Antigen binding protein and application thereof

Abstract

The invention provides an antigen binding protein specifically binding to EGFR (Epidermal Growth Factor Receptor). The invention also provides a nucleic acid molecule encoding the antigen binding protein, a vector and a cell comprising the same, and application of the nucleic acid molecule and the vector and the cell in disease treatment.

Description

Technical Field

[0001] This application relates to the field of biomedicine, specifically to an antigen-binding protein and its applications. Background Technology

[0002] Immunotherapy, which mobilizes the body's immune system to eradicate tumors, has revolutionized cancer treatment and achieved significant efficacy in clinical trials. However, these successes have been largely limited to certain types of hematologic malignancies, with less than ideal results for most solid tumors. The reasons for this are varied, but primarily related to the tumor-immune microenvironment (TIME). In the tumor-immune microenvironment, multiple immune components interact, collectively influencing the efficacy of immunotherapy. Increasing research evidence suggests that intervention targeting specific immune cell types within the TIME can reactivate suppressed anti-tumor immune responses, thereby enhancing the effectiveness of immunotherapy; however, this activation has not yet reached its optimal state.

[0003] To further improve efficacy, researchers have explored combination therapies using multiple drugs, including simultaneous or sequential use of different drugs, or the simultaneous delivery of multiple therapies using biomaterials. These approaches offer the possibility of simultaneously targeting multiple immune cells in the TIME (Time-Induced Therapy). However, these strategies often come with increased toxicity risks, and most combination therapies do not produce synergistic therapeutic effects.

[0004] To address the challenges of combination immunotherapy, researchers have begun exploring strategies to integrate multiple drugs into a single molecule. For example, bispecific or multispecific antibodies have been designed, containing two or more modules, such as tumor-targeting components, T-cell recruiters, checkpoint inhibitors, and immune agonists, to improve specificity, reduce toxicity, and enhance anti-tumor efficacy. Despite significant progress, existing strategies still have some limitations: First, most strategies are limited to bispecificity, activating only one type of immune cell (such as T cells or NK cells), which is insufficient when dealing with the complexities of TIME, especially when targeting multiple immune cells simultaneously; second, existing strategies lack sufficient modularity to integrate a wide variety of therapeutic modules to target different immune subsets within TIME.

[0005] While some reports have demonstrated multispecific or modular approaches, no system currently integrates these characteristics onto a single platform. Therefore, there is an urgent need for a platform that can integrate multiple therapeutic modules into a single drug, thereby enabling simultaneous targeting of multiple immune factors in TIME. Summary of the Invention

[0006] This application provides a multispecific conjugate formed by programmatically integrating multiple therapeutic modules into a single drug via a triple bioorthogonal reaction using a multispecific conjugate arm. The resulting multispecific conjugate can simultaneously activate multiple immune cells in the tumor-immune microenvironment. The multispecific conjugate of this application, through a multispecific conjugate arm with three orthogonal groups, can precisely connect three different payloads and exhibits virtually undetectable toxicity in vivo. The multispecific conjugate of this application integrates therapeutic modules including a tumor-targeting component, an immune activator, and a stimulant to obtain various multimodal targeting chimeras.

[0007] As an example, when nanobody modules are attached, these modules are programmable and, due to their small size, are advantageous for penetrating solid tumors. The multispecific conjugates of this application can recruit various cells, including tumor cells and immune cells, through a unique backbone, placing these cells in suitable spatial locations to interact and alter the tumor microenvironment, thereby exhibiting an enhanced anti-tumor immune response. The multispecific conjugates of this application can also open new avenues for immunotherapy and other fields.

[0008] On one hand, this application provides an antigen-binding protein that specifically binds to EGFR. In some embodiments, the antigen-binding protein that specifically binds to EGFR includes an EGFR antibody or an antigen-binding fragment thereof. The EGFR antibody is preferably a nanobody. For example, the EGFR antibody may comprise HCDR1, HCDR2, and HCDR3, and HCDR1, HCDR2, and HCDR3 contain amino acid sequences selected from any one or more of the following groups:

[0009] (1) HCDR1: SEQ ID NO: 2, HCDR2: SEQ ID NO: 3, and HCDR3: SEQ ID NO: 4,

[0010] (2) HCDR1: SEQ ID NO: 6, HCDR2: SEQ ID NO: 7, and HCDR3: SEQ ID NO: 8,

[0011] (3) HCDR1: SEQ ID NO: 10, HCDR2: SEQ ID NO: 11, and HCDR3: SEQ ID NO: 12,

[0012] (4) HCDR1: SEQ ID NO: 2, HCDR2: SEQ ID NO: 3, and HCDR3: SEQ ID NO: 4,

[0013] (5) HCDR1: SEQ ID NO: 2, HCDR2: SEQ ID NO: 3, and HCDR3: SEQ ID NO: 15,

[0014] (6) HCDR1: SEQ ID NO: 17, HCDR2: SEQ ID NO: 3, and HCDR3: SEQ ID NO: 18,

[0015] (7) HCDR1: SEQ ID NO: 2, HCDR2: SEQ ID NO: 20, and HCDR3: SEQ ID NO: 21,

[0016] (8) HCDR1: SEQ ID NO: 23, HCDR2: SEQ ID NO: 3, and HCDR3: SEQ ID NO: 24,

[0017] (9) HCDR1: SEQ ID NO: 2, HCDR2: SEQ ID NO: 3, and HCDR3: SEQ ID NO: 15,

[0018] (10) HCDR1: SEQ ID NO: 27, HCDR2: SEQ ID NO: 3, and HCDR3: SEQ ID NO: 28,

[0019] (11) HCDR1: SEQ ID NO: 30, HCDR2: SEQ ID NO: 31, and HCDR3: SEQ ID NO: 32, 1

[0021] (17) HCDR1: SEQ ID NO: 49, HCDR2: SEQ ID NO: 50, and HCDR3: SEQ ID NO: 51.

[0022] In some embodiments, the antigen-binding protein that specifically binds to EGFR includes a heavy chain variable region comprising an amino acid sequence represented by any one of SEQ ID NO: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61, and 65.

[0023] In some embodiments, the antigen-binding protein that specifically binds to EGFR is an antibody or its antigen-binding fragment.

[0024] In some embodiments, the antigen-binding protein that specifically binds to EGFR is a monoclonal antibody, a chimeric antibody, a humanized antibody, or a fully human antibody.

[0025] In some embodiments, the antigen-binding protein that specifically binds to EGFR is a monospecific antibody or a multispecific antibody.

[0026] In some embodiments, the antigen-binding protein that specifically binds to EGFR is a nanobody VHH, wherein the VHH contains the amino acid sequence shown in any one of SEQ ID NO: 1, 5, 9, 13, 14, 16, 19, 22, 25, 26, 29, 33, 36, 39, 42, 46 and 48.

[0027] On the other hand, this application provides a multispecific conjugate having a structure as shown in Formula I: (Equation I), where,

[0028] W is or ,in, Representing the connection site, W passes through Connect to La, Lb, and Lc respectively;

[0029] La is -C(=O)-X 1 -(K 1 ) n1 -(Y 1 ) p1 -(L 1 ) q1 -,

[0030] Lb is -C(=O)-X 2 -(K 2 ) n2 -(Y 2 ) p2 -(L 2 ) q2 -,

[0031] Lc is -C(=O)-X 3 -(K 3 ) n3 -(Y 3 ) p3 -(L 3 ) q3 -,

[0032] in,

[0033] X 1 X 2 , and X 3 Each is independently selected from the following groups: -NH- and -O-.

[0034] (K 1 ) n1 , (K 2) n2 , and (K 3 ) n3 Each of the following groups is independently selected: -CH2-(CH2-O-CH2)3-(CH2)2-, -(CH2)2-(CH2-O-CH2)3-CH2-, -CH2-(CH2-O-CH2)3-CH2-, and -(CH2)2-(CH2-O-CH2)3-(CH2)2-, or n1, n2, or n3 is independently 0.

[0035] (Y 1 ) p1 , (Y 2 ) p2 , and (Y 3 ) p3 Each independently as -NR 1 -C(=O)-,R 1 Selected from the following groups: hydrogen, protium, deuterium, and tritium; or p1, p2, or p3 each independently equals 0.

[0036] (L 1 ) q1 , (L 2 ) q2 , and (L 3 ) q3 Each is independently selected from the group consisting of -CH2-aryl-, -(CH2)2-, and -CH2-, or q1, q2, and q3 are each independently 0, wherein the aryl group is a 6-membered to 14-membered aryl group.

[0037] A 2 B 2 and C 2 Each is independently selected from the following groups: , , , , , , , , , , and covalent bonds, where R 12 R 13 and R 14 Each is independently selected from the following groups: hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxyl, alkoxy, amino, amide, ester, sulfonamide, urea, C1-C 12 Alkyl groups, 3- to 12-membered cycloalkyl groups, 3- to 12-membered heterocycloalkyl groups, C2-C 12 alkenyl, C2-C 12 Alkynyl, 6- to 14-membered aryl and 6- to 14-membered heteroaryl, among which, Represents the connection site, and A 2 B 2 and C 2 pass With La, Lb, Lc and P respectively 1 P 2 and P 3 connect,

[0038] P 1 P 2 and P 3 Each is independently selected from the following groups: nucleic acids, polypeptides, sugars, fats, proteins, and small molecules, and P 1 P 2 and P 3 Cells can be recruited. In some embodiments, the recruited cells may refer to P... 1 P 2 and / or P 3 By acting on receptors on the cell surface or on targets inside the cell, cells are attracted or mobilized to a specific region. In some embodiments, P 1 P 2 and P 3 At least one of them can regulate immune cells (including, activate and / or suppress immune cells), P 1 P 2 and P 3 At least one of the other components can bind to tumor cells.

[0039] In some embodiments, W in the multispecific conjugate is .

[0040] In some embodiments, X in the multispecific conjugate 1 X 2 and X 3 All are -NH-.

[0041] In some embodiments, (K) in the multispecific conjugate 1 ) n1 , (K 2 ) n2 , and (K 3 ) n3 All are -(CH2)2-(CH2-O-CH2)3-(CH2)2-.

[0042] In some embodiments, the (Y) in the multispecific conjugate 1 ) p1 , (Y 2 ) p2 , and (Y 3 ) p3Each independently as -NR 1 -C(=O)-,R 1 Selected from the following group: hydrogen, protium, deuterium and tritium.

[0043] In some embodiments, (L) in the multispecific conjugate 1 ) q1 , (L 2 ) q2 , and (L 3 ) q3 Each is independently selected from: -CH2- and -CH2-aryl-.

[0044] In some embodiments, A in the multispecific conjugate 2 Selected from the following group: , and .

[0045] In some embodiments, B in the multispecific conjugate 2 Selected from the following group: , and .

[0046] In some embodiments, C in the multispecific conjugate 2 Selected from the following group: , and .

[0047] In some embodiments, A in the multispecific conjugate 2 B 2 and C 2 They are respectively: , and .

[0048] In some embodiments, the multispecific conjugate has a structure as shown in formula IIa-1 or IIa-2: ,

[0049] , where P 1 P 2 and P 3 As defined in Formula I. For example, the multispecific conjugate has a structure as shown in Formula IIa-1.

[0050] Furthermore, regarding the linker backbone of multispecific conjugates, those skilled in the art can refer to the PCT international application publication text WO2022 / 100696A1, which lists those. Besides IIa-1 (unlinked P) used in the embodiments of this application... 1 P2 and P 3 (Referring to Ia-1 at the time), those skilled in the art will know that other connecting skeletons in WO2022 / 100696A1, such as the structures of formulas Ia-2 to Ia-10, connect the P of this application. 1 P 2 and P 3 Afterwards, the objective of this invention can also be achieved:

[0051] ,

[0052] , , , , ,

[0053] ,and

[0054] .

[0055] In some implementations, P 1 P 2 and P 3 At least one of them contains a tumor cell binding portion, and at least one contains an immune cell regulation portion.

[0056] In some embodiments, the tumor cell binding portion comprises a tumor antigen-binding protein. In some embodiments, the tumor cell binding portion comprises an antibody or an antigen-binding fragment thereof that targets a tumor antigen.

[0057] In some embodiments, the tumor antigen includes any one or more selected from the group consisting of: CEA (carcinoembryonic antigen), HER2 (human epidermal growth factor receptor 2), EGFR (epidermal growth factor receptor), VEGF (vascular endothelial growth factor), PSMA (prostate-specific membrane antigen), CA125 (cancer antigen 125), CA19-9 (carbohydrate antigen 19-9), CA15-3 (carbohydrate antigen 15-3), AFP (alpha-fetoprotein), PSA (prostate-specific antigen), MUC1 (membrane protein 1), and Mesothelioma. n (mesothelin), CD20 (B cell surface antigen), CD30 (Hodgkin's lymphoma-associated antigen), CD33 (myeloid cell surface antigen), CD56 (neural cell adhesion molecule), CD117 (c-Kit), CD138 (plasma cell marker), BCMA (B cell maturation antigen), CSF1R (macrophage colony-stimulating factor 1 receptor), DLL3 (delta-like protein 3), GPC3 (glial cell protein 3), ROR1 (receptor tyrosine kinase-like orphan receptor 1), Tn-Antigen (tumor-associated glycoprotein antigen), Globo H (tumor-associated antigen), EpCAM (epithelial cell adhesion molecule), WT1 (Wilms tumor protein), NY-ESO-1 (New York esophageal squamous cell carcinoma 1), Survivin (apoptosis inhibitor protein), hTERT (human telomerase reverse transcriptase), Galectin-3 (galactolectin 3), PD-L1 (programmed death-ligand 1), Claudin 18.2 (tight junction protein 18.2), Trop-2 (tumor-associated calcium signaling protein 2), 5T4 (tumor-associated antigen 5T4), KRAS (Kirsten rat sarcoma virus oncogene homolog), IDO1 (indoleamine 2,3-dioxygenase 1), B7-H3 (immune checkpoint protein), GPNMB (glycoprotein NMB), FAP (fibroblast activator protein), HSP90 (heat shock protein 90), LAMP1 (lysosome-associated membrane protein 1), NRG1 (neuroregulatory protein 1), NRP-1 (neuroregulatory protein receptor 1), SLC46A3 (solute carrier family 46 member 3), SPP2 (secretory phosphoprotein 2), TAAs (tumor-associated antigens), TRP-2 (tyrosinase-associated protein 2), and TTK (serine / threonine kinase).

[0058] In some embodiments, the tumor antigen includes any one or more selected from the group consisting of: EGFR, HER2, HER3, TROP-2, tissue factor (TF), Nectin-4, c-Met, B7-H3, CLDN18.2, MUC-1, PSCA / PSMA / PSA, FRα, CD19, CD20, CD22, CD33, CD123, CD38, CEA, CD25, CD46, CD79B, and BCMA.

[0059] In some embodiments, the tumor cell binding portion is an EGFR antibody. The EGFR antibody is preferably a nanobody.

[0060] For example, the EGFR antibody includes HCDR1, HCDR2, and HCDR3, wherein HCDR1 includes the amino acid sequence shown in SEQ ID NO: 2, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 3, and HCDR3 includes the amino acid sequence shown in SEQ ID NO: 4. For example, the EGFR antibody includes a variable region, which may contain the amino acid sequence shown in SEQ ID NO: 1.

[0061] For example, the EGFR antibody includes HCDR1, HCDR2, and HCDR3, wherein HCDR1 includes the amino acid sequence shown in SEQ ID NO: 6, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 7, and HCDR3 includes the amino acid sequence shown in SEQ ID NO: 8. For example, the EGFR antibody includes a variable region, which may contain the amino acid sequence shown in SEQ ID NO: 5.

[0062] For example, the EGFR antibody includes HCDR1, HCDR2, and HCDR3, wherein HCDR1 includes the amino acid sequence shown in SEQ ID NO: 10, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 11, and HCDR3 includes the amino acid sequence shown in SEQ ID NO: 12. For example, the EGFR antibody includes a variable region, which may contain the amino acid sequence shown in SEQ ID NO: 9.

[0063] For example, the EGFR antibody includes HCDR1, HCDR2, and HCDR3, wherein HCDR1 includes the amino acid sequence shown in SEQ ID NO: 2, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 3, and HCDR3 includes the amino acid sequence shown in SEQ ID NO: 4. For example, the EGFR antibody includes a variable region, which may contain the amino acid sequence shown in SEQ ID NO: 13.

[0064] For example, the EGFR antibody includes HCDR1, HCDR2, and HCDR3, wherein HCDR1 includes the amino acid sequence shown in SEQ ID NO: 2, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 3, and HCDR3 includes the amino acid sequence shown in SEQ ID NO: 15. For example, the EGFR antibody includes a variable region, which may contain the amino acid sequence shown in SEQ ID NO: 14.

[0065] For example, the EGFR antibody includes HCDR1, HCDR2, and HCDR3, wherein HCDR1 includes the amino acid sequence shown in SEQ ID NO: 17, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 3, and HCDR3 includes the amino acid sequence shown in SEQ ID NO: 18. For example, the EGFR antibody includes a variable region, which may contain the amino acid sequence shown in SEQ ID NO: 16.

[0066] For example, the EGFR antibody includes HCDR1, HCDR2, and HCDR3, wherein HCDR1 includes the amino acid sequence shown in SEQ ID NO: 2, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 20, and HCDR3 includes the amino acid sequence shown in SEQ ID NO: 21. For example, the EGFR antibody includes a variable region, which may contain the amino acid sequence shown in SEQ ID NO: 19.

