Multi-specific conjugate and application thereof

Abstract

The invention relates to a multispecific conjugate which has a structure as shown in a formula I: # imgabs0 #. The multispecific conjugate disclosed by the invention can be accurately connected with three different payloads through a connecting arm with three orthogonal groups, so that recruitment of various cells is realized. The present application also provides the use of multispecific conjugates in the treatment of disease, particularly immunotherapy.

Description

Technical Field

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

[0002] Immunotherapy has revolutionized cancer treatment by mobilizing the body's immune system to eradicate tumors, and has achieved remarkable results in clinical trials. However, these successes are mainly limited to certain types of blood tumors, while the treatment effects on most solid tumors are not ideal. The reasons for this are varied, but mainly related to the tumor-immune microenvironment (TIME). In the tumor immune microenvironment, multiple immune components interact with each other and jointly affect the efficacy of immunotherapy. Increasing research evidence shows that intervention targeting specific immune cell types in TIME can reactivate suppressed anti-tumor immune responses, thereby improving the effectiveness of immunotherapy, but this activation effect has not yet reached its optimal state.

[0003] To further improve the efficacy, researchers have tried combination therapies of multiple drugs, including the simultaneous or sequential use of different drugs, or the use of biomaterials to deliver multiple therapies simultaneously. These methods provide the possibility of simultaneously targeting multiple immune cells in TIME. However, these strategies are often accompanied by increased toxicity risks, and most combination therapies do not 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 are designed, which contain two or more modules, such as tumor targeting parts, 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 cells (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 integration of a variety of therapeutic modules to target different immune subsets within TIME.

[0005] Although some reports have demonstrated examples of multispecificity or modularity, there is currently no system that can 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 multi-specific conjugate, which is formed by programming multiple therapeutic modules into a single drug through a multi-specific coupling arm through a triple bioorthogonal reaction. The resulting multi-specific 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 multi-specific coupling arm with three orthogonal groups, and almost no toxicity is detected in vivo. The multi-specific conjugate of the present application integrates therapeutic modules including tumor targeting parts, immune activators and stimulators to obtain various multi-modal targeting chimeras.

[0007] As an example, when nano antibody modules are connected, these modules are programmable and, due to their small size, are conducive to 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, changing the tumor microenvironment, and thus showing 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 following group: -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 is a 6- 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, hydroxyl, 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 Each is 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: 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 the following can regulate immune cells (including, activate and / or inhibit immune cells), 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 group:

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

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

[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] Where P 1 , P 2 and P 3 They are respectively defined as 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 may 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, structures of formulas Ia-2 to Ia-10, 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 peptides 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 antigen includes any one or more selected from the following group: 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 (apoptosis inhibitory protein), hTERT (human telomerase reverse transcriptase), Galectin-3 (galactose lectin 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 includes HCDR1, HCDR2 and HCDR3, and 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: 4. For example, the EGFR antibody includes a variable region, and the variable region may include the amino acid sequence shown in SEQ ID NO: 1.

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

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

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

[0046] For example, the EGFR antibody includes HCDR1, HCDR2 and HCDR3, and 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: 14.

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

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

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

[0050] For example, the EGFR antibody includes HCDR1, HCDR2 and HCDR3, and 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 includes HCDR1, HCDR2 and HCDR3, and the HCDR1 includes the amino acid sequence shown in SEQ ID NO: 27, 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: 28. For example, the EGFR antibody includes a variable region, and the variable region may include the amino acid sequence shown in SEQ ID NO: 26.

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

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

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

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

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

[0057] For example, the EGFR antibody includes HCDR1, HCDR2 and HCDR3, and 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 includes HCDR1, HCDR2 and HCDR3, and the HCDR1 includes the amino acid sequence shown in SEQ ID NO: 49, the HCDR2 includes the amino acid sequence shown in SEQ ID NO: 50, and the HCDR3 includes the amino acid sequence shown in SEQ ID NO: 51. For example, the EGFR antibody includes a variable region, and the variable region may include 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 some embodiments, the immune cells include myeloid immune cells and lymphoid immune cells. In some embodiments, the immune cells are myeloid immune cells. In some embodiments, the immune cells are lymphoid immune cells.

[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 regulating portion targets immune cell surface receptors. Examples of immune cell surface receptors that can be used for the purposes of the present application are as follows, including but 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 regulating part targets an immune cell intracellular receptor. Examples of immune cell intracellular receptors that can be used for the purposes of the present application are as follows, including but 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 portion 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 regulating portion comprises a CD3 antibody. The CD3 antibody is preferably a nanobody. For example, the CD3 antibody may comprise HCDR1, HCDR2 and HCDR3, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:54, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:55, and the HCDR3 comprises the amino acid sequence shown 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 shown in SEQ ID NO:53.

[0069] In certain embodiments, the immune cell regulation portion includes a PD-L1 antibody. The PD-L1 antibody is preferably a nanobody. For example, the PD-L1 antibody may include HCDR1, HCDR2 and HCDR3, 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.

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

[0071] In certain embodiments, the immune cell regulation portion includes a CD16 antibody. The CD16 antibody is preferably a nanobody. For example, the CD16 antibody may include HCDR1, HCDR2 and HCDR3, and the HCDR1 includes the amino acid sequence shown in SEQ ID NO:58, the HCDR2 includes the amino acid sequence shown in SEQ ID NO:59, and the HCDR3 includes the amino acid sequence shown 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 shown in SEQ ID NO:57.

[0072] In certain embodiments, the immune cell regulating portion comprises an NKG2D antibody. The NKG2D antibody is preferably the nanobody. For example, the NKG2D antibody comprises HCDR1, HCDR2 and HCDR3, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:62, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:63, and the HCDR3 comprises the amino acid sequence shown 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 shown 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 modulating portion targeting an intracellular receptor of an immune cell may be an immunosuppressant.

[0076] In certain embodiments, the immune agonist or immunosuppressant comprises any one or more selected from the following groups: Toll-like receptor (TLR) agonists (e.g., CpG oligonucleotides, lipopolysaccharide LPS, IMDQ), interferon gene stimulator receptor (STING) agonists (e.g., Cyclic dinucleotides, diABZI), retinoic acid-inducible gene factor I (RIG-I)-like receptor (RLR) agonists, leucine-rich nucleotide binding domain repeats (NLR), C-type lectin receptor (CLR agonists, protein kinase agonists (e.g., JAK kinase agonists), phosphokinase agonists (e.g., SHP-1 / SHP-2 phosphatase inhibitors), transcription factor activators (e.g., NF-κB activators, NFAT activators), intracellular signaling molecules (e.g., interleukins, such as IL-2, IL-12, IL-15), immunomodulatory small molecules (e.g., imidazoline compounds), PKC agonists, apoptosis pathway regulators (e.g., Bcl-2 family protein inhibitors), Interferons (e.g., IFN-α, IFN-β, IFN-γ), co-stimulatory molecule agonists (e.g., anti-CD28 antibodies, anti-OX40 antibodies, anti-4-1BB antibodies), chemoimmunostimulators (e.g., imatinib), cell metabolism regulators (mTOR agonists), signal transduction and transcription activator (STAT) 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, 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 transduction and transcription activator 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 immune suppressor 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 linker that can achieve the purpose of the present invention includes, but is not limited to: a peptide bond linker, an acid-sensitive linker, a reduction-sensitive linker, an enzyme-sensitive linker, a light-sensitive linker, a bioorthogonal reaction linker, a pH-sensitive linker and / or a temperature-sensitive linker. For example, the cleavable linker may include a disulfide bond (SS), which can be broken under reducing conditions to release P 1 , P 2 and / or P 3 .

