DLL3 targeted binding proteins and uses thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a DLL3 targeted binding protein and application. The DLL3 targeted binding protein has an amino acid sequence as shown in SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3. The DLL3 targeted binding protein disclosed by the invention can be specifically bound with DLL3, the binding affinity reaches nM level, the molecular weight is about 18kDa which is far less than that of a traditional antibody molecule, and the DLL3 targeted binding protein has stronger tissue penetrability and lower immunogenicity. Related tests show that the DLL3 targeted binding protein disclosed by the invention can be used for diagnosis, treatment and drug delivery of DLL3 positive tumors, and has a wide clinical application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and in particular relates to a DLL3 targeted binding protein and use thereof. BACKGROUND

[0002] DLL3 (Delta-like ligand 3) is an inhibitory ligand in the Notch signaling pathway, mainly expressed in small cell lung cancer and neuroendocrine tumors (such as gastrointestinal pancreatic, bladder, prostate and cervical neuroendocrine cancer, etc.). In small cell lung cancer, the positive rate of DLL3 expression is as high as 85%, and its expression is mainly located in the cell membrane and cytoplasm, showing high tumor selectivity. In contrast, the expression of DLL3 in normal tissues is low, and only weakly expressed in the Golgi membrane and cytoplasmic vesicles of some specific tissues (such as pancreatic islets, pituitary, testis). This differential expression and localization in tumor tissues and normal tissues makes DLL3 a highly potential tumor treatment target.

[0003] The high expression of DLL3 in neuroendocrine tumors is closely related to its important role in tumor occurrence, development and metastasis. DLL3 promotes the occurrence of neuroendocrine tumors by inhibiting the activation of the Notch signaling pathway, and enhances the migration and invasion ability of tumor cells. Therefore, the treatment strategy targeting DLL3 has important clinical significance in inhibiting tumor growth and metastasis.

[0004] At present, the targeting treatment strategies for DLL3 mainly include antibody conjugated drugs (ADC), bispecific T cell engager (BiTE), chimeric antigen receptor T cells (CAR-T) and radionuclide labeled drugs, etc. A DLL3xCD3 bispecific antibody Tarlatamab shows good efficacy and safety in the treatment of small cell lung cancer in phase III clinical trials, and has been accelerated for marketing approval. However, the existing DLL3 targeted therapeutic drugs still have certain limitations, such as large molecular weight, poor tissue penetration, high production cost, etc. Therefore, it is of great clinical value to develop a DLL3 targeted binding protein with small molecular weight, high affinity and strong specificity. SUMMARY

[0005] The purpose of the present application is to provide a protein capable of specifically binding to DLL3, a coding nucleic acid thereof, an expression vector containing the nucleic acid, a host cell and the use thereof in the preparation of a drug for treating DLL3 positive tumors. The present application screens a DLL3 targeted binding protein from a phage anchor protein library by phage display technology, which has high affinity and specificity, and can provide new tools and strategies for the diagnosis and treatment of DLL3 positive tumors.

[0006] To this end, the present application provides the following technical solutions.

[0007] The first aspect of the present application provides a DLL3 targeting binding protein selected from the group consisting of:

[0008] (1) a targeting binding protein having an amino acid sequence shown in SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3; or

[0009] (2) a protein homologous to or having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% amino acid sequence identity to the amino acid sequence shown in SEQ ID NO. 1, SEQ ID NO. 2 or SEQ ID NO. 3; or

[0010] (3) a targeting binding protein derived from (1) or (2) with one or several amino acids substituted, deleted or added in the amino acid sequence of (1) or (2).

[0011] In a preferred embodiment of the present application, the amino acid sequence of SEQ ID NO. 1 at positions 31, 33, 34, 36, 44, 45, 64, 66, 67, 69, 77, 78, 97, 99, 100, 102, 110 and 111 comprises amino acid changes, i.e. the skeleton sequence of the four DLL3 targeting binding protein sequences is consistent, only the amino acids at the above-mentioned positions are different.

[0012] The second aspect of the present application provides a fusion protein comprising:

[0013] (1) a DLL3 targeting binding protein as described above;

[0014] (2) a CD16A targeting binding protein; and

[0015] (3) a bridging intermediate.