[0067] For example, the EGFR antibody includes HCDR1, HCDR2, and HCDR3, wherein HCDR1 includes the amino acid sequence shown in SEQ ID NO: 23, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 3, and HCDR3 includes the amino acid sequence shown in SEQ ID NO: 24. For example, the EGFR antibody includes a variable region, which may contain the amino acid sequence shown in SEQ ID NO: 22.

[0068] For example, the EGFR antibody includes HCDR1, HCDR2, and HCDR3, wherein HCDR1 includes the amino acid sequence shown in SEQ ID NO: 2, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 3, and HCDR3 includes the amino acid sequence shown in SEQ ID NO: 15. For example, the EGFR antibody includes a variable region, which may contain the amino acid sequence shown in SEQ ID NO: 25.

[0069] For example, the EGFR antibody includes HCDR1, HCDR2, and HCDR3, wherein HCDR1 includes the amino acid sequence shown in SEQ ID NO: 27, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 3, and HCDR3 includes the amino acid sequence shown in SEQ ID NO: 28. For example, the EGFR antibody includes a variable region, which may contain the amino acid sequence shown in SEQ ID NO: 26.

[0070] For example, the EGFR antibody includes HCDR1, HCDR2, and HCDR3, wherein HCDR1 includes the amino acid sequence shown in SEQ ID NO: 30, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 31, and HCDR3 includes the amino acid sequence shown in SEQ ID NO: 32. For example, the EGFR antibody includes a variable region, which may contain the amino acid sequence shown in SEQ ID NO: 29.

[0071] For example, the EGFR antibody includes HCDR1, HCDR2, and HCDR3, wherein HCDR1 includes the amino acid sequence shown in SEQ ID NO: 2, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 34, and HCDR3 includes the amino acid sequence shown in SEQ ID NO: 35. For example, the EGFR antibody includes a variable region, which may contain the amino acid sequence shown in SEQ ID NO: 33.

[0072] For example, the EGFR antibody includes HCDR1, HCDR2, and HCDR3, wherein HCDR1 includes the amino acid sequence shown in SEQ ID NO: 27, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 37, and HCDR3 includes the amino acid sequence shown in SEQ ID NO: 38. For example, the EGFR antibody includes a variable region, which may contain the amino acid sequence shown in SEQ ID NO: 36.

[0073] For example, the EGFR antibody includes HCDR1, HCDR2, and HCDR3, wherein HCDR1 includes the amino acid sequence shown in SEQ ID NO: 40, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 3, and HCDR3 includes the amino acid sequence shown in SEQ ID NO: 41. For example, the EGFR antibody includes a variable region, which may contain the amino acid sequence shown in SEQ ID NO: 39.

[0074] For example, the EGFR antibody includes HCDR1, HCDR2, and HCDR3, wherein HCDR1 includes the amino acid sequence shown in SEQ ID NO: 43, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 44, and HCDR3 includes the amino acid sequence shown in SEQ ID NO: 45. For example, the EGFR antibody includes a variable region, which may contain the amino acid sequence shown in SEQ ID NO: 42.

[0075] For example, the EGFR antibody includes HCDR1, HCDR2, and HCDR3, wherein HCDR1 includes the amino acid sequence shown in SEQ ID NO: 6, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 3, and HCDR3 includes the amino acid sequence shown in SEQ ID NO: 47. For example, the EGFR antibody includes a variable region, which may contain the amino acid sequence shown in SEQ ID NO: 46.

[0076] For example, the EGFR antibody includes HCDR1, HCDR2, and HCDR3, wherein HCDR1 includes the amino acid sequence shown in SEQ ID NO: 49, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 50, and HCDR3 includes the amino acid sequence shown in SEQ ID NO: 51. For example, the EGFR antibody includes a variable region, which may contain the amino acid sequence shown in SEQ ID NO: 48.

[0077] In some embodiments, the tumor cell binding portion is a HER2 antibody. The HER2 antibody is preferably an affinity derivative.

[0078] For example, the HER2 affinity comprises the amino acid sequence shown in SEQ ID NO: 52.

[0079] In some embodiments, the immune cells include myeloid immune cells and lymphoid immune cells. In some embodiments, the immune cells are myeloid immune cells. In some embodiments, the immune cells are lymphoid immune cells.

[0080] In some embodiments, the immune cells include T cells (e.g., γδ T cells, regulatory T cells, memory T cells), natural killer cells, dendritic cells, macrophages, monocytes, B cells, mast cells, eosinophils, basophils, neutrophils, and / or MAIT cells.

[0081] In some embodiments, the immune cells include T cells, natural killer cells, dendritic cells, macrophages, monocytes, neutrophils, and / or B cells.

[0082] In some embodiments, the immune cell regulation portion targets immune cell surface receptors. Examples of immune cell surface receptors that can be used for the purposes of this application include, but are not limited to: CD3, CD4, CD8, CD28, CTLA-4, CD2, CD5, CD6, CD7, CD10, CD16, CD27, CD30, CD40, CD40L (CD154), CD80, CD86, CD95 (Fas), CD95L (FasL), OX40 (CD134), PD-1, PD-L1, ICOS (CD278), NKG2D, KIR, LFA-1 (CD11a / CD18), CLEC9A, and CR3.

[0083] In some embodiments, the immune cell regulatory portion targets intracellular receptors of immune cells. Examples of intracellular receptors of immune cells that can be used for the purposes of this application include, but are not limited to: NF-κB (nuclear factor κB), STAT (signal transduction and transcription activator), STING, PI3K (phosphatidylinositol 3-kinase), AKT (protein kinase B), mTOR (mammalian target of rapamycin), MAPK (mitogen-activated protein kinase), PLCγ (phospholipase Cγ), SYK (splenic tyrosine kinase), JAK (Janus kinase), SHP-1 (Src homology region phosphatase 1), IRF (interferon regulatory factor), AP-1 (activator protein 1), NFAT (nuclear factor activated T cell), FoxP3, GATA-3, T-bet, and RORγt.

[0084] In some embodiments, the immune cell regulation portion includes an antibody or an antigen-binding fragment thereof that targets a receptor on the surface of immune cells.

[0085] In some embodiments, the immune cell surface receptors include any one or more selected from the group consisting of: CD3, PD-L1, CLEC9A, CD16, NKG2D, antigen peptide-MHC molecular complex (pMHC), CD45, CD4, CD8, PD1, CD25, CD69, CD28, CD80, CD86, LAG3, IL-2R, IL-10R, IL-12R, IL-4R, CD47, CD40, and CD40L.

[0086] In some embodiments, the immune cell modulation portion includes a CD3 antibody. The CD3 antibody is preferably a nanobody. For example, the CD3 antibody may include HCDR1, HCDR2, and HCDR3, wherein HCDR1 includes the amino acid sequence shown in SEQ ID NO: 54, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 55, and HCDR3 includes the amino acid sequence shown in SEQ ID NO: 56. For example, the CD3 antibody may include a variable region, and the variable region may include the amino acid sequence shown in SEQ ID NO: 53.

[0087] In some embodiments, the immune cell modulation portion includes a PD-L1 antibody. The PD-L1 antibody is preferably a nanobody. For example, the PD-L1 antibody may include HCDR1, HCDR2, and HCDR3, wherein HCDR1 includes the amino acid sequence shown in SEQ ID NO: 66, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 67, and HCDR3 includes the amino acid sequence shown in SEQ ID NO: 68. For example, the PD-L1 antibody may include a variable region, and the variable region may include the amino acid sequence shown in SEQ ID NO: 65.

[0088] In some embodiments, the immune cell modulation portion includes a CLEC9A antibody. The CLEC9A antibody is preferably a nanobody. For example, the CLEC9A antibody may include HCDR1, HCDR2, and HCDR3, wherein HCDR1 includes the amino acid sequence shown in SEQ ID NO: 70, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 71, and HCDR3 includes the amino acid sequence shown in SEQ ID NO: 72. For example, the CLEC9A antibody may include a variable region, which may include the amino acid sequence shown in SEQ ID NO: 69.

[0089] In some embodiments, the immune cell modulation portion includes a CD16 antibody. The CD16 antibody is preferably a nanobody. For example, the CD16 antibody may include HCDR1, HCDR2, and HCDR3, wherein HCDR1 includes the amino acid sequence shown in SEQ ID NO: 58, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 59, and HCDR3 includes the amino acid sequence shown in SEQ ID NO: 60. For example, the CD16 antibody may include a variable region, which may include the amino acid sequence shown in SEQ ID NO: 57.

[0090] In some embodiments, the immune cell modulation portion includes an NKG2D antibody. The NKG2D antibody is preferably a nanobody. For example, the NKG2D antibody includes HCDR1, HCDR2, and HCDR3, wherein HCDR1 includes the amino acid sequence shown in SEQ ID NO: 62, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 63, and HCDR3 includes the amino acid sequence shown in SEQ ID NO: 64. For example, the NKG2D antibody may include a variable region, which may include the amino acid sequence shown in SEQ ID NO: 61.

[0091] In some embodiments, the immune cell regulation portion targets intracellular receptors of immune cells.

[0092] The immune cell regulatory component of the target immune cell intracellular receptor can be an immune agonist.

[0093] The immune cell regulatory component of the target immune cell intracellular receptor can be an immunosuppressant.

[0094] In some embodiments, the immune agonist or immunosuppressant includes any one or more selected from the group consisting of: Toll-like receptor (TLR) agonists (e.g., CpG oligonucleotides, lipopolysaccharide LPS, IMDQ), interferon gene-stimulated receptor (STING) agonists (e.g., Cyclic acid receptor agonists). dinucleotides, diABZI), retinoic acid-induced gene gene kinase I (RIG-I)-like receptor (RLR) agonists, leucine-rich nucleotide-binding domain repeats (NLRs), C-type lectin receptor (CLR) agonists, protein kinase agonists (e.g., JAK kinase agonists), phosphokinase agonists (e.g., SHP-1 / SHP-2 phosphatase inhibitors), transcription factor activators (e.g., NF-κB activators, NFAT activators), intracellular signaling molecules (e.g., interleukins, such as IL-2, IL-12, IL-15), immunomodulatory small molecules (e.g., imidazoline compounds), PKC agonists, apoptosis pathway regulators (e.g., Bcl-2 family protein inhibitors), interferons (e.g., IFN-α, IFN-β, IFN-γ), co-stimulatory molecule agonists (e.g., anti-CD28 antibodies, anti-OX40 antibodies, anti-4-1BB antibodies), chemoimmunostimulators (e.g., imatinib), cell metabolism regulators (mTOR agonists), signal transduction and transcription activator (STAT) protein inhibitors, indoleamine 2, 3-dioxygenase (IDO) inhibitors, glucocorticoids (e.g., prednisone), calcineurin inhibitors (e.g., cyclosporine, tacrolimus), antimetabolites (e.g., azathioprine, mycophenolate mofetil) mofetil), mTOR inhibitors (e.g., sirolimus, everolimus), T-cell depletion monoclonal antibodies (e.g., alemtuzumab), Janus kinase inhibitors (e.g., tofacitinib), tripterygium glycosides, leflunomide, mizoribine, polyclonal antibodies (e.g., anti-lymphocyte immunoglobulin (ATG)), infliximab, adalimumab, golimumab, and FTY720 derivatives (e.g., fingolimod).In some embodiments, the immune agonist or inhibitor includes any one or more selected from the group consisting of: Toll-like receptor (TLR) agonists, interferon gene-stimulated receptor (STING) agonists, retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), leucine-rich nucleotide-binding domain repeats (NLRs), C-type lectin receptors (CLRs), signal transduction and transcription activating proteins (STATs), and indoleamine 2,3-dioxygenase (IDO).

[0095] In some embodiments, the immune agonist or inhibitor includes any one or more selected from the group consisting of IMDQ, diABZI, CPG, STAT3 ASO, cGAMP, and IDO inhibitors.

[0096] In some embodiments, the immune agonist includes any one or more selected from the group consisting of IMDQ, diABZI, CPG, and cGAMP.

[0097] In some embodiments, the immunosuppressant includes any one or more selected from the group consisting of STAT3ASO, the IDO inhibitor Indoximod, GDC-0919, and Epacadostat.

[0098] In some implementations, P 1 P 2 and P 3 The tumor cell-targeting portion and / or immune cell-regulating portion of the protein can be released, thereby binding to tumor cells or regulating immune cells. At this time, P... 1 P 2 and P 3 It may further include cleavable or cleavable linkers. Cleavable linkers that can achieve the objectives of this invention include, but are not limited to: peptide linkers, acid-sensitive linkers, reduction-sensitive linkers, enzyme-sensitive linkers, light-sensitive linkers, bioorthogonal reaction linkers, pH-sensitive linkers, and / or temperature-sensitive linkers. For example, the cleavable linker may contain a disulfide bond (SS), which can be cleaved under reducing conditions to release P. 1 P 2 and / or P 3 .

[0099] In some implementations, P 1 P 2 and P 3 Any one of them is the tumor cell binding portion, any one is the T cell regulatory portion, and any one is a portion selected from the group consisting of: dendritic cell regulatory portion, natural killer cell regulatory portion, and myeloid immune cell regulatory portion.

[0100] In one specific implementation, P 1 P 2 and P 3 One of them contains a tumor antigen-binding protein, one contains a T cell surface receptor-binding protein, and one contains a dendritic cell surface receptor-binding protein. For example, P 1 P 2 and P 3 One of them contains the EGFR antibody, one contains the CD3 antibody, and one contains the PD-L1 antibody. For example, P 1 P 2 and P 3 One of them contains the HER2 antibody, one contains the CD3 antibody, and one contains the PD-L1 antibody.

[0101] In one specific implementation, P 1 P 2 and P 3 One of them contains a tumor antigen-binding protein, one contains a T cell surface receptor-binding protein, and one contains a natural killer cell surface receptor-binding protein. For example, P 1 P 2 and P 3 One of them contains the EGFR antibody, one contains the CD3 antibody, and one contains the CD16 antibody. For example, P 1 P 2 and P 3 One of them contains the HER2 antibody, one contains the CD3 antibody, and one contains the CD16 antibody.

[0102] In one specific implementation, P 1 P 2 and P 3 One of them contains a tumor antigen-binding protein, one contains a T cell surface receptor-binding protein, and one contains an intracellular receptor modulator of myeloid immune cells. For example, P 1 P 2 and P 3 One of them contains the EGFR antibody, one contains the CD3 antibody, and one contains a TLR agonist (e.g., IMDQ). For example, P 1 P 2 and P 3 One of them contains the HER2 antibody, one contains the CD3 antibody, and one contains a TLR agonist (e.g., IMDQ).

[0103] On the other hand, this application provides an antigen-binding protein that specifically binds to HER2. In some embodiments, the antigen-binding protein that specifically binds to HER2 is an affinity protein.

[0104] An affinity protein is typically a small, robust protein that binds to a target protein or peptide with high affinity. Its properties are similar to those of a monoclonal antibody, thus classifying it as an antibody mimic. Affinities are characterized by their small molecular weight, rapid folding rate, structural stability, and tolerance to chemical modifications.

[0105] For example, the antigen-binding protein that specifically binds to HER2 contains the amino acid sequence shown in SEQ ID NO: 52.

[0106] On the other hand, this application provides an antigen-binding protein that specifically binds to CD3. In some embodiments, the antigen-binding protein that specifically binds to CD3 includes a CD3 antibody or an antigen-binding fragment thereof. The CD3 antibody is preferably a nanobody. For example, the CD3 antibody may include HCDR1, HCDR2, and HCDR3, wherein HCDR1 includes the amino acid sequence shown in SEQ ID NO: 54, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 55, and HCDR3 includes the amino acid sequence shown in SEQ ID NO: 56. For example, the CD3 antibody may include a variable region, and the variable region may include the amino acid sequence shown in SEQ ID NO: 53.

[0107] On the other hand, this application provides an antigen-binding protein that specifically binds to PD-L1. In some embodiments, the antigen-binding protein that specifically binds to PD-L1 includes a PD-L1 antibody or an antigen-binding fragment thereof. The PD-L1 antibody is preferably a nanobody. For example, the PD-L1 antibody may include HCDR1, HCDR2, and HCDR3, wherein HCDR1 includes the amino acid sequence shown in SEQ ID NO: 66, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 67, and HCDR3 includes the amino acid sequence shown in SEQ ID NO: 68. For example, the PD-L1 antibody may include a variable region, and the variable region may include the amino acid sequence shown in SEQ ID NO: 65.

[0108] On the other hand, this application provides an antigen-binding protein that specifically binds to CLEC9A. In some embodiments, the antigen-binding protein that specifically binds to CLEC9A includes a CLEC9A antibody or an antigen-binding fragment thereof. The CLEC9A antibody is preferably a nanobody. For example, the CLEC9A antibody may include HCDR1, HCDR2, and HCDR3, wherein HCDR1 includes the amino acid sequence shown in SEQ ID NO: 70, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 71, and HCDR3 includes the amino acid sequence shown in SEQ ID NO: 72. For example, the CLEC9A antibody may include a variable region, which may include the amino acid sequence shown in SEQ ID NO: 69.

