set forth in SEQ ID NO: 49; Preferably, the anti-TROP2 antibody comprises the amino acid sequence of SEQ ID NO:46, or an amino acid sequence having 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 thereto.

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