Multispecific conjugates and their applications

Abstract

This application relates to a multispecific conjugate having the structure shown in Formula I: #imgabs0#. The multispecific conjugate of this application, through a linker arm having three orthogonal groups, can precisely link three different payloads, thereby recruiting a variety of cells. This application also provides the use of the multispecific conjugate in treating diseases, particularly immunotherapy.

Description

Technical Field

[0001] The present application relates to the field of biomedicine, and specifically to a multi-specific drug conjugate and its application. Background Art

[0002] Immunotherapy, which mobilizes the body's immune system to eradicate tumors, has revolutionized cancer treatment and achieved remarkable efficacy in clinical trials. However, these successes have been primarily limited to certain types of hematologic malignancies, with suboptimal results for most solid tumors. The reasons for this are diverse, but primarily relate to the tumor-immune microenvironment (TIME). Within the TIME, multiple immune components interact and collectively influence the efficacy of immunotherapy. A growing body of research evidence suggests that intervening in 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 optimal levels.

[0003] To further improve efficacy, researchers have attempted multi-drug combination therapies, including the simultaneous or sequential use of different drugs or the simultaneous delivery of multiple therapies using biomaterials. These approaches offer the potential for simultaneously targeting multiple immune cells within TIME. However, these strategies are often accompanied by increased toxicity risks, and most combination therapies fail to produce synergistic therapeutic effects.

[0004] In order to solve the problems encountered in combination immunotherapy, researchers have begun to explore strategies to integrate multiple drugs into a single molecule. For example, bispecific or multispecific antibodies have been designed, which contain two or more modules, such as tumor targeting moieties, T cell recruiters, checkpoint inhibitors, and immune agonists, to improve specificity, reduce toxicity, and enhance anti-tumor effects. Despite remarkable achievements, existing strategies still have some limitations: First, most strategies are limited to bispecificity and can only activate one type of immune cell (such as T cells or NK cells), which is not enough when facing the complexity of TIME, especially when it is necessary to target multiple immune cells at the same time; second, existing strategies lack sufficient modularity and cannot meet the needs of integrating a variety of therapeutic modules to target different immune subpopulations within TIME.

[0005] Although some reports have demonstrated multispecific or modular examples, no system has yet been able to integrate these features into a single platform. Therefore, a platform that can integrate multiple therapeutic modules into a single drug is urgently needed to simultaneously target multiple immune factors in TIME. Summary of the Invention

[0006] The present application provides a multispecific conjugate, which is formed by programming multiple therapeutic modules into a single drug through a multispecific coupling arm through a triple bioorthogonal reaction. The resulting multispecific conjugate can simultaneously activate multiple immune cells in the tumor-immune microenvironment. The specific conjugate of the present application can accurately connect three different payloads through a multispecific coupling arm with three orthogonal groups, and almost no toxicity is detected in vivo. The multispecific conjugate of the present application integrates therapeutic modules including tumor targeting moieties, immune activators and stimulators to obtain various multimodal targeting chimeras.

[0007] As an example, when nanoantibody modules are connected, these modules are programmable and, due to their small size, are advantageous for penetrating solid tumors. The multispecific conjugates of the present application can recruit a variety of cells, including tumor cells and immune cells, through a unique skeleton, so that these cells are in a suitable spatial position, thereby interacting with each other and changing the tumor microenvironment, thereby demonstrating an enhanced anti-tumor immune response. The multispecific conjugates of the present application can also open up new avenues for immunotherapy and other fields.

[0008] In one aspect, the present application provides a multispecific conjugate having a structure as shown in Formula I: in,

[0009] W is in, Represents the connection site, W is Connected to La, Lb and Lc respectively;

[0010] La is -C(=O)-X 1 -(K 1 ) n1 -(Y 1 ) p1 -(L 1 ) q1 -,

[0011] Lb is -C(=O)-X 2 -(K 2 ) n2 -(Y 2 ) p2 -(L 2 ) q2 -,

[0012] Lc is -C(=O)-X 3 -(K 3 ) n3 -(Y 3 ) p3 -(L 3 ) q3 -,

[0013] in,

[0014] X 1 , X 2 , and X 3 are each independently selected from the group consisting of: -NH- and -O-,

[0015] (K 1 ) n1 , (K 2 ) n2 , and (K 3 ) n3 are each independently selected from the group consisting of -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 are each independently 0,

[0016] (Y 1 ) p1 ,(Y 2 ) p2 , and (Y 3 ) p3 Each independently is -NR 1 -C(=O)-,R 1 is selected from the group consisting of hydrogen, protium, deuterium and tritium; or p1, p2 or p3 are each independently 0,

[0017] (L 1 ) q1 ,(L 2 ) q2 , and (L 3 ) q3 are each independently selected from the following group: -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,

[0018] A 2 , B 2 and C 2 Each independently selected from the following group: and covalent bonds, where R 12 , R 13 and R 14 Each is independently selected from the following group: hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, C1-C 12Alkyl, 3- to 12-membered cycloalkyl, 3- to 12-membered heterocycloalkyl, C2-C 12 Alkenyl, C2-C 12 Alkynyl, 6- to 14-membered aryl, and 6- to 14-membered heteroaryl, wherein represents the attachment site, and A 2 、B 2 and C 2 pass Respectively with La, Lb, Lc and P 1 、P 2 and P 3 connect,

[0019] P 1 、P 2 and P 3 are independently selected from the group consisting of nucleic acids, polypeptides, sugars, fats, proteins and small molecules, and P 1 、P 2 and P 3 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, the cells acted on are attracted or mobilized to a specific area. 1 、P 2 and P 3 At least one of which can regulate immune cells (including activating and / or inhibiting immune cells), P 1 、P 2 and P 3 At least one of the other can bind to tumor cells.

[0020] In certain embodiments, W in the multispecific conjugate is

[0021] In certain embodiments, X in the multispecific conjugate 1 , X 2 and X 3 All are -NH-.

[0022] In certain embodiments, the multispecific conjugate (K 1 ) n1 , (K 2 ) n2 , and (K 3 ) n3 They are all -(CH2)2-(CH2-O-CH2)3-(CH2)2-.

[0023] In certain embodiments, the multispecific conjugate (Y 1 ) p1 ,(Y 2 ) p2 , and (Y 3 ) p3 Each independently is -NR 1 -C(=O)-,R 1 Selected from the group consisting of hydrogen, protium, deuterium and tritium.

[0024] In certain embodiments, the multispecific conjugate (L 1 ) q1 ,(L 2 ) q2 , and (L 3 ) q3 Each is independently selected from: -CH2- and -CH2-aryl-.

[0025] In certain embodiments, A in the multispecific conjugate 2 Select from the following groups:

[0026] In certain embodiments, B in the multispecific conjugate 2 Select from the following groups:

[0027] In certain embodiments, the C 2 Select from the following groups:

[0028] In certain embodiments, A in the multispecific conjugate 2 、B 2 and C 2 They are:

[0029] In certain embodiments, the multispecific conjugate has a structure as shown in Formula IIa-1 or IIa-2:

[0030]

[0031] Among them, P 1 、P 2 and P 3 are respectively as defined in Formula I. For example, the multispecific conjugate has a structure as shown in Formula IIa-1.

[0032] In addition, regarding the connection backbone of the multispecific conjugate, those skilled in the art can refer to those listed in PCT International Application Publication WO2022 / 100696A1. 1、P 2 and P 3 It should be known to those skilled in the art that other connecting skeletons in WO2022 / 100696A1, for example, the structures of formulas Ia-2 to Ia-10, can connect the P 1 、P 2 and P 3 Afterwards, the purpose of the present invention can also be achieved:

[0033]

[0034]

[0035]

[0036]

[0037] In certain embodiments, P 1 、P 2 and P 3 At least one of the proteins comprises a tumor cell binding portion, and at least one comprises an immune cell modulating portion.

[0038] In certain embodiments, the tumor cell binding moiety comprises a tumor antigen binding protein. In certain embodiments, the tumor cell binding moiety comprises an antibody or antigen binding fragment thereof that targets a tumor antigen.

[0039] In certain embodiments, the tumor antigens include 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), Mesotheli n (mesothelin), CD20 (B cell surface antigen), CD30 (Hodgkin 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 (inhibitor of apoptosis protein), hTERT (human telomerase reverse transcriptase), Galectin-3 (galectin 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 viral oncogene homolog), IDO1 (indoleamine 2,3-dioxygenase 1), B7-H3 (immune checkpoint protein), GPNMB (glycoprotein NMB), FAP (fibroblast activation protein), HSP90 (heat shock protein 90), LAMP1 (lysosomal-associated membrane protein 1), NRG1 (neuregulin 1), NRP-1 (neuregulin receptor 1), SLC46A3 (solute carrier family 46 member 3), SPP2 (secreted phosphoprotein 2), TAAs (tumor-associated antigens), TRP-2 (tyrosinase-related protein 2), and TTK (serine / threonine kinase).

[0040] In certain embodiments, the tumor antigens include any one or more selected from the following groups: 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.

[0041] In certain embodiments, the tumor cell binding moiety is an EGFR antibody. The EGFR antibody is preferably a nanobody.

[0042] For example, the EGFR antibody comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 2, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 4. For example, the EGFR antibody comprises a variable region, and the variable region may comprise the amino acid sequence shown in SEQ ID NO: 1.

[0043] For example, the EGFR antibody comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 6, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 7, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 8. For example, the EGFR antibody comprises a variable region, and the variable region may comprise the amino acid sequence shown in SEQ ID NO: 5.

[0044] For example, the EGFR antibody comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 10, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 11, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 12. For example, the EGFR antibody comprises a variable region, and the variable region may comprise the amino acid sequence shown in SEQ ID NO: 9.

[0045] For example, the EGFR antibody comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 2, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 4. For example, the EGFR antibody comprises a variable region, and the variable region may comprise the amino acid sequence shown in SEQ ID NO: 13.

[0046] For example, the EGFR antibody comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 2, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 15. For example, the EGFR antibody comprises a variable region, and the variable region may comprise the amino acid sequence shown in SEQ ID NO: 14.

[0047] For example, the EGFR antibody comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 17, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 18. For example, the EGFR antibody comprises a variable region, and the variable region may comprise the amino acid sequence shown in SEQ ID NO: 16.

[0048] For example, the EGFR antibody comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 2, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 20, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 21. For example, the EGFR antibody comprises a variable region, and the variable region may comprise the amino acid sequence shown in SEQ ID NO: 19.

[0049] For example, the EGFR antibody comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 23, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 24. For example, the EGFR antibody comprises a variable region, and the variable region may comprise the amino acid sequence shown in SEQ ID NO: 22.

[0050] For example, the EGFR antibody includes HCDR1, HCDR2, and HCDR3, wherein the HCDR1 includes the amino acid sequence shown in SEQ ID NO: 2, the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 3, and the HCDR3 includes the amino acid sequence shown in SEQ ID NO: 15. For example, the EGFR antibody includes a variable region, and the variable region may include the amino acid sequence shown in SEQ ID NO: 25.

[0051] For example, the EGFR antibody comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 27, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 28. For example, the EGFR antibody comprises a variable region, and the variable region may comprise the amino acid sequence shown in SEQ ID NO: 26.

[0052] For example, the EGFR antibody comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 30, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 31, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 32. For example, the EGFR antibody comprises a variable region, and the variable region may comprise the amino acid sequence shown in SEQ ID NO: 29.

[0053] For example, the EGFR antibody comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 2, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 34, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 35. For example, the EGFR antibody comprises a variable region, and the variable region may comprise the amino acid sequence shown in SEQ ID NO: 33.

[0054] For example, the EGFR antibody comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 27, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 37, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 38. For example, the EGFR antibody comprises a variable region, and the variable region may comprise the amino acid sequence shown in SEQ ID NO: 36.

[0055] For example, the EGFR antibody comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 40, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 3, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 41. For example, the EGFR antibody comprises a variable region, and the variable region may comprise the amino acid sequence shown in SEQ ID NO: 39.

[0056] For example, the EGFR antibody comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 43, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 44, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 45. For example, the EGFR antibody comprises a variable region, and the variable region may comprise the amino acid sequence shown in SEQ ID NO: 42.

[0057] For example, the EGFR antibody includes HCDR1, HCDR2, and HCDR3, wherein the HCDR1 includes the amino acid sequence shown in SEQ ID NO: 6, the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 3, and the HCDR3 includes the amino acid sequence shown in SEQ ID NO: 47. For example, the EGFR antibody includes a variable region, and the variable region may include the amino acid sequence shown in SEQ ID NO: 46.

[0058] For example, the EGFR antibody comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 49, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 50, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 51. For example, the EGFR antibody comprises a variable region, and the variable region may comprise the amino acid sequence shown in SEQ ID NO: 48.

[0059] In certain embodiments, the tumor cell binding moiety is a HER2 antibody. The HER2 antibody is preferably an affibody.

[0060] For example, the HER2 affinity body includes the amino acid sequence shown in SEQ ID NO:52.

[0061] In certain embodiments, the immune cells include myeloid immune cells and lymphoid immune cells. In certain embodiments, the immune cells are myeloid immune cells. In certain embodiments, the immune cells are lymphoid immune cells.

[0062] In certain 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.

[0063] In certain embodiments, the immune cells include T cells, natural killer cells, dendritic cells, macrophages, monocytes, neutrophils and / or B cells.

[0064] In certain embodiments, the immune cell modulating portion targets an immune cell surface receptor. 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.

[0065] In certain embodiments, the immune cell modulating portion targets an immune cell intracellular receptor. Examples of immune cell intracellular receptors that can be used for the purposes of the present application include, but are not limited to, NF-κB (nuclear factor κB), STAT (signal transducer and activator of transcription), STING, PI3K (phosphatidylinositol 3-kinase), AKT (protein kinase B), mTOR (mammalian target of rapamycin), MAPK (mitogen-activated protein kinase), PLCγ (phospholipase Cγ), SYK (spleen 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 cells), FoxP3, GATA-3, T-bet, and RORγt.

[0066] In certain embodiments, the immune cell modulating moiety comprises an antibody or antigen-binding fragment thereof that targets an immune cell surface receptor.

[0067] In certain embodiments, the immune cell surface receptors include any one or more selected from the following groups: CD3, PD-L1, CLEC9A, CD16, NKG2D, antigen peptide-MHC molecule complex (pMHC), CD45, CD4, CD8, PD1, CD25, CD69, CD28, CD80, CD86, LAG3, IL-2R, IL-10R, IL-12R, IL-4R, CD47, CD40 and CD40L.