[0109] On the other hand, this application provides an antigen-binding protein that specifically binds to CD16. In some embodiments, the antigen-binding protein that specifically binds to CD16 includes a CD16 antibody or an antigen-binding fragment thereof. The CD16 antibody is preferably a nanobody. For example, the CD16 antibody may include HCDR1, HCDR2, and HCDR3, wherein HCDR1 includes the amino acid sequence shown in SEQ ID NO: 58, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 59, and HCDR3 includes the amino acid sequence shown in SEQ ID NO: 60. For example, the CD16 antibody may include a variable region, which may include the amino acid sequence shown in SEQ ID NO: 57.

[0110] On the other hand, this application provides an antigen-binding protein that specifically binds to NKG2D. In some embodiments, the antigen-binding protein that specifically binds to NKG2D includes an NKG2D antibody or an antigen-binding fragment thereof. The NKG2D antibody is preferably the nanobody described above. For example, the NKG2D antibody includes HCDR1, HCDR2, and HCDR3, wherein HCDR1 includes the amino acid sequence shown in SEQ ID NO: 62, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 63, and HCDR3 includes the amino acid sequence shown in SEQ ID NO: 64. For example, the NKG2D antibody may include a variable region, which may include the amino acid sequence shown in SEQ ID NO: 61.

[0111] On the other hand, this application provides isolated nucleic acids that encode the antigen-binding protein.

[0112] On the other hand, this application provides a vector containing the isolated nucleic acid.

[0113] On the other hand, this application provides cells containing the vector and / or the isolated nucleic acid.

[0114] On the other hand, this application provides an immunoconjugate comprising the antigen-binding protein.

[0115] On the other hand, this application provides a pharmaceutical composition comprising the multispecific conjugate described in this application and / or the antigen-binding protein. The pharmaceutical composition may further comprise pharmaceutically acceptable excipients or carriers.

[0116] On the other hand, this application provides a method for treating and / or preventing tumors, the method comprising administering to a subject in need an effective amount of the multispecific conjugate, the antigen-binding protein, the isolated nucleic acid, the carrier, the cell, the immunoconjugate, and / or the pharmaceutical composition described in this application.

[0117] On the other hand, this application provides the use of the multispecific conjugate, the antigen-binding protein, the isolated nucleic acid, the carrier, the cell, the immune conjugate and / or the pharmaceutical composition described in this application in the preparation of a medicament for the treatment and / or prevention of tumors.

[0118] On the other hand, this application provides the multispecific conjugate, the antigen-binding protein, the isolated nucleic acid, the carrier, the cell, the immunoconjugate and / or the pharmaceutical composition described in this application for the treatment and / or prevention of tumors.

[0119] In some embodiments, the administration includes injection, preferably intravenous injection.

[0120] In some embodiments, the dosage of the multispecific conjugate is 0-200 mg / kg body weight. For example, the dosage of the multispecific conjugate is 0-150 mg / kg body weight, 0-100 mg / kg body weight, 0-50 mg / kg body weight, 0-30 mg / kg body weight, 0-20 mg / kg body weight, 0-15 mg / kg body weight, or 1-12 mg / kg body weight.

[0121] In some embodiments, the dosing interval of the multispecific conjugate is 12 hours to 2 weeks. In some embodiments, the dosing interval of the multispecific conjugate is 12 hours to 2 weeks, 12 hours to 10 days, 12 hours to 1 week, 12 hours to 5 days, 12 hours to 3 days, 24 hours to 2 weeks, 24 hours to 10 days, 24 hours to 1 week, 24 hours to 5 days, 24 hours to 3 days, or 24 hours to 48 hours.

[0122] In some embodiments, the tumor includes a solid tumor. In some embodiments, the tumor includes a hemangioma.

[0123] In some embodiments, the tumor includes lung cancer, breast cancer, colorectal cancer, prostate cancer, stomach cancer, liver cancer, pancreatic cancer, ovarian cancer, cervical cancer, thyroid cancer, melanoma, lymphoma, leukemia, brain tumor, bone cancer, kidney cancer, bladder cancer, esophageal cancer, nasopharyngeal carcinoma, melanoma, skin cancer, laryngeal cancer, oral cancer, tongue cancer, gallbladder cancer, bile duct cancer, skin cancer, testicular cancer, uterine cancer, endometrial cancer, renal pelvis cancer, renal cell carcinoma, bladder cancer, glioma, neuroblastoma, multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma, myeloid leukemia, myelodysplastic syndrome, soft tissue sarcoma, osteosarcoma, liposarcoma, neurofibrosarcoma, angiosarcoma, gastrointestinal stromal tumor, thymoma, thymic carcinoma, pituitary adenoma, retinoblastoma / or glioblastoma.

[0124] In some embodiments, the tumor is selected from one or more of the following groups: breast cancer, colorectal cancer, lung cancer, ovarian cancer, bladder cancer, endometrial cancer, sarcoma, and pancreatic cancer.

[0125] On the other hand, this application provides a method for treating and / or preventing immune system-related diseases, the method comprising administering to a subject in need an effective amount of the multispecific conjugate, the antigen-binding protein, the isolated nucleic acid, the carrier, the cell, the immunoconjugate, and / or the pharmaceutical composition described in this application.

[0126] On the other hand, this application provides the use of the multispecific conjugate, the antigen-binding protein, the isolated nucleic acid, the carrier, the cell, the immune conjugate and / or the pharmaceutical composition described in this application in the preparation of a medicament for the treatment and / or prevention of immune system-related diseases.

[0127] On the other hand, this application provides the multispecific conjugate, the antigen-binding protein, the isolated nucleic acid, the carrier, the cell, the immune conjugate and / or the pharmaceutical composition described in this application for the treatment and / or prevention of immune system-related diseases.

[0128] On the other hand, this application provides a method for treating, preventing, or improving immune diseases or conditions in cells, tissues, organs, or animals, the method comprising administering to the cells, tissues, organs, or animals an effective amount of the multispecific conjugate, the antigen-binding protein, the isolated nucleic acid, the carrier, the cells, the immune conjugate, and / or the pharmaceutical composition described in this application.

[0129] On the other hand, this application provides a method for enhancing or stimulating an immune response or function, the method comprising administering to an individual (e.g., cells, tissues, organs, or animals) an effective amount of the multispecific conjugate described in this application, the antigen-binding protein, the isolated nucleic acid, the carrier, the cells, the immunoconjugate, and / or the pharmaceutical composition described herein. The method may be an in vivo method, an in vitro method, an ex vivo method, or a method in living cells. In some embodiments, the enhancement or stimulation of the immune response or function includes promoting the release of cytokines (e.g., IL12, IL1β, and / or IFNγ) from T cells. In some embodiments, the enhancement or stimulation of the immune response or function includes promoting the expression of Perforin, PD1, and / or Ki67 by T cells. In some embodiments, the enhancement or stimulation of the immune response or function includes promoting the expression of CD86 and / or HLA-DR by dendritic cells. In some embodiments, the enhancement or stimulation of the immune response or function includes promoting the release of cytokines (e.g., IFNγ and / or MIP-1β) from NK cells. In some embodiments, the enhanced or stimulated immune response or function includes promoting the expression of CD83 and / or CD40 on myeloid dendritic cells. In some embodiments, the enhanced or stimulated immune response or function includes altering the ratio of M1 and M2 macrophages in myeloid macrophages. In some embodiments, the enhanced or stimulated immune response or function includes promoting the expression of CD14, CD40, and / or CD80 on myeloid monocytes. In some embodiments, the enhanced or stimulated immune response or function includes inducing immune memory.

[0130] On the other hand, this application provides a method for activating immune cells, the method comprising administering to immune cells an effective amount of the multispecific conjugate described in this application, the antigen-binding protein, the isolated nucleic acid, the carrier, the cells, the immune conjugate, and / or the pharmaceutical composition described herein. The method may be an in vivo method, an in vitro method, an ex vivo method, or a method in living cells. In some embodiments, activating immune cells includes promoting the release of cytokines (e.g., IL12, IL1β, and / or IFNγ) from T cells. In some embodiments, activating immune cells includes promoting the expression of Perforin, PD1, and / or Ki67 by T cells. In some embodiments, activating immune cells includes promoting the expression of CD86 and / or HLA-DR by dendritic cells. In some embodiments, activating immune cells includes promoting the release of cytokines (e.g., IFNγ and / or MIP-1β) from NK cells. In some embodiments, activating immune cells includes promoting the expression of CD83 and / or CD40 by myeloid dendritic cells. In some embodiments, activating immune cells includes altering the ratio of M1 and M2 macrophages in myeloid macrophages. In some embodiments, activating immune cells includes promoting the expression of CD14, CD40, and / or CD80 on myeloid monocytes. Other aspects and advantages of this application will readily become apparent to those skilled in the art from the detailed description below. The following detailed description shows and describes only exemplary embodiments of this application. As those skilled in the art will recognize, the content of this application enables them to make modifications to the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application pertains. Accordingly, the descriptions in the accompanying drawings and specification of this application are merely exemplary and not restrictive. Attached Figure Description

[0131] The specific features of the invention involved in this application are shown in the appended claims. The features and advantages of the invention can be better understood by referring to the exemplary embodiments and drawings described in detail below. A brief description of the drawings is as follows:

[0132] Figure 1 The image shows LC-MS characterization of anti-EGFR-LPETG and anti-HER2-LPETG.

[0133] Figure 2 The image shows LC-MS characterization of anti-CD3-DBCO and anti-CD3-BCN.

[0134] Figure 3 The image shows LC-MS characterization of anti-CD16-BCN and anti-NKG2D-BCN.

[0135] Figure 4 The image shows LC-MS characterization of anti-PDL1-BCN and anti-CLEC9A-BCN.

[0136] Figure 5 The image shows LC-MS characterization of (IMDQ)6-BCN and (diABZI)6-BCN.

[0137] Figure 6 The image shows LC-MS characterization of CPG-DBCO and STAT3-DBCO.

[0138] Figure 7 The image shows the LC-MS characterization of (CMV)2-BCN.

[0139] Figure 8 The image shows the LC-MS and SDS-PAGE characterization of the multispecific conjugate EGFR-CD3-PDL1. The left image is a schematic diagram of EGFR-CD3-PDL1, the middle image is the LC-MS characterization image, and the right image is the SDS-PAGE characterization image.

[0140] Figure 9 The diagram shows how EGFR-CD3-PDL1 simultaneously recruits T cells and dendritic cells (DCs) to target tumors.

[0141] Figure 10 The image shows the LC-MS and SDS-PAGE characterization of the multispecific conjugate EGFR-CD3-CLEC9A. The left image is a schematic diagram of EGFR-CD3-CLEC9A, the middle image is the LC-MS characterization image, and the right image is the SDS-PAGE characterization image.

[0142] Figure 11 The diagram shows how EGFR-CD3-CLEC9A simultaneously recruits T cells and dendritic cells (DCs) to target tumors.

[0143] Figure 12 The image shows the LC-MS and SDS-PAGE characterization of the multispecific conjugate EGFR-CD3-CD16. The left image is a schematic diagram of EGFR-CD3-CD16, the middle image is the LC-MS characterization image, and the right image is the SDS-PAGE characterization image.

[0144] Figure 13 The diagram shows how EGFR-CD3-CD16 simultaneously recruits T cells and natural killer (NK) cells to target tumors.

[0145] Figure 14The image shows the LC-MS and SDS-PAGE characterization of the multispecific conjugate EGFR-CD3-NKG2D. The left image is a schematic diagram of EGFR-CD3-NKG2D, the middle image is the LC-MS characterization image, and the right image is the SDS-PAGE characterization image.

[0146] Figure 15 The diagram shows how EGFR-CD3-NKG2D simultaneously recruits T cells and natural killer (NK) cells to target tumors.

[0147] Figure 16 The image shows the LC-MS and SDS-PAGE characterization of the multispecific conjugate HER2-CD3-(IMDQ)6. The left image is a schematic diagram of HER2-CD3-(IMDQ)6, the middle image is the LC-MS characterization image, and the right image is the SDS-PAGE characterization image.

[0148] Figure 17 The diagram shows how HER2-CD3-(IMDQ)6 simultaneously recruits T cells and myeloid immune cells to target tumors.

[0149] Figure 18 The image shows the LC-MS and SDS-PAGE characterization of the multispecific conjugate HER2-CD3-(diABZI)6. The left image is a schematic diagram of HER2-CD3-(diABZI)6, the middle image is the LC-MS characterization image, and the right image is the SDS-PAGE characterization image.

[0150] Figure 19 The diagram shows how HER2-CD3-(diABZI)6 simultaneously recruits T cells and myeloid immune cells to target tumors.

[0151] Figure 20 The image shows the LC-MS and SDS-PAGE characterizations of the multispecific conjugate EGFR-CD3-CPG. The left image is a schematic diagram of EGFR-CD3-CPG, the middle image is the LC-MS characterization, and the right image is the SDS-PAGE characterization.

[0152] Figure 21 The diagram shows how EGFR-CD3-CPG simultaneously recruits T cells and myeloid immune cells to target tumors.

[0153] Figure 22 The image shows the LC-MS and SDS-PAGE characterization of the multispecific conjugate EGFR-CD3-STAT3. The left image is a schematic diagram of EGFR-CD3-STAT3, the middle image is the LC-MS characterization image, and the right image is the SDS-PAGE characterization image.

[0154] Figure 23The diagram shows how EGFR-CD3-STAT3 simultaneously recruits T cells and myeloid immune cells to target tumors.

[0155] Figure 24 The image shows the LC-MS and SDS-PAGE characterization of the multispecific conjugate EGFR-CD3-(CMV)2. The left image is a schematic diagram of EGFR-CD3-(CMV)2, the middle image is the LC-MS characterization image, and the right image is the SDS-PAGE characterization image.

[0156] Figure 25 The diagram shows EGFR-CD3-(CMV)2 simultaneously recruiting T cells and CMV antigen-specific T cells to target tumors.

[0157] Figure 26A The image shows the evaluation of the multispecific conjugate EGFR-CD3-PDL1 in a tumor cell-T cell-dendritic cell co-culture system.

[0158] Figure 26B This demonstrates tumor cell killing mediated by the multispecific conjugate EGFR-CD3-PDL1 in a tumor cell-T cell-dendritic cell co-culture system.

[0159] Figure 26C This shows T cell activation mediated by the multispecific conjugate EGFR-CD3-PDL1 (CD69) in a tumor cell-T cell-dendritic cell co-culture system. + T cells).

[0160] Figure 26D This shows T cell activation mediated by the multispecific conjugate EGFR-CD3-PDL1 (IFNγ and Granzyme B) in a tumor cell-T cell-dendritic cell co-culture system.

[0161] Figure 26E This demonstrates dendritic cell (DC) activation mediated by the multispecific conjugate EGFR-CD3-PDL1 (CD83) in a tumor cell-T cell-dendritic cell co-culture system. + and CD86 + Dendritic cells)

[0162] Figure 26F This demonstrates dendritic cell (DC) activation mediated by the multispecific conjugate EGFR-CD3-PDL1 (IL12 and IL1β cytokines) in a tumor cell-T cell-dendritic cell co-culture system.

[0163] Figure 27A This shows the evaluation of the therapeutic effect of EGFR-CD3-PDL1 in the PBMC humanized mouse model.

[0164] Figure 27B The curve shown is the tumor suppression curve of EGFR-CD3-PDL1 in the PBMC humanized mouse model.

[0165] Figure 27C The results show that EGFR-CD3-PDL1 activates intratumoral T cells and increases perforin in the PBMC humanized mouse model. + PD1 + and Ki67 + percentage of T cells.

[0166] Figure 28A This shows the evaluation of the therapeutic effect of EGFR-CD3-PDL1 in the HSC humanized mouse model.

[0167] Figure 28B The curve shown is the tumor suppression curve of EGFR-CD3-PDL1 in the HSC humanized mouse model.

[0168] Figure 28C The results show that EGFR-CD3-PDL1 activates intratumoral T cells and increases the expression levels of Perforin, Granzyme B, and PD1 on T cells in the HSC humanized mouse model.

[0169] Figure 28D The results show that EGFR-CD3-PDL1 activates intratumoral dendritic cells and increases CD86 in the HSC humanized mouse model. + Percentage of dendritic cells and proportion of HLA-DR-overexpressing dendritic cells.

[0170] Figure 29A The image shows an evaluation of the therapeutic effect of EGFR-CD3-PDL1 in a transgenic humanized mouse model.

[0171] Figure 29B The image shows the tumor suppression curve of EGFR-CD3-PDL1 in a transgenic humanized mouse model.

[0172] Figure 29C The results show that EGFR-CD3-PDL1 activates intratumoral T cells and increases perforin in a transgenic humanized mouse model. + Granzyme B + and Ki67 + percentage of T cells.

[0173] Figure 29D The results show that EGFR-CD3-PDL1 activates intratumoral dendritic cells and increases CD80 in a transgenic humanized mouse model. + CD86 +Percentage of dendritic cells and proportion of dendritic cells with high MHC-II expression.

[0174] Figure 29E The results show that EGFR-CD3-PDL1 increases the proportion of OVA antigen-specific T cells in tumors and spleen in a transgenic humanized mouse model.

[0175] Figure 29F The image shows that EGFR-CD3-PDL1 enhances tumor-specific immunity in a transgenic humanized mouse model. The left image shows the results of the ELISPOT experiment, and the right image shows the corresponding statistical results.

[0176] Figure 30A This image shows an evaluation of the therapeutic effect of EGFR-CD3-PDL1 in a patient's tumor tissue model.