[0081] In certain embodiments, P 1 , P 2 and P 3 Any one of them is the tumor cell binding portion, any one of them is a T cell regulating portion, and any one of them 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 T cell surface receptor binding protein, and one comprises a dendritic cell surface receptor binding protein. For example, P 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 T cell surface receptor binding protein, and one contains a natural killer cell surface receptor binding protein. 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 modulator. 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 includes an EGFR antibody or an antigen binding fragment thereof. The EGFR antibody is preferably a nanobody. For example, the EGFR antibody may include HCDR1, HCDR2 and HCDR3, and the HCDR1, HCDR2 and HCDR3 include 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, strong proteins that can bind to target proteins or peptides with high affinity, similar in nature to monoclonal antibodies, and are therefore antibody mimics. Affibodies have the characteristics of small relative molecular mass, fast folding rate, stable structure, and tolerance 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] On the other hand, 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 includes a CD3 antibody or an antigen binding fragment thereof. The CD3 antibody is preferably a nanobody. For example, the CD3 antibody may include HCDR1, HCDR2 and HCDR3, and the HCDR1 includes the amino acid sequence shown in SEQ ID NO:54, the HCDR2 includes the amino acid sequence shown in SEQ ID NO:55, and the HCDR3 includes the amino acid sequence shown in SEQ ID NO:56. For example, the CD3 antibody may include a variable region, and the variable region may include the amino acid sequence shown in SEQ ID NO:53.

[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 includes a PD-L1 antibody or an antigen binding fragment thereof. The PD-L1 antibody is preferably a nanobody. For example, the PD-L1 antibody may include HCDR1, HCDR2 and HCDR3, 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] On the other hand, 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 includes a CLEC9A antibody or an antigen binding fragment thereof. The CLEC9A antibody is preferably a nanobody. For example, the CLEC9A antibody may include HCDR1, HCDR2 and HCDR3, and the HCDR1 includes the amino acid sequence shown in SEQ ID NO:70, the HCDR2 includes the amino acid sequence shown in SEQ ID NO:71, and the HCDR3 includes the amino acid sequence shown in SEQ ID NO:72. For example, the CLEC9A antibody may include a variable region, and the variable region may include the amino acid sequence shown 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, and the HCDR1 includes the amino acid sequence shown in SEQ ID NO:58, the HCDR2 includes the amino acid sequence shown in SEQ ID NO:59, and the HCDR3 includes the amino acid sequence shown 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 shown 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 includes an NKG2D antibody or an antigen binding fragment thereof. The NKG2D antibody is preferably the nanobody. For example, the NKG2D antibody includes HCDR1, HCDR2 and HCDR3, and the HCDR1 includes the amino acid sequence shown in SEQ ID NO:62, the HCDR2 includes the amino acid sequence shown in SEQ ID NO:63, and the HCDR3 includes the amino acid sequence shown in SEQID NO:64. For example, the NKG2D antibody may include a variable region, and the variable region may include the amino acid sequence shown in SEQID 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 in the present application. 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 an effective amount of the multi-specific conjugate, the antigen-binding protein, the isolated nucleic acid, the vector, the cell, the immunoconjugate and / or the pharmaceutical composition described in the present application to a subject in need.

[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 in the present application in the preparation of a drug 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 in the present application, 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, the method comprising administering an effective amount of the multi-specific conjugate, the antigen-binding protein, the isolated nucleic acid, the vector, the cell, the immunoconjugate and / or the pharmaceutical composition described in the present application to a subject in need.

[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 in the present application in the preparation of a drug 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 in the present application, 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 multi-specific conjugate, the antigen-binding protein, the isolated nucleic acid, the vector, the cell, the immunoconjugate and / or the pharmaceutical composition described in the present application.

[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, a tissue, an organ, or an animal) an effective amount of the multispecific conjugate described in the present application, 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 a living cell. 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 enhanced 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 myeloid dendritic cells to express CD83 and / or CD40. 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 myeloid monocytes to express CD14, CD40 and / or CD80. 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 multi-specific conjugate described in the present application, 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 may 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 will be appreciated by those skilled in the art, the content of the present application enables those skilled in the art to modify the disclosed specific embodiments without departing from the spirit and scope of the invention to which the present application relates. Accordingly, the descriptions in the drawings and specification of the present application are merely exemplary and not restrictive. BRIEF DESCRIPTION OF THE DRAWINGS

[0136] The specific features of the invention involved in this application are shown in the attached claims. The features and advantages of the invention involved in this application can be better understood by referring to the exemplary embodiments and drawings described in detail below. The drawings are briefly described as follows:

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

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

[0139] Figure 3 Shown is the 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 is the LC-MS characterization of (IMDQ)6-BCN and (diABZI)6-BCN.

[0142] Figure 6 Shown is the 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 is the LC-MS and SDS-PAGE characterization of the multispecific conjugate EGFR-CD3-PDL1. The left is a schematic diagram of EGFR-CD3-PDL1, the middle is the LC-MS characterization diagram, and the right is the SDS-PAGE characterization diagram.

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

[0146] Fig.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] Fig.11 Shown is a schematic diagram of EGFR-CD3-CLEC9A simultaneously recruiting T cells and dendritic cells (DCs) to target tumors.

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

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

[0150] Fig.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] Fig.15 Shown is a schematic diagram of EGFR-CD3-NKG2D simultaneously recruiting T cells and natural killer (NK) cells to target tumors.

[0152] Fig.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] Fig.17 Shown is a schematic diagram of HER2-CD3-(IMDQ)6 simultaneously recruiting T cells and myeloid immune cells to target tumors.

[0154] Fig.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] Fig.19 Shown is a schematic diagram of HER2-CD3-(diABZI)6 simultaneously recruiting T cells and myeloid immune cells to target tumors.

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

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

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

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

[0160] Fig.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] Fig.25 Shown is a schematic diagram of simultaneous recruitment of T cells by EGFR-CD3-(CMV)2 and targeting of CMV antigen-specific T cells to tumors.