[0068] In certain embodiments, the immune cell modulating portion comprises a CD3 antibody. The CD3 antibody is preferably a nanobody. For example, the CD3 antibody may comprise HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:54, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:55, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:56. For example, the CD3 antibody may comprise a variable region, and the variable region may comprise the amino acid sequence set forth in SEQ ID NO:53.

[0069] In certain embodiments, the immune cell modulating portion comprises a PD-L1 antibody. The PD-L1 antibody is preferably a nanobody. For example, the PD-L1 antibody may comprise HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO: 66, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO: 67, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO: 68. For example, the PD-L1 antibody may comprise a variable region, and the variable region may comprise the amino acid sequence set forth in SEQ ID NO: 65.

[0070] In certain embodiments, the immune cell modulating portion comprises a CLEC9A antibody. The CLEC9A antibody is preferably a nanobody. For example, the CLEC9A antibody may comprise HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:70, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:71, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:72. For example, the CLEC9A antibody may comprise a variable region, wherein the variable region may comprise the amino acid sequence set forth in SEQ ID NO:69.

[0071] In certain embodiments, the immune cell modulating portion comprises a CD16 antibody. The CD16 antibody is preferably a nanobody. For example, the CD16 antibody may comprise HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:58, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:59, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:60. For example, the CD16 antibody may comprise a variable region, wherein the variable region may comprise the amino acid sequence set forth in SEQ ID NO:57.

[0072] In certain embodiments, the immune cell modulating portion comprises an NKG2D antibody. The NKG2D antibody is preferably the aforementioned Nanobody. For example, the NKG2D antibody comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:62, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:63, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:64. For example, the NKG2D antibody may comprise a variable region, wherein the variable region may comprise the amino acid sequence set forth in SEQ ID NO:61.

[0073] In certain embodiments, the immune cell modulating moiety targets an immune cell intracellular receptor.

[0074] The immune cell modulating portion that targets an intracellular receptor of an immune cell may be an immune agonist.

[0075] The immune cell regulating portion targeting an intracellular receptor of an immune cell may be an immunosuppressive agent.

[0076] In certain embodiments, the immune agonist or immunosuppressant comprises any one or more selected from the following groups: Toll-like receptor (TLR) agonist (e.g., CpG oligonucleotides, lipopolysaccharide LPS, IMDQ), interferon gene stimulator receptor (STING) agonist (e.g., Cyclic dinucleotides, diABZI), retinoic acid-inducible gene factor I (RIG-I)-like receptor (RLR) agonist, leucine-rich nucleotide binding domain repeat sequence (NLR), C-type lectin receptor (CLR agonist, protein kinase agonist (e.g., JAK kinase agonist), phosphokinase agonist (e.g., SHP-1 / SHP-2 phosphatase inhibitor), transcription factor activator (e.g., NF-κB activator, NFAT activator), intracellular signaling molecule (e.g., interleukin, such as IL-2, IL-12, IL-15), immunomodulatory small molecules (e.g., imidazoline compounds), PKC agonist, apoptosis pathway regulator (e.g., Bcl-2 family protein inhibitor), 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 transducer and activator of transcription (STAT) 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-depleting monoclonal antibodies (e.g., alemtuzumab), Janus kinase inhibitors (e.g., tofacitinib), tripterygium wilfordii polyglycosides, leflunomide, mizoribine, polyclonal antibodies (e.g., anti-lymphocyte immunoglobulin (ATG)), infliximab, adalimumab, golimumab, and FTY720 derivatives (e.g., fingolimod).In certain embodiments, the immune agonist or inhibitor comprises any one or more selected from the following groups: Toll-like receptor (TLR) agonist, interferon gene stimulator receptor (STING) agonist, retinoic acid-inducible gene I (RIG-I)-like receptor (RLR), leucine-rich nucleotide binding domain repeat sequence (NLR), C-type lectin receptor (CLR), signal transducer and activator of transcription protein (STAT) and indoleamine 2,3-dioxygenase (IDO).

[0077] In certain embodiments, the immune agonist or inhibitor comprises any one or more selected from the group consisting of IMDQ, diABZI, CPG, STAT3 ASO, cGAMP, and IDO inhibitor.

[0078] In certain embodiments, the immune agonist comprises any one or more selected from the group consisting of IMDQ, diABZI, CPG, and cGAMP.

[0079] In certain embodiments, the immunosuppressant comprises any one or more selected from the group consisting of STAT3ASO, IDO inhibitor Indoximod, GDC-0919, and Epacadostat.

[0080] In certain embodiments, P 1 、P 2 and P 3 The tumor cell targeting portion and / or immune cell modulating portion in the P can be released to bind to tumor cells or modulate immune cells. 1 、P 2 and P 3 It may further include a cleavable linker, or a cleavable linker. The cleavable linkers that can achieve the purpose of the present invention include, but are not limited to: peptide bond 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 include a disulfide bond (SS), which can be broken under reducing conditions, thereby releasing P 1 、P 2 and / or P 3 .

[0081] In certain embodiments, P 1 、P 2 and P 3 Any one of the above is the tumor cell binding portion, any one of the above is a T cell regulating portion, and any one of the above is a portion selected from the group consisting of a dendritic cell regulating portion, a natural killer cell regulating portion, and a myeloid immune cell regulating portion.

[0082] In a specific embodiment, P 1 、P 2 and P 3 One of the proteins comprises a tumor antigen binding protein, one comprises a binding protein for a T cell surface receptor, and one comprises a binding protein for a dendritic cell surface receptor. 1 、P 2 and P 3 One of the two comprises the EGFR antibody, one comprises the CD3 antibody, and one comprises the PD-L1 antibody. 1 、P 2 and P 3 One of the two comprises the HER2 antibody, one comprises the CD3 antibody, and one comprises the PD-L1 antibody.

[0083] In a specific embodiment, P 1 、P 2 and P 3 One of them contains a tumor antigen binding protein, one contains a binding protein for a T cell surface receptor, and one contains a binding protein for a natural killer cell surface receptor. 1 、P 2 and P 3 One of the two comprises the EGFR antibody, one comprises the CD3 antibody, and one comprises the CD16 antibody. 1 、P 2 and P 3 One of the two comprises the HER2 antibody, one comprises the CD3 antibody, and one comprises the CD16 antibody.

[0084] In a specific embodiment, P 1 、P 2 and P 3 One of them contains a tumor antigen binding protein, one contains a binding protein for a T cell surface receptor, and one contains a myeloid immune cell intracellular receptor regulator. 1 、P 2 and P 3 One of the two comprises the EGFR antibody, one comprises the CD3 antibody, and one comprises a TLR agonist (e.g., IMDQ). 1 、P 2 and P 3 One of the two comprises the HER2 antibody, one comprises the CD3 antibody, and one comprises a TLR agonist (eg, IMDQ).

[0085] On the other hand, the present application provides an antigen-binding protein that specifically binds to EGFR. In certain embodiments, the antigen-binding protein that specifically binds to EGFR comprises 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 the HCDR1, HCDR2, and HCDR3 comprise an amino acid sequence selected from any one or more of the following groups:

[0086] (1) HCDR1: SEQ ID NO:2, HCDR2: SEQ ID NO:3, and HCDR3: SEQ ID NO:4,

[0087] (2) HCDR1: SEQ ID NO: 6, HCDR2: SEQ ID NO: 7, and HCDR3: SEQ ID NO: 8,

[0088] (3) HCDR1: SEQ ID NO: 10, HCDR2: SEQ ID NO: 11, and HCDR3: SEQ ID NO: 12,

[0089] (4) HCDR1: SEQ ID NO:2, HCDR2: SEQ ID NO:3, and HCDR3: SEQ ID NO:4,

[0090] (5) HCDR1: SEQ ID NO:2, HCDR2: SEQ ID NO:3, and HCDR3: SEQ ID NO:15,

[0091] (6) HCDR1: SEQ ID NO: 17, HCDR2: SEQ ID NO: 3, and HCDR3: SEQ ID NO: 18,

[0092] (7) HCDR1: SEQ ID NO:2, HCDR2: SEQ ID NO:20, and HCDR3: SEQ ID NO:21,

[0093] (8) HCDR1: SEQ ID NO: 23, HCDR2: SEQ ID NO: 3, and HCDR3: SEQ ID NO: 24,

[0094] (9) HCDR1: SEQ ID NO: 2, HCDR2: SEQ ID NO: 3, and HCDR3: SEQ ID NO: 15,

[0095] (10) HCDR1: SEQ ID NO: 27, HCDR2: SEQ ID NO: 3, and HCDR3: SEQ ID NO: 28,

[0096] (11) HCDR1: SEQ ID NO:30, HCDR2: SEQ ID NO:31, and HCDR3: SEQ ID NO:32,

[0097] (12) HCDR1: SEQ ID NO:2, HCDR2: SEQ ID NO:34, and HCDR3: SEQ ID NO:35,

[0098] (13) HCDR1: SEQ ID NO: 27, HCDR2: SEQ ID NO: 37, and HCDR3: SEQ ID NO: 38,

[0099] (14) HCDR1: SEQ ID NO: 40, HCDR2: SEQ ID NO: 3, and HCDR3: SEQ ID NO: 41,

[0100] (15) HCDR1: SEQ ID NO: 43, HCDR2: SEQ ID NO: 44, and HCDR3: SEQ ID NO: 45,

[0101] (16) HCDR1: SEQ ID NO: 6, HCDR2: SEQ ID NO: 3, and HCDR3: SEQ ID NO: 47, and

[0102] (17) HCDR1: SEQ ID NO:49, HCDR2: SEQ ID NO:50, and HCDR3: SEQ ID NO:51.

[0103] In certain embodiments, the antigen binding protein that specifically binds to EGFR comprises a heavy chain variable region, which comprises the amino acid sequence shown in any one of SEQ ID NO: 1, 5, 9, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49, 53, 57, 61 and 65.

[0104] In certain embodiments, the antigen binding protein that specifically binds to EGFR is an antibody or an antigen binding fragment thereof.

[0105] In certain 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.

[0106] In certain embodiments, the antigen binding protein that specifically binds to EGFR is a monospecific antibody or a multispecific antibody.

[0107] In certain embodiments, the antigen binding protein that specifically binds to EGFR is a nanobody VHH, which comprises 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.

[0108] On the other hand, the present application provides an antigen binding protein that specifically binds to HER2. In certain embodiments, the antigen binding protein that specifically binds to HER2 is an Affibody.

[0109] Affibodies are small, robust proteins that can bind to target proteins or peptides with high affinity, similar in properties to monoclonal antibodies and therefore serve as antibody mimics. Affibodies are characterized by their small molecular weight, rapid folding rate, and stable structure, making them resistant to chemical modification.

[0110] For example, the antigen binding protein that specifically binds to HER2 comprises the amino acid sequence shown in SEQ ID NO:52.

[0111] In another aspect, the present application provides an antigen-binding protein that specifically binds to CD3. In certain embodiments, the antigen-binding protein that specifically binds to CD3 comprises a CD3 antibody or an antigen-binding fragment thereof. The CD3 antibody is preferably a nanobody. For example, the CD3 antibody may comprise a HCDR1, a HCDR2, and a HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:54, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:55, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:56. For example, the CD3 antibody may comprise a variable region, and the variable region may comprise the amino acid sequence set forth in SEQ ID NO:53.

[0112] On the other hand, the present application provides an antigen-binding protein that specifically binds to PD-L1. In certain embodiments, the antigen-binding protein that specifically binds to PD-L1 comprises 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, and the HCDR1 includes the amino acid sequence shown in SEQ ID NO: 66, the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 67, and the 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.

[0113] In another aspect, the present application provides an antigen-binding protein that specifically binds to CLEC9A. In certain embodiments, the antigen-binding protein that specifically binds to CLEC9A comprises a CLEC9A antibody or an antigen-binding fragment thereof. The CLEC9A antibody is preferably a nanobody. For example, the CLEC9A antibody may comprise a HCDR1, a HCDR2, and a HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:70, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:71, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:72. For example, the CLEC9A antibody may comprise a variable region, and the variable region may comprise the amino acid sequence set forth in SEQ ID NO:69.

[0114] On the other hand, the present application provides an antigen-binding protein that specifically binds to CD16. In certain embodiments, the antigen-binding protein that specifically binds to CD16 comprises 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 the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:58, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:59, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:60. For example, the CD16 antibody may include a variable region, and the variable region may include the amino acid sequence set forth in SEQ ID NO:57.

[0115] On the other hand, the present application provides an antigen-binding protein that specifically binds to NKG2D. In certain embodiments, the antigen-binding protein that specifically binds to NKG2D comprises an NKG2D antibody or an antigen-binding fragment thereof. The NKG2D antibody is preferably the aforementioned Nanobody. For example, the NKG2D antibody comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:62, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:63, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:64. For example, the NKG2D antibody may comprise a variable region, and the variable region may comprise the amino acid sequence set forth in SEQ ID NO:61.

[0116] In another aspect, the present application provides an isolated nucleic acid encoding the antigen binding protein.

[0117] In another aspect, the present application provides a vector comprising the isolated nucleic acid.

[0118] In another aspect, the present application provides a cell comprising the vector and / or the isolated nucleic acid.

[0119] In another aspect, the present application provides an immunoconjugate comprising the antigen binding protein.

[0120] In another aspect, the present application provides a pharmaceutical composition comprising the multispecific conjugate and / or the antigen-binding protein described herein. The pharmaceutical composition may further comprise a pharmaceutically acceptable excipient or carrier.

[0121] On the other hand, the present application provides a method for treating and / or preventing tumors, comprising administering to a subject in need thereof an effective amount of the multispecific conjugate, the antigen-binding protein, the isolated nucleic acid, the vector, the cell, the immunoconjugate and / or the pharmaceutical composition described herein.

[0122] On the other hand, the present application provides the use of the multispecific conjugate, the antigen-binding protein, the isolated nucleic acid, the vector, the cell, the immunoconjugate and / or the pharmaceutical composition described herein in the preparation of a medicament for treating and / or preventing tumors.

[0123] On the other hand, the present application provides the multispecific conjugate, the antigen-binding protein, the isolated nucleic acid, the vector, the cell, the immunoconjugate and / or the pharmaceutical composition described herein, which are used for treating and / or preventing tumors.

[0124] In certain embodiments, the administering comprises injection, preferably intravenous injection.