[0177] Figure 30B The results show that EGFR-CD3-PDL1 activates T cells and dendritic cells in a patient tumor tissue model, promoting the secretion of IFNγ, TNFα, IL1β and IL12 cytokines in PTC.

[0178] Figure 30C The results show that EGFR-CD3-PDL1 treatment inhibits the growth of PTC derived from the patient's tumor tissue.

[0179] Figure 31A The image shows the evaluation of the multispecific conjugate EGFR-CD3-CD16 in a tumor cell-T cell co-culture system.

[0180] Figure 31B The image shows tumor cell killing mediated by the multispecific conjugate EGFR-CD3-CD16 in a tumor cell-T cell co-culture system.

[0181] Figure 31C This shows T cell activation mediated by the multispecific conjugate EGFR-CD3-CD16 (CD69) in a tumor cell-T cell co-culture system. + T cells).

[0182] Figure 31D The image shows T cell activation (IFNγ cytokine) mediated by the multispecific conjugate EGFR-CD3-CD16 in a tumor cell-T cell co-culture system.

[0183] Figure 32A The image shows the evaluation of the multispecific conjugate EGFR-CD3-CD16 in a tumor cell-natural killer (NK) co-culture system.

[0184] Figure 32BThis demonstrates tumor cell killing mediated by the multispecific conjugate EGFR-CD3-CD16 in a tumor cell-natural killer cell co-culture system.

[0185] Figure 32C This shows the activation of natural killer cells (CD107) mediated by the multispecific conjugate EGFR-CD3-CD16 in a tumor cell-natural killer cell co-culture system. + Natural killer cells).

[0186] Figure 32D The image shows natural killer cell activation mediated by the multispecific conjugate EGFR-CD3-CD16 (IFNγ and MIP-1β cytokines) in a tumor cell-natural killer cell co-culture system.

[0187] Figure 33A The image shows the release of IMDQ molecules from the multispecific conjugate HER2-CD3-(IMDQ)6 in the tumor reducing microenvironment.

[0188] Figure 33B The image shows LC-MS detection of HER2-CD3-(IMDQ)6 releasing IMDQ molecules in the tumor reducing microenvironment.

[0189] Figure 34A The image shows the evaluation of the multispecific conjugate HER2-CD3-(IMDQ)6 in a tumor cell-T cell co-culture system.

[0190] Figure 34B This demonstrates tumor cell killing mediated by the multispecific conjugate HER2-CD3-(IMDQ)6 in a tumor cell-T cell co-culture system.

[0191] Figure 34C This shows T cell activation (CD69) mediated by the multispecific conjugate HER2-CD3-(IMDQ)6 in a tumor cell-T cell co-culture system. + T cells).

[0192] Figure 34D The image shows T cell activation (IFNγ cytokine) mediated by the multispecific conjugate HER2-CD3-(IMDQ)6 in a tumor cell-T cell co-culture system.

[0193] Figure 35A The display shows an assessment of tumor-targeting myeloid immune cell activation mediated by HER2-CD3-(IMDQ)6.

[0194] Figure 35B This shows HER2-CD3-(IMDQ)6-mediated activation of myeloid dendritic cells (CD83). + CD40+ Dendritic cells).

[0195] Figure 35C The display shows HER2-CD3-(IMDQ)6-mediated activation of myeloid macrophages (M1 and M2 macrophage ratio).

[0196] Figure 35D The display shows HER2-CD3-(IMDQ)6-mediated myeloid monocyte activation (CD14, CD40, CD80 expression levels). Detailed Implementation

[0197] The following specific embodiments illustrate the implementation of the invention. Those skilled in the art can easily understand other advantages and effects of the invention from the content disclosed in this specification.

[0198] Terminology Definition

[0199] In this application, the term "antigen-binding protein" generally refers to a protein containing a portion that binds to an antigen, and optionally, a scaffold or backbone portion that allows the antigen-binding portion to adopt a conformation that promotes the binding of the antigen-binding protein to the antigen. Examples of antigen-binding proteins include, but are not limited to, antibodies, antigen-binding fragments (Fab, Fab', F(ab)2, Fv fragments, F(ab')2, VHH, scFv, di-scFv, and / or dAb), immunoconjugates, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs, or fusion proteins, provided they exhibit the desired antigen-binding activity.

[0200] In this application, the term "antibody" generally refers to an immunoglobulin capable of specifically binding to a corresponding antigen. The antibody may be secreted by immune cells (e.g., effector B cells). The antibody may be a monoclonal antibody (including a full-length monoclonal antibody comprising two light chains and two heavy chains), a polyclonal antibody, a multispecific antibody (e.g., a bispecific antibody), a humanized antibody, a fully human antibody, a chimeric antibody, and / or a camelified single-domain antibody. An "antibody" may generally comprise a protein, or an antigen-binding fragment thereof, consisting of at least two heavy chains (HC) and two light chains (LC) linked together by disulfide bonds. Each heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region. In some naturally occurring IgG, IgD, and IgA antibodies, the heavy chain constant region comprises three domains: CH1, CH2, and CH3. In some naturally occurring antibodies, each light chain comprises a light chain variable region (VL) and a light chain constant region. The light chain constant region comprises one domain: CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), which alternate with more conserved regions called framework regions (FRs). Each VH and VL contains three CDRs and four framework regions (FRs), arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable domains of the natural heavy and light chains each contain four FR regions (H-FR1, H-FR2, H-FR3, H-FR4, L-FR1, L-FR2, L-FR3, L-FR4), mostly in a β-sheet configuration, linked by three CDRs to form loops, and in some cases forming part of a β-sheet structure. The CDRs in each chain are closely clustered together by the FR regions and, together with CDRs from the other chain, form the antigen-binding site of the antibody.

[0201] In this application, the term "VHH" may also be referred to as a nanobody or heavy chain single-domain antibody, generally referring to a variable region of a heavy chain antibody that naturally lacks the antibody light chain. The term "VHH" is used to distinguish between the heavy chain variable domain (VH) and light chain variable domain (VL) present in conventional four-chain antibodies. Further description of VHHs or nanobodies can be found in Muyldermans' article (Reviews in Molecular Biotechnology 74: 277-302, 2001). VHHs or nanobodies are characterized by the presence of one or more "Hallmark residues" in one or more framework sequences. The VHH may also include its humanized or camelified form, as well as other modifications, portions or fragments, derivatives or "nanobody fusions," multivalent or multispecific constructs, and modifications to improve the VHH's half-life. VHHs form minimal antigen-binding fragments while retaining the binding affinity and specificity of full-length antibodies. VHHs have long CDR3 loops and convex complementary sites, enabling them to enter the internal lumen of the target antigen.

[0202] The terms “protein,” “polypeptide,” and “peptide” are used interchangeably herein and generally refer to polymers of amino acid residues and their variants and synthetic analogs. “Peptide” can also refer to a portion of the amino acid sequence derived from its original protein, for example, after trypsin digestion. One or more amino acid residues in the polymer can be synthetic, non-naturally occurring amino acids, such as chemical analogs of corresponding naturally occurring amino acids, as well as polymers of naturally occurring amino acids. The term also includes post-translational modifications of polypeptides, such as glycosylation, phosphorylation, and acetylation. In this application, the term “isolated” antigen-binding protein generally refers to an antigen-binding protein that has been identified, isolated, and / or recovered from components of its production environment (e.g., natural or recombinant). Contaminating components of its production environment are often substances that interfere with its research, diagnostic, or therapeutic use and can include enzymes, hormones, and other protein or non-protein solutes. Isolated antigen-binding proteins or antibodies are typically prepared via at least one purification step.

[0203] The protein and / or amino acid sequences involved in this application should also be understood to include at least the following range: variants or homologs having the same or similar functions as the protein. In this application, the variant can be a protein or polypeptide that has undergone substitution, deletion, or addition of one or more amino acids in the amino acid sequence of the protein (e.g., the multispecific binding protein or antigen-binding protein described in this application). For example, the functional variant may comprise a protein or polypeptide that has undergone amino acid alterations through substitution, deletion, and / or insertion of at least one, such as 1-30, 1-20, or 1-10, or even 1, 2, 3, 4, or 5 amino acids. The functional variant can substantially retain the biological properties of the protein or polypeptide before the alteration (e.g., substitution, deletion, or addition). For example, the functional variant can retain at least 60%, 70%, 80%, 90%, or 100% of the biological activity (e.g., antigen-binding capacity) of the protein or polypeptide before the alteration. For example, the substitution can be a conserved substitution.

[0204] In this application, a portion of the amino acid sequence of the multispecific binding protein or antigen-binding protein may be homologous to the corresponding amino acid sequence in an antibody from a specific species, or belong to a specific category. For example, both the variable region and the constant region of an antibody may originate from the variable region and the constant region of an antibody from an animal species (such as a human).

[0205] In this application, the homolog can be a protein or polypeptide that has at least about 85% (e.g., at least about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or higher) sequence homology with the amino acid sequence of the protein and / or the polypeptide (e.g., the antigen-binding protein described in this application).

[0206] In this application, homology generally refers to the similarity, resemblance, or association between two or more sequences. The "sequence homology percentage" can be calculated by comparing two sequences to be aligned within a comparison window, determining the number of positions in the two sequences that contain the same nucleic acid bases (e.g., A, T, C, G) or the same amino acid residues (e.g., Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys, and Met) to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window (i.e., the window size), and multiplying the result by 100 to produce the sequence homology percentage. Alignments performed to determine the sequence homology percentage can be performed in various ways known in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, or Megalign (DNASTAR) software. Those skilled in the art can determine suitable parameters for sequence alignment, including any algorithm required to achieve maximum alignment across the full-length sequence being compared or within the target sequence region. Homology can also be determined using FASTA and BLAST. A description of the FASTA algorithm can be found in W. R. Pearson and D. J. Lipman, “An Improved Tool for Biological Sequence Alignment,” Proceedings of the National Academy of Sciences (Proc. Natl. Acad. Sci.), 85: 2444-2448, 1988; and D. J. Lipman and W. R. Pearson, “A Fast and Sensitive Search for Protein Similarity,” Science, 227: 1435-1441, 1989. A description of the BLAST algorithm can be found in S. Altschul, W. Gish, W. Miller, E. W. Myers, and D. Lipman, “A Basic Tool for Local Alignment Search,” Journal of Molecular Biology, 215: 403-410, 1990.

[0207] In this application, the term "halogen" generally refers to fluorine, chlorine, bromine and iodine.

[0208] In this application, the term "urea" generally refers to -(HN-CO-)2N-.

[0209] In this application, the term "alkyl" generally includes saturated aliphatic groups, including straight-chain alkyl groups (e.g., methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, etc.) and branched-chain alkyl groups (e.g., isopropyl, tert-butyl, isobutyl, etc.). The term alkyl may further include alkyl groups in which the main chain carbon atom is substituted by one or more oxygen, nitrogen, sulfur, or phosphorus atoms of carbon. The alkyl groups described in this application may contain 1-20, 1-12, 1-10, 1-8, or 1-6 carbon atoms.

[0210] In this application, the term "alkenyl" generally refers to any cyclic or acyclic branched or unbranched unsaturated carbon chain portion having one or more double bonds. In this application, the term "alkenyl" generally refers to any cyclic or acyclic branched or unbranched unsaturated carbon chain portion having one or more triple bonds. The alkenyl group described in this application may contain 2-20, 2-12, 2-10, 2-8, or 2-6 carbon atoms.

[0211] In this application, the term "alkynyl" generally refers to an unsaturated straight-chain or branched alkynyl group, such as ethynyl, 1-propynyl, propynyl, butynyl, etc. The alkynyl group can be substituted or unsubstituted. The alkynyl group described in this application may contain 2-20, 2-12, 2-10, 2-8, or 2-6 carbon atoms.

[0212] In this application, as those skilled in the art will know, terms such as "alkyl," "alkenyl," and "cycloalkyl" may be preceded by an identifier indicating the number of atoms present in the group under specific conditions, for example, C1-C6 alkyl, etc., and the subscript number following "C" indicates the number of carbon atoms present in the group. For example, C3 alkyl refers to an alkyl group having three carbon atoms (e.g., n-propyl, isopropyl). For example, the term "C 1~6 "Alkyl" includes alkyl groups containing 1 to 6 carbon atoms.

[0213] In this application, the term "alkyl chain" generally refers to a residue derived from an alkyl chain by removing a hydrogen atom. Alkyl chains can be substituted or unsubstituted, substituted or non-substituted. The term "alkyl chain" generally refers to a saturated straight-chain or branched aliphatic hydrocarbon group having residues derived from the removal of hydrogen atoms from the same carbon atom or two different carbon atoms of a parent alkane, and can be a straight-chain or branched group containing 1 to 20 carbon atoms, for example, containing 1 to 12 carbon atoms, such as an alkyl chain containing 1 to 6 carbon atoms. Non-limiting examples of alkyl chains include, but are not limited to, methyl, ethyl, propyl, propyl, butyl, etc. The alkyl group can be substituted or unsubstituted, alternative or non-alternative. For example, when substituted, the substituent can be replaced at any usable linking point. The substituent can be independently selected from one or more substituents chosen from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocyclic, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, and oxo, for example, it can be hydrogen, protium, deuterium, tritium, halogen, -NO2, -CN, -OH, -SH, -NH2, -C(O)H, -CO2H, -C(O)C(O)H, -C(O)CH2C(O)H, -S(O)H, -S(O)2H, -C(O)NH2, -SO2NH2, -OC(O)H, -N(H)SO2H or C 1-6 Aliphatic groups.

[0214] In this application, the term "cycloalkyl" generally refers to residues derived by removing hydrogen atoms from the same or multiple different carbon atoms of a carbide ring. The term "cycloalkane" generally refers to saturated or partially unsaturated monocyclic or polycyclic hydrocarbons, the carbide ring containing 3 to 20 carbon atoms, 3 to 12 carbon atoms, 3 to 10 carbon atoms, or 3 to 8 carbon atoms. Non-limiting examples of monocyclic carbocyclic rings include cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane, cyclohexene, cyclohexadiene, cycloheptane, cycloheptane, cyclooctane, etc.; polycyclic carbocyclic rings may include spirocyclic, fused, and bridged rings. Cycloalkyl groups may be substituted or unsubstituted.

[0215] In this application, the term "heterocyclic alkyl" generally refers to stable, non-aromatic 3-7 membered monocyclic structures, fused 7-10 membered bicyclic heterocyclic structures, or bridged 6-10 membered bicyclic heterocyclic structures. These cyclic structures can be saturated or partially saturated, and in addition to carbon atoms, they contain one or more heteroatoms selected from the group consisting of oxygen, sulfur, and nitrogen. For example, it may contain 1-4 heteroatoms as defined above. When used to refer to atoms in a heterocyclic structure, the term "nitrogen" may include nitrogen that has undergone a substitution reaction. Heterocyclic alkyl groups can be substituted or unsubstituted.

[0216] In this application, the term "aryl" generally refers to a monocyclic aromatic group comprising 3 to 12 substituted or unsubstituted members, wherein each atom of the ring may be carbon (i.e., carbocyclic aryl), or one or more of the atoms may be heteroatoms (i.e., heteroaryl). The aryl group may be substituted or unsubstituted, and when substituted, the substituent may be one or more of the following groups, independently selected from the group consisting of: alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycloalkylthio.

[0217] The term "heteroatom" is generally accepted in the art and refers to an atom of any element other than carbon or hydrogen. Exemplary heteroatoms include boron, nitrogen, oxygen, phosphorus, sulfur, and selenium.

[0218] In this application, the term "heteroaryl" generally refers to a residue derived from the removal of hydrogen atoms from the same carbon atom or multiple different carbon atoms of a heteroaryl ring. The term "heteroaryl ring" refers to a heteroaryl system comprising 1 to 4 heteroatoms and 5 to 14 ring atoms, wherein the heteroatoms may be selected from the group consisting of oxygen, sulfur, and nitrogen. The heteroaryl group may be 5 to 10-membered, or 5- or 6-membered, such as furanyl, thiophene, pyridinyl, pyrroleyl, N-alkylpyrroleyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl, etc. The heteroaryl ring may be fused to an aryl, heterocyclic, or cycloalkyl ring, wherein the ring connected to the parent structure is the heteroaryl ring. The heteroaryl group can be optionally substituted or unsubstituted. When substituted, the substituent can be one or more of the following groups, independently selected from the group consisting of: alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycloalkylthio.

[0219] In this application, the term "alkoxy" generally refers to an alkyl group to which an oxygen group is attached. Representative alkoxy groups include methoxy, ethoxy, propoxy, tert-butoxy, etc.

[0220] In this application, the terms “optional” or “optionally” generally refer to an event or environment described subsequently that may but does not have to occur, and the description includes situations in which the event or environment occurs or does not occur. For example, “optionally alkyl-substituted heterocyclic group” means that an alkyl group may but does not have to be present, and the description can include cases where the heterocyclic group is substituted with an alkyl group and cases where the heterocyclic group is not substituted with an alkyl group.

[0221] In this application, the compounds or conjugates comprise tautomers, meso compounds, racemic compounds, enantiomers, and / or diastereomers of the compounds. In this application, the term "diastereomer" generally refers to a stereoisomer having two or more chiral centers and whose molecules are not mirror images of each other. Diastereomers can have different physical properties, such as melting point, boiling point, spectral properties, and reactivity. In this application, the term "meso compound" generally refers to a molecule containing asymmetric atoms but possessing symmetry factors that result in zero total optical rotation within the molecule. The terms "racemic compound" or "racemic mixture" refer to a composition consisting of two enantiomers in equimolar amounts.