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

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

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

[0165] Fig.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] Fig.26E The results show the activation of dendritic cells (DCs) mediated by the multispecific conjugate EGFR-CD3-PDL1 in the tumor cell-T cell-dendritic cell co-culture system (CD83 + and CD86 + Dendritic cells

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

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

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

[0170] Fig.27C Shown is EGFR-CD3-PDL1 activation of intratumoral T cells and increased Perforin in a PBMC humanized mouse model + PD1 + and Ki67 + T cell percentage.

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

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

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

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

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

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

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

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

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

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

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

[0182] Fig. 30B Shown is that EGFR-CD3-PDL1 activates T cells and dendritic cells in the patient tumor tissue model and promotes the secretion of IFNγ, TNFα, IL1β and IL12 cytokines in PTC.

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

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

[0185] Fig.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] Fig.31C The figure shows the activation of T cells mediated by the multispecific conjugate EGFR-CD3-CD16 in the tumor cell-T cell co-culture system (CD69 + T cells).

[0187] Fig.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] Fig.32A Shown is the evaluation of the multispecific conjugate EGFR-CD3-CD16 in a tumor cell-natural killer cell (NK) co-culture system.

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

[0190] Fig.32C Shown is 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] Fig.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] Fig.33A Shown is the release of IMDQ molecules from the multispecific conjugate HER2-CD3-(IMDQ)6 in the reducing microenvironment of a tumor.

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

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

[0195] Fig.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] Fig.34C Shown is the activation of T cells (CD69 + T cells).

[0197] Fig.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] Fig.35A Shown is the assessment of HER2-CD3-(IMDQ)6-mediated tumor-targeted myeloid immune cell activation.

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

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

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

[0202] The following is an explanation of the implementation of the present invention by means of specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0203] Definition of terms

[0204] In the present application, the term "antigen binding protein" generally refers to a protein comprising a portion that binds an antigen, and optionally a scaffold or skeleton portion that allows the portion that binds the antigen to adopt a conformation that promotes the antigen binding protein to bind 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 show the desired antigen binding activity.

[0205] In the present application, the term "antibody" generally refers to an immunoglobulin that can specifically bind to a 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 include a protein of at least two heavy chains (HC) and two light chains (LC) interconnected by disulfide bonds, or an antigen-binding fragment thereof. Each heavy chain includes 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 includes three domains CH1, CH2, and CH3. In some naturally occurring antibodies, each light chain includes a light chain variable region (VL) and a light chain constant region. The light chain constant region includes one domain CL. The VH and VL regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), which alternate with more conservative regions called framework regions (FRs). Each VH and VL comprises three CDRs and four framework regions (FRs), arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3 and FR4. The variable domains of natural 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-folded configuration, connected by three CDRs, forming loops connected, and in some cases forming part of the β-folded structure. The CDRs in each chain are closely together through the FR region, 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 the light chain variable domain region (VL) present in conventional 4-chain antibodies. For further description of VHH or nano antibody, reference may be made to the article (Reviews in Molecular Biotechnology 74:277-302, 2001) of Muyldermans. The feature of VHH or nano antibody is that they may 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 the smallest antigen binding fragment, retaining the binding affinity and specificity of full-length antibodies. VHH has a longer CDR3 loop and a convex paratope, which enables them to enter the internal 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. "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 a synthetic non-naturally occurring amino acid, such as a chemical analog of a corresponding naturally occurring amino acid, and a naturally occurring amino acid polymer. The term also includes post-translational modifications of the polypeptide, such as glycosylation, phosphorylation and acetylation. In the present application, the term "isolated" antigen-binding protein generally refers to an antigen-binding protein that has been identified, separated and / or recovered from a component of its production environment (e.g., natural or recombinant). The contaminating components of its production environment are generally substances that interfere with its research, diagnosis 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 protein and / or amino acid sequence involved in the present application should also be understood to include at least the following scope: variants or homologs with the same or similar functions as the protein. In the present application, the variant may be a protein or polypeptide in which one or more amino acids are substituted, deleted or added in the amino acid sequence of the protein (e.g., the multispecific binding protein or antigen binding protein described in the present application). For example, the functional variant may include a protein or polypeptide that has been substituted, deleted and / or inserted with at least 1, such as 1-30, 1-20 or 1-10, and for example 1, 2, 3, 4 or 5 amino acids and has an amino acid change. The functional variant may substantially maintain the biological properties of the protein or polypeptide before the change (e.g., substitution, deletion or addition). For example, the functional variant may maintain 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 may 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 the corresponding amino acid sequence in an antibody from a specific species, or belong to a specific class. For example, the variable region and constant region of an antibody may both be from the variable region and constant region of an antibody of an animal species (such as human).

[0210] In the present application, the homolog can be a protein or polypeptide having at least about 85% (for example, 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 (for example, the antigen-binding protein described in the present application).

[0211] In the present application, described homology generally refers to the similarity, similarity or association between two or more sequences.Can calculate " sequence homology percentage ratio " in the following manner: two sequences to be compared are compared in a 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 of position to obtain the number of matching positions, the number of matching positions is divided by the total number of positions (that is, window size) in the comparison window, and the result is multiplied by 100, to produce sequence homology percentage ratio.Comparison carried out in order to determine the sequence homology percentage ratio, can realize by multiple 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 the sequence of interest. The homology can also be determined by the following methods: FASTA and BLAST. A description of the FASTA algorithm can be found in W. R. Earson and DJ. Lipman, "Improved tools for biological sequence comparisons," Proc. Natl. Acad. Sci., 85: 2444-2448, 1988; and DJ. Lipman and W. R. Earson, "Rapid and sensitive protein similarity searches," Science, 227: 1435-1441, 1989. A description of the BLAST algorithm can be found in S. Altschul, W. Gish, W. Miller, E. W. Myers and D. Lipman, "A basic local alignment search tool," Journal of Molecular Biology, 215: 403-410, 1990.

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

[0215] In the present 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 the present 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 described in the present application may contain 2-20, 2-12, 2-10, 2-8 or 2-6 carbon atoms.

[0216] In the present application, the term "alkynyl" generally refers to an unsaturated straight or branched chain alkynyl, such as ethynyl, 1-propynyl, propargyl, butynyl, etc. The alkynyl group may be substituted or unsubstituted. The alkynyl group described herein may 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., and 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, which has a residue derived from removing a hydrogen atom from the same carbon atom or two different carbon atoms of a parent alkane, 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. The alkyl radical may be substituted or unsubstituted, substituted or unsubstituted, for example, when substituted, the substituent may be substituted at any available point of attachment, and the substituent may be independently selected from one or more of 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 the present 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, and the carbocyclic ring contains 3 to 20 carbon atoms, can contain 3 to 12 carbon atoms, can contain 3 to 10 carbon atoms, and can contain 3 to 8 carbon atoms. Non-limiting examples of monocyclic carbocyclic rings include cyclopropane, cyclobutane, cyclopentane, cyclopentene, cyclohexane, cyclohexene, cyclohexadiene, cycloheptane, cycloheptatriene, cyclooctane, etc.; polycyclic carbocyclic rings can include spirocyclic, condensed ring and bridged ring carbocyclic rings. Cycloalkyl can be substituted or unsubstituted.