[0125] In certain embodiments, the multispecific conjugate is administered at a dose of 0-200 mk / kg body weight, for example, the multispecific conjugate is administered at a dose of 0-150 mk / kg body weight, 0-100 mk / kg body weight, 0-50 mk / kg body weight, 0-30 mk / kg body weight, 0-20 mk / kg body weight, 0-15 mk / kg body weight, or 1-12 mk / kg body weight.

[0126] In certain embodiments, the dosing interval of the multispecific conjugate is 12 hours to 2 weeks. In certain 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.

[0127] In certain embodiments, the tumor comprises a solid tumor. In certain embodiments, the tumor comprises a hematological tumor.

[0128] In certain embodiments, the tumor comprises 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 cancer, 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 tumor, retinoblastoma and / or glioblastoma.

[0129] In certain 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.

[0130] On the other hand, the present application provides a method for treating and / or preventing immune system-related diseases, comprising administering to a subject in need thereof an effective amount of the multispecific conjugate, the antigen-binding protein, the isolated nucleic acid, the vector, the cell, the immunoconjugate and / or the pharmaceutical composition described herein.

[0131] On the other hand, the present application provides the use of the multispecific conjugate, the antigen-binding protein, the isolated nucleic acid, the vector, the cell, the immunoconjugate and / or the pharmaceutical composition described herein in the preparation of a medicament for treating and / or preventing immune system-related diseases.

[0132] On the other hand, the present application provides the multispecific conjugate, the antigen-binding protein, the isolated nucleic acid, the vector, the cell, the immunoconjugate and / or the pharmaceutical composition described herein, which are used to treat and / or prevent immune system-related diseases.

[0133] On the other hand, the present application provides a method for treating, preventing or ameliorating an immune disease or condition in a cell, tissue, organ or animal, the method comprising administering to the cell, tissue, organ or animal an effective amount of the multispecific conjugate, the antigen-binding protein, the isolated nucleic acid, the vector, the cell, the immunoconjugate and / or the pharmaceutical composition described herein.

[0134] On the other hand, the present application provides a method for enhancing or stimulating an immune response or function, the method comprising administering to an individual (e.g., a cell, tissue, organ, or animal) an effective amount of the multispecific conjugate described herein, the antigen-binding protein, the isolated nucleic acid, the vector, the cell, the immunoconjugate, and / or the pharmaceutical composition. The method may be an in vivo method, an in vitro method, an ex vivo method, or a method in living cells. In certain embodiments, the enhancing or stimulating immune response or function comprises promoting T cells to release cytokines (e.g., IL12, IL1β, and / or IFNγ). In certain embodiments, the enhancing or stimulating immune response or function comprises promoting T cells to express Perforin, PD1, and / or Ki67. In certain embodiments, the enhancing or stimulating immune response or function comprises promoting dendritic cells to express CD86 and / or HLA-DR. In certain embodiments, the enhancing or stimulating immune response or function comprises promoting NK cells to release cytokines (e.g., IFNγ and / or MIP-1β). In certain embodiments, the enhancing or stimulating immune response or function comprises promoting the expression of CD83 and / or CD40 by myeloid dendritic cells. In certain embodiments, the enhancing or stimulating immune response or function comprises changing the ratio of M1 and M2 macrophages in myeloid macrophages. In certain embodiments, the enhancing or stimulating immune response or function comprises promoting the expression of CD14, CD40 and / or CD80 by myeloid monocytes. In certain embodiments, the enhancing or stimulating immune response or function comprises inducing immune memory.

[0135] On the other hand, the present application provides a method for activating immune cells, the method comprising administering an effective amount of the multispecific conjugate described herein, the antigen-binding protein, the isolated nucleic acid, the vector, the cell, the immunoconjugate and / or the pharmaceutical composition to the immune cells. The method can be an in vivo method, an in vitro method, an ex vivo method or a method in living cells. In certain embodiments, the activated immune cells include promoting T cells to release cytokines (e.g., IL12, IL1β and / or IFNγ). In certain embodiments, the activated immune cells include promoting T cells to express Perforin, PD1 and / or Ki67. In certain embodiments, the activated immune cells include promoting dendritic cells to express CD86 and / or HLA-DR. In certain embodiments, the activated immune cells include promoting NK cells to release cytokines (e.g., IFNγ and / or MIP-1β). In certain embodiments, the activated immune cells include promoting myeloid dendritic cells to express CD83 and / or CD40. In certain embodiments, the activated immune cells include changing the ratio of M1 and M2 macrophages in myeloid macrophages. In certain embodiments, the activated immune cells include promoting myeloid mononuclear cells to express CD14, CD40 and / or CD80. Those skilled in the art can easily gain insight into other aspects and advantages of the present application from the detailed description below. The detailed description below only shows and describes exemplary embodiments of the present application. As those skilled in the art will appreciate, the content of this application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention involved in this application. Accordingly, the descriptions in the drawings and specification of this application are merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0136] The specific features of the inventions of this application are set forth in the appended claims. The features and advantages of the inventions of this application can be better understood by referring to the exemplary embodiments described in detail below and the accompanying drawings. A brief description of the drawings is as follows:

[0137] Figure 1 Shown is LC-MS characterization of anti-EGFR-LPETG and anti-HER2-LPETG.

[0138] Figure 2 Shown is LC-MS characterization of anti-CD3-DBCO and anti-CD3-BCN.

[0139] Figure 3 Shown is LC-MS characterization of anti-CD16-BCN and anti-NKG2D-BCN.

[0140] Figure 4 Shown is LC-MS characterization of anti-PDL1-BCN and anti-CLEC9A-BCN.

[0141] Figure 5 Shown are LC-MS characterizations of (IMDQ)6-BCN and (diABZI)6-BCN.

[0142] Figure 6 Shown is LC-MS characterization of CPG-DBCO and STAT3-DBCO.

[0143] Figure 7 Shown is the LC-MS characterization of (CMV)2-BCN.

[0144] Figure 8 Shown are LC-MS and SDS-PAGE characterizations of the multispecific conjugate EGFR-CD3-PDL1. The left is a schematic diagram of EGFR-CD3-PDL1, the middle is an LC-MS characterization diagram, and the right is an SDS-PAGE characterization diagram.

[0145] Figure 9 Shown is a schematic diagram of EGFR-CD3-PDL1 simultaneously recruiting T cells and dendritic cells (DCs) to target tumors.

[0146] Figure 10 Shown are LC-MS and SDS-PAGE characterizations of the multispecific conjugate EGFR-CD3-CLEC9A. The left is a schematic diagram of EGFR-CD3-CLEC9A, the middle is an LC-MS characterization diagram, and the right is an SDS-PAGE characterization diagram.

[0147] Figure 11 Shown is a schematic diagram of EGFR-CD3-CLEC9A simultaneously recruiting T cells and dendritic cells (DCs) to target tumors.

[0148] Figure 12 Shown are LC-MS and SDS-PAGE characterizations of the multispecific conjugate EGFR-CD3-CD16. The left is a schematic diagram of EGFR-CD3-CD16, the middle is an LC-MS characterization diagram, and the right is an SDS-PAGE characterization diagram.

[0149] Figure 13 Shown is a schematic diagram of EGFR-CD3-CD16 simultaneously recruiting T cells and natural killer (NK) cells to target tumors.

[0150] Figure 14Shown are LC-MS and SDS-PAGE characterizations of the multispecific conjugate EGFR-CD3-NKG2D. The left is a schematic diagram of EGFR-CD3-NKG2D, the middle is an LC-MS characterization diagram, and the right is an SDS-PAGE characterization diagram.

[0151] Figure 15 Shown is a schematic diagram of EGFR-CD3-NKG2D simultaneously recruiting T cells and natural killer (NK) cells to target tumors.

[0152] Figure 16 Shown are LC-MS and SDS-PAGE characterizations of the multispecific conjugate HER2-CD3-(IMDQ)6. The left is a schematic diagram of HER2-CD3-(IMDQ)6, the middle is an LC-MS characterization diagram, and the right is an SDS-PAGE characterization diagram.

[0153] Figure 17 Shown is a schematic diagram of HER2-CD3-(IMDQ)6 simultaneously recruiting T cells and myeloid immune cells to target tumors.

[0154] Figure 18 Shown are LC-MS and SDS-PAGE characterizations of the multispecific conjugate HER2-CD3-(diABZI)6. The left is a schematic diagram of HER2-CD3-(diABZI)6, the middle is an LC-MS characterization diagram, and the right is an SDS-PAGE characterization diagram.

[0155] Figure 19 Shown is a schematic diagram of HER2-CD3-(diABZI)6 simultaneously recruiting T cells and myeloid immune cells to target tumors.

[0156] Figure 20 Shown are LC-MS and SDS-PAGE characterizations of the multispecific conjugate EGFR-CD3-CPG. The left is a schematic diagram of EGFR-CD3-CPG, the middle is an LC-MS characterization diagram, and the right is an SDS-PAGE characterization diagram.

[0157] Figure 21 Shown is a schematic diagram of EGFR-CD3-CPG simultaneously recruiting T cells and myeloid immune cells to target tumors.

[0158] Figure 22 Shown are LC-MS and SDS-PAGE characterizations of the multispecific conjugate EGFR-CD3-STAT3. The left is a schematic diagram of EGFR-CD3-STAT3, the middle is an LC-MS characterization diagram, and the right is an SDS-PAGE characterization diagram.

[0159] Figure 23 Shown is a schematic diagram of EGFR-CD3-STAT3 simultaneously recruiting T cells and myeloid immune cells to target tumors.

[0160] Figure 24 Shown are LC-MS and SDS-PAGE characterizations of the multispecific conjugate EGFR-CD3-(CMV)2. The left is a schematic diagram of EGFR-CD3-(CMV)2, the middle is an LC-MS characterization diagram, and the right is an SDS-PAGE characterization diagram.

[0161] Figure 25 Shown is a schematic diagram of the simultaneous recruitment of T cells by EGFR-CD3-(CMV)2 and the targeting of CMV antigen-specific T cells to tumors.

[0162] Figure 26A Shown is the evaluation of the multispecific conjugate EGFR-CD3-PDL1 in a tumor cell-T cell-dendritic cell co-culture system.

[0163] Figure 26B Shown is the multispecific conjugate EGFR-CD3-PDL1-mediated tumor cell killing in a tumor cell-T cell-dendritic cell co-culture system.

[0164] Figure 26C The figure shows the activation of T cells (CD69 + T cells).

[0165] Figure 26D Shown is T cell activation (IFNγ and Granzyme B) mediated by the multispecific conjugate EGFR-CD3-PDL1 in a tumor cell-T cell-dendritic cell co-culture system.

[0166] Figure 26E The results show that the multispecific conjugate EGFR-CD3-PDL1 mediated DC activation (CD83 + and CD86 + dendritic cells)

[0167] Figure 26F Shown is the activation of dendritic cells (DCs) (IL12 and IL1β cytokines) mediated by the multispecific conjugate EGFR-CD3-PDL1 in a tumor cell-T cell-dendritic cell co-culture system.

[0168] Figure 27A Shown is the evaluation of the therapeutic effect of EGFR-CD3-PDL1 in a PBMC humanized mouse model.

[0169] Figure 27BShown are the EGFR-CD3-PDL1 tumor inhibition curves in the PBMC humanized mouse model.

[0170] Figure 27C Shown is the activation of intratumoral T cells by EGFR-CD3-PDL1 in a PBMC humanized mouse model, which increases Perforin + PD1 + and Ki67 + T cell percentage.

[0171] Figure 28A Shown is the evaluation of EGFR-CD3-PDL1 therapeutic efficacy in a humanized HSC mouse model

[0172] Figure 28B Shown are the EGFR-CD3-PDL1 tumor inhibition curves in the HSC humanized mouse model.

[0173] Figure 28C The results show that EGFR-CD3-PDL1 activates intratumoral T cells in the HSC humanized mouse model and increases the expression of T cell Perforin, Granzyme B, and PD1.

[0174] Figure 28D Shown is the activation of intratumoral dendritic cells by EGFR-CD3-PDL1 in a humanized HSC mouse model, which increases CD86 + The percentage of dendritic cells and the proportion of dendritic cells with high HLA-DR expression.

[0175] Figure 29A Shown is the evaluation of the therapeutic effect of EGFR-CD3-PDL1 in a transgenic humanized mouse model.

[0176] Figure 29B Shown are the tumor inhibition curves of EGFR-CD3-PDL1 in a transgenic humanized mouse model.

[0177] Figure 29C Shown is a transgenic humanized mouse model in which EGFR-CD3-PDL1 activates intratumoral T cells and increases Perforin + 、Granzyme B + and Ki67 + T cell percentage.

[0178] Figure 29D Shown is a transgenic humanized mouse model in which EGFR-CD3-PDL1 activates intratumoral dendritic cells and increases CD80 + 、CD86 + The percentage of dendritic cells and the proportion of dendritic cells with high MHC-II expression.

[0179] Figure 29E Shown is that EGFR-CD3-PDL1 increases the proportion of OVA antigen-specific T cells in tumors and spleens in a transgenic humanized mouse model.

[0180] Figure 29F Shown is the enhanced tumor-specific immunity of EGFR-CD3-PDL1 in a transgenic humanized mouse model. The left is the photographic results of the ELISPOT experiment, and the right is the corresponding statistical results.

[0181] Figure 30A Shown is the evaluation of EGFR-CD3-PDL1 therapeutic efficacy in a patient tumor tissue model.

[0182] Figure 30B Shown is 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.

[0183] Figure 30C It is shown that EGFR-CD3-PDL1 treatment inhibits the growth of PTC derived from patient tumor tissue.

[0184] Figure 31A Shown is the evaluation of the multispecific conjugate EGFR-CD3-CD16 in a tumor cell-T cell co-culture system.

[0185] Figure 31B Shown is the tumor cell killing mediated by the multispecific conjugate EGFR-CD3-CD16 in a tumor cell-T cell co-culture system.

[0186] Figure 31C The figure shows the activation of T cells (CD69) mediated by the multispecific conjugate EGFR-CD3-CD16 in the tumor cell-T cell co-culture system. + T cells).

[0187] Figure 31D Shown is T cell activation (IFNγ cytokine) mediated by the multispecific conjugate EGFR-CD3-CD16 in a tumor cell-T cell co-culture system.