[0222] In this application, certain atoms of the compounds may appear in more than one isotopic form. For example, hydrogen may appear as protium (…). 1 H), deuterium ( 2 H) and tritium ( 3 Carbon exists in the form of H, and it may exist in three different isotopes (H). 12 C 13 C and 14 C) Naturally occurring. Examples of isotopes that may be incorporated into the compounds of this application include, but are not limited to, those that exist naturally. 15 N、 18 O、 17 O、 18 F, 32 P, 33 P, 129 I, 131 I, 123 I, 124 I, 125 I, or similar isotopes. Unless otherwise specified, the structures described in this application may also include compounds that differ only in the presence or absence of one or more isotopically enriched atoms. For example, compounds that are otherwise identical in structure to those described in this application, except that hydrogen atoms are replaced by deuterium or tritium, or carbon atoms are replaced by carbon-13 or carbon-14, are within the scope of this application.

[0223] In this application, the term "immune cell" generally refers to cells that can participate in or are associated with an immune response. Immune cells may include all leukocytes and non-leukocyte antigen-presenting cells. The term includes individual cells, cell lines, or cell cultures. The cells may include not only a specific type of cell but also the progeny of those cells.

[0224] In this application, the term "pharmaceuticalally acceptable adjuvant" generally includes pharmaceutically acceptable carriers, excipients, or stabilizers that are non-toxic to the cells or mammals exposed to them at the doses and concentrations used. Typically, physiologically acceptable carriers are pH-buffered aqueous solutions.

[0225] As used herein, the term "treatment" refers to the administration of an oral or topical therapeutic agent, such as a cell comprising any of the modified cells of this application or a pharmaceutical composition comprising modified cells, to a patient who has one or more symptoms of a disease, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Typically, the therapeutic agent is administered to the patient in an amount that effectively relieves one or more symptoms of the disease (therapeutic effective amount). The desired effects of treatment include reducing the rate of disease progression, improving or alleviating the disease state, and resolving or improving prognosis. For example, an individual is successfully "treated" if one or more cancer-related symptoms are reduced or eliminated, including but not limited to, reducing (or destroying) the proliferation of cancer cells, reducing symptoms originating from the disease, improving the quality of life of individuals with the disease, reducing the dosage of other medications needed to treat the disease, delaying disease progression, and / or prolonging individual survival.

[0226] In this application, the term "specificity" generally refers to the selective recognition of a specific epitope of an antigen by an antibody. For example, natural antibodies are monospecific. As in this application, the term "multispecific" refers to selectivity with two or more antigen-binding sites, wherein at least two bind to different antigens or different epitopes of the same antigen. For example, it can be multispecific for at least two different antigens (i.e., EGFR as the first antigen and CD3 as the second antigen). In one embodiment of the invention, the multispecific antibody according to the invention can be bispecific. In another embodiment of the invention, the multispecific antibody according to the invention can be trispecific.

[0227] In this application, the term "coupling" generally refers to the connection of two compounds by a covalent bond or by a strong non-covalent interaction, such as a covalent bond. For example, this application provides a connection in which the N3 group of one compound reacts with the alkynyl group of another compound to form a covalent bond, and thus a coupling of the two compounds can be formed.

[0228] In this application, the term "connecting arm" generally refers to a central compound that can connect to different other compounds. The central compound can connect to one or more other compounds via covalent bonds or strong non-covalent interactions to form another, larger compound. For example, the connecting arm of this application can be connected to one or more compounds comprising lipids, proteins, nucleic acids, small molecules, or polysaccharides, or any combination thereof, to form a conjugate.

[0229] In this application, the term "administration" generally refers to the delivery of a substance to a subject in need via any route known in the art. Pharmaceutical carriers and formulations or compositions are also well known in the art. Routes of administration may include intravenous, intramuscular, intradermal, subcutaneous, transdermal, mucosal, intratumoral, and / or mucosal administration.

[0230] In this application, the term "comprising" generally means including the explicitly specified features, but does not exclude other elements. The terms "above" and "below" generally refer to situations that include the stated number.

[0231] In this application, the term "about" generally refers to a variation within a range of 0.5% to 10% above or below a specified value, such as a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value. Invention Details

[0233] Multispecific conjugates

[0234] On the one hand, this application provides a multispecific conjugate that, through a trivalent multispecific conjugate arm, combines P... 1 P 2 or P 3 Three different or identical active molecules with properties of interest are integrated into a single molecular backbone. For the structure, properties or preparation methods of multispecific coupling arms, please refer to the international publication text of the PCT application WO2022 / 100696A1. All multispecific coupling arms mentioned in that international application can be used in this application to achieve the purpose of this application.

[0235] In one embodiment, the multi-specific coupling arm has a structure as shown in Formula I: (Equation I), where,

[0236] W is or ,in, Representing the connection site, W passes through Connect to La, Lb, and Lc respectively;

[0237] La is -C(=O)-X 1 -(K 1 ) n1 -(Y 1 ) p1 -(L 1 ) q1 -, Lb is -C(=O)-X 2 -(K 2 ) n2 -(Y 2 ) p2 -(L 2 ) q2 -, Lc is -C(=O)-X 3 -(K3 ) n3 -(Y 3 ) p3 -(L 3 ) q3 -,in,

[0238] X 1 X 2 , and X 3 Each is independently selected from the following groups: -NH- and -O-.

[0239] (K 1 ) n1 , (K 2 ) n2 , and (K 3 ) n3 Each of the following groups is independently selected: -CH2-(CH2-O-CH2)3-(CH2)2-, -(CH2)2-(CH2-O-CH2)3-CH2-, -CH2-(CH2-O-CH2)3-CH2-, and -(CH2)2-(CH2-O-CH2)3-(CH2)2-, or n1, n2, or n3 is independently 0.

[0240] (Y 1 ) p1 , (Y 2 ) p2 , and (Y 3 ) p3 Each independently as -NR 1 -C(=O)-,R 1 Selected from the following groups: hydrogen, protium, deuterium, and tritium; or p1, p2, or p3 each independently equals 0.

[0241] (L 1 ) q1 , (L 2 ) q2 , and (L 3 ) q3 Each is independently selected from the group consisting of -CH2-aryl-, -(CH2)2-, and -CH2-, or q1, q2, and q3 are each independently 0, wherein the aryl group is a 6-membered to 14-membered aryl group.

[0242] A 2 B 2 and C 2 Each is independently selected from functional groups capable of undergoing the following reactions: cycloaddition reactions involving azide groups, Diels-Alder reactions involving tetrazine groups, and / or transpeptidation reactions of SrtA.

[0243] P 1 P 2 and P3 Each is independently selected from the following groups: sugars, lipids, nucleic acids, peptides, proteins, and small molecules, as well as combinations thereof, and P 1 P 2 and P 3 It can recruit cells.

[0244] Building upon WO2022 / 100696A1, the inventors of this application further discovered that multispecific conjugates can recruit cells, not just interact with free molecules. By selecting appropriate P... 1 P 2 and P 3 The multispecific conjugates of this application can recruit at least three cells to a suitable spatial location, or can interact with at least three cells. This interaction can be cell binding or modulation of cell activity.

[0245] In this application, A 2 B 2 and C 2 Each can be independently selected from the following groups: , , , , , , , , , , and covalent bonds, where R 12 R 13 and R 14 Each is independently selected from the following groups: hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxyl, alkoxy, amino, amide, ester, sulfonamide, urea, C1-C 12 Alkyl groups, 3- to 12-membered cycloalkyl groups, 3- to 12-membered heterocycloalkyl groups, C2-C 12 alkenyl, C2-C 12 Alkynyl, 6- to 14-membered aryl and 6- to 14-membered heteroaryl, among which, Represents the connection site, and A 2 B 2 and C 2 pass With La, Lb, Lc and P respectively 1 P 2 and P 3 Connection, as long as A 2 B 2 and C 2 Each P can be connected through a connection reaction. 1 P 2 and P 3 And they do not interfere with each other.

[0246] In this application, W in the multispecific conjugate can be... .

[0247] In this application, X in the multispecific conjugate 1 X 2 and X 3 Both can be -NH-.

[0248] In this application, X in the multispecific conjugate 1 X 2 and X 3 Both can be -NH-.

[0249] In this application, the (K) in the multispecific conjugate 1 ) n1 , (K 2 ) n2 , and (K 3 ) n3 All of them can be -(CH2)2-(CH2-O-CH2)3-(CH2)2-.

[0250] In this application, the (Y) in the multispecific conjugate 1 ) p1 , (Y 2 ) p2 , and (Y 3 ) p3 They can be independently designated as -NR 1 -C(=O)-,R 1 Selected from the following group: hydrogen, protium, deuterium and tritium.

[0251] In this application, the (L) in the multispecific conjugate 1 ) q1 , (L 2 ) q2 , and (L 3 ) q3 They can be independently selected from: -CH2- and -CH2-aryl-.

[0252] In this application, A in the multispecific conjugate 2 You can choose from the following groups: , and .

[0253] In this application, B in the multispecific conjugate 2 You can choose from the following groups: , and .

[0254] In this application, C in the multispecific conjugate 2 You can choose from the following groups: , and .

[0255] In this application, A in the multispecific conjugate 2 B 2 and C 2 They can be: , and .

[0256] In this application, the multispecific conjugate may have a structure as shown in formula IIa-1 or IIa-2: ,

[0257] , where P 1 P 2 and P 3 As defined in this application.

[0258] For example, the multispecific conjugate can have a structure as shown in Formula IIa-1, wherein P 1 P 2 and P 3 As defined in this application.

[0259] P 1 P 2 and P 3

[0260] In this application, P 1 P 2 and / or P 3 It can be a molecule that recruits cells, and its molecular types can be nucleic acids, peptides, sugars, fats, proteins, and small molecules, or combinations thereof.

[0261] In some implementations, P 1 P 2 and / or P 3 At least one of them can act on tumor cells, recruit tumor cells, bind to tumor cells, bind to tumor cell surface proteins, bind to tumor antigens, or bind to intracellular targets of tumor cells.

[0262] In some implementations, P 1 P 2 and / or P 3 At least one of them can act on immune cells, recruit immune cells, bind to immune cells, bind to the surface proteins of immune cells, or bind to intracellular targets of immune cells.

[0263] Those skilled in the art can select appropriate P based on the type of tumor cells or immune cells to which they wish to act. 1 P 2 and / or P 3 The target site for action, and the selection of appropriate P 1 P 2 and / or P 3 Molecular types.

[0264] In one specific implementation, P 1 P 2 and / or P 3 Both can be antigen-binding proteins.

[0265] In another specific implementation, P 1 P 2 and / or P 3 It can contain antigen-binding proteins and small molecules.

[0266] In this application, P 1 P 2 and P 3 At least one of them may include a tumor cell binding portion, and at least one may include an immune cell regulating portion.

[0267] In this application, the tumor cell binding portion may contain a tumor antigen-binding protein. In this application, the tumor cell binding portion may contain an antibody or an antigen-binding fragment targeting a tumor antigen.

[0268] In this application, the immune cells may include myeloid immune cells and lymphoid immune cells. In some embodiments, the immune cells are myeloid immune cells. In some embodiments, the immune cells are lymphoid immune cells.

[0269] In this application, the immune cells may include T cells (e.g., γδ T cells, regulatory T cells, memory T cells), natural killer cells, dendritic cells, macrophages, monocytes, B cells, mast cells, eosinophils, basophils, neutrophils and / or MAIT cells.

[0270] In this application, the immune cells may include T cells, natural killer cells, dendritic cells, macrophages, and / or monocytes.

[0271] In this application, the immune cell regulation portion may include an antibody or an antigen-binding fragment thereof that targets proteins on the surface of immune cells.

[0272] In this application, the immune cell regulation portion can target intracellular receptors of immune cells.

[0273] antigen-binding proteins

[0274] On the other hand, this application provides an antigen-binding protein that specifically binds to EGFR. In some embodiments, the antigen-binding protein that specifically binds to EGFR includes an EGFR antibody or an antigen-binding fragment thereof. The EGFR antibody is preferably a nanobody. For example, the EGFR antibody may comprise HCDR1, HCDR2, and HCDR3, and HCDR1, HCDR2, and HCDR3 may comprise amino acid sequences selected from any one or more of the following groups: (1) HCDR1: SEQ ID NO: 2, HCDR2: SEQ ID NO: 3, and HCDR3: SEQ ID NO: 4, (2) HCDR1: SEQ ID NO: 6, HCDR2: SEQ ID NO: 7, and HCDR3: SEQ ID NO: 8, (3) HCDR1: SEQ ID NO: 10, HCDR2: SEQ ID NO: 11, and HCDR3: SEQ ID NO: 12, (4) HCDR1: SEQ ID NO: 2, HCDR2: SEQ ID NO: 3, and HCDR3: SEQ ID NO: 4, (5) HCDR1: SEQ ID NO: 2, HCDR2: SEQ ID NO: 3, and HCDR3: SEQ ID NO: 15, (6) HCDR1: SEQ ID NO: 17, HCDR2: SEQ ID NO: 3, and HCDR3: SEQ ID NO: 18, (7) HCDR1: SEQ ID NO: 2, HCDR2: SEQ ID NO: 20, and HCDR3: SEQ ID NO: 21, (8) HCDR1: SEQ ID NO: 23, HCDR2: SEQ ID NO: 3, and HCDR3: SEQ ID NO: 24, (9) HCDR1: SEQ ID NO: 2, HCDR2: SEQ ID NO: 3, and HCDR3: SEQ ID NO: 15, (10) HCDR1: SEQ ID NO: 27, HCDR2: SEQ ID NO: 3, and HCDR3: SEQ ID NO: 28, (11) HCDR1: SEQ ID NO: 30, HCDR2: SEQ ID NO: 31, and HCDR3: SEQ ID NO: 32, (12) HCDR1: SEQ ID NO: 2, HCDR2: SEQ ID NO: 34, and HCDR3: SEQ ID NO: 35, (13) HCDR1: SEQ ID NO: 27, HCDR2: SEQ ID NO: 37, and HCDR3: SEQ ID NO: 38, (14) HCDR1: SEQ ID NO: 40,HCDR2: SEQ ID NO: 3, and HCDR3: SEQ ID NO: 41, (15) HCDR1: SEQ ID NO: 43, HCDR2: SEQ ID NO: 44, and HCDR3: SEQ ID NO: 45, (16) HCDR1: SEQ ID NO: 6, HCDR2: SEQ ID NO: 3, and HCDR3: SEQ ID NO: 47, and (17) HCDR1: SEQ ID NO: 49, HCDR2: SEQ ID NO: 50, and HCDR3: SEQ ID NO: 51.

[0275] On the other hand, this application provides an antigen-binding protein that specifically binds to HER2. The amino acid sequence of the antigen-binding protein that specifically binds to HER2 is shown in SEQ ID NO: 52.

[0276] On the other hand, this application provides an antigen-binding protein that specifically binds to CD3. In some embodiments, the antigen-binding protein that specifically binds to CD3 includes a CD3 antibody or an antigen-binding fragment thereof. The CD3 antibody is preferably a nanobody. For example, the CD3 antibody may include HCDR1, HCDR2, and HCDR3, wherein HCDR1 includes the amino acid sequence shown in SEQ ID NO: 54, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 55, and HCDR3 includes the amino acid sequence shown in SEQ ID NO: 56. For example, the CD3 antibody may include a variable region, and the variable region may include the amino acid sequence shown in SEQ ID NO: 53.

[0277] On the other hand, this application provides an antigen-binding protein that specifically binds to PD-L1. In some embodiments, the antigen-binding protein that specifically binds to PD-L1 includes a PD-L1 antibody or an antigen-binding fragment thereof. The PD-L1 antibody is preferably a nanobody. For example, the PD-L1 antibody may include HCDR1, HCDR2, and HCDR3, wherein HCDR1 includes the amino acid sequence shown in SEQ ID NO: 66, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 67, and HCDR3 includes the amino acid sequence shown in SEQ ID NO: 68. For example, the PD-L1 antibody may include a variable region, and the variable region may include the amino acid sequence shown in SEQ ID NO: 65.

[0278] On the other hand, this application provides an antigen-binding protein that specifically binds to CLEC9A. In some embodiments, the antigen-binding protein that specifically binds to CLEC9A includes a CLEC9A antibody or an antigen-binding fragment thereof. The CLEC9A antibody is preferably a nanobody. For example, the CLEC9A antibody may include HCDR1, HCDR2, and HCDR3, wherein HCDR1 includes the amino acid sequence shown in SEQ ID NO: 70, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 71, and HCDR3 includes the amino acid sequence shown in SEQ ID NO: 72. For example, the CLEC9A antibody may include a variable region, which may include the amino acid sequence shown in SEQ ID NO: 69.

[0279] On the other hand, this application provides an antigen-binding protein that specifically binds to CD16. In some embodiments, the antigen-binding protein that specifically binds to CD16 includes a CD16 antibody or an antigen-binding fragment thereof. The CD16 antibody is preferably a nanobody. For example, the CD16 antibody may include HCDR1, HCDR2, and HCDR3, wherein HCDR1 includes the amino acid sequence shown in SEQ ID NO: 58, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 59, and HCDR3 includes the amino acid sequence shown in SEQ ID NO: 60. For example, the CD16 antibody may include a variable region, which may include the amino acid sequence shown in SEQ ID NO: 57.