[0220] In the present application, the term "heterocycloalkyl" generally refers to a stable 3-7-membered monocyclic structure without aromaticity, a fused 7-10-membered bicyclic heterocyclic structure or a bridged 6-10-membered bicyclic heterocyclic structure, which can be saturated or partially saturated. In addition to carbon atoms, these cyclic structures also contain one or more heteroatoms, wherein the heteroatoms can be selected from the following groups: oxygen, sulfur and nitrogen. For example, it contains 1-4 heteroatoms as defined above. When used to represent atoms on a heterocyclic ring structure, the term "nitrogen" can include nitrogen that has undergone a substitution reaction. Heterocycloalkyl can be substituted or unsubstituted.

[0221] In the present application, the term "aryl" generally refers to a 3-12 membered substituted or unsubstituted monocyclic aromatic group, wherein each atom of the ring may be carbon (i.e., carbocyclic aryl), or wherein one or more atoms may be heteroatoms (i.e., heteroaryl). The aryl group may be substituted or unsubstituted, and when substituted, the substituent may be one or more of the following groups independently selected from the following group: alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxyl, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, 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] In the present application, the term "heteroaryl" generally refers to a residue derived from the same carbon atom or multiple different carbon atoms of a heteroaromatic ring by removing hydrogen atoms. The term "heteroaromatic ring" refers to a heteroaromatic system containing 1 to 4 heteroatoms, 5 to 14 ring atoms, wherein the heteroatoms can be selected from the following groups: oxygen, sulfur and nitrogen. The heteroaryl group can be 5 to 10 yuan, and can be 5 yuan or 6 yuan, such as furanyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyrimidyl, pyrazinyl, imidazolyl, tetrazolyl, etc. The heteroaryl ring can be fused to an aryl, heterocyclyl or cycloalkyl ring, wherein the ring connected to the parent structure is a 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 following groups: alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkyloxy, heterocycloalkyloxy, cycloalkylthio and heterocycloalkylthio.

[0224] In the present 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 may but need not occur, and the description includes occasions 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 may but need not be present, and the description may include situations where the heterocyclic group is substituted with an alkyl group and situations where the heterocyclic group is not substituted with an alkyl group.

[0226] In the present application, the compound or conjugate of the present application comprises its tautomer, mesomorph, racemate, enantiomer and / or diastereomer of the compound. In the present application, the term "diastereomer" generally refers to a stereoisomer with two or more chiral centers and whose molecules are not mirror images of each other. Diastereomers can have different physical properties, such as, melting point, boiling point, spectral properties and reactivity. In the present application, the term "mesomorph" generally refers to an atom containing asymmetry in a molecule, but has a symmetry factor that makes the total optical rotation in the molecule zero. The term "racemate" or "racemic mixture" refers to a composition consisting of two enantiomeric substances in equimolar amounts.

[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) exists naturally. 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 consistent with the structures of the present application except that hydrogen atoms are replaced by deuterium or tritium, or carbon atoms are replaced by carbon 13 or carbon 14, are within the scope of the present application.

[0228] In this application, the term "immune cell" generally refers to a cell that can participate in an immune response or is associated with an immune response. Immune cells can include all leukocytes and non-leukocyte antigen presenting cells. The term includes individual cells, cell lines or cell cultures. The cells include not only certain 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 doses and concentrations employed. Typically, the physiologically acceptable carrier is a pH buffered aqueous solution.

[0230] As used herein, the term "treatment" refers to administering an internal or external therapeutic agent to a patient, such as a pharmaceutical composition comprising any of the modified cells of the present application or comprising modified cells, the patient having one or more symptoms of a disease, and the therapeutic agent is known to have 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 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 the present application, the term "specificity" generally refers to the selective recognition of a specific epitope of an antigen by an antibody. For example, a natural antibody is monospecific. As in the present 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 present invention can be bispecific. In another embodiment of the invention, the multispecific antibody according to the present invention can be trispecific.

[0232] In the present application, the term "coupling" generally refers to connecting two compounds by a covalent bond or by a strong non-covalent interaction, such as a connection by a covalent bond. For example, the present application provides a connection in which N3 of one compound can react with an alkynyl group of another compound to form a covalent bond, and a conjugate of the two compounds can be formed.

[0233] In the present application, the term "tether" generally refers to a central compound to which different other compounds can be connected. The central compound can be connected to one or more other compounds to form another larger compound by covalent bonds or by strong non-covalent interactions. For example, the tether of the present application can be connected to one or more compounds having lipids, proteins, nucleic acids, small molecules or polysaccharides, or any combination thereof to form a conjugate.

[0234] In this application, the term "administering" 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 may include: intravenous, intramuscular, intradermal, subcutaneous, transdermal, mucosal, intratumoral and / or mucosal.

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

[0236] In this application, the term "about" generally refers to variations within a range of 0.5%-10% above or below a specified value, for example, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5% or 10% above or below a specified value. 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 that binds P 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 multi-specific coupling arm, reference can be made to the international publication text WO2022 / 100696A1 of the PCT application. All multi-specific coupling arms mentioned in this international application can be used in the present application to achieve the purpose of the present 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 is 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 is a 6- 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 the present 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 binding to cells, or regulating the activity of cells.

[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, hydroxyl, 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 Connection, as long as A 2 , B 2 and C 2 P can be connected to each other through a 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 be independently -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 each be independently selected from: -CH2- and -CH2-aryl-.

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

[0261] In the present application, A in the multispecific conjugate2 , B 2 and C 2 They 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] Where 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 of the above.

[0268] In certain embodiments, P 1 , P 2 and / or P 3 At least one of the above 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 an immune cell, recruit an immune cell, bind to an immune cell, bind to a surface protein of an immune cell, or bind to an intracellular target of an immune cell.

[0270] A person skilled in the art can select a suitable P according to the type of tumor cells or immune cells that 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 The molecular type.

[0271] In a specific embodiment, P 1 , P 2 and / 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 peptides may comprise a tumor cell binding portion, and at least one of the peptides 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 an antigen binding fragment thereof targeting a tumor antigen.

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

[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 part 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 regulating portion can target immune cell intracellular receptors.

[0280] Antigen Binding Protein

[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 includes an EGFR antibody or an antigen binding fragment thereof. The EGFR antibody is preferably a nanobody. For example, the EGFR antibody may comprise HCDR1, HCDR2 and HCDR3, and 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 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:32 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] On the other hand, the present application provides an antigen binding protein that specifically binds to HER2. The antigen binding protein that specifically binds to HER2 has an amino acid sequence as shown in SEQ ID NO:52.