[0188] Figure 32A Shown is the evaluation of the multispecific conjugate EGFR-CD3-CD16 in a tumor cell-natural killer (NK) cell co-culture system.

[0189] Figure 32B Shown is the multispecific conjugate EGFR-CD3-CD16-mediated tumor cell killing in a tumor cell-natural killer cell co-culture system.

[0190] Figure 32C The figure 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).

[0191] Figure 32D Shown is the activation of natural killer cells (IFNγ and MIP-1β cytokines) mediated by the multispecific conjugate EGFR-CD3-CD16 in a tumor cell-natural killer cell co-culture system.

[0192] Figure 33A Shown is the release of IMDQ molecules from the multispecific conjugate HER2-CD3-(IMDQ)6 in the reducing microenvironment of a tumor.

[0193] Figure 33B Shown is the LC-MS detection of IMDQ molecules released by HER2-CD3-(IMDQ)6 in the tumor reducing microenvironment

[0194] Figure 34A Shown is the evaluation of the multispecific conjugate HER2-CD3-(IMDQ)6 in a tumor cell-T cell co-culture system.

[0195] Figure 34B Shown is the tumor cell killing mediated by the multispecific conjugate HER2-CD3-(IMDQ)6 in a tumor cell-T cell co-culture system.

[0196] Figure 34C Shown is the activation of T cells (CD69) mediated by the multispecific conjugate HER2-CD3-(IMDQ)6 in a tumor cell-T cell co-culture system. + T cells).

[0197] Figure 34D Shown is T cell activation (IFNγ cytokine) mediated by the multispecific conjugate HER2-CD3-(IMDQ)6 in a tumor cell-T cell co-culture system.

[0198] Figure 35A Shown is the assessment of HER2-CD3-(IMDQ)6-mediated tumor-targeted myeloid immune cell activation.

[0199] Figure 35B Shown is HER2-CD3-(IMDQ)6-mediated activation of myeloid dendritic cells (CD83 + CD40 + dendritic cells).

[0200] Figure 35CShown is HER2-CD3-(IMDQ)6-mediated myeloid macrophage activation (ratio of M1 to M2 macrophages).

[0201] Figure 35D Shown is HER2-CD3-(IMDQ)6-mediated myeloid monocyte activation (CD14, CD40, and CD80 expression). DETAILED DESCRIPTION

[0202] The following describes the implementation of the present invention through specific embodiments. People familiar with this technology can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0203] Definition of terms

[0204] In this application, the term "antigen binding protein" generally refers to a protein comprising a portion that binds to an antigen, and optionally a scaffold or backbone portion that allows the portion that binds to the antigen to adopt a conformation that promotes 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, etc., as long as they exhibit the desired antigen binding activity.

[0205] In this application, the term "antibody" generally refers to an immunoglobulin that can undergo a specific binding reaction with the corresponding antigen. The antibody can be secreted by an immune cell (e.g., an effector B cell). The antibody can 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 camelized single domain antibody. An "antibody" can generally comprise a protein comprising at least two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds, or an antigen-binding fragment thereof. 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 regions of hypervariability, called complementarity determining regions (CDRs), which alternate with more conserved regions called framework regions (FRs). Each VH and VL comprises three CDRs and four framework regions (FRs), arranged in the following order from amino-terminus to carboxyl-terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable domains of native heavy and light chains each comprise four FR regions (H-FR1, H-FR2, H-FR3, H-FR4, L-FR1, L-FR2, L-FR3, L-FR4), most of which adopt a β-sheet configuration and are connected by three CDRs, forming loops connecting and, in some cases, forming part of the β-sheet structure. The CDRs in each chain are brought together in close proximity by the FR regions and, together with the CDRs from the other chain, form the antigen binding site of the antibody.

[0206] In the present application, the term "VHH" may also be referred to as nano antibody, heavy chain single domain antibody, generally refers to a heavy chain antibody variable region of a natural missing antibody light chain. The term "VHH" is used to distinguish between the heavy chain variable domain (VH) and light chain variable domain region (VL) present in conventional 4-chain antibodies. Further description of VHH or nano antibody can refer to the article (Reviews in Molecular Biotechnology 74:277-302,2001) of Muyldermans. VHH or nano antibody is characterized in that they can have one or more "Hallmark residues" in one or more framework sequences. The VHH may also include its humanized or camelized form, as well as other modifications, parts or fragments, derivatives or "nano antibody fusions", multivalent or multispecific constructs, and modifications that improve the VHH half-life. VHH forms a minimum antigen-binding fragment, retaining the binding affinity and specificity of a full-length antibody. VHH has a longer CDR3 loop and a convex paratope, which enables them to enter the inner cavity of the target antigen.

[0207] The terms "protein", "polypeptide" and "peptide" are used interchangeably herein and generally refer to polymers of amino acid residues and variants and synthetic analogs thereof. A "peptide" may also refer to a partial amino acid sequence derived from its original protein, for example after trypsin digestion. One or more amino acid residues in the polymer may be synthetic non-naturally occurring amino acids, for example chemical analogs of the corresponding naturally occurring amino acids, as well as naturally occurring amino acid polymers. The term also includes post-translational modifications of the polypeptide, 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, separated and / or recovered from components of its production environment (e.g., natural or recombinant). Contaminating components of its production environment are generally substances that interfere with its research, diagnostic or therapeutic use, and may include enzymes, hormones and other proteins or non-protein solutes. An isolated antigen-binding protein or antibody will generally be prepared by at least one purification step.

[0208] The proteins and / or amino acid sequences referred to in this application should also be understood to include at least the following scope: variants or homologs that have the same or similar functions as the protein. In this application, the variant can be a protein or polypeptide that has one or more amino acids substituted, deleted, or added 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 can include a protein or polypeptide that has amino acid changes through at least 1, such as 1-30, 1-20, or 1-10, and for example 1, 2, 3, 4, or 5 amino acid substitutions, deletions, and / or insertions. The functional variant can substantially retain the biological properties of the protein or polypeptide before the change (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 ability) of the protein or polypeptide before the change. For example, the substitution can be a conservative substitution.

[0209] In the present application, a portion of the amino acid sequence of the multispecific binding protein or antigen-binding protein may be homologous to a corresponding amino acid sequence in an antibody from a particular species, or belong to a particular class. For example, both the variable region and the constant region of an antibody may be derived from the variable region and constant region of an antibody from a single animal species (e.g., human).

[0210] In the present application, the homolog can be a protein or polypeptide having 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 more) sequence homology with the amino acid sequence of the protein and / or the polypeptide (e.g., the antigen-binding protein described herein).

[0211] In the present application, described homology generally refers to the similarity, similarity or association between two or more sequences.Can calculate " sequence homology per-cent " in the following manner: two sequences to be compared are compared in comparison window, determine that there is identical nucleic acid base (for example, A, T, C, G) or identical amino acid residue (for example, Ala, Pro, Ser, Thr, Gly, Val, Leu, Ile, Phe, Tyr, Trp, Lys, Arg, His, Asp, Glu, Asn, Gln, Cys and Met) number in the position to obtain the number of matching positions, with the number of matching positions divided by the total number of positions (that is, window size) in the comparison window, and result is multiplied by 100, to produce sequence homology per-cent.Comparison carried out in order to determine the sequence homology per-cent, can realize by several ways known in the art, for example, use publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximum alignment over the full length of the sequences being compared or within a region of interest. Homology can also be determined by the following methods: FASTA and BLAST. A description of the FASTA algorithm can be found in W. R. Earson and D. J. Lipman, "Improved tools for biological sequence comparison," Proc. Natl. Acad. Sci., 85: 2444-2448, 1988; and D. J. Lipman and W. R. Earson, "Rapid and sensitive protein similarity search," Science, 227: 1435-1441, 1989. A description of the BLAST algorithm can be found in S. Altschul, W. Gish, W. Miller, E. W. Myers, and D. Lipman, "A basic local alignment search tool," J. Mol. Biol., 215: 403-410, 1990.

[0212] In this application, the term "halogen" is generally meant to include fluorine, chlorine, bromine and iodine.

[0213] In this application, the term "urea" generally refers to -(HN-CO-)2N-.

[0214] 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.), branched-chain alkyl groups (e.g., isopropyl, tert-butyl, isobutyl, etc.). The term alkyl may further include alkyl groups whose main chain carbon atoms are substituted with one or more carbon atoms of oxygen, nitrogen, sulfur, or phosphorus. The alkyl groups described herein may contain 1-20, 1-12, 1-10, 1-8, or 1-6 carbon atoms.

[0215] In this application, the term "alkenyl" generally refers to any cyclic or acyclic branched or unbranched unsaturated carbon chain portion, which has 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, which has one or more triple bonds. The alkenyl groups described herein may contain 2-20, 2-12, 2-10, 2-8 or 2-6 carbon atoms.

[0216] In this application, the term "alkynyl" generally refers to an unsaturated straight or branched chain alkynyl group, such as ethynyl, 1-propynyl, propargyl, butynyl, etc. The alkynyl group can be substituted or unsubstituted. The alkynyl group described herein can contain 2-20, 2-12, 2-10, 2-8, or 2-6 carbon atoms.

[0217] In this application, as known to those skilled in the art, terms such as "alkyl", "alkenyl", "cycloalkyl", etc. may be preceded by an identifier to indicate the number of atoms present in the group in a particular case, for example, C1-C6 alkyl, etc. The subscript number after "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 The term "alkyl" includes alkyl groups containing 1 to 6 carbon atoms.

[0218] In the present application, the term "alkanyl" generally refers to a residue derived from an alkanyl group by removing a hydrogen atom. Alkanyl groups can be substituted or non-substituted, substituted or non-substituted. The term "alkanyl" generally refers to a saturated straight or branched aliphatic hydrocarbon group having a residue derived from the same carbon atom or two different carbon atoms of a parent alkane by removing a hydrogen atom, which can be a straight or branched group containing 1 to 20 carbon atoms, for example, containing 1 to 12 carbon atoms, for example, an alkanyl group containing 1 to 6 carbon atoms. Non-limiting examples of alkanyl groups include, but are not limited to, methyl, ethyl, propyl, propyl, butyl, etc. Alkyl groups may be substituted or unsubstituted, substituted or unsubstituted, for example, when substituted, the substituents may be substituted at any available point of attachment, and the substituents may be independently selected from one or more substituents selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio, heterocycloalkylthio, and oxo, for example, 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 group.

[0219] In this application, the term "cycloalkyl" generally refers to a residue derived from the same carbon atom or multiple different carbon atoms of a carbocyclic ring by removing hydrogen atoms. The term "cycloalkane" generally refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon, the carbocyclic ring containing 3 to 20 carbon atoms, can contain 3 to 12 carbon atoms, can contain 3 to 10 carbon atoms, can contain 3 to 8 carbon atoms. Non-limiting examples of monocyclic carbocycles include cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane, cyclohexene, cyclohexadiene, cycloheptane, cycloheptatriene, cyclooctane, etc.; polycyclic carbocycles can include spirocyclic, fused ring and bridged ring carbocycles. Cycloalkyl groups can be substituted or unsubstituted.

[0220] In this application, the term "heterocycloalkyl" generally refers to a stable non-aromatic 3- to 7-membered monocyclic ring structure, a fused 7- to 10-membered bicyclic heterocyclic ring structure, or a bridged 6- to 10-membered bicyclic heterocyclic ring structure. These ring structures may be saturated or partially saturated. In addition to carbon atoms, these ring structures may contain one or more heteroatoms, wherein the heteroatoms may be selected from the following group: oxygen, sulfur, and nitrogen. For example, the heteroatoms may contain 1 to 4 heteroatoms as defined above. When used to refer to atoms in a heterocyclic ring structure, the term "nitrogen" may include nitrogen that has undergone substitution reactions. Heterocycloalkyl groups may be substituted or unsubstituted.

[0221] In this application, the term "aryl" generally refers to a 3- to 12-membered, substituted or unsubstituted monocyclic aromatic group, wherein each atom of the ring can be carbon (i.e., a carbocyclic aryl group), or one or more atoms can be a heteroatom (i.e., a heteroaryl group). The aryl group can be 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, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycloalkylthio.

[0222] The term "heteroatom" is art-recognized and refers to an atom of any element other than carbon or hydrogen. Exemplary heteroatoms include boron, nitrogen, oxygen, phosphorus, sulfur, and selenium.

[0223] As used herein, 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 heteroaromatic ring. The term "heteroaromatic ring" refers to a heteroaromatic system containing 1 to 4 heteroatoms and 5 to 14 ring atoms, wherein the heteroatoms can be selected from the group consisting of oxygen, sulfur, and nitrogen. A heteroaryl group can be 5 to 10-membered, and can be 5- or 6-membered, such as furanyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl, and the like. The heteroaryl ring can be fused to an aryl, heterocyclyl, or cycloalkyl ring, wherein the ring attached to the parent structure is the heteroaryl ring. The heteroaryl group may be optionally 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, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, and heterocycloalkylthio.

[0224] 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, and the like.

[0225] In this application, the term "optional" or "optionally" generally means that the subsequently described event or circumstance can but need not occur, and the description includes instances where the event or circumstance occurs or does not occur. For example, "a heterocyclic group optionally substituted with an alkyl group" means that the alkyl group can but need not be present, and the description includes instances where the heterocyclic group is substituted with an alkyl group and instances where the heterocyclic group is not substituted with an alkyl group.

[0226] In the present application, the compounds or conjugates of the present application include their tautomers, mesomorphs, racemates, enantiomers, and / or diastereomers of the compound. In the present application, the term "diastereomer" generally refers to stereoisomers 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 points, boiling points, spectral properties and reactivity. In the present application, the term "mesomorph" generally refers to an asymmetric atom in the molecule, but has symmetry factors that make the total optical rotation in the molecule zero. The term "racemate" or "racemic mixture" refers to a composition consisting of equimolar amounts of two enantiomeric substances.

[0227] In the present application, some atoms of the compounds of the present application may appear in more than one isotopic form. For example, hydrogen may appear in the form of protium ( 1 H), deuterium ( 2 H) and tritium ( 3 H), carbon can exist in three different isotopes ( 12 C. 13 C and 14 C) naturally occurring. Examples of isotopes that can be incorporated into the compounds of the present application also include, but are not limited to 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 indicated, the structures described herein may also include compounds that differ only in the presence or absence of one or more isotopically enriched atoms. For example, compounds that are identical to the structures described herein except for the replacement of hydrogen atoms by deuterium or tritium, or the replacement of carbon atoms by carbon 13 or carbon 14 are within the scope of this application.