[0280] On the other hand, this application provides an antigen-binding protein that specifically binds to NKG2D. In some embodiments, the antigen-binding protein that specifically binds to NKG2D includes an NKG2D antibody or an antigen-binding fragment thereof. The NKG2D antibody is preferably the nanobody described above. For example, the NKG2D antibody includes HCDR1, HCDR2, and HCDR3, wherein HCDR1 includes the amino acid sequence shown in SEQ ID NO: 62, HCDR2 includes the amino acid sequence shown in SEQ ID NO: 63, and HCDR3 includes the amino acid sequence shown in SEQ ID NO: 64. For example, the NKG2D antibody may include a variable region, which may include the amino acid sequence shown in SEQ ID NO: 61.

[0281] The antigen-binding protein of this application can be prepared using techniques well-known in the art, such as hybridoma methods, recombinant DNA technology, phage display technology, synthetic techniques, or combinations thereof, or other techniques known in the art. Variants can refer to amino acid sequence mutants of antibodies, as well as covalent derivatives of natural polypeptides, provided they retain biological activity equivalent to that of the natural polypeptide. The difference between amino acid sequence mutants and natural amino acid sequences generally lies in the substitution of one or more amino acids in the natural amino acid sequence, or the deletion and / or insertion of one or more amino acids in the polypeptide sequence. Deletion mutants include fragments of the natural polypeptide and N-terminal and / or C-terminal truncated mutants. Typically, amino acid sequence mutants have at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% homology to the natural sequence.

[0282] The aforementioned antigen-binding proteins can all be used in the multispecific conjugates of this application. Those skilled in the art can also select appropriate antigen-binding protein sequences to prepare multispecific conjugates as needed.

[0283] Drug compositions, treatment methods

[0284] On the other hand, this application provides a method for treating and / or alleviating tumors, the method comprising administering to a subject in need a multispecific conjugate, antigen-binding protein, nucleic acid, cell, pharmaceutical composition, kit, or immunoconjugate according to this application. An appropriate P can be selected based on the type of tumor to be treated and / or alleviated. 1 P 2 or P 3 Then, according to the method of this application, a multispecific conjugate targeting specific cells is prepared.

[0285] For example, the tumor may be a tumor that highly expresses EGFR. For example, the tumor may be an EGFR-positive tumor. For example, EGFR-positive tumors may include, but are not limited to: non-small cell lung cancer (NSCLC), glioblastoma, head and neck squamous cell carcinoma, breast cancer, gastric cancer, colorectal cancer, bladder cancer, liver cancer, ovarian cancer, and pancreatic cancer.

[0286] For example, the tumor may be a tumor that highly expresses HER2. For example, the tumor may be a HER2-positive tumor. For example, HER2-positive tumors may include, but are not limited to: breast cancer, gastric cancer, esophageal cancer, lung cancer, ovarian cancer, bladder cancer, endometrial cancer, glioblastoma, prostate cancer, and bile duct cancer.

[0287] The tumor can be a primary tumor or a metastatic tumor.

[0288] The multispecific conjugates, antigen-binding proteins, nucleic acids, cells, pharmaceutical compositions, kits, or immunoconjugates described in this application can be administered alone or as a pharmaceutical composition in combination with a pharmaceutically acceptable adjuvant. For example, they can be administered in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients. Such compositions may include buffers such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol; proteins; peptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

[0289] The multispecific conjugates, antigen-binding proteins, nucleic acids, cells, pharmaceutical compositions, kits, or immunoconjugates of this application may be administered to patients subcutaneously, intradermally, intratumorally, intranodally, intraspinally, intramuscularly, intravenously (iv), or intraperitoneally. Preferably, the pharmaceutical compositions of this application can be formulated for intravenous administration.

[0290] The multispecific conjugates, antigen-binding proteins, nucleic acids, cells, pharmaceutical compositions, kits, or immune conjugates described in this application can be used in combination with one or more other anti-tumor therapies (e.g., surgery, chemotherapy, radiotherapy, targeted therapy, or immune checkpoint inhibitors).

[0291] The dosage and frequency of cell administration in this application will be determined by practical factors, such as the patient's condition, the type and severity of the patient's disease, and taking into account the patient's age, weight, tumor size, degree of infection or metastasis, and individual differences in the condition.

[0292] The embodiments described below are not intended to be limited by any theory, but are merely for illustrating the multispecific conjugates, preparation methods and uses of this application, and are not intended to limit the scope of the invention.

[0293] The embodiments described below are not intended to be limited by any theory, but are merely for illustrating the fusion protein, preparation method and use of this application, and are not intended to limit the scope of the invention.

[0294] Example

[0295] Example 1: Preparation and LC-MS characterization of the coupling module

[0296] (1) Tumor cell EGFR / HER2 receptor targeting module

[0297] Nanobodies against the EGFR receptor of human tumor cells, with amino acid sequences SEQ ID NO: 1-51. An affinity variant against the HER2 receptor of human tumor cells, with an amino acid sequence SEQ ID NO: 52. Both nanobodies were expressed in *Escherichia coli* (E. coli) under IPTG induction and overnight expression at 30 °C. The resulting nanobodies were purified sequentially by nickel column chromatography and size exclusion chromatography. The N-terminus of each nanobodies was linked to an LPETG sequence (SEQ ID NO: 76) and a 6His tag for purification, and they were named anti-EGFR-LPETG and anti-HER2-LPETG, respectively, for subsequent synthesis of multispecific conjugates.

[0298] Anti-EGFR-LPETG and anti-HER2-LPETG were characterized by LC-MS, and the results are as follows: Figure 1 As shown, anti-EGFR-LPETG and anti-HER2-LPETG were successfully prepared.

[0299] (2) T cell CD3 receptor targeting module

[0300] A nanobody targeting the human T-cell CD3 receptor, with the amino acid sequence SEQ ID NO: 53-56, was expressed in *E. coli* under the same expression and purification conditions as described above. The purified nanobody also contained the LPETG sequence (SEQ ID NO: 76) at its N-terminus, and was linked via a linker (SEQ ID NO: 77), and named anti-CD3-LPETG.

[0301] 100 μM anti-CD3-LPETG was dissolved in PBS solution, and then 10 μM Sortase A transpeptidase and 1 mM DBCO-PEG3-Gly were added. After reacting at room temperature for 2 hours, the product anti-CD3-DBCO was purified by size exclusion chromatography and used for subsequent synthesis of multispecific conjugates.

[0302] 100 μM anti-CD3-LPETG was dissolved in PBS solution, and then 10 μM Sortase A transpeptidase and 1 mM BCN-PEG3-Gly were added. After reacting at room temperature for 2 hours, the product anti-CD3-BCN was purified by size exclusion chromatography and used for subsequent synthesis of multispecific conjugates.

[0303] Anti-CD3-DBCO and anti-CD3-BCN were characterized by LC-MS, and the results are as follows: Figure 2 As shown, anti-CD3-DBCO and anti-CD3-BCN were successfully prepared.

[0304] (3) Natural killer cell CD16 / NKG2D receptor targeting module

[0305] Nanobodies against human natural killer cell CD16 receptor and NKG2D receptor, with amino acid sequences SEQ ID NO: 57-64, were obtained. The expression and purification of these nanobodies were similar to those described above, yielding products containing the LPETG sequence (SEQ ID NO: 76) at the N-terminus, named anti-CD16-LPETG and anti-NKG2D-LPETG, respectively.

[0306] 100 μM anti-CD16-LPETG was dissolved in PBS solution, and then 10 μM Sortase A transpeptidase and 1 mM BCN-PEG3-Gly were added. After reacting at room temperature for 2 hours, the product anti-CD16-BCN was purified by size exclusion chromatography and used for subsequent synthesis of multispecific conjugates.

[0307] 100 μM anti-NKG2D-LPETG was dissolved in PBS solution, and then 10 μM Sortase A transpeptidase and 1 mM BCN-PEG3-Gly were added. After reacting at room temperature for 2 hours, the product anti-NKG2D-BCN was purified by size exclusion chromatography and used for subsequent synthesis of multispecific conjugates.

[0308] Anti-CD16-BCN and anti-NKG2D-BCN were characterized by LC-MS, and the results are as follows: Figure 3 As shown, anti-CD16-BCN and anti-NKG2D-BCN were successfully prepared.

[0309] (4) Dendritic cell PDL1 / CLEC9A receptor targeting module

[0310] Nanobodies against human dendritic cell PDL1 and CLEC9A receptors, with amino acid sequences SEQ ID NO: 65-72, were expressed and purified using the same method described above, ultimately yielding products containing the LPETG sequence (SEQ ID NO: 76) at the N-terminus, named anti-PDL1-LPETG and anti-CLEC9A-LPETG.

[0311] 100 μM anti-PDL1-LPETG was dissolved in PBS solution, and then 10 μM Sortase A transpeptidase and 1 mM BCN-PEG3-Gly were added. After reacting at room temperature for 2 hours, the product anti-PDL1-BCN was purified by size exclusion chromatography and used for subsequent synthesis of multispecific conjugates.

[0312] 100 μM anti-CLEC9A-LPETG was dissolved in PBS solution, and then 10 μM Sortase A transpeptidase and 1 mM BCN-PEG3-Gly were added. After reacting at room temperature for 2 hours, the product anti-CLEC9A-BCN was purified by size exclusion chromatography and used for subsequent synthesis of multispecific conjugates.

[0313] Anti-PDL1-BCN and anti-CLEC9A-BCN were characterized by LC-MS, and the results are as follows: Figure 4 As shown, anti-PDL1-BCN and anti-CLEC9A-BCN were successfully prepared.

[0314] (5) Myeloid immune cell-targeted IMDQ / diABZI module

[0315] The small molecule agonist IMDQ (CAS: 1258457-59-8) and the small molecule agonist diABZI (CAS: 2137975-93-8) of myeloid immune cell TLR7 / 8 receptors were both purchased from MCE. IMDQ or diABZI (70 mM), small molecule DBCO-SS-NHS (50 mM), and a catalytic amount of triethylamine (50 μM) were mixed and reacted at room temperature for 2 hours to produce the intermediate DBCO-SS-IMDQ or DBCO-SS-diABZI. This molecule contains cleavable disulfide bonds (SS), so it can break in the tumor microenvironment and release the IMDQ or diABZI agonist without leaving a trace.

[0316] The intermediate DBCO-SS-IMDQ or DBCO-SS-diABZI (10 mM) was reacted with the branched peptide (N3)6-LPETG (1 mM) with the LPETG sequence and 6 azide (N3) functional groups at room temperature for 2 hours to produce the intermediate (IMDQ)6-LPETG or (diABZI)6-LPETG.

[0317] 0.2 mM intermediate (IMDQ)6-LPETG or (diABZI)6-LPETG and 0.2 mM Sortase A transpeptidase were mixed in PBS, and then 2 mM BCN-PEG3-Gly was added. The reaction system was stirred at room temperature for 2 hours, and then the final product (IMDQ)6-BCN or (diABZI)6-BCN was obtained by HPLC purification, which was used for subsequent synthesis of multispecific conjugates.

[0318] (IMDQ)6-BCN and (diABZI)6-BCN were characterized by LC-MS, and the results are as follows: Figure 5 As shown, (IMDQ)6-BCN and (diABZI)6-BCN were successfully prepared.

[0319] (6) CPG / STAT3 module targeted by myeloid immune cells

[0320] CPG, a nucleic acid agonist of the TLR9 receptor for myeloid immune cells, has an amino group at its 3' end, and the corresponding nucleic acid sequence is SEQ ID NO: 73 (5'-T*C*G*T*C*G*T*T*T*T*G*T*C*G*T*T*T*G*T*C*G*T*T-3', where * represents a thiolated backbone. STAT3 ASO, an oligonucleotide inhibitor of the STAT3 receptor for myeloid immune cells, also has an amino group at its 3' end, and the corresponding nucleic acid sequence is SEQ ID NO: 74 (5'-MeC*T*A*T*T*T*G*G*A*T*G*T*MeC*A*G*MeC-3', where * represents a thiolated backbone and Me represents methylation). 100 μM CPG or STAT3 ASO was dissolved in PBS, and then 1 mM DDBCO-SS-NHS was added. After reacting at room temperature for 2 hours, the products CPG-DBCO and STAT3-DBCO were obtained by ethanol precipitation for subsequent synthesis of multispecific conjugates. The disulfide bond (SS) contained in this module is responsive to reducing conditions, thus enabling the traceless release of CPG and STAT3 ASO in the tumor microenvironment.

[0321] CPG-DBCO and STAT3-DBCO were characterized by LC-MS, and the results are as follows: Figure 6 As shown, CPG-DBCO and STAT3-DBCO were successfully prepared.

[0322] (7) Antigen-specific T cell-targeted CMV module

[0323] A branched peptide containing two CMV antigen peptide sequences (SEQ ID NO: 75), two MMP enzyme cleavage sequences, and an N-terminal LPETG sequence (SEQ ID NO: 76) was synthesized by GenScript Biotech and named (CMV)2-LPETG. This branched peptide can therefore respond to the specific upregulation of MMP enzymes in the tumor microenvironment, achieving in situ release of the CMV antigen peptide. The released CMV peptide will be recognized and presented by dendritic cells in the environment, further activating CMV antigen-specific T cells in the tumor microenvironment. 100 μM (CMV)2-LPETG, 1 mM BCN-PEG3-Gly, and 10 μM Sortase A enzyme were mixed in PBS solution. The reaction system was incubated at room temperature for 2 hours, and then the product (CMV)2-BCN was purified by HPLC for subsequent construction of multispecific conjugates.

[0324] (CMV)2-BCN was characterized by LC-MS, and the results are as follows: Figure 7 As shown, (CMV)2-BCN was successfully prepared.

[0325] Example 2: Characterization of the binding force of the coupling module

[0326] (1) Binding force of Anti-EGFR-LPETG nanobody to EGFR receptor in tumor cells

[0327] The binding affinity of the anti-EGFR-LPETG nanobody to the EGFR receptor was determined using a Fortebio® Octet RED96 instrument. First, a biotin-tagged EGFR receptor protein (purchased from Sinocare) was prepared and dissolved in equilibration buffer (PBS, 0.05% Tween-20, 0.5% BSA) to a final concentration of 100 ug / ml. Then, nanobody solutions of different concentrations were prepared. Binding affinity was determined using a streptavidin-conjugated sensor, employing the general binding affinity measurement method of the Fortebio® Octet RED96. The determination mainly included three processes: immobilization, binding, and dissociation. After the determination, the binding constant K between the nanobody and the EGFR receptor was obtained by fitting the data using the instrument's built-in multi-concentration kinetic analysis software. on dissociation constant K dis The final binding affinity KD values ​​are shown in Table 1 below. The results show that the nanobody of this application can bind to the EGFR receptor with high binding affinity.

[0328] Table 1

[0329]

[0330] (2) Binding affinity of Anti-HER2-LPETG affinity to HER2 receptor on tumor cells

[0331] Similarly, the binding affinity of the anti-HER2-LPETG affinity antibody to the HER2 receptor was measured using a Fortebio® Octet RED96. The results are shown in Table 2 below. The results indicate that anti-HER2-LPETG can bind to the HER2 receptor with high binding affinity.

[0332] Table 2

[0333]

[0334] (3) Binding affinity of Anti-CD3-LPETG nanobody to T cell CD3 receptor

[0335] Similarly, the binding affinity of the anti-CD3-LPETG nanobody to the CD3 receptor was measured using a Fortebio® Octet RED96. The results are shown in Table 3 below. The results indicate that anti-CD3-LPETG can bind to the CD3 receptor with high binding affinity.

[0336] Table 3

[0337]

[0338] (4) Binding affinity of Anti-CD16-LPETG nanobody to CD16 receptor on natural killer cells

[0339] Similarly, the binding affinity between the anti-CD16-LPETG nanobody and the CD16 receptor was determined using a Fortebio® Octet RED96. The results are shown in Table 4 below. The results indicate that anti-CD16-LPETG can bind to the CD16 receptor with high binding affinity.

[0340] Table 4

[0341]

[0342] (5) Binding affinity between Anti-NKG2D-LPETG nanobody and NKG2D receptor on natural killer cells

[0343] Similarly, the binding affinity between the anti-NKG2D-LPETG nanobody and the NKG2D receptor was determined using a Fortebio® Octet RED96. The results are shown in Table 5 below. The results indicate that anti-NKG2D-LPETG can bind to the NKG2D receptor with high binding affinity.

[0344] Table 5

[0345]

[0346] (6) Binding affinity of Anti-PDL1-LPETG nanobody to PDL1 receptor on dendritic cells

[0347] Similarly, the binding affinity between the anti-PDL1-LPETG nanobody and the PDL1 receptor was determined using a Fortebio® Octet RED96. The results are shown in Table 6 below. The results indicate that anti-PDL1-LPETG can bind to the PDL1 receptor with high binding affinity.

[0348] Table 6

[0349]

[0350] (7) Binding affinity of Anti-CLEC9A-LPETG nanobody to CLEC9A receptor on dendritic cells

[0351] Similarly, the binding affinity between the anti-CLEC9A-LPETG nanobody and the CLEC9A receptor was determined using a Fortebio® Octet RED96. The results are shown in Table 6 below. The results indicate that anti-CLEC9A-LPETG can bind to the CLEC9A receptor with high binding affinity.