[0283] On the other hand, 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 includes a CD3 antibody or an antigen binding fragment thereof. The CD3 antibody is preferably a nanobody. For example, the CD3 antibody may include HCDR1, HCDR2 and HCDR3, and the HCDR1 includes the amino acid sequence shown in SEQ ID NO:54, the HCDR2 includes the amino acid sequence shown in SEQ ID NO:55, and the HCDR3 includes the amino acid sequence shown in SEQ ID NO:56. For example, the CD3 antibody may include a variable region, and the variable region may include the amino acid sequence shown in SEQ ID NO:53.

[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 includes a PD-L1 antibody or an antigen binding fragment thereof. The PD-L1 antibody is preferably a nanobody. For example, the PD-L1 antibody may include HCDR1, HCDR2 and HCDR3, 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] On the other hand, 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 includes a CLEC9A antibody or an antigen binding fragment thereof. The CLEC9A antibody is preferably a nanobody. For example, the CLEC9A antibody may include HCDR1, HCDR2 and HCDR3, and the HCDR1 includes the amino acid sequence shown in SEQ ID NO:70, the HCDR2 includes the amino acid sequence shown in SEQ ID NO:71, and the HCDR3 includes the amino acid sequence shown in SEQ ID NO:72. For example, the CLEC9A antibody may include a variable region, and the variable region may include the amino acid sequence shown 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, and the HCDR1 includes the amino acid sequence shown in SEQ ID NO:58, the HCDR2 includes the amino acid sequence shown in SEQ ID NO:59, and the HCDR3 includes the amino acid sequence shown 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 shown 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 includes an NKG2D antibody or an antigen binding fragment thereof. The NKG2D antibody is preferably the nanobody. For example, the NKG2D antibody includes HCDR1, HCDR2 and HCDR3, and the HCDR1 includes the amino acid sequence shown in SEQ ID NO:62, the HCDR2 includes the amino acid sequence shown in SEQ ID NO:63, and the HCDR3 includes the amino acid sequence shown in SEQID NO:64. For example, the NKG2D antibody may include a variable region, and the variable region may include the amino acid sequence shown in SEQID NO:61.

[0288] The antigen-binding proteins of the present application can be prepared using techniques widely known in the art, such as hybridoma methods, recombinant DNA technology, phage display technology, synthetic technology or a combination of such technologies, or other technologies known in the art. Variants can refer to amino acid sequence mutants of antibodies, as well as covalent derivatives of natural polypeptides, provided that the biological activity equivalent to that of the natural polypeptide is retained. The difference between the amino acid sequence mutant and the natural amino acid sequence is generally that one or more amino acids in the natural amino acid sequence are replaced or one or more amino acids are deleted and / or inserted in the polypeptide sequence. Deletion mutants include fragments of natural polypeptides and N-terminal and / or C-terminal truncation mutants. Generally, the amino acid sequence mutant has at least 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99% homology compared to the natural 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 methods of treatment

[0291] In another aspect, the present application provides a method for treating and / or alleviating a tumor, the method comprising administering the multispecific conjugate, antigen binding protein, nucleic acid, cell, pharmaceutical composition, kit or immunoconjugate described herein to a subject in need thereof. The appropriate P can be selected according to 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 with positive EGFR. For example, the tumor with positive EGFR can include but is 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 with high expression of 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 are administered in combination with one or more pharmaceutically or physiologically acceptable carriers, diluents or excipients. Such compositions may include buffers such as neutral buffered saline, sulfate buffered saline, etc.; carbohydrates such as glucose, mannose, sucrose or dextran, mannitol; proteins; 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 patients subcutaneously, intradermally, intratumorally, intranodally, intraspinal, intramuscularly, by intravenous (iv) injection or intraperitoneally. Preferably, the pharmaceutical composition of the present application can be formulated to be suitable 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 human tumor cell EGFR receptor, whose amino acid sequence is SEQ ID NO: 1-51. Affinibody against human tumor cell HER2 receptor, whose amino acid sequence is SEQ ID NO: 52. All of the above nanobodies were expressed in E. coli, and the expression conditions were IPTG induction and overnight expression at 30°C. The expressed nanobodies were purified by nickel column purification and size exclusion chromatography in turn, and the N-termini of the obtained nanobodies were all connected to the LPETG sequence (SEQ ID NO: 76), and the 6His tag was used for purification, named anti-EGFR-LPETG and anti-HER2-LPETG, and used 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] The nanobody against human T cell CD3 receptor has an amino acid sequence of SEQ ID NO: 53-56. The CD3 nanobody is expressed in Escherichia coli, and the expression and purification conditions are the same as above. The purified nanobody N-terminus also contains the LPETG sequence (SEQ ID NO: 76), which is connected by a linker (SEQ ID NO: 77) and named 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] The nanobodies against human natural killer cell CD16 receptor and NKG2D receptor have amino acid sequences of SEQ ID NO: 57-64 respectively. The expression and purification of the nanobodies are similar to the above, and the obtained products contain LPETG sequence (SEQ ID NO: 76) at the N-terminus, which 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] The nanobodies against human dendritic cell PDL1 receptor and CLEC9A receptor have amino acid sequences of SEQ ID NO: 65-72 respectively. The nanobodies were expressed and purified in the same manner as above, and finally products containing LPETG sequence (SEQ ID NO: 76) at the N-terminus were obtained, which were 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] Myeloid immune cell TLR7 / 8 receptor small molecule agonist IMDQ (CAS: 1258457-59-8) and STING receptor small molecule agonist diABZI (CAS: 2137975-93-8) were purchased from MCE. IMDQ or diABZI (70mM), small molecule DBCO-SS-NHS (50mM) and catalytic triethylamine (50μM) were mixed and reacted at room temperature for 2 hours to produce the intermediate DBCO-SS-IMDQ or DBCO-SS-diABZI, which contains a cleavable disulfide bond (SS), so it can be broken in the tumor microenvironment and release the IMDQ or diABZI agonist without a trace.

[0323] The intermediate DBCO-SS-IMDQ or DBCO-SS-diABZI (10 mM) was reacted with the branched peptide (N3)6-LPETG (1 mM) having 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) CPG / STAT3 module targeting myeloid immune cells

[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*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). 100μM CPG or STAT3 ASO was dissolved in PBS, and then 1mMDBCO-SS-NHS small molecule 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 can respond to reducing conditions, so CPG and STAT3 ASO can be released without trace 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 enzyme cleavage sequences, and an N-terminal LPETG sequence (SEQ ID NO: 76) was synthesized by GenScript and named (CMV) 2-LPETG. The branched peptide can therefore respond to the MMP enzyme specifically upregulated in the tumor microenvironment to achieve in situ release of the CMV antigen peptide. The released CMV peptide will be recognized and presented by dendritic cells in the environment, further activating CMV antigen-specific T cells in the tumor microenvironment. 100 μM (CMV) 2-LPETG, 1 mM BCN-PEG3-Gly small molecule, and 10 μM Sortase A enzyme were mixed in PBS solution. 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 the protein was dissolved in a balanced solution (PBS, 0.05% Tween-20, 0.5% BSA) to a final concentration of 100ug / ml for use. Then, nanoantibody solutions of different concentrations were prepared for use. The binding ability was measured using a streptavidin-coupled sensor. OctetRED96 universal binding force determination method. The determination mainly includes three main processes: solidification, binding, and dissociation. After the determination, the binding constant K of the nanoantibody 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 higher binding affinity:

[0335]

[0336] (2) Binding ability of Anti-HER2-LPETG affinity body 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 a higher binding affinity:

[0338]

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

[0340] Similarly, using Octet RED96 was used to measure the binding affinity of anti-CD3-LPETG nanobody to CD3 receptor. The results are as follows. The results show that anti-CD3-LPETG can bind to CD3 receptor with higher binding 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 measure the binding affinity of anti-CD16-LPETG nanoantibody to CD16 receptor. The results are as follows. The results show that anti-CD16-LPETG can bind to CD16 receptor with a higher binding affinity:

[0344]

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

[0346] Similarly, using Octet RED96 was used to measure the binding affinity of anti-NKG2D-LPETG nanoantibody to NKG2D receptor. The results are as follows. The results show that anti-NKG2D-LPETG can bind to NKG2D receptor with higher binding affinity:

[0347]

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

[0349] Similarly, using Octet RED96 was used to measure the binding affinity of anti-PDL1-LPETG nanoantibody to PDL1 receptor. The results are as follows. The results show that anti-PDL1-LPETG can bind to PDL1 receptor with a higher binding 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 measure the binding affinity of anti-CLEC9A-LPETG nanobody to CLEC9A receptor. The results are as follows. The results show that anti-CLEC9A-LPETG can bind to CLEC9A receptor with higher binding 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 multispecific conjugate coupling arm Ia-1 (from Chinese patent application No. 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 the intermediate EGFR-N3-TZ was purified by size exclusion chromatography. 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 the EGFR-CD3-TZ intermediate was purified by size exclusion chromatography after the reaction. Finally, EGFR-CD3-TZ was reacted with the anti-PDL1-BCN module prepared above in equal amounts, the reaction concentration was also 100 μM, the reaction conditions were room temperature for 2 hours, and then size exclusion chromatography was performed for purification 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 Fig. 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. Fig.10 shown.

[0362] like Fig.11 As shown, the multispecific conjugate EGFR-CD3-CLEC9A achieves co-activation of tumor-targeted T cells and dendritic cells by simultaneously binding to tumor cell EGFR, T cell CD3 and dendritic cell CLEC9A receptors, thereby 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. Fig.12 shown.

[0365] like Fig.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. Fig.14 shown.

[0368] like Fig.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 coupling 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. The results are shown in Fig.16 shown.

[0371] like Fig.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 the 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 above, we finally synthesized the multispecific conjugate HER2-CD3-(diABZI)6 by reacting the multispecific conjugate coupling 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. The results are as follows Fig.18 shown.

[0374] like Fig.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 the 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. Fig. 20 shown.

[0377] like Fig.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 the myeloid immune cells to activate the TLR9 receptor, 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. Fig. 22 shown.

[0380] like Fig.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 the myeloid immune cells to act on the STAT3 receptor, thereby hindering 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 coupling 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 as follows Fig.24 shown.

[0383] like Fig.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 label were dissolved in equilibrium solution (PBS, 0.05% Tween-20, 0.5% BSA) for use, with a final concentration of 100ug / ml. EGFR-CD3-PDL1 protein of different concentrations was also dissolved in equilibrium solution for use. The binding capacity was measured by streptavidin-coupled sensor, using Octet RED96 universal binding force determination method. The determination mainly includes three main processes: solidification, binding, and dissociation. After the determination, the binding force 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 Fig.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% carbon dioxide, and the culture medium was RPMI-1640. Then, different concentrations of multispecific conjugates EGFR-CD3-PDL1 or bispecific conjugate controls (EGFR-CD3, PDL1-CD3, and EGFR-PDL1) were added and cultured for 24 hours to detect EGFR-CD3-PDL1-mediated tumor cell killing, T cell activation, and dendritic cell activation.

[0389] The firefly luciferase assay kit (Promega, Cat#E1500) was used to measure the EGFR-CD3-PDL1-mediated tumor cell killing. Specifically, after the above co-culture, the cell supernatant was removed from the 96-well plate, PBS was added to wash once, and then the substrate of the kit was added to incubate for 5-30 minutes, and the cell killing test was performed according to the instructions. The results of EGFR-CD3-PDL1-mediated tumor cell killing are shown in Fig.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 for staining at 4°C for 30 minutes, and then flow cytometry analysis was performed. The analysis results are shown in Fig.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: Fig.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 for staining at 4°C for 1 hour, and then flow cytometry analysis was performed. The analysis results are shown in Fig.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: Fig.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. Fig.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 for the tumor to grow to a volume > 100mm 3 After 1 day (set as day 1), human PBMC (10 7 Each mouse) was humanized for immune reconstitution. Subsequently, EGFR-CD3-PDL1, bispecific conjugate controls (EGFR-CD3, PDL1-CD3 and EGFR-PDL1) and an equal volume of PBS were injected into the tail vein of the mice. The injection time was 2, 5, 8, 11, 14 and 17 days, and the injection dose was 2 mg / kg EGFR-CD3-PDL1. The other groups were injected according to the equimolar amount.

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

[0397] After the 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. Fig.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. Fig.28A As shown, HSC humanized mice huHSC-NCG-hIL15 were purchased from Jicui Yaokang. A549 tumor cells (10 6 Each mouse), when the tumor grew to a volume >100mm 3 After that, drug administration began (set as day 1). EGFR-CD3-PDL1, bispecific conjugate controls (EGFR-CD3, PDL1-CD3 and EGFR-PDL1) and an equal volume of PBS were injected into the tail vein of mice. The injection time was 2, 5, 8, 11, 14, 17, 20 and 23 days, and the injection dose was 2 mg / kg EGFR-CD3-PDL1. The other groups were injected according to the equimolar amount.

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

[0401] After the 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 Fig.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, with CD86 and HLA-DR on dendritic cells as the analysis markers. The results are shown in Fig.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. Fig.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 dose of 5 × 10 5 cells per mouse. When the tumor grows to 100 mm 3 The mice were injected with 2 mg / kg EGFR-CD3-PDL1 or an equal volume of PBS for treatment on days 2, 5, 8, and 11.

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

[0405] After the 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 shown in Figure 2. Fig.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 markers for analysis were CD80, CD86 and MHC-II on dendritic cells. The results are shown in Fig.29D shown.

[0406] After the treatment was completed (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. Fig.29E shown.