[0228] In this application, the term "immune cell" generally refers to cells that can participate in or are associated with an immune response. Immune cells can include all white blood cells and non-leukocyte antigen-presenting cells. The term includes individual cells, cell lines, or cell cultures. The cells include not only specific cells but also the progeny of these cells.

[0229] In this application, the term "pharmaceutically acceptable adjuvant" generally includes pharmaceutically acceptable carriers, excipients or stabilizers that are non-toxic to cells or mammals exposed thereto at the dosages and concentrations employed. Typically, a physiologically acceptable carrier is a pH buffered aqueous solution.

[0230] As used herein, the term "treatment" refers to administering to a patient an internal or external therapeutic agent, such as a cell comprising any modification of the present application or a pharmaceutical composition comprising modified cells, wherein the patient has one or more symptoms of the disease, and it is known that the therapeutic agent has a therapeutic effect on these symptoms. Typically, the patient is administered an amount of the therapeutic agent that effectively alleviates one or more symptoms of the disease (therapeutically effective amount). The desired effects of treatment include reducing the rate of disease progression, improving or alleviating the disease state, and regressing or improving the prognosis. For example, if one or more symptoms associated with cancer are alleviated or eliminated, including but not limited to, reducing (or destroying) cancer cell proliferation, reducing symptoms from the disease, improving the quality of life of those individuals with the disease, reducing the dosage of other drugs needed to treat the disease, delaying the progression of the disease, and / or prolonging individual survival, the individual is successfully "treated".

[0231] 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 used in this application, the term "multispecific" refers to the selectivity of having two or more antigen binding sites, at least two of which 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 a first antigen and CD3 as a 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.

[0232] 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 connection by a covalent bond. For example, this application provides a connection in which an N3 group of one compound can react with an alkynyl group of another compound to form a covalent bond, thereby forming a conjugate of the two compounds.

[0233] As used herein, the term "linker" generally refers to a central compound to which various other compounds can be attached. The central compound can be linked to one or more other compounds via covalent bonds or strong non-covalent interactions to form a larger compound. For example, the linker herein can be linked to one or more compounds comprising lipids, proteins, nucleic acids, small molecules, or polysaccharides, or any combination thereof, to form a conjugate.

[0234] In this application, the term "administer" generally refers to delivering a substance to a subject in need thereof by any route known in the art. Pharmaceutical carriers and formulations or compositions are also well known in the art. Routes of administration can include intravenous, intramuscular, intradermal, subcutaneous, transdermal, mucosal, intratumoral, and / or mucosal.

[0235] In this application, the term "comprising" generally refers to including the features specified but not excluding other elements. The terms "above" and "below" generally refer to including the number.

[0236] In this application, the term "about" generally refers to a variation within a range of 0.5%-10% above or below the specified value, for example, 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 the specified value. Detailed Description of the Invention

[0238] Multispecific conjugates

[0239] On the one hand, the present application provides a multispecific conjugate, which is a trivalent multispecific conjugate arm. 1 、P 2 or P 3 Three different or identical active molecules with properties of interest are integrated into one molecular skeleton. For the structure, properties or preparation method of the multispecific coupling arm, reference can be made to the international publication text WO2022 / 100696A1 of the PCT application. All multispecific coupling arms mentioned in this international application can be used in this application to achieve the purpose of this application.

[0240] In one embodiment, the multispecific coupling arm has a structure as shown in Formula I: (Formula I), wherein

[0241] W is in, Represents the connection site, W is Connected to La, Lb and Lc respectively;

[0242] 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 -(K 3 )n3 -(Y 3 ) p3 -(L 3 ) q3 -,in,

[0243] X 1 , X 2 , and X 3 are each independently selected from the group consisting of: -NH- and -O-,

[0244] (K 1 ) n1 , (K 2 ) n2 , and (K 3 ) n3 Each independently selected from the following group: -CH2-(CH2-O-CH2)3-(CH2)2-, -

[0245] (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 are each independently 0,

[0246] (Y 1 ) p1 ,(Y 2 ) p2 , and (Y 3 ) p3 Each independently is -NR 1 -C(=O)-,R 1 is selected from the group consisting of hydrogen, protium, deuterium and tritium; or p1, p2 or p3 are each independently 0,

[0247] (L 1 ) q1 ,(L 2 ) q2 , and (L 3 ) q3 are each independently selected from the following group: -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,

[0248] A 2 , B 2 and C 2 Each is independently selected from functional groups that can undergo the following reactions: cycloaddition reaction involving an azide group, Diels-Alder reaction involving a tetrazine group, and / or transpeptidation reaction of SrtA,

[0249] P 1 、P2 and P 3 Each is independently selected from the group consisting of sugars, lipids, nucleic acids, polypeptides, proteins and small molecules, and combinations thereof, and P 1 、P 2 and P 3 Can recruit cells.

[0250] Based on WO2022 / 100696A1, the inventors of this application further discovered that multispecific conjugates can recruit cells, not just interact with free molecules. 1 、P 2 and P 3 The multispecific conjugate of the present application can recruit at least three cells to a suitable spatial location, or can interact with at least three cells. The interaction can be to bind to cells or to modulate cell activity.

[0251] In this application, A 2 , B 2 and C 2 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 group: hydrogen, protium, deuterium, tritium, halogen, nitro, cyano, hydroxy, alkoxy, amino, amide, ester, sulfonamide, urea, C1-C 12 Alkyl, 3- to 12-membered cycloalkyl, 3- to 12-membered heterocycloalkyl, C2-C 12 Alkenyl, C2-C 12 Alkynyl, 6- to 14-membered aryl, and 6- to 14-membered heteroaryl, wherein represents the attachment site, and A 2 、B 2 and C 2 pass Respectively with La, Lb, Lc and P 1 、P 2 and P 3 Connect, as long as A 2 , B 2 and C 2 P can be connected to each other through ligation reaction 1 、P 2 and P 3 Without interfering with each other.

[0252] In the present application, W in the multispecific conjugate can be

[0253] In the present application, X in the multispecific conjugate is 1 , X 2 and X 3 They may all be -NH-.

[0254] In the present application, X in the multispecific conjugate is 1 , X 2 and X 3 They may all be -NH-.

[0255] In the present application, the multispecific conjugate (K 1 ) n1 , (K 2 ) n2 , and (K 3 ) n3 They can all be -(CH2)2-(CH2-O-CH2)3-(CH2)2-.

[0256] In the present application, the multispecific conjugate (Y 1 ) p1 ,(Y 2 ) p2 , and (Y 3 ) p3 Can each independently be -NR 1 -C(=O)-,R 1 Selected from the group consisting of hydrogen, protium, deuterium and tritium.

[0257] In the present application, the multispecific conjugate (L 1 ) q1 ,(L 2 ) q2 , and (L 3 ) q3 They may be each independently selected from: -CH2- and -CH2-aryl-.

[0258] In the present application, the A in the multispecific conjugate 2 Can be selected from the following groups:

[0259] In the present application, B in the multispecific conjugate 2 Can be selected from the following groups:

[0260] In the present application, the C 2 Can be selected from the following groups:

[0261] In the present application, the A in the multispecific conjugate 2 、B 2 and C 2They can be:

[0262] In the present application, the multispecific conjugate may have a structure as shown in Formula IIa-1 or IIa-2:

[0263]

[0264] Among them, P 1 、P 2 and P 3 Respectively as defined in this application.

[0265] For example, the multispecific conjugate may have a structure as shown in Formula IIa-1, wherein P 1 、P 2 and P 3 Respectively as defined in this application.

[0266] P 1 、P 2 and P 3

[0267] In this application, P 1 、P 2 and / or P 3 It can be a molecule that recruits cells, and its molecule type can be nucleic acid, polypeptide, sugar, fat, protein and small molecule, or a combination thereof.

[0268] In certain embodiments, P 1 、P 2 and / or P 3 At least one of the compounds can act on tumor cells, recruit tumor cells, bind to tumor cells, bind to surface proteins of tumor cells, bind to tumor antigens, or bind to intracellular targets of tumor cells.

[0269] In certain embodiments, P 1 、P 2 and / or P 3 At least one of the above can act on immune cells, recruit immune cells, bind to immune cells, bind to surface proteins of immune cells, or bind to intracellular targets of immune cells.

[0270] Those skilled in the art can select appropriate P according to the type of tumor cells or immune cells they wish to act on. 1 、P 2 and / or P 3 The target of action and the selection of appropriate P 1 、P 2 and / or P 3 molecular type.

[0271] In a specific embodiment, P 1 、P 2and / or P 3 Both may be antigen binding proteins.

[0272] In another specific embodiment, P 1 、P 2 and / or P 3 Antigen binding proteins and small molecules may be included.

[0273] In this application, P 1 、P 2 and P 3 At least one of the proteins may comprise a tumor cell binding portion, and at least one of the proteins may comprise an immune cell modulating portion.

[0274] In the present application, the tumor cell binding moiety may comprise a tumor antigen binding protein. In the present application, the tumor cell binding moiety may comprise an antibody or antigen binding fragment thereof that targets a tumor antigen.

[0275] In the present application, the immune cells may include myeloid immune cells and lymphoid immune cells. In certain embodiments, the immune cells are myeloid immune cells. In certain embodiments, the immune cells are lymphoid immune cells.

[0276] In the present application, the immune cells may include T cells (e.g., γδT 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.

[0277] In the present application, the immune cells may include T cells, natural killer cells, dendritic cells, macrophages and / or monocytes.

[0278] In the present application, the immune cell regulating portion may include an antibody or an antigen-binding fragment thereof that targets an immune cell surface protein.

[0279] In the present application, the immune cell regulatory portion can target immune cell intracellular receptors.

[0280] Antigen binding proteins

[0281] On the other hand, the present application provides an antigen-binding protein that specifically binds to EGFR. In certain embodiments, the antigen-binding protein that specifically binds to EGFR comprises 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 the 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: 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: 18, and HCDR3: SEQ ID NO: 29. 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: 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.

[0282] In another aspect, the present application provides an antigen-binding protein that specifically binds to HER2, wherein the amino acid sequence of the antigen-binding protein that specifically binds to HER2 is shown in SEQ ID NO: 52.

[0283] In another aspect, the present application provides an antigen-binding protein that specifically binds to CD3. In certain embodiments, the antigen-binding protein that specifically binds to CD3 comprises a CD3 antibody or an antigen-binding fragment thereof. The CD3 antibody is preferably a nanobody. For example, the CD3 antibody may comprise a HCDR1, a HCDR2, and a HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:54, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:55, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:56. For example, the CD3 antibody may comprise a variable region, and the variable region may comprise the amino acid sequence set forth in SEQ ID NO:53.

[0284] On the other hand, the present application provides an antigen-binding protein that specifically binds to PD-L1. In certain embodiments, the antigen-binding protein that specifically binds to PD-L1 comprises 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, and the HCDR1 includes the amino acid sequence shown in SEQ ID NO: 66, the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 67, and the 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.

[0285] In another aspect, the present application provides an antigen-binding protein that specifically binds to CLEC9A. In certain embodiments, the antigen-binding protein that specifically binds to CLEC9A comprises a CLEC9A antibody or an antigen-binding fragment thereof. The CLEC9A antibody is preferably a nanobody. For example, the CLEC9A antibody may comprise a HCDR1, a HCDR2, and a HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:70, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:71, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:72. For example, the CLEC9A antibody may comprise a variable region, and the variable region may comprise the amino acid sequence set forth in SEQ ID NO:69.

[0286] On the other hand, the present application provides an antigen-binding protein that specifically binds to CD16. In certain embodiments, the antigen-binding protein that specifically binds to CD16 comprises 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 the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:58, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:59, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:60. For example, the CD16 antibody may include a variable region, and the variable region may include the amino acid sequence set forth in SEQ ID NO:57.

[0287] On the other hand, the present application provides an antigen-binding protein that specifically binds to NKG2D. In certain embodiments, the antigen-binding protein that specifically binds to NKG2D comprises an NKG2D antibody or an antigen-binding fragment thereof. The NKG2D antibody is preferably the aforementioned Nanobody. For example, the NKG2D antibody comprises HCDR1, HCDR2, and HCDR3, wherein the HCDR1 comprises the amino acid sequence set forth in SEQ ID NO:62, the HCDR2 comprises the amino acid sequence set forth in SEQ ID NO:63, and the HCDR3 comprises the amino acid sequence set forth in SEQ ID NO:64. For example, the NKG2D antibody may comprise a variable region, and the variable region may comprise the amino acid sequence set forth in SEQ ID NO:61.

[0288] The antigen-binding proteins of the present 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 of such techniques, or other techniques known in the art. Variants can refer to amino acid sequence mutants of antibodies, as well as covalent derivatives of native polypeptides, provided that they retain biological activity equivalent to that of the native polypeptide. Amino acid sequence mutants generally differ from the native amino acid sequence in that one or more amino acids in the native amino acid sequence are substituted, or one or more amino acids are deleted and / or inserted into the polypeptide sequence. Deletion mutants include fragments of native polypeptides and N-terminal and / or C-terminal truncation mutants. Typically, amino acid sequence mutants have at least 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% homology to the native sequence.

[0289] The above antigen binding proteins can all be used in the multispecific conjugates of the present application. Those skilled in the art can also select appropriate antigen binding protein sequences to prepare multispecific conjugates as needed.

[0290] Pharmaceutical compositions and treatment methods

[0291] In another aspect, the present application provides a method for treating and / or alleviating tumors, comprising administering to a subject in need thereof a multispecific conjugate, antigen-binding protein, nucleic acid, cell, pharmaceutical composition, kit, or immunoconjugate as described herein. An appropriate P can be selected based on the type of tumor to be treated and / or alleviated. 1 、P 2 or P 3 , and then a multispecific conjugate targeting specific cells is prepared according to the method of the present application.

[0292] For example, the tumor can be a tumor with high expression of EGFR. For example, the tumor can be a tumor that is EGFR positive. For example, EGFR positive tumors can 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.

[0293] For another example, the tumor may be a tumor that overexpresses 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.

[0294] The tumor may be a primary tumor or a metastatic tumor.

[0295] The multispecific conjugates, antigen-binding proteins, nucleic acids, cells, pharmaceutical compositions, kits, or immunoconjugates described herein 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 can include buffers such as neutral buffered saline, sulfate-buffered saline, and the like; carbohydrates such as glucose, mannose, sucrose, or dextran, mannitol; proteins; polypeptides or amino acids such as glycine; antioxidants; chelating agents such as EDTA or glutathione; adjuvants (e.g., aluminum hydroxide); and preservatives.