[0352] Table 7

[0353]

[0354] Example 3: Preparation, LC-MS / SDS-PAGE characterization, and mechanism of action of multispecific conjugates

[0355] (1) Preparation, characterization and mechanism of action of EGFR-CD3-PDL1

[0356] EGFR-CD3-PDL1 multispecific conjugates were synthesized using multispecific conjugate conjugate arm Ia-1 (from Chinese patent application No. 202011270000.5). First, the multispecific conjugate conjugate arm Ia-1 was reacted with the previously prepared anti-EGFR-LPETG module. Specifically, 1 mM of the multispecific conjugate conjugate arm Ia-1 and 100 μM of Sortase A were mixed and reacted at room temperature for 2 hours. The mixture was then purified by size exclusion chromatography to obtain the intermediate product EGFR-N3-TZ. Next, EGFR-N3-TZ and the previously prepared anti-CD3-DBCO module were reacted at a 1:1 ratio at a concentration of 100 μM at room temperature for 2 hours. After the reaction, the EGFR-CD3-TZ intermediate was purified by size exclusion chromatography. Finally, EGFR-CD3-TZ was reacted with the prepared anti-PDL1-BCN module in equivalence at a concentration of 100 μM for 2 hours at room temperature. Size exclusion chromatography was then used to purify the multispecific conjugate EGFR-CD3-PDL1.

[0357] EGFR-CD3-PDL1 was characterized by LC-MS and SDS-PAGE, and the results are as follows: Figure 8 As shown.

[0358] like Figure 9 As shown, the multispecific conjugate EGFR-CD3-PDL1 achieves tumor-targeted co-activation of T cells and dendritic cells by simultaneously binding to the EGFR receptor of tumor cells, CD3 receptor of T cells, and PDL1 receptor of dendritic cells.

[0359] (2) Preparation, characterization and mechanism of action of EGFR-CD3-CLEC9A

[0360] Using a similar method, we synthesized the multispecific conjugate EGFR-CD3-CLEC9A through the reaction of the multispecific conjugate coupling arm Ia-1, anti-EGFR-LPETG, anti-CD3-DBCO, and anti-CLEC9A-BCN modules. EGFR-CD3-CLEC9A was characterized by LC-MS and SDS-PAGE, with results as follows: Figure 10 As shown.

[0361] like Figure 11 As shown, the multispecific conjugate EGFR-CD3-CLEC9A simultaneously binds to the EGFR receptor on tumor cells, the CD3 receptor on T cells, and the CLEC9A receptor on dendritic cells, thereby achieving the co-activation of tumor-targeting T cells and dendritic cells and improving the efficacy of immunotherapy.

[0362] (3) Preparation, characterization and mechanism of action of EGFR-CD3-CD16

[0363] Using a similar method, we synthesized the multispecific conjugate EGFR-CD3-CD16 via the reactions of the conjugate coupling arm Ia-1, anti-EGFR-LPETG, anti-CD3-DBCO, and anti-CD16-BCN modules. EGFR-CD3-CD16 was characterized by LC-MS and SDS-PAGE, with results as follows: Figure 12 As shown.

[0364] like Figure 13 As shown, the multispecific conjugate EGFR-CD3-CD16 is expected to simultaneously bind to tumor EGFR, T cell CD3, and natural killer cell CD16 receptors, thereby enabling experimental co-recruitment of tumor-targeted T cells and natural killer cells.

[0365] (4) Preparation, characterization and mechanism of action of EGFR-CD3-NKG2D

[0366] Using a similar method, we synthesized the multispecific conjugate EGFR-CD3-NKG2D through the reaction of the conjugate coupling arm Ia-1, anti-EGFR-LPETG, anti-CD3-DBCO, and anti-NKG2D-BCN modules. EGFR-CD3-NKG2D was characterized by LC-MS and SDS-PAGE, with results as follows: Figure 14 As shown.

[0367] like Figure 15 As shown, the multispecific conjugate EGFR-CD3-NKG2D is expected to simultaneously bind to tumor EGFR, T cell CD3, and natural killer cell NKG2D receptors, thereby achieving tumor-targeted co-recruitment of T cells and natural killer cells.

[0368] (5) Preparation, characterization and mechanism of action of HER2-CD3-(IMDQ)6

[0369] Using a similar method as described above, we synthesized the multispecific conjugate HER2-CD3-(IMDQ)6 through the reaction of the multispecific conjugate conjugate conjugate arm Ia-1, anti-HER2-LPETG, anti-CD3-DBCO, and (IMDQ)6-BCN module. HER2-CD3-(IMDQ)6 was characterized by LC-MS and SDS-PAGE, with results as follows: Figure 16 As shown.

[0370] like Figure 17As shown, the multispecific conjugate HER2-CD3-(IMDQ)6 achieves tumor-targeted T cell recruitment by binding to tumor HER2 and T cell CD3 receptors. Simultaneously, under reductive conditions in the tumor microenvironment, the IMDQ agonist in HER2-CD3-(IMDQ)6 is released, subsequently entering myeloid immune cells to activate TLR7 / 8 receptors, thereby activating the immune system.

[0371] (6) Preparation, characterization and mechanism of action of HER2-CD3-(diABZI)6

[0372] Using a similar method as described above, we synthesized the multispecific conjugate HER2-CD3-(diABZI)6 through the reaction of the multispecific conjugate coupling arm Ia-1, anti-HER2-LPETG, anti-CD3-DBCO, and (diABZI)6-BCN module. HER2-CD3-(diABZI)6 was characterized by LC-MS and SDS-PAGE, with results as follows: Figure 18 As shown.

[0373] like Figure 19 As shown, the multispecific conjugate HER2-CD3-(diABZI)6 achieves tumor-targeted T-cell recruitment by binding to tumor HER2 and T-cell CD3 receptors. Simultaneously, under reductive conditions in the tumor microenvironment, the diABZI agonist in HER2-CD3-(diABZI)6 is released, subsequently entering myeloid immune cells to activate the STING receptor, thereby activating immunity.

[0374] (7) Preparation, characterization and mechanism of action of EGFR-CD3-CPG

[0375] Using a similar method as described above, we synthesized the multispecific conjugate EGFR-CD3-CPG through the reaction of the multispecific conjugate coupling arm Ia-1, anti-EGFR-LPETG, anti-CD3-BCN, and CPG-DBCO module. EGFR-CD3-CPG was characterized by LC-MS and SDS-PAGE, and the results are as follows: Figure 20 As shown.

[0376] like Figure 21 As shown, the multispecific conjugate EGFR-CD3-CPG will achieve tumor-targeted T cell recruitment by binding to tumor EGFR and T cell CD3 receptors. Simultaneously, under the reducing conditions of the tumor microenvironment, CPG molecules in EGFR-CD3-CPG will be released, subsequently entering myeloid immune cells to activate TLR9 receptors, thereby activating immunity.

[0377] (8) Preparation, characterization and mechanism of action of EGFR-CD3-STAT3

[0378] Using a similar method as described above, we synthesized the multispecific conjugate EGFR-CD3-STAT3 through the reaction of the conjugate coupling arm Ia-1, anti-EGFR-LPETG, anti-CD3-BCN, and STAT3-DBCO module. EGFR-CD3-STAT3 was characterized by LC-MS and SDS-PAGE, and the results are as follows: Figure 22 As shown.

[0379] like Figure 23 As shown, the multispecific conjugate EGFR-CD3-STAT3 will achieve tumor-targeted T cell recruitment by binding to tumor EGFR and T cell CD3 receptors. Simultaneously, under reductive conditions in the tumor microenvironment, STAT3 molecules in EGFR-CD3-STAT3 will be released and subsequently enter myeloid immune cells to act on STAT3 receptors, thereby inhibiting the proliferation of immunosuppressive cells such as MDSCs and TAMs.

[0380] (9) Preparation, characterization and mechanism of action of EGFR-CD3-(CMV)2

[0381] Using a similar method as described above, we synthesized the multispecific conjugate EGFR-CD3-(CMV)2 through reactions involving the conjugate conjugate arm Ia-1, anti-EGFR-LPETG, anti-CD3-DBCO, and (CMV)2-BCN module. EGFR-CD3-(CMV)2 was characterized by LC-MS and SDS-PAGE, with results as follows: Figure 24 As shown.

[0382] like Figure 25 As shown, the multispecific conjugate EGFR-CD3-(CMV)2 achieves tumor-targeted T cell recruitment by binding to tumor EGFR and T cell CD3 receptors. Simultaneously, under the action of MMP enzymes in the tumor microenvironment, CMV antigenic peptides in EGFR-CD3-(CMV)2 are released and then recognized and presented by surrounding antigen-presenting cells (APCs), further activating CMV-specific T cells.

[0383] Example 4: EGFR-CD3-PDL1 binding to EGFR receptors on tumor cells, CD3 receptors on T cells, and PDL1 receptors on dendritic cells

[0384] The binding of EGFR-CD3-PDL1 to EGFR, CD3, and PDL1 receptors was determined using a Fortebio® Octet RED96 instrument. Biotin-tagged EGFR, CD3, and PDL1 proteins were dissolved in equilibration buffer (PBS, 0.05% Tween-20, 0.5% BSA) at a final concentration of 100 μg / ml. Different concentrations of EGFR-CD3-PDL1 protein were also dissolved in equilibration buffer. Binding affinity was measured using a streptavidin-coupled sensor, employing the general binding affinity measurement method of the Fortebio® Octet RED96. The measurement mainly included three processes: solidification, binding, and dissociation. After the measurement, the binding affinity K of EGFR-CD3-PDL1 to different receptors was obtained by fitting the data using the instrument's built-in multi-concentration kinetic analysis software. D The values ​​are shown in Table 8 below:

[0385] Table 8

[0386]

[0387] Example 5: EGFR-CD3-PDL1-mediated co-recruitment of tumor-targeting T cells and dendritic cells (DCs)

[0388] like Figure 26A As shown, EGFR-positive A549 tumor cells (10 4 ), CD3-positive human T cells (2×10) 5 ) and PDL1-positive human dendritic cells (10 4 The cells were co-cultured in 96-well plates at 37 °C, 5% CO2, and RPMI-1640 medium. Different concentrations of the multispecific conjugate EGFR-CD3-PDL1 or bispecific conjugate controls (EGFR-CD3, PDL1-CD3, and EGFR-PDL1) were then added and cultured for 24 hours. EGFR-CD3-PDL1-mediated tumor cell killing, T cell activation, and dendritic cell activation were then detected.

[0389] EGFR-CD3-PDL1-mediated tumor cell killing was measured using a firefly luciferase assay kit (Promega, Cat#E1500). Specifically, after the co-culture was completed, the cell supernatant was removed from the 96-well plate, the cells were washed once with PBS, and then the kit substrate was added and incubated for 5-30 min. Cell killing was then detected according to the instructions. The results of EGFR-CD3-PDL1-mediated tumor cell killing are shown below. Figure 26B As shown.

[0390] EGFR-CD3-PDL1-mediated T cell activation was detected by flow cytometry. Specifically, after the co-culture was completed, all cells were collected and resuspended in flow cytometry analysis solution (PBS containing 1% FBS and 2% EDTA). Anti-CD3 and anti-CD69 flow cytometry fluorescent antibodies were added, and staining was performed at 4 °C for 30 min, followed by flow cytometry analysis. The results are shown below. Figure 26C As shown.

[0391] EGFR-CD3-PDL1-mediated T cell activation was detected using an ELISA kit (Thermo Fisher Scientific). Specifically, after the co-culture was completed, all supernatants were collected for IFNγ cytokine and Granzyme B assays. The assay procedure followed the kit's standard instructions, and the results are as follows: Figure 26D As shown.

[0392] EGFR-CD3-PDL1-mediated dendritic cell activation was detected by flow cytometry. Specifically, after the co-culture was completed, all cells were collected and resuspended in flow cytometry analysis solution (PBS containing 1% FBS and 2% EDTA). Anti-CD11c, anti-CD83, and anti-CD86 flow cytometry fluorescent antibodies were added, and staining was performed at 4°C for 1 hour. Flow cytometry analysis was then performed, and the results are shown below. Figure 26E As shown.

[0393] EGFR-CD3-PDL1-mediated dendritic cell activation was detected using an ELISA kit (Thermo Fisher Scientific). Specifically, after the co-culture was completed, all supernatants were collected for IL12 and IL1β cytokine assays. The assay procedure followed the kit's standard instructions, and the results are as follows: Figure 26F As shown.

[0394] Example 6: EGFR-CD3-PDL1 activates T cells and inhibits tumors in a PBMC humanized mouse model.

[0395] Evaluate the therapeutic effect of EGFR-CD3-PDL1 in a PBMC humanized mouse model. Figure 27A As shown, immunodeficient M-NSG mice were purchased from Shanghai Southern Model Biotechnology Co., Ltd., and then subcutaneously inoculated with A549 tumor cells (10... 6 (per mouse). Wait for the tumor to grow to a volume > 100 mm. 3 Afterwards (set as day 1), human PBMCs (10) were injected via the tail vein. 7Each mouse underwent humanized immune remodeling. Subsequently, mice were injected via tail vein with EGFR-CD3-PDL1, bispecific conjugate controls (EGFR-CD3, PDL1-CD3, and EGFR-PDL1), and an equal volume of PBS. Injections were administered on days 2, 5, 8, 11, 14, and 17 at a dose of 2 mg / kg EGFR-CD3-PDL1. Other groups received equimolar doses.

[0396] Tumor volume changes in mice were continuously recorded after drug administration, and tumor growth curves were plotted as follows: Figure 27B As shown.

[0397] After treatment (day 30), all mice were sacrificed. Tumor tissue from the mice was collected and prepared into single-cell suspensions for later use. These cell suspensions were stained with flow cytometry antibodies against anti-CD45, anti-CD3, anti-CD4, anti-CD8, anti-Perforin, anti-PD1, and anti-Ki67, following the antibody requirements. EGFR-CD3-PDL1-mediated T cell activation in this animal model was then analyzed by flow cytometry. The main biomarkers analyzed were T cell perforin, PD1, and Ki67. The results are shown below. Figure 27C As shown.

[0398] Example 7: EGFR-CD3-PDL1 activates T cells / dendritic cells and inhibits tumors in HSC humanized mice.

[0399] Evaluate the therapeutic effect of EGFR-CD3-PDL1 in a humanized HSC mouse model. Figure 28A As shown, the HSC humanized mouse huHSC-NCG-hIL15 was purchased from Jicui Pharmaceutical. First, A549 tumor cells (10...) were subcutaneously inoculated. 6 (per mouse), when the tumor grows to a volume > 100 mm 3 Drug administration was then initiated (day 1). Mice were administered EGFR-CD3-PDL1, bispecific conjugate controls (EGFR-CD3, PDL1-CD3, and EGFR-PDL1), and an equal volume of PBS via tail vein injection. Drug administration occurred on days 2, 5, 8, 11, 14, 17, 20, and 23, at a dose of 2 mg / kg EGFR-CD3-PDL1. Other groups received equimolar doses.

[0400] Tumor volume changes in mice were continuously recorded after drug administration, and tumor growth curves were plotted as follows: Figure 28B As shown.

[0401] After treatment (day 25), all mice were sacrificed. Tumor tissue from the mice was collected and prepared into single-cell suspensions for later use. These cell suspensions were stained with flow cytometry antibodies against anti-CD45, anti-CD3, anti-CD4, anti-CD8, anti-Perforin, anti-Granzyme B, and anti-PD1, following the antibody requirements. EGFR-CD3-PDL1-mediated T cell activation was then analyzed by flow cytometry, with the markers being Perforin, Granzyme B, and PD1 on T cells. The results are as follows: Figure 28C As shown. Similarly, cells were stained with anti-CD45, anti-CD3, anti-CD14, anti-CD123, anti-HLA-DR, anti-CD1c, anti-CD141, and anti-CD86, and then flow cytometry was performed to analyze EGFR-CD3-PDL1-mediated dendritic cell activation. The markers analyzed were CD86 and HLA-DR on dendritic cells. The results are shown in the figure. Figure 28D As shown.

[0402] Example 8: EGFR-CD3-PDL1 activates T cells / dendritic cells, inhibits tumor growth, and induces antigen-specific immunity in transgenic humanized mice.

[0403] Evaluate the therapeutic effect of EGFR-CD3-PDL1 in a transgenic humanized mouse model. Figure 29A As shown, the CD3EDG and PDL1 dual-target humanized mouse hCD3EDG / hPDL1 C57 was purchased from Shanghai Southern Model Biotechnology Co., Ltd. MC38 tumor cells overexpressing human EGFR receptor and OVA antigen protein (MC38hEGFR) were also used. + OVA + ) was inoculated into hCD3EDG / hPDL1 C57 mice at a dose of 5 × 10⁻⁶. 5 Cells per mouse. When the tumor grew to 100 mm 3 Dosing began on day 1. Mice were treated with 2 mg / kg EGFR-CD3-PDL1 or an equal volume of PBS on days 2, 5, 8, and 11.

[0404] Tumor volume changes in mice were continuously recorded after drug administration, and tumor growth curves were plotted as follows: Figure 29B As shown.