[0407] After the treatment (day 15), all mice were killed. The spleen of the mice was taken to prepare a single cell suspension, and then the mouse IFNγ ELISPOT kit (Dakoway) was used to detect whether immune memory was generated in the mice 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 generated. The results are shown in Fig.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). Fig. 30AAs shown, the obtained tumor tissue was digested and cultured to form tumor microspheres (PTCs), and the culture method was referred to the reported literature (Sci. Transl. Med. 12, eaaz1723.). When the cultured PTC size was >40 μm, EGFR-CD3-PDL1 multispecific conjugate, bispecific conjugate control (EGFR-CD3, PDL1-CD3 and EGFR-PDL1) or an equal volume of PBS was added for continued culture, and then EGFR-CD3-PDL1-mediated T cell and dendritic cell activation, as well as PTC growth inhibition were 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. Fig. 30B shown.

[0411] After adding the multispecific conjugate for 7 days, we detected the growth of PTC in the system. The detection method was based on the reported literature (Sci. Transl. Med. 12, eaaz1723.). The results are as follows: Fig. 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 label were dissolved in equilibrium solution (PBS, 0.05% Tween-20, 0.5% BSA) for use, with a final concentration of 100ug / ml. EGFR-CD3-CD16 protein of different concentrations was also dissolved in equilibrium solution for use. The binding force was measured by a streptavidin-coupled sensor using Octet RED96 universal binding force determination method. The determination mainly includes three main processes: solidification, binding, and dissociation. After the determination, the binding force 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 Fig.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% carbon dioxide, and the culture medium was RPMI-1640. Then, different concentrations of multispecific conjugates EGFR-CD3-CD16 or bispecific conjugate controls (EGFR-CD3 and EGFR-CD16) were added and cultured for 24 hours to detect EGFR-CD3-CD16-mediated tumor cell killing and T cell activation.

[0418] The 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, PBS was added for washing once, and then the substrate of the kit was added for incubation for 5-30 minutes, and the cell killing test was performed according to the instructions. The results of EGFR-CD3-CD16-mediated tumor cell killing are shown in Fig.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 for staining at 4°C for 30 minutes, and then flow cytometry analysis was performed. The analysis results are shown in Fig.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: Fig.31D shown.

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

[0422] like Fig.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% carbon dioxide, and the culture medium was RPMI-1640. Then, different concentrations of multispecific conjugates EGFR-CD3-CD16 or bispecific conjugate controls (EGFR-CD3 and EGFR-CD16) were added and cultured for 24 hours to detect EGFR-CD3-CD16-mediated tumor cell killing and natural killer cell activation.

[0423] The 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, PBS was added for washing once, and then the substrate of the kit was added for incubation for 5-30 minutes, and the cell killing test was performed according to the instructions. The results of EGFR-CD3-CD16-mediated tumor cell killing are shown in Fig.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 for staining at 4°C for 30 minutes, and then flow cytometry analysis was performed. The analysis results are shown in Fig.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: Fig.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) for use at a final concentration of 100ug / ml. HER2-CD3-(IMDQ)6 proteins of different concentrations were also dissolved in equilibrium solution for use. The binding capacity was determined by a streptavidin-coupled sensor using Octet RED96 universal binding force determination method. The determination mainly includes three main processes: solidification, binding, and dissociation. After the determination, the binding force 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 reductive microenvironment

[0431] like Fig.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 a 96-well plate at 37°C, 5% carbon dioxide, and the culture medium was RPMI-1640. In order to simulate the tumor reductive 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 shown in Fig.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 Fig.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% carbon dioxide, and the culture medium was RPMI-1640. Then, different concentrations of multispecific conjugates HER2-CD3-(IMDQ)6 or bispecific conjugate controls (HER2-CD3 and HER2-(IMDQ)6) were added and cultured for 24 hours to detect HER2-CD3-(IMDQ)6-mediated tumor cell killing and T cell activation.

[0436] The firefly luciferase assay kit (Promega, Cat#E1500) was used to measure HER2-CD3-(IMDQ)6-mediated tumor cell killing. Specifically, after the above co-culture, the cell supernatant was removed from the 96-well plate, PBS was added for washing once, and then the substrate of the kit was added for incubation for 5-30 minutes, and the cell killing test was performed according to the instructions. The results of HER2-CD3-(IMDQ)6-mediated tumor cell killing are shown in Figure 2. Fig.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 for staining at 4°C for 30 minutes, and then flow cytometry analysis was performed. The analysis results are shown in Fig.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 referred to the standard instructions of the kit, and the test results were as follows: Fig.34D shown.

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

[0440] like Fig.35A , HER2-positive SK-BR3 tumor cells (10 4 ) and CD3 positive human T cells (2×10 5) were co-cultured in a 96-well plate 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 reductive microenvironment. Subsequently, 100, 10 or 1 nM HER2-CD3-(IMDQ)6 multispecific conjugate was added and incubated for 48 hours. The supernatant after incubation was collected and co-cultured with the separated human myeloid immune cells for 24 hours, and the activation of myeloid immune cells mediated by HER2-CD3-(IMDQ)6 multispecific conjugate was detected by flow cytometry.

[0441] The myeloid immune cells after the above culture were collected and resuspended for use. First, the activation of myeloid dendritic cells mediated by HER2-CD3-(IMDQ)6 was determined, and the cells were stained with anti-CD11c, anti-HLA-DR, anti-CD83 and anti-CD40, and then flow cytometry was performed. Then, the activation of myeloid macrophages mediated by HER2-CD3-(IMDQ)6 was determined, and the cells were stained with anti-CD11c, anti-HLA-DR, anti-CD68, anti-CD206 and anti-CD86 flow antibodies, and then flow cytometry was performed. Finally, the activation of myeloid monocytes mediated by HER2-CD3-(IMDQ)6 was determined, and the cells were stained with anti-HLA-DR, anti-CD206, anti-CD14, anti-CD40 and anti-CD80 flow antibodies, and then flow cytometry was performed. Among them, antibody staining and flow cytometric analysis were performed according to conventional procedures. For activation of myeloid dendritic cells, CD83 and CD40 markers were mainly evaluated, and the test results were as follows Fig.35B As shown in Figure 2, the activation of myeloid macrophages was mainly evaluated by the ratio of M1 and M2 macrophages. Fig.35C As shown in Figure 2, the activation of myeloid monocytes was evaluated by mainly evaluating CD14, CD40, and CD80 markers. Fig.35D shown.

Claims

1. A multispecific conjugate having a structure as shown in Formula IIa-1: in, P 1 , P 2 and P 3 are each independently selected from the group consisting of nucleic acids, polypeptides, proteins and small molecules, and P 1 , P 2 and P 3 Can recruit cells.

2. The multispecific conjugate of claim 1, wherein P 1 , P 2 and P 3 At least one of the peptides comprises a tumor cell binding portion and at least one comprises an immune cell modulating portion.

3. The multispecific conjugate of claim 2, wherein the tumor cell binding portion comprises an antibody or antigen binding fragment thereof that targets a tumor antigen.