[0296] The multispecific conjugates, antigen-binding proteins, nucleic acids, cells, pharmaceutical compositions, kits or immunoconjugates of the present application can be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intraspinal, intramuscularly, by intravenous (iv) injection or intraperitoneally. Preferably, the pharmaceutical compositions of the present application can be formulated for intravenous administration.

[0297] The multispecific conjugates, antigen-binding proteins, nucleic acids, cells, pharmaceutical compositions, kits, or immunoconjugates described herein can be co-administered with one or more other anti-tumor therapies (e.g., surgery, chemotherapy, radiotherapy, targeted therapy, or immune checkpoint inhibitors).

[0298] The dosage and frequency of administration of the cells of the present 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 individual differences in the patient's age, weight, tumor size, degree of infection or metastasis, and condition.

[0299] Without intending to be bound by any theory, the following examples are merely intended to illustrate the multispecific conjugates, preparation methods, and uses of the present application, and are not intended to limit the scope of the present invention.

[0300] Without intending to be bound by any theory, the following examples are merely intended to illustrate the fusion protein, preparation method, and use of the present application, and are not intended to limit the scope of the present invention.

[0301] Example

[0302] Example 1 Preparation of coupling module and LC-MS characterization

[0303] (1) Tumor cell EGFR / HER2 receptor targeting module

[0304] Nanobodies against the EGFR receptor of human tumor cells, whose amino acid sequence is SEQ ID NO: 1-51. Affibodies against the HER2 receptor of human tumor cells, whose amino acid sequence is SEQ ID NO: 52. The above nanobodies were all expressed in E. coli, with IPTG induction and overnight expression at 30°C. The expressed nanobodies were purified by nickel column purification and size exclusion chromatography in turn. The N-termini of the obtained nanobodies were all connected to the LPETG sequence (SEQ ID NO: 76) and the 6His tag for purification. They were named anti-EGFR-LPETG and anti-HER2-LPETG for the subsequent synthesis of multispecific conjugates.

[0305] Anti-EGFR-LPETG and anti-HER2-LPETG were characterized by LC-MS. Figure 1 As shown, anti-EGFR-LPETG and anti-HER2-LPETG were successfully prepared.

[0306] (2) T cell CD3 receptor targeting module

[0307] A nanobody against the human T cell CD3 receptor, having the amino acid sequence of SEQ ID NOs: 53-56. This CD3 nanobody was expressed in Escherichia 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, which was linked via a linker (SEQ ID NO: 77) and designated anti-CD3-LPETG.

[0308] 100 μM anti-CD3-LPETG was dissolved in PBS solution, and then 10 μM Sortase A transpeptidase and 1 mM DBCO-PEG3-Gly small molecule were added. After reacting at room temperature for 2 hours, the product anti-CD3-DBCO was purified by size exclusion chromatography for subsequent synthesis of multispecific conjugates.

[0309] 100 μM anti-CD3-LPETG was dissolved in PBS solution, and then 10 μM Sortase A transpeptidase and 1 mM BCN-PEG3-Gly small molecule were added. After reacting at room temperature for 2 hours, the product anti-CD3-BCN was purified by size exclusion chromatography for subsequent synthesis of multispecific conjugates.

[0310] Anti-CD3-DBCO and anti-CD3-BCN were characterized by LC-MS. Figure 2 As shown, anti-CD3-DBCO and anti-CD3-BCN were successfully prepared.

[0311] (3) Natural killer cell CD16 / NKG2D receptor targeting module

[0312] Nanobodies against human natural killer cell CD16 receptor and NKG2D receptor, whose amino acid sequences are SEQ ID NOs: 57-64, respectively. The expression and purification of these nanobodies are similar to those described above, and the resulting products contain the LPETG sequence (SEQ ID NO: 76) at the N-terminus, and are named anti-CD16-LPETG and anti-NKG2D-LPETG.

[0313] 100 μM anti-CD16-LPETG was dissolved in PBS solution, and then 10 μM Sortase A transpeptidase and 1 mM BCN-PEG3-Gly small molecule were added. After reacting at room temperature for 2 hours, the product anti-CD16-BCN was purified by size exclusion chromatography for subsequent synthesis of multispecific conjugates.

[0314] 100 μM anti-NKG2D-LPETG was dissolved in PBS solution, and then 10 μM Sortase A transpeptidase and 1 mM BCN-PEG3-Gly small molecule were added. After reacting at room temperature for 2 hours, the product anti-NKG2D-BCN was purified by size exclusion chromatography for subsequent synthesis of multispecific conjugates.

[0315] Anti-CD16-BCN and anti-NKG2D-BCN were characterized by LC-MS. Figure 3 As shown, anti-CD16-BCN and anti-NKG2D-BCN were successfully prepared.

[0316] (4) Dendritic cell PDL1 / CLEC9A receptor targeting module

[0317] Nanobodies against human dendritic cell PDL1 receptor and CLEC9A receptor, whose amino acid sequences are SEQ ID NOs: 65-72, respectively. The nanobodies were expressed and purified in the same manner as above, and finally products containing the LPETG sequence (SEQ ID NO: 76) at the N-terminus were obtained, named anti-PDL1-LPETG and anti-CLEC9A-LPETG.

[0318] 100 μM anti-PDL1-LPETG was dissolved in PBS solution, and then 10 μM Sortase A transpeptidase and 1 mM BCN-PEG3-Gly small molecule were added. After reacting at room temperature for 2 hours, the product anti-PDL1-BCN was purified by size exclusion chromatography for subsequent synthesis of multispecific conjugates.

[0319] 100 μM anti-CLEC9A-LPETG was dissolved in PBS solution, and then 10 μM Sortase A transpeptidase and 1 mM BCN-PEG3-Gly small molecule were added. After reacting at room temperature for 2 hours, the product anti-CLEC9A-BCN was purified by size exclusion chromatography for subsequent synthesis of multispecific conjugates.

[0320] Anti-PDL1-BCN and anti-CLEC9A-BCN were characterized by LC-MS. Figure 4 As shown, anti-PDL1-BCN and anti-CLEC9A-BCN were successfully prepared.

[0321] (5) IMDQ / diABZI module targeting myeloid immune cells

[0322] IMDQ (CAS: 1258457-59-8), a small molecule agonist for TLR7 / 8 receptors on myeloid immune cells, and diABZI (CAS: 2137975-93-8), a small molecule agonist for the STING receptor, were purchased from MCE. IMDQ or diABZI (70 mM), the small molecule DBCO-SS-NHS (50 mM), and a catalytic amount of triethylamine (50 μM) were reacted at room temperature for 2 hours to produce the intermediate DBCO-SS-IMDQ or DBCO-SS-diABZI. This molecule contains a cleavable disulfide bond (SS), allowing it to break in the tumor microenvironment, releasing the IMDQ or diABZI agonist seamlessly.

[0323] 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 product (IMDQ)6-LPETG or (diABZI)6-LPETG.

[0324] 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 small molecule was added. The reaction system was stirred at room temperature for 2 hours and then purified by HPLC to obtain the final product (IMDQ)6-BCN or (diABZI)6-BCN for subsequent synthesis of multispecific conjugates.

[0325] (IMDQ)6-BCN and (diABZI)6-BCN were characterized by LC-MS. Figure 5As shown, (IMDQ)6-BCN and (diABZI)6-BCN were successfully prepared.

[0326] (6) Myeloid immune cell-targeted CPG / STAT3 module

[0327] The myeloid immune cell TLR9 receptor nucleic acid agonist CPG has an amino modification 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*T*G*T*C*G*T*T*T*T*G*T*C*G*T*T-3', * represents a thio-modified backbone). The myeloid immune cell STAT3 receptor oligonucleotide inhibitor STAT3 ASO also has an amino modification 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', * represents a thio-modified backbone, and Me represents a methylated modification). 100 μM CPG or STAT3 ASO was dissolved in PBS, and then 1 mM DBCO-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 achieving traceless release of CPG and STAT3 ASO in the tumor microenvironment.

[0328] CPG-DBCO and STAT3-DBCO were characterized by LC-MS. Figure 6 As shown, CPG-DBCO and STAT3-DBCO were successfully prepared.

[0329] (7) CMV module targeted by antigen-specific T cells

[0330] A branched peptide containing two CMV antigen peptide sequences (SEQ ID NO:75), two MMP cleavage sequences, and an N-terminal LPETG sequence (SEQ ID NO:76) was synthesized by GenScript and named (CMV)2-LPETG. This branched peptide can respond to MMP enzymes specifically upregulated in the tumor microenvironment, achieving in situ release of CMV antigen peptides. The released CMV peptides are 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 small molecule, and 10 μM Sortase A enzyme were mixed in PBS. The reaction system was allowed to stand at room temperature for 2 hours, and then the product (CMV)2-BCN was purified by HPLC for subsequent construction of multispecific conjugates.

[0331] (CMV)2-BCN was characterized by LC-MS. Figure 7 As shown, (CMV)2-BCN was successfully prepared.

[0332] Example 2 Characterization of the binding force of the coupling module

[0333] (1) Binding ability of Anti-EGFR-LPETG nanoantibody to EGFR receptor of tumor cells

[0334] use Octet RED96 was used to measure the binding of anti-EGFR-LPETG nanoantibodies to EGFR receptors. First, EGFR receptor protein with a Biotin tag (purchased from Sino Biological) was prepared and dissolved in a balancing solution (PBS, 0.05% Tween-20, 0.5% BSA) to a final concentration of 100ug / ml. Nanoantibody solutions of different concentrations were then prepared for use. Binding was measured using a streptavidin-coupled sensor. OctetRED96 universal binding capacity determination method. The determination mainly includes three main processes: solidification, binding, and dissociation. After the determination, the binding constant K of the nanobody binding to the EGFR receptor is obtained by fitting the multi-concentration kinetic analysis software provided by the instrument. on , dissociation constant K dis The final binding affinity KD value is as follows. The results show that the nanobody of the present application can bind to the EGFR receptor with a high binding affinity:

[0335]

[0336] (2) Binding ability of Anti-HER2-LPETG affinity to HER2 receptor of tumor cells

[0337] Similarly, using Octet RED96 was used to measure the binding affinity of anti-HER2-LPETG antibody to HER2 receptor. The results are as follows, showing that anti-HER2-LPETG can bind to HER2 receptor with high affinity:

[0338]

[0339] (3) Binding ability of Anti-CD3-LPETG nanobody to T cell CD3 receptor

[0340] Similarly, using Octet RED96 was used to determine the binding affinity of anti-CD3-LPETG nanobody to the CD3 receptor. The results are as follows, showing that anti-CD3-LPETG can bind to the CD3 receptor with a high affinity:

[0341]

[0342] (4) Binding ability of Anti-CD16-LPETG nanoantibody to natural killer cell CD16 receptor

[0343] Similarly, using Octet RED96 was used to determine the binding affinity of anti-CD16-LPETG nanobody to the CD16 receptor. The results are as follows, showing that anti-CD16-LPETG can bind to the CD16 receptor with a high affinity:

[0344]

[0345] (5) Binding ability of Anti-NKG2D-LPETG nanoantibody to natural killer cell NKG2D receptor

[0346] Similarly, using Octet RED96 was used to determine the binding affinity of the anti-NKG2D-LPETG nanobody to the NKG2D receptor. The results are as follows, showing that anti-NKG2D-LPETG can bind to the NKG2D receptor with a high affinity:

[0347]

[0348] (6) Binding ability of Anti-PDL1-LPETG nanobody to PDL1 receptor on dendritic cells

[0349] Similarly, using Octet RED96 was used to determine the binding affinity of the anti-PDL1-LPETG nanobody to the PDL1 receptor. The results are as follows, showing that anti-PDL1-LPETG can bind to the PDL1 receptor with high affinity:

[0350]

[0351]

[0352] (7) Binding ability of Anti-CLEC9A-LPETG nanobody to dendritic cell CLEC9A receptor

[0353] Similarly, using Octet RED96 was used to determine the binding affinity of the anti-CLEC9A-LPETG nanobody to the CLEC9A receptor. The results are as follows, showing that anti-CLEC9A-LPETG can bind to the CLEC9A receptor with high affinity:

[0354]

[0355] Example 3 Preparation, LC-MS / SDS-PAGE Characterization and Mechanism of Action of Multispecific Conjugates

[0356] (1) Preparation, characterization and mechanism of action of EGFR-CD3-PDL1

[0357] The EGFR-CD3-PDL1 multispecific conjugate was synthesized by the multispecific conjugate coupling arm Ia-1 (from the Chinese patent application with application number 202011270000.5). First, the multispecific conjugate coupling arm Ia-1 was reacted with the anti-EGFR-LPETG module prepared above. Specifically, 1mM multispecific conjugate coupling arm Ia-1 and 100μM Sortase A were mixed and reacted at room temperature for 2 hours, and then purified by size exclusion chromatography to obtain the intermediate product EGFR-N3-TZ. Then, EGFR-N3-TZ and the anti-CD3-DBCO module prepared above were reacted in a 1:1 ratio, the reaction concentration was 100μM, the reaction conditions were room temperature for 2 hours, and after the reaction was completed, the EGFR-CD3-TZ intermediate was purified by size exclusion chromatography. Finally, EGFR-CD3-TZ was reacted with the anti-PDL1-BCN module prepared above in equal amounts, with the same reaction concentration of 100 μM, at room temperature for 2 hours, and then purified by size exclusion chromatography to obtain the multispecific conjugate EGFR-CD3-PDL1.

[0358] EGFR-CD3-PDL1 was characterized by LC-MS and SDS-PAGE. Figure 8 shown.

[0359] 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 tumor cell EGFR, T cell CD3 and dendritic cell PDL1 receptors.

[0360] (2) Preparation, characterization and mechanism of action of EGFR-CD3-CLEC9A

[0361] Using a similar method as above, we finally synthesized the multispecific conjugate EGFR-CD3-CLEC9A by reacting the multispecific conjugate 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. Figure 10 shown.

[0362] like Figure 11 As shown, the multispecific conjugate EGFR-CD3-CLEC9A simultaneously binds to the tumor cell EGFR, T cell CD3 and dendritic cell CLEC9A receptors, thereby achieving co-activation of tumor-targeted T cells and dendritic cells and improving the effect of immunotherapy.