[0405] After treatment (day 15), all mice were sacrificed. Tumor tissue from the mice was collected and prepared into single-cell suspensions for later use. These cell suspensions were stained with flow cytometry antibodies against anti-CD45, anti-CD3, anti-CD4, anti-CD8, anti-Perforin, anti-Granzyme B, and anti-Ki67, following the antibody requirements. EGFR-CD3-PDL1-mediated T cell activation was then analyzed by flow cytometry, with Perforin, Granzyme B, and Ki67 on T cells as markers. Results are as follows: Figure 29C As shown. Cells were stained with anti-CD45, anti-Lin, anti-CD11c, anti-MHC-II, anti-XCR1, anti-CD11b, anti-CD80, and anti-CD86. Flow cytometry analysis was then performed to analyze EGFR-CD3-PDL1-mediated dendritic cell activation. The markers analyzed were CD80, CD86, and MHC-II on dendritic cells. The results are shown below. Figure 29D As shown.

[0406] After treatment (day 15), all mice were sacrificed. Spleens and tumor tissues were collected and prepared into single-cell suspensions for later use. Cells were stained with anti-CD45, anti-Lin, anti-CD11c, anti-MHC-II, anti-XCR1, anti-CD11b, and OVA-tetramer. Flow cytometry analysis was then performed to analyze changes in OVA antigen-specific T cells after EGFR-CD3-PDL1 treatment. The results are as follows: Figure 29E As shown.

[0407] All mice were sacrificed on day 15 after treatment. Mouse spleens were harvested and prepared into single-cell suspensions. The presence of immune memory in mice treated with EGFR-CD3-PDL1 was then detected using a mouse IFNγ ELISPOT kit (Dakow). The detection was performed using a standardized kit procedure, and qualitative and quantitative analyses were conducted based on the number of spots produced. Results are as follows: Figure 29F As shown.

[0408] Example 9: EGFR-CD3-PDL1 activates T cells / dendritic cells and inhibits tumors in lung cancer patient tissues.

[0409] Tumor tissue from non-small cell lung cancer patients was obtained from Peking University Cancer Hospital (ethics code: 2019KT41). For example... Figure 30AAs shown, the obtained tumor tissue was digested and cultured to form tumor microspheres (PTCs), following a previously reported method (Sci. Transl. Med. 12, eaaz1723.). When the cultured PTC size > 40 μm, an EGFR-CD3-PDL1 multispecific conjugate, a bispecific conjugate control (EGFR-CD3, PDL1-CD3, and EGFR-PDL1), or an equal volume of PBS was added for further culture. Subsequently, the activation of T cells and dendritic cells mediated by EGFR-CD3-PDL1, as well as the inhibition of PTC growth, were measured.

[0410] After culturing for 24 hours with the addition of the multispecific conjugate, the supernatant of the culture system was collected. EGFR-CD3-PDL1-mediated T cell and dendritic cell activation was detected using an ELISA kit (ThermoFisher Scientific). The assay was performed using a standardized kit procedure. For T cell activation, we measured IFNγ and TNFα cytokines. For dendritic cell activation, we measured IL12 and IL1β cytokines. The results are shown below. Figure 30B As shown.

[0411] Seven days after treatment with the multispecific conjugate, we detected the growth of PTC in the system using a method reported in the literature (Sci. Transl. Med. 12, eaaz1723.). The results are as follows. Figure 30C As shown.

[0412] Example 10: EGFR-CD3-CD16 binding to EGFR receptors on tumor cells, CD3 receptors on T cells, and CD16 receptors on natural killer cells.

[0413] The binding of EGFR-CD3-CD16 to EGFR, CD3, and CD16 receptors was determined using a Fortebio® Octet RED96 instrument. Biotin-tagged EGFR, CD3, and CD16 proteins were dissolved in equilibration buffer (PBS, 0.05% Tween-20, 0.5% BSA) at a final concentration of 100 μg / ml. Different concentrations of EGFR-CD3-CD16 protein were also dissolved in equilibration buffer. Binding affinity was determined using a streptavidin-coupled sensor and the general binding affinity assay method of the Fortebio® Octet RED96. The assay mainly included three processes: solidification, binding, and dissociation. After the assay, the binding affinity KB of EGFR-CD3-CD16 to different receptors was obtained by fitting the data using the instrument's built-in multi-concentration kinetic analysis software. D The values ​​are shown in Table 9 below:

[0414] Table 9

[0415]

[0416] Example 11: EGFR-CD3-CD16-mediated co-recruitment of tumor-targeting T cells and natural killer cells

[0417] (1) EGFR-CD3-CD16-mediated tumor-targeting T cell recruitment

[0418] like Figure 31A EGFR-positive A549 tumor cells (10 4 ) and CD3-positive human T cells (2×10 5 The cells were co-cultured in 96-well plates at 37 °C, 5% CO2, in RPMI-1640 medium. Different concentrations of the multispecific conjugate EGFR-CD3-CD16 or the bispecific conjugate control (EGFR-CD3 and EGFR-CD16) were then added and cultured for 24 hours. EGFR-CD3-CD16-mediated tumor cell killing and T cell activation were then detected.

[0419] EGFR-CD3-CD16-mediated tumor cell killing was measured using a firefly luciferase assay kit (Promega, Cat#E1500). Specifically, after the co-culture process described above, the cell supernatant was removed from the 96-well plates, the cells were washed once with PBS, and then the kit substrate was added and incubated for 5-30 min. Cell killing was then detected according to the instructions. The results of EGFR-CD3-CD16-mediated tumor cell killing are shown below. Figure 31B As shown.

[0420] EGFR-CD3-CD16-mediated T cell activation was detected by flow cytometry. Specifically, after the co-culture was completed, all cells were collected and resuspended in flow cytometry analysis solution (PBS containing 1% FBS and 2% EDTA). Anti-CD3 and anti-CD69 flow cytometry fluorescent antibodies were added, and staining was performed at 4 °C for 30 min, followed by flow cytometry analysis. The results are shown below. Figure 31C As shown.

[0421] EGFR-CD3-CD16-mediated T cell activation was detected using an ELISA kit (Thermo Fisher Scientific). Specifically, after the co-culture was completed, all supernatants were collected for IFNγ cytokine detection. The detection procedure followed the kit's standard instructions, and the results are as follows: Figure 31D As shown.

[0422] (2) EGFR-CD3-CD16-mediated tumor-targeting natural killer cell recruitment

[0423] likeFigure 32A EGFR-positive A549 tumor cells (10 4 ) and CD16-positive human natural killer cells (10 5 The cells were co-cultured in 96-well plates at 37 °C, 5% CO2, in RPMI-1640 medium. Different concentrations of the multispecific conjugate EGFR-CD3-CD16 or the bispecific conjugate control (EGFR-CD3 and EGFR-CD16) were then added and cultured for 24 hours. EGFR-CD3-CD16-mediated tumor cell killing and natural killer cell activation were then detected.

[0424] EGFR-CD3-CD16-mediated tumor cell killing was measured using a firefly luciferase assay kit (Promega, Cat#E1500). Specifically, after the co-culture process described above, the cell supernatant was removed from the 96-well plates, the cells were washed once with PBS, and then the kit substrate was added and incubated for 5-30 min. Cell killing was then detected according to the instructions. The results of EGFR-CD3-CD16-mediated tumor cell killing are shown below. Figure 32B As shown.

[0425] EGFR-CD3-CD16-mediated natural killer cell activation was detected by flow cytometry. Specifically, after the co-culture was completed, all cells were collected and resuspended in flow cytometry analysis solution (PBS containing 1% FBS and 2% EDTA). Anti-CD56 and anti-CD107 flow cytometry fluorescent antibodies were added, and staining was performed at 4 °C for 30 min, followed by flow cytometry analysis. The results are shown below. Figure 32C As shown.

[0426] EGFR-CD3-CD16-mediated natural killer cell activation was detected using an ELISA kit (Thermo Fisher Scientific). Specifically, after the co-culture was completed, all supernatants were collected for IFNγ and MIP-1β cytokine detection. The detection procedure followed the kit's standard instructions, and the results are as follows: Figure 32D As shown.

[0427] Example 12: HER2-CD3-(IMDQ)6 binds to HER2 receptors on tumor cells and CD3 receptors on T cells.

[0428] The binding of HER2-CD3-(IMDQ)6 to HER2 and CD3 receptors was determined using a Fortebio® Octet RED96 instrument. Biotin-tagged HER2 and CD3 proteins were dissolved in equilibration buffer (PBS, 0.05% Tween-20, 0.5% BSA) to a final concentration of 100 μg / ml. Different concentrations of HER2-CD3-(IMDQ)6 protein were also dissolved in equilibration buffer. Binding affinity was measured using a streptavidin-conjugated sensor, employing the general binding affinity measurement method of the Fortebio® Octet RED96. The measurement mainly included three processes: solidification, binding, and dissociation. After the measurement, the binding affinity KB of HER2-CD3-(IMDQ)6 to different receptors was obtained by fitting the data using the instrument's built-in multi-concentration kinetic analysis software. D The values ​​are shown in Table 10 below:

[0429] Table 10

[0430]

[0431] Example 13: HER2-CD3-(IMDQ)6 releases IMDQ agonists in a tumor-reducing microenvironment.

[0432] like Figure 33A As shown, the multispecific conjugate HER2-CD3-(IMDQ)6, due to the presence of disulfide bonds (SS) in its molecule, can respond to reducing substances (such as GSH, Cys, etc.) in the tumor microenvironment, and can target and release IMDQ agonists in situ after tumor resection, thereby further activating the immune system.

[0433] HER2-positive SKBR3 tumor cells (10 4 ) and CD3-positive human T cells (2×10 5 The cells were co-cultured in 96-well plates at 37 °C, 5% CO2, using RPMI-1640 medium. To simulate a tumor-reducing microenvironment, 150 μM GSH and 20 μM Cys were added to the system for co-culturing. Subsequently, 20 μM of the multispecific conjugate HER2-CD3-(IMDQ)6 was added, and the cells were cultured for another 48 hours. The supernatant was collected, concentrated, and then subjected to LC-MS to detect the release of the IMDQ agonist. The results are shown below. Figure 33B As shown.

[0434] Example 14: HER2-CD3-(IMDQ)6-mediated co-recruitment of tumor-targeting T cells and myeloid immune cells

[0435] (1) HER2-CD3-(IMDQ)6-mediated tumor-targeting T cell recruitment

[0436] likeFigure 34A HER2-positive SK-BR3 tumor cells (10 4 ) and CD3-positive human T cells (2×10 5 The cells were co-cultured in 96-well plates at 37 °C, 5% CO2, in RPMI-1640 medium. Different concentrations of the multispecific conjugate HER2-CD3-(IMDQ)6 or the bispecific conjugate control (HER2-CD3 and HER2-(IMDQ)6) were then added and cultured for 24 hours. HER2-CD3-(IMDQ)6-mediated tumor cell killing and T cell activation were then detected.

[0437] HER2-CD3-(IMDQ)6-mediated tumor cell killing was measured using a firefly luciferase assay kit (Promega, Cat#E1500). Specifically, after the co-culture was completed, the cell supernatant was removed from the 96-well plate, the cells were washed once with PBS, and then the kit substrate was added and incubated for 5-30 min. Cell killing was then detected according to the instructions. The results of HER2-CD3-(IMDQ)6-mediated tumor cell killing are shown below. Figure 34B As shown.

[0438] HER2-CD3-(IMDQ)6-mediated T cell activation was detected by flow cytometry. After co-culture, all cells were collected and resuspended in flow cytometry analysis solution (PBS containing 1% FBS and 2% EDTA). Anti-CD3 and anti-CD69 flow cytometry fluorescent antibodies were added, and staining was performed at 4°C for 30 min, followed by flow cytometry analysis. The results are shown below. Figure 34C As shown.

[0439] HER2-CD3-(IMDQ)6-mediated T cell activation was detected using an ELISA kit (Thermo Fisher Scientific). After the co-culture process, all supernatants were collected for IFNγ cytokine detection. The assay procedure followed the kit's standard instructions, and the results are as follows: Figure 34D As shown.

[0440] (2) HER2-CD3-(IMDQ)6-mediated tumor-targeting myeloid immune cell recruitment

[0441] like Figure 35A HER2-positive SK-BR3 tumor cells (10 4 ) and CD3-positive human T cells (2×10 5Human myeloid immune cells were co-cultured in 96-well plates at 37 °C with 5% CO2 in RPMI-1640 medium. 150 μM GSH and 20 μM Cys were added to simulate a tumor-reducing microenvironment. Subsequently, 100, 10, or 1 nM HER2-CD3-(IMDQ)6 multispecific conjugate was added and incubated for another 48 hours. The supernatant was collected and co-cultured with isolated human myeloid immune cells for 24 hours. Flow cytometry was used to detect HER2-CD3-(IMDQ)6 multispecific conjugate-mediated activation of myeloid immune cells.

[0442] After collecting and resuspending the cultured myeloid immune cells, the following steps were performed: First, HER2-CD3-(IMDQ)6-mediated activation of myeloid dendritic cells was measured by staining with anti-CD11c, anti-HLA-DR, anti-CD83, and anti-CD40, followed by flow cytometry analysis. Next, HER2-CD3-(IMDQ)6-mediated activation of myeloid macrophages was measured by staining with anti-CD11c, anti-HLA-DR, anti-CD68, anti-CD206, and anti-CD86 flow cytometry antibodies, followed by flow cytometry analysis. Finally, HER2-CD3-(IMDQ)6-mediated activation of myeloid monocytes was measured by staining with anti-HLA-DR, anti-CD206, anti-CD14, anti-CD40, and anti-CD80 flow cytometry antibodies, followed by flow cytometry analysis. All antibody staining and flow cytometry analysis were performed according to standard procedures. For the activation of myeloid dendritic cells, the CD83 and CD40 biomarkers were mainly evaluated, and the detection results are as follows: Figure 35B As shown in the figure. For the activation of myeloid macrophages, the proportions of M1 and M2 macrophages were primarily assessed, and the results are as follows: Figure 35C As shown in the figure. For the activation of myeloid monocytes, the main biomarkers assessed were CD14, CD40, and CD80. The detection results are shown in the figure. Figure 35D As shown.

Claims

1. An antigen-binding protein capable of specifically binding to EGFR, said antigen-binding protein comprising HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 comprise an amino acid sequence selected from any one or more of the following groups: (1) HCDR1: SEQ ID NO: 2, HCDR2: SEQ ID NO: 3, and HCDR3: SEQ ID NO: 15, (2) HCDR1: SEQ ID NO: 2, HCDR2: SEQ ID NO: 3, and HCDR3: SEQ ID NO: 4, (3) HCDR1: SEQ ID NO: 43, HCDR2: SEQ ID NO: 44, and HCDR3: SEQ ID NO: 45, (4) HCDR1: SEQ ID NO: 6, HCDR2: SEQ ID NO: 7, and HCDR3: SEQ ID NO: 8, (5) HCDR1: SEQ ID NO: 10, HCDR2: SEQ ID NO: 11, and HCDR3: SEQ ID NO: 12, (6) HCDR1: SEQ ID NO: 17, HCDR2: SEQ ID NO: 3, and HCDR3: SEQ ID NO: 18, (7) HCDR1: SEQ ID NO: 2, HCDR2: SEQ ID NO: 20, and HCDR3: SEQ ID NO: 21, (8) HCDR1: SEQ ID NO: 23, HCDR2: SEQ ID NO: 3, and HCDR3: SEQ ID NO: 24, (9) HCDR1: SEQ ID NO: 27, HCDR2: SEQ ID NO: 3, and HCDR3: SEQ ID NO: 28, (10) HCDR1: SEQ ID NO: 30, HCDR2: SEQ ID NO: 31, and HCDR3: SEQ ID NO: 32, (11) HCDR1: SEQ ID NO: 2, HCDR2: SEQ ID NO: 34, and HCDR3: SEQ ID NO: 35, (12) HCDR1: SEQ ID NO: 27, HCDR2: SEQ ID NO: 37, and HCDR3: SEQ ID NO: 38, (13) HCDR1: SEQ ID NO: 40, HCDR2: SEQ ID NO: 3, and HCDR3: SEQ ID NO: 41, (14) HCDR1: SEQ ID NO: 6, HCDR2: SEQ ID NO: 3, and HCDR3: SEQ ID NO: 47, and (15) HCDR1: SEQ ID NO: 49, HCDR2: SEQ ID NO: 50, and HCDR3: SEQ ID NO:

51.

2. The antigen-binding protein according to claim 1, wherein it is an antibody or its antigen-binding fragment.

3. The antigen-binding protein according to any one of claims 1-2, comprising an antibody heavy chain variable region, said heavy chain variable region comprising an amino acid sequence represented by any one of SEQ ID NO: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61 and 65.

4. The antigen-binding protein according to any one of claims 1-3, wherein it is a nanobody.

5. An isolated nucleic acid that encodes the antigen-binding protein according to any one of claims 1-4.

6. A vector comprising the isolated nucleic acid as described in claim 5.

7. A cell comprising the vector of claim 6 and / or the isolated nucleic acid of claim 5.

8. An immunoconjugate comprising the antigen-binding protein of any one of claims 1-4.

9. A pharmaceutical composition comprising the antigen-binding protein of any one of claims 1-4, and optionally a pharmaceutically acceptable excipient or carrier.

10. Use in the preparation of a medicament for treating and / or preventing tumors, comprising the antigen-binding protein of any one of claims 1-4 and / or the pharmaceutical composition of claim 9.

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