4. The multispecific conjugate according to any one of claims 2 to 3, wherein the tumor antigen comprises any one or more selected from the group consisting of EGFR, HER2, HER3, TROP-2, tissue factor (TF), Nectin-4, c- Met, B7-H3, CLDN18.2, MUC-1, PSCA / PSMA / PSA, FRα, CD19, CD20, CD22, CD33, CD123, CD38, CEA, CD25, CD46, CD79B and BCMA.

5. The multispecific conjugate according to any one of claims 2 to 4, wherein the tumor cell binding portion is an EGFR antibody, preferably a nanobody, more preferably the EGFR antibody comprises HCDR1, HCDR2 and HCDR3, and the HCDR1 comprises the amino acid sequence shown in any one of SEQ ID NOs: 2, 6, 10, 17, 23, 27, 30, 40, 43 and 49, the HCDR2 comprises the amino acid sequence shown in any one of SEQ ID NOs: 3, 7, 11, 20, 31, 34, 37, 44 and 50, and the HCDR3 comprises the amino acid sequence shown in any one of SEQ ID NOs: 4, 8, 12, 15, 18, 21, 24, 28, 32, 35, 38, 41, 45, 47 and 51.

6. The multispecific conjugate according to claims 2-5, wherein the tumor cell binding portion is a HER2 antibody, preferably a HER2 affibody, and more preferably the HER2 affibody comprises the amino acid sequence shown in SEQ ID NO:

52.

7. The multispecific conjugate according to any one of claims 2-6, wherein the immune cells include myeloid immune cells and lymphoid immune cells.

8. The multispecific conjugate of any one of claims 2-7, wherein the immune cells comprise T cells, natural killer cells, dendritic cells, macrophages, monocytes, neutrophils and / or B cells.

9. The multispecific conjugate of any one of claims 2-8, wherein the immune cell modulating portion targets an immune cell surface receptor or targets an immune cell intracellular receptor.

10. The multispecific conjugate of any one of claims 2-9, wherein the immune cell modulating portion comprises an antibody or antigen binding fragment thereof that targets an immune cell surface receptor.

11. The multispecific conjugate of claim 10, wherein the immune cell surface receptor comprises any one or more selected from the group consisting of 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.

12. The multispecific conjugate according to any one of claims 10-11, wherein the immune cell modulating portion comprises a CD3 antibody, preferably a nanobody, more preferably the CD3 antibody comprises HCDR1, HCDR2 and HCDR3, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 54, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 55, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 56, Or, the immune cell regulating portion comprises a PD-L1 antibody, preferably a nanobody, more preferably, the PD-L1 antibody comprises HCDR1, HCDR2 and HCDR3, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 66, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 67, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 68, Or, the immune cell regulating portion comprises a CLEC9A antibody, preferably a nanobody, more preferably, the CLEC9A antibody comprises HCDR1, HCDR2 and HCDR3, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO: 70, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO: 71, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: 72, Or, the immune cell regulating portion comprises a CD16 antibody, preferably a nanobody, more preferably, the CD16 antibody comprises HCDR1, HCDR2 and HCDR3, and the HCDR1 comprises the amino acid sequence shown in SEQ ID NO:58, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:59, and the HCDR3 comprises the amino acid sequence shown in SEQ ID NO: The amino acid sequence shown in ID NO:60, Or, the immune cell regulating portion includes a NKG2D antibody, preferably a nanobody, more preferably, the NKG2D antibody includes HCDR1, HCDR2 and HCDR3, and the HCDR1 includes the amino acid sequence shown in SEQ ID NO:62, the HCDR2 includes the amino acid sequence shown in SEQ ID NO:63, and the HCDR3 includes the amino acid sequence shown in SEQ ID NO:

64.

13. The multispecific conjugate according to claim 12, wherein the immune cell regulatory portion comprises a CMV antigen peptide, preferably the CMV antigen peptide comprises the amino acid sequence shown in SEQ ID NO:

75.

14. The multispecific conjugate of any one of claims 2-13, wherein the immune cell modulating portion comprises an agonist or inhibitor targeting an intracellular receptor of an immune cell.

15. The multispecific conjugate of claim 14, wherein the immune agonist or inhibitor comprises any one or more selected from the group consisting of: Toll-like receptor (TLR) agonists, interferon gene stimulator receptor (STING) agonists, retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs), leucine-rich nucleotide binding domain repeats (NLRs), C-type lectin receptors (CLRs), signal transduction and transcription activator proteins (STATs), and indoleamine 2,3-dioxygenase (IDO), and the immune agonist preferably comprises any one or more selected from the group consisting of: IMDQ, diABZI, CPG and cGAMP, the immunosuppressant preferably includes any one or more selected from the following group: STAT3 ASO, IDO inhibitor Indoximod, GDC-0919 and Epacadostat.

16. The multispecific conjugate of any one of claims 1 to 15, wherein P 1 , P 2 and P 3 Further included is a cleavable linker.

17. The multispecific conjugate of any one of claims 1 to 16, wherein P 1 , P 2 and P 3 They are respectively the tumor cell binding portion, the 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.

18. An antigen binding protein capable of specifically binding to EGFR, wherein the antigen binding protein comprises 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: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:3 NO:24, (9) HCDR1: SEQ ID NO:2, HCDR2: SEQ ID NO:3, and HCDR3: SEQ ID NO:15, (10) HCDR1: SEQ ID NO:27, HCDR2: SEQ ID NO:3, and HCDR3: SEQ ID NO:28, (11) HCDR1: SEQ ID NO:30, HCDR2: SEQ ID NO:31, and HCDR3: SEQ ID NO:32, (12) HCDR1: SEQ ID NO:2, HCDR2: SEQ ID NO:34, and HCDR3: SEQ ID NO:34 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. The antigen-binding protein according to claim 18 , which is an antibody or an antigen-binding fragment thereof.

20. The antigen binding protein according to any one of claims 18-19, which is a nanobody.

21. A pharmaceutical composition comprising the multispecific conjugate of any one of claims 1-17, the antigen binding protein of any one of claims 18-20, and optionally a pharmaceutically acceptable excipient or carrier.

22. A method for treating and / or preventing tumors, comprising administering to a subject in need thereof an effective amount of the multispecific conjugate of any one of claims 1-17, the antigen binding protein of any one of claims 18-20, and / or the pharmaceutical composition of claim 21.

23. The method of claim 22, wherein the tumor is selected from one or more of the group consisting of breast cancer, colorectal cancer, lung cancer, ovarian cancer, bladder cancer, endometrial cancer, sarcoma, and pancreatic cancer.

24. 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 of any one of claims 1-17, the antigen binding protein of any one of claims 18-20 and / or the pharmaceutical composition of claim 21.

25. A method for enhancing or stimulating an immune response or function, the method comprising administering to an individual an effective amount of the multispecific of any one of claims 1-17, the antigen-binding protein conjugate of any one of claims 18-20, and / or the pharmaceutical composition of claim 21.

Images