[0363] (3) Preparation, characterization and mechanism of action of EGFR-CD3-CD16

[0364] Using a similar method as above, we finally synthesized the multispecific conjugate EGFR-CD3-CD16 by reacting the multispecific conjugate 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. Figure 12 shown.

[0365] 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 experimentally co-recruiting tumor-targeted T cells and natural killer cells.

[0366] (4) Preparation, characterization and mechanism of action of EGFR-CD3-NKG2D

[0367] Using a similar method as above, we finally synthesized the multispecific conjugate EGFR-CD3-NKG2D by reacting the multispecific conjugate 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. Figure 14 shown.

[0368] like Figure 15As 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 T cell and natural killer cell co-recruitment.

[0369] (5) Preparation, characterization and mechanism of action of HER2-CD3-(IMDQ)6

[0370] Using a similar method as above, we finally synthesized the multispecific conjugate HER2-CD3-(IMDQ)6 by reacting the multispecific conjugate arm Ia-1, anti-HER2-LPETG, anti-CD3-DBCO and (IMDQ)6-BCN modules. HER2-CD3-(IMDQ)6 was characterized by LC-MS and SDS-PAGE. Figure 16 shown.

[0371] like Figure 17 As shown, the multispecific conjugate HER2-CD3-(IMDQ)6 achieves tumor-targeted T cell recruitment by binding to tumor HER2 and T cell CD3 receptors. At the same time, under the reducing conditions of the tumor microenvironment, the IMDQ agonist in HER2-CD3-(IMDQ)6 will be released and then enter myeloid immune cells to activate TLR7 / 8 receptors, thereby activating immunity.

[0372] (6) Preparation, characterization and mechanism of action of HER2-CD3-(diABZI)6

[0373] Using a similar method as described above, we synthesized the multispecific conjugate HER2-CD3-(diABZI)6 by reacting the multispecific conjugate arm Ia-1, anti-HER2-LPETG, anti-CD3-DBCO and (diABZI)6-BCN modules. HER2-CD3-(diABZI)6 was characterized by LC-MS and SDS-PAGE. Figure 18 shown.

[0374] like Figure 19 As shown, the multispecific conjugate HER2-CD3-(diABZI)6 will achieve tumor-targeted T cell recruitment by binding to tumor HER2 and T cell CD3 receptors. At the same time, under the reducing conditions of the tumor microenvironment, the diABZI agonist in HER2-CD3-(diABZI)6 will be released and then enter myeloid immune cells to activate the STING receptor, thereby activating immunity.

[0375] (7) Preparation, characterization and mechanism of action of EGFR-CD3-CPG

[0376] Using a similar method as above, we finally synthesized the multispecific conjugate EGFR-CD3-CPG by reacting the multispecific conjugate arm Ia-1, anti-EGFR-LPETG, anti-CD3-BCN and CPG-DBCO modules. EGFR-CD3-CPG was characterized by LC-MS and SDS-PAGE. Figure 20 shown.

[0377] 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 receptor. At the same time, under the reducing conditions of the tumor microenvironment, the CPG molecules in EGFR-CD3-CPG will be released and then enter myeloid immune cells to activate TLR9 receptors, thereby activating immunity.

[0378] (8) Preparation, characterization and mechanism of action of EGFR-CD3-STAT3

[0379] Using a similar method as above, we finally synthesized the multispecific conjugate EGFR-CD3-STAT3 by reacting the multispecific conjugate arm Ia-1, anti-EGFR-LPETG, anti-CD3-BCN and STAT3-DBCO modules. EGFR-CD3-STAT3 was characterized by LC-MS and SDS-PAGE. Figure 22 shown.

[0380] 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 receptor. At the same time, under the reducing conditions of the tumor microenvironment, the STAT3 molecule in EGFR-CD3-STAT3 will be released and then enter myeloid immune cells to act on the STAT3 receptor, thereby inhibiting the proliferation of immunosuppressive cells such as MDSCs and TAMs.

[0381] (9) Preparation, characterization and mechanism of action of EGFR-CD3-(CMV)2

[0382] Using a similar method as above, we finally synthesized the multispecific conjugate EGFR-CD3-(CMV)2 by reacting the multispecific conjugate arm Ia-1, anti-EGFR-LPETG, anti-CD3-DBCO and (CMV)2-BCN modules. EGFR-CD3-(CMV)2 was characterized by LC-MS and SDS-PAGE. The results are shown in Figure 2. Figure 24 shown.

[0383] like Figure 25 As shown, the multispecific conjugate EGFR-CD3-(CMV)2 will achieve tumor-targeted T cell recruitment by binding to tumor EGFR and T cell CD3 receptor. At the same time, under the action of MMP enzymes in the tumor microenvironment, CMV antigen peptides in EGFR-CD3-(CMV)2 will be released and then recognized and presented by surrounding antigen-presenting cells (APCs), further activating CMV-specific T cells.

[0384] Example 4 EGFR-CD3-PDL1 binds to tumor cell EGFR, T cell CD3, and dendritic cell PDL1 receptors

[0385] use Octet RED96 was used to measure the binding of EGFR-CD3-PDL1 to EGFR, CD3, and PDL1 receptors. EGFR protein, CD3 protein, and PDL1 protein with Biotin tags were dissolved in equilibrium solution (PBS, 0.05% Tween-20, 0.5% BSA) and used at a final concentration of 100 μg / ml. EGFR-CD3-PDL1 protein at different concentrations was also dissolved in equilibrium solution and used. Binding was determined using a streptavidin-coupled sensor. Octet RED96 universal binding capacity determination method. The determination mainly includes three main processes: solidification, binding, and dissociation. After the determination, the binding capacity K of EGFR-CD3-PDL1 with different receptors is obtained by fitting the multi-concentration kinetic analysis software provided by the instrument. D The values ​​are as follows:

[0386]

[0387] Example 5 EGFR-CD3-PDL1-mediated tumor-targeted T cell-dendritic cell (DC) co-recruitment

[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 ) were co-cultured in 96-well plates at 37°C, 5% CO2, and RPMI-1640 medium. Subsequently, different concentrations of the multispecific conjugate EGFR-CD3-PDL1 or bispecific conjugate controls (EGFR-CD3, PDL1-CD3, and EGFR-PDL1) were added and cultured for 24 hours. EGFR-CD3-PDL1-mediated tumor cell killing, T cell activation, and dendritic cell activation were measured.

[0389] EGFR-CD3-PDL1-mediated tumor cell killing was measured using a firefly luciferase assay kit (Promega, Cat#E1500). Specifically, after the above co-culture, the cell supernatant was removed from the 96-well plate, washed once with PBS, and then the substrate of the kit was added and incubated for 5-30 minutes. Cell killing was then detected according to the instructions. The results of EGFR-CD3-PDL1-mediated tumor cell killing are shown in Figure 2. Figure 26B shown.

[0390] EGFR-CD3-PDL1 mediated T cell activation was detected by flow cytometry. Specifically, after the above 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 stained at 4°C for 30 minutes, and then flow cytometric analysis was performed. The analysis results are shown in Figure 2. Figure 26C shown.

[0391] EGFR-CD3-PDL1-mediated T cell activation was detected using an ELISA kit (Thermo Fisher Scientific). Specifically, after the above co-culture was completed, all supernatants were collected for IFNγ cytokine and Granzyme B detection. The detection process was referred to the standard instructions of the kit, and the detection results were as follows: Figure 26D shown.

[0392] EGFR-CD3-PDL1-mediated dendritic cell activation was detected by flow cytometry. Specifically, after the above 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 stained at 4°C for 1 hour, and then flow cytometry analysis was performed. The analysis results are shown in Figure 2. Figure 26E shown.

[0393] EGFR-CD3-PDL1-mediated dendritic cell activation was detected using an ELISA kit (Thermo Fisher Scientific). Specifically, after the above co-culture was completed, all supernatants were collected for IL12 and IL1β cytokine detection. The detection process was referred to the standard instructions of the kit, and the detection results were as follows: Figure 26F shown.

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

[0395] The therapeutic effect of EGFR-CD3-PDL1 was evaluated in a PBMC humanized mouse model. Figure 27AImmunodeficient M-NSG mice were purchased from Shanghai Model Organisms Technology Co., Ltd. and then subcutaneously inoculated with A549 tumor cells (10 6 Each mouse). Wait until the tumor grows to a volume >100mm 3 After 1 day (set as day 1), human PBMC (10 7 Humanized immune reconstitution was performed with 100 mice per day (100 mg / kg). Subsequently, mice were injected with EGFR-CD3-PDL1, bispecific conjugate controls (EGFR-CD3, PDL1-CD3, and EGFR-PDL1), and an equal volume of PBS via the tail vein. Drug injections were administered on days 2, 5, 8, 11, 14, and 17 at a dose of 2 mg / kg EGFR-CD3-PDL1. Other groups were injected with equimolar doses.

[0396] After the start of drug administration, the changes in the tumor volume of mice were continuously recorded and the tumor growth curve was drawn. Figure 27B shown.

[0397] After the end of treatment (day 30), all mice were killed. The mouse tumor tissue was prepared into a single cell suspension for later use. The cell suspension was stained with anti-CD45, anti-CD3, anti-CD4, anti-CD8, anti-Perforin, anti-PD1 and anti-Ki67 flow cytometry antibodies, and the staining process was carried out according to the antibody requirements. Then, the EGFR-CD3-PDL1-mediated T cell activation in this animal model was analyzed by flow cytometry. The main analysis markers were T cell Perforin, PD1 and Ki67. The analysis results are shown in Figure 2. Figure 27C shown.

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

[0399] The therapeutic effect of EGFR-CD3-PDL1 was evaluated in a humanized HSC mouse model. Figure 28A As shown, HSC humanized mice huHSC-NCG-hIL15 were purchased from Jicui Yaokang. First, A549 tumor cells (10 6 Each mouse), when the tumor grew to a volume >100mm 3 Dosing began after 48 hours (designated as day 1). Mice were injected via the tail vein with EGFR-CD3-PDL1, bispecific conjugate controls (EGFR-CD3, PDL1-CD3, and EGFR-PDL1), and an equal volume of PBS. Drug injections were administered on days 2, 5, 8, 11, 14, 17, 20, and 23 at a dose of 2 mg / kg EGFR-CD3-PDL1. Other groups were injected with equimolar doses.

[0400] After the start of drug administration, the changes in the tumor volume of mice were continuously recorded and the tumor growth curve was drawn. Figure 28B shown.

[0401] After the end of treatment (day 25), all mice were killed. The mouse tumor tissue was prepared into a single cell suspension for later use. The cell suspension was stained with anti-CD45, anti-CD3, anti-CD4, anti-CD8, anti-Perforin, anti-Granzyme B and anti-PD1 flow cytometry antibodies. The staining process was carried out according to the antibody requirements. Then, EGFR-CD3-PDL1-mediated T cell activation was analyzed by flow cytometry. The analysis markers were Perforin, Granzyme B and PD1 on T cells. The results are as follows: Figure 28C 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 analysis markers were CD86 and HLA-DR on dendritic cells. The results are shown in Figure 28D 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] The therapeutic efficacy of EGFR-CD3-PDL1 was evaluated 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 Model Organisms Technology Co., Ltd. MC38 tumor cells overexpressing human EGFR receptor and OVA antigen protein (MC38hEGFR + OVA + ) were inoculated into hCD3EDG / hPDL1 C57 mice at a volume of 5 × 10 5 cells per mouse. When the tumor grows to 100mm 3 Mice were treated with 2 mg / kg EGFR-CD3-PDL1 or an equal volume of PBS on days 2, 5, 8, and 11.

[0404] After the start of drug administration, the changes in the tumor volume of mice were continuously recorded and the tumor growth curve was drawn. Figure 29B shown.

[0405] After the end of treatment (day 15), all mice were killed. The mouse tumor tissue was prepared into a single cell suspension for later use. The cell suspension was stained with anti-CD45, anti-CD3, anti-CD4, anti-CD8, anti-Perforin, anti-Granzyme B and anti-Ki67 flow cytometry antibodies. The staining process was carried out according to the antibody requirements. Then, EGFR-CD3-PDL1-mediated T cell activation was analyzed by flow cytometry. The analysis markers were Perforin, Granzyme B and Ki67 on T cells. The results are as follows Figure 29C The cells were stained with anti-CD45, anti-Lin, anti-CD11c, anti-MHC-II, anti-XCR1, anti-CD11b, anti-CD80, and anti-CD86, and then flow cytometry was performed to analyze EGFR-CD3-PDL1-mediated dendritic cell activation. The analysis markers were CD80, CD86, and MHC-II on dendritic cells. The results are shown in Figure 2. Figure 29D shown.

[0406] After the end of treatment (day 15), all mice were killed. The spleen and tumor tissues of the mice were prepared into single cell suspensions for later use. Similarly, the cells were stained with anti-CD45, anti-Lin, anti-CD11c, anti-MHC-II, anti-XCR1, anti-CD11b and OVA-tetramer, and then flow cytometry was performed to analyze the changes in OVA antigen-specific T cells after EGFR-CD3-PDL1 treatment. The analysis results are shown in Figure 2. Figure 29E shown.

[0407] After the end of treatment (day 15), all mice were killed. The spleen of the mice was prepared into a single cell suspension, and then the mouse IFNγ ELISPOT kit (Dakoway) was used to detect whether the mice produced immune memory after EGFR-CD3-PDL1 treatment. The standardized process of the kit was used for detection, and qualitative and quantitative analysis was performed based on the number of spots produced. The results are as follows Figure 29F shown.

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

[0409] Tumor tissues from patients with non-small cell lung cancer were obtained from Peking University Cancer Hospital (Ethics No.: 2019KT41). Figure 30AAs shown, the obtained tumor tissue was digested and cultured to form tumor microspheres (PTCs). The culture method was similar to that reported in the literature (Sci. Transl. Med. 12, eaaz1723). When the cultured PTCs were >40 μm in size, EGFR-CD3-PDL1 multispecific conjugates, bispecific conjugate controls (EGFR-CD3, PDL1-CD3, and EGFR-PDL1), or an equal volume of PBS were added and culture continued. EGFR-CD3-PDL1-mediated T cell and dendritic cell activation, as well as PTC growth inhibition, were then measured.