[0016] In a preferred embodiment of the present application, the bridging intermediate is SpyCatcher-SpyTag, which comprises SpyCatcher and SpyTag; wherein the SpyCatcher is modified on the DLL3 targeting binding protein, the SpyTag is modified on the CD16A targeting binding protein, and the SpyCatcher and SpyTag react via an amide bond to generate the bridging intermediate SpyCatcher SpyTag coupling the DLL3 targeting binding protein and the CD16A targeting binding protein.

[0017] In a preferred embodiment of the application, the fusion protein is formed by the DLL3 N-terminal fusion protein of the amino acid sequence set forth in SEQ ID NO. 4 linked to the CD16A C-terminal fusion protein of the amino acid sequence set forth in SEQ ID NO. 5.

[0018] A third aspect of the application provides a chimeric antigen receptor comprising an extracellular domain, a transmembrane domain, and an intracellular domain; wherein the extracellular domain comprises a DLL3 antigen binding domain that specifically binds to DLL3, and wherein the antigen binding domain is selected from a DLL3-targeting binding protein or fusion protein as previously described.

[0019] A fourth aspect of the application provides a nucleic acid molecule comprising a sequence selected from:

[0020] (i) encoding a DLL3-targeting binding protein, fusion protein, or chimeric antigen receptor as previously described;

[0021] (ii) a variant having at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to (i); or

[0022] (iii) a complement of (i) or (ii).

[0023] A fifth aspect of the application provides a vector comprising a nucleic acid molecule as previously described.

[0024] In a preferred embodiment of the application, the vector comprises one or a combination of a retroviral vector, a DNA vector, a plasmid, an RNA vector, an adenoviral vector, an adenovirus-associated vector, a lentiviral vector.

[0025] A sixth aspect of the application provides an engineered immune cell expressing a chimeric antigen receptor, a nucleic acid molecule, or a vector as previously described.

[0026] In a preferred embodiment of the application, the immune cell is one or a combination of a T cell, a tumor infiltrating lymphocyte, an NK cell, a TCR-expressing cell, a dendritic cell, or an NK-T cell.

[0027] A seventh aspect of the application provides use of a DLL3-targeting binding protein, fusion protein, chimeric antigen receptor, nucleic acid molecule, vector, or engineered immune cell as previously described in the manufacture of a medicament for treating a tumor and / or a disease and / or a symptom associated with a tumor.

[0028] An eighth aspect of the application provides a pharmaceutical composition comprising:

[0029] (1) a therapeutically effective amount of a DLL3-targeting binding protein, fusion protein, chimeric antigen receptor, nucleic acid molecule, vector or engineered immune cell as previously described;

[0030] (2) a pharmaceutically or immunologically acceptable carrier or excipient.

[0031] In a preferred embodiment of the present application, the medicament is used for treating an autoimmune disease or a proliferative disease;

[0032] The autoimmune disease includes rheumatoid arthritis, ankylosing spondylitis, systemic lupus erythematosus, cutaneous lupus erythematosus, lupus nephritis, IgA nephropathy, Sjogren's syndrome, polymyositis, dermatomyositis, scleroderma, psoriasis, plaque psoriasis, alopecia areata, multiple sclerosis, amyotrophic lateral sclerosis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, graft-versus-host disease, organ transplant rejection, autoimmune hepatitis, type I diabetes, autoimmune vasculitis, eczema or asthma;

[0033] The proliferative disease includes a neoplasm, a solid tumor, a hematological tumor, a malignant ascites or a malignant pleural effusion; wherein the solid tumor can be benign or malignant, primary or metastatic, the malignant solid tumor can be a carcinoma or a sarcoma, for example, an epithelial cell carcinoma, an endothelial cell carcinoma, a squamous cell carcinoma, a teratoma, a lung tumor, a papillomavirus-induced carcinoma, an adenocarcinoma, a carcinoma, a melanoma, an angiosarcoma, a neuroblastoma, a metastatic lung cancer, a non-small cell lung cancer, a small cell lung cancer, a breast cancer, a Merkel cell carcinoma, an ovarian cancer, a renal cell carcinoma, a metastatic renal cell carcinoma, a head and neck cancer, a bladder cancer, a non-muscular invasive bladder cancer; the hematological tumor can be selected from a leukemia, a lymphoma, a multiple myeloma, for example, a B-cell lymphoma, a T-cell lymphoma, a cutaneous T-cell lymphoma, a T-cell large granular lymphocyte leukemia

[0034] A ninth aspect of the present application provides a pharmaceutical preparation comprising the pharmaceutical composition as previously described.

[0035] By means of the above technical solution, the present application has at least the following advantages:

[0036] 1. High affinity and specificity: the DLL3-targeting binding protein of the present application can specifically bind to DLL3, and the binding affinity reaches the nM level.

[0037] 2. Small molecular weight: the molecular weight of the DLL3-targeting binding protein of the present application is about 18 kDa, which is much smaller than that of a traditional antibody molecule, and has stronger tissue penetration and lower immunogenicity.

[0038] 3. Simple production process: the DLL3 targeting binding protein of the present application can be expressed by a prokaryotic expression system such as E. coli, has low production cost, and is suitable for large-scale preparation.

[0039] 4. Multifunctional application: the DLL3 targeting binding protein of the present application can be used for the diagnosis, treatment and drug delivery of DLL3 positive tumors, and has a wide clinical application prospect.

[0040] The above description is only a summary of the technical solutions of the present application. In order to more clearly understand the technical means of the present application, and to implement the content of the description, the following will be described in detail with the preferred embodiments of the present application. BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 is the binding condition of the DLL3 targeting binding proteins 1, 2 and 3 and the DLL3 molecule detected by the biofilm interference technology; wherein A is the affinity determination of the DLL3 targeting binding protein 1, B is the affinity determination of the DLL3 targeting binding protein 2, and C is the affinity determination of the DLL3 targeting binding protein 3.

[0042] Figure 2 is the SDS-PAGE result of the DLL3 targeting binding protein expressed by E. coli.

[0043] Figure 3 is the binding condition of the DLL3 targeting binding protein and the DLL3 expression positive cell line SHP-77 and the DLL3 expression negative cell line A549 detected by flow cytometry; D3DP refers to the DLL3 targeting binding protein, and Ctrl DP refers to the same type of control protein which does not bind to DLL3; D3DP binds to the SHP-77 cells which express DLL3, and does not bind to the A549 cells which do not express DLL3.

[0044] Figure 4 is the binding condition of the DLL3 targeting binding protein and the DLL3 expression positive cell line SHP-77 and the DLL3 expression negative cell line A549 detected by cell immunofluorescence; D3DP binds to the SHP-77 cells which express DLL3, and does not bind to the A549 cells which do not express DLL3.

[0045] Figure 5 is the SDS-PAGE result of the DLL3 targeting binding protein-CD16A targeting binding protein fusion protein.

[0046] Figure 6is the promotion effect of the DLL3-targeting binding protein-CD16A-targeting binding protein fusion protein on NK activation by flow detection; wherein the fusion protein D316A can promote the secretion of IFN-γ, TNF-α and CD107a by NK cells, and has statistical difference compared with the simple NK group, the NK+SHP-77 group and the NK+SHP-77+Ctrl16A group.

[0047] Figure 7 is the detection of the DLL3-targeting binding protein-CD16A-targeting binding protein fusion protein by luciferase reporter gene method to promote the killing effect of NK cells on SHP-77 tumor cells; wherein at any ratio of effector to target of 10:1, 5:1, 2:1 and 1:1, the fusion protein D316A has obvious killing effect on SHP-77 tumor cells, and has statistical difference compared with the control.

[0048] Figure 8 is the anti-tumor effect of the DLL3-targeting binding protein-CD16A-targeting binding protein fusion protein in a mouse small cell lung cancer subcutaneous tumor model; wherein compared with the simple NK group and the PBS group, the fusion protein D316A prolongs the survival of mice, and nearly 40% of mice still have survival rate on the 60th day. DETAILED DESCRIPTION

[0049] In order to make the technical means, creative characteristics, purposes and effects realized by the present application easy to understand, the technical solutions in the embodiments of the present application will be described clearly and completely in combination with the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor belong to the scope of protection of the present application.

[0050] As used herein, "containing", "having" or "including" includes "comprising", "consisting essentially of", "consisting essentially of" and "consisting of"; "consisting essentially of", "consisting