[0410] After adding the multispecific conjugate and culturing for 24 hours, the culture supernatant was collected. EGFR-CD3-PDL1-mediated T cell and dendritic cell activation was detected using an ELISA kit (ThermoFisher Scientific). The test was performed using the standardized procedure of the kit. For T cell activation, we detected IFNγ and TNFα cytokines. For dendritic cell activation, we detected IL12 and IL1β cytokines. The test results are shown in Figure 2. Figure 30B shown.

[0411] After 7 days of treatment with the multispecific conjugate, we detected the growth of PTC in the system. The detection method was based on the literature (Sci. Transl. Med. 12, eaaz1723.). The results are as follows: Figure 30C shown.

[0412] Example 10 EGFR-CD3-CD16 binds to tumor cell EGFR, T cell CD3, and natural killer cell CD16 receptors

[0413] use Octet RED96 was used to measure the binding of EGFR-CD3-CD16 to EGFR, CD3, and CD16 receptors. EGFR protein, CD3 protein, and CD16 protein with Biotin tags were dissolved in equilibrium solution (PBS, 0.05% Tween-20, 0.5% BSA) and used at a final concentration of 100 μg / ml. EGFR-CD3-CD16 protein at different concentrations was also dissolved in equilibrium solution and used. Binding was determined using a streptavidin-coupled sensor. Octet RED96 universal binding capacity determination method. The determination mainly includes three main processes: solidification, binding, and dissociation. After the determination, the binding capacity K of EGFR-CD3-CD16 with different receptors is obtained by fitting the multi-concentration kinetic analysis software provided by the instrument. D The values ​​are as follows:

[0414]

[0415] Example 11EGFR-CD3-CD16-mediated tumor-targeted T cell-natural killer cell co-recruitment

[0416] (1) EGFR-CD3-CD16-mediated tumor-targeted T cell recruitment

[0417] like Figure 31A , EGFR-positive A549 tumor cells (10 4 ) and CD3-positive human T cells (2×10 5 ) were co-cultured in 96-well plates at 37°C, 5% CO2, and RPMI-1640 medium. Subsequently, different concentrations of the multispecific conjugate EGFR-CD3-CD16 or a bispecific control conjugate (EGFR-CD3 and EGFR-CD16) were added and cultured for 24 hours. EGFR-CD3-CD16-mediated tumor cell killing and T cell activation were measured, respectively.

[0418] EGFR-CD3-CD16-mediated tumor cell killing was measured using a firefly luciferase assay kit (Promega, Cat#E1500). Specifically, after the above co-culture, the cell supernatant was removed from the 96-well plate, washed once with PBS, and then the substrate of the kit was added and incubated for 5-30 minutes. Cell killing was then detected according to the instructions. The results of EGFR-CD3-CD16-mediated tumor cell killing are shown in Figure 2. Figure 31B shown.

[0419] EGFR-CD3-CD16 mediated T cell activation was detected by flow cytometry. Specifically, after the above 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 stained at 4°C for 30 minutes, and then flow cytometric analysis was performed. The analysis results are shown in Figure 2. Figure 31C shown.

[0420] EGFR-CD3-CD16 mediated T cell activation was detected using an ELISA kit (Thermo Fisher Scientific). Specifically, after the above co-culture was completed, all supernatants were collected for IFNγ cytokine detection. The detection process was referred to the standard instructions of the kit, and the detection results were as follows: Figure 31D shown.

[0421] (2) EGFR-CD3-CD16-mediated tumor-targeted natural killer cell recruitment

[0422] like Figure 32A , EGFR-positive A549 tumor cells (10 4) and CD16-positive human natural killer cells (10 5 ) were co-cultured in 96-well plates at 37°C, 5% CO2, and RPMI-1640 medium. Subsequently, different concentrations of the multispecific conjugate EGFR-CD3-CD16 or a bispecific control conjugate (EGFR-CD3 and EGFR-CD16) were added and cultured for 24 hours. EGFR-CD3-CD16-mediated tumor cell killing and natural killer cell activation were measured, respectively.

[0423] EGFR-CD3-CD16-mediated tumor cell killing was measured using a firefly luciferase assay kit (Promega, Cat#E1500). Specifically, after the above co-culture, the cell supernatant was removed from the 96-well plate, washed once with PBS, and then the substrate of the kit was added and incubated for 5-30 minutes. Cell killing was then detected according to the instructions. The results of EGFR-CD3-CD16-mediated tumor cell killing are shown in Figure 2. Figure 32B shown.

[0424] EGFR-CD3-CD16 mediated natural killer cell activation was detected by flow cytometry. Specifically, after the above 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 stained at 4°C for 30 minutes, and then flow cytometric analysis was performed. The analysis results are shown in Figure 2. Figure 32C shown.

[0425] EGFR-CD3-CD16-mediated natural killer cell activation was detected using an ELISA kit (Thermo Fisher Scientific). Specifically, after the above co-culture was completed, all supernatants were collected for IFNγ and MIP-1β cytokine detection. The detection process was referred to the standard instructions of the kit, and the detection results were as follows: Figure 32D shown.

[0426] Example 12 HER2-CD3-(IMDQ)6 binds to tumor cell HER2 and T cell CD3 receptors

[0427] use Octet RED96 was used to measure the binding of HER2-CD3-(IMDQ)6 to HER2 and CD3 receptors. HER2 and CD3 proteins with Biotin tags were dissolved in equilibrium solution (PBS, 0.05% Tween-20, 0.5% BSA) at a final concentration of 100 μg / ml. HER2-CD3-(IMDQ)6 proteins at different concentrations were also dissolved in equilibrium solution. Binding was determined using a streptavidin-coupled sensor. Octet RED96 universal binding capacity determination method. The determination mainly includes three main processes: solidification, binding, and dissociation. After the determination, the binding capacity K of HER2-CD3-(IMDQ)6 with different receptors is obtained by fitting the multi-concentration kinetic analysis software provided by the instrument. D The values ​​are as follows:

[0428]

[0429]

[0430] Example 13 HER2-CD3-(IMDQ)6 releases IMDQ agonist in the tumor reducing microenvironment

[0431] like Figure 33A As shown, the multispecific conjugate HER2-CD3-(IMDQ)6 can respond to reducing substances (such as GSH, Cys, etc.) in the tumor microenvironment due to the presence of disulfide bonds (SS) in the molecule, and can release IMDQ agonists in situ after targeting the tumor, thereby further activating immunity.

[0432] HER2-positive SKBR3 tumor cells (10 4 ) and CD3-positive human T cells (2×10 5 ) were co-cultured in 96-well plates at 37°C, 5% carbon dioxide, and RPMI-1640 medium. To simulate the tumor's reducing microenvironment, 150 μM GSH and 20 μM Cys were added to the system for co-culture. Subsequently, 20 μM of the multispecific conjugate HER2-CD3-(IMDQ)6 was added and cultured for 48 hours. The supernatant was collected and concentrated, and the release of the IMDQ agonist was detected by LC-MS. The test results are as follows: Figure 33B shown.

[0433] Example 14 HER2-CD3-(IMDQ)6-mediated tumor-targeted T cell-myeloid immune cell co-recruitment

[0434] (1) HER2-CD3-(IMDQ)6-mediated tumor-targeted T cell recruitment

[0435] like Figure 34A , HER2-positive SK-BR3 tumor cells (10 4 ) and CD3-positive human T cells (2×10 5) were co-cultured in 96-well plates at 37°C, 5% CO2, in RPMI-1640 medium. Subsequently, different concentrations of the multispecific conjugate HER2-CD3-(IMDQ)6 or a bispecific control conjugate (HER2-CD3 and HER2-(IMDQ)6) were added and cultured for 24 hours. HER2-CD3-(IMDQ)6-mediated tumor cell killing and T cell activation were measured, respectively.

[0436] HER2-CD3-(IMDQ)6-mediated tumor cell killing was measured using a firefly luciferase assay kit (Promega, Cat#E1500). Specifically, after the above co-culture, the cell supernatant was removed from the 96-well plate, washed once with PBS, and then the substrate of the kit was added and incubated for 5-30 minutes. Cell killing was then detected according to the instructions. The results of HER2-CD3-(IMDQ)6-mediated tumor cell killing are shown in Figure 2. Figure 34B shown.

[0437] 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 stained at 4°C for 30 minutes, and then flow cytometric analysis was performed. The analysis results are shown in Figure 2. Figure 34C shown.

[0438] HER2-CD3-(IMDQ)6-mediated T cell activation was detected using an ELISA kit (Thermo Fisher Scientific). After the above co-culture was completed, all supernatants were collected for IFNγ cytokine detection. The detection process was based on the kit standard instructions. The test results were as follows: Figure 34D shown.

[0439] (2) HER2-CD3-(IMDQ)6-mediated tumor-targeted myeloid immune cell recruitment

[0440] like Figure 35A , HER2-positive SK-BR3 tumor cells (10 4 ) and CD3-positive human T cells (2×10 5) were co-cultured in 96-well plates at 37°C, 5% carbon dioxide, and the culture medium was RPMI-1640. 150 μM GSH and 20 μM Cys were added to the system to simulate the tumor's reductive microenvironment. Subsequently, 100, 10, or 1 nM HER2-CD3-(IMDQ)6 multispecific conjugate was added and incubated for a further 48 hours. The supernatant after incubation was collected and co-cultured with isolated human myeloid immune cells for 24 hours, and the activation of myeloid immune cells mediated by the HER2-CD3-(IMDQ)6 multispecific conjugate was detected by flow cytometry.

[0441] The cultured myeloid immune cells were collected and resuspended for later use. First, HER2-CD3-(IMDQ)6-mediated myeloid dendritic cell activation was measured by staining the cells with anti-CD11c, anti-HLA-DR, anti-CD83, and anti-CD40, and then performing flow cytometric analysis. Then, HER2-CD3-(IMDQ)6-mediated myeloid macrophage activation was measured by staining the cells with anti-CD11c, anti-HLA-DR, anti-CD68, anti-CD206, and anti-CD86 flow cytometric antibodies, and then performing flow cytometric analysis. Finally, HER2-CD3-(IMDQ)6-mediated myeloid monocyte activation was measured by staining the cells with anti-HLA-DR, anti-CD206, anti-CD14, anti-CD40, and anti-CD80 flow cytometric antibodies, and then performing flow cytometric analysis. Among them, antibody staining and flow cytometric analysis were performed according to conventional procedures. For the activation of myeloid dendritic cells, the main markers were evaluated, CD83 and CD40, and the test results were as follows: Figure 35B As shown in Figure 2. For the activation of myeloid macrophages, the proportion of M1 and M2 macrophages was mainly evaluated. The test results are shown in Figure 2. Figure 35C As shown in Figure 2. For the activation of myeloid monocytes, the main markers evaluated were CD14, CD40, and CD80. The test results are shown in Figure 2. Figure 35D shown.

Claims

1. A multispecific conjugate having the structure shown in Formula IIa-1: (Formula IIa-1), wherein P 1 、P 2 and P 3 A tumor cell binding portion, a T cell regulating portion, and a portion selected from the group consisting of a dendritic cell regulating portion, a natural killer cell regulating portion, and a myeloid immune cell regulating portion, wherein The tumor cell binding portion comprises an EGFR nanobody, which comprises HCDR1, HCDR2 and HCDR3, and the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 2, the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 3, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 15; The T cell regulatory portion includes a CD3 nanobody, which includes HCDR1, HCDR2 and HCDR3, and the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 54, the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 55, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO:

56. The dendritic cell regulating portion includes a PD-L1 nanobody or a CLEC9A nanobody, wherein the PD-L1 nanobody includes HCDR1, HCDR2 and HCDR3, and the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 66, the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 67, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 68; the CLEC9A nanobody includes HCDR1, HCDR2 and HCDR3, and the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 70, the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 71, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO:

72. The natural killer cell regulating portion comprises a CD16 nanobody or a NKG2D nanobody, wherein the CD16 nanobody comprises HCDR1, HCDR2 and HCDR3, and the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 58, the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 59, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 60; the NKG2D nanobody comprises HCDR1, HCDR2 and HCDR3, and the amino acid sequence of the HCDR1 is shown in SEQ ID NO: 62, the amino acid sequence of the HCDR2 is shown in SEQ ID NO: 63, and the amino acid sequence of the HCDR3 is shown in SEQ ID NO: 64, and The myeloid immune cell regulating portion includes CPG or STAT3 ASO; Or, P 1 、P 2 and P 3 They are respectively a tumor cell binding portion, a T cell regulatory portion, and a myeloid immune cell regulatory portion, wherein the tumor cell binding portion includes a HER2 affinity body, the amino acid sequence of the HER2 affinity body is as shown in SEQ ID NO: 52, the T cell regulatory portion includes a CD3 nanobody, which includes HCDR1, HCDR2 and HCDR3, and the amino acid sequence of the HCDR1 is as shown in SEQ ID NO: 54, the amino acid sequence of the HCDR2 is as shown in SEQ ID NO: 55, the amino acid sequence of the HCDR3 is as shown in SEQ ID NO: 56, and the myeloid immune cell regulatory portion includes IMDQ or diABZI.

2. The multispecific conjugate according to claim 1, wherein the amino acid sequence of the EGFR nanobody is as shown in SEQ ID NO:

14.

3. The multispecific conjugate according to claim 1, wherein the amino acid sequence of the CD3 Nanobody is as shown in SEQ ID NO:

53.

4. The multispecific conjugate according to claim 1, wherein the amino acid sequence of the PD-L1 nanobody is as shown in SEQ ID NO:

65.

5. The multispecific conjugate according to claim 1, wherein the amino acid sequence of the CLEC9A Nanobody is as shown in SEQ ID NO:

69.

6. The multispecific conjugate according to claim 1, wherein the amino acid sequence of the CD16 nanobody is as shown in SEQ ID NO:

57.

7. The multispecific conjugate according to claim 1, wherein the amino acid sequence of the NKG2D nanobody is as shown in SEQ ID NO:

61.

8. The multispecific conjugate of claim 1, wherein P 1 、P 2 and P 3 Further comprising a cleavable linker.

9. A pharmaceutical composition comprising the multispecific conjugate according to any one of claims 1 to 8, and optionally a pharmaceutically acceptable excipient or carrier.

10. Use of the multispecific conjugate according to any one of claims 1 to 8 and / or the pharmaceutical composition according to claim 9 in the preparation of a medicament for treating and / or preventing tumors. The use according to claim 10 , wherein the tumor is a tumor with high expression of EGFR.

12. The use according to claim 10, wherein 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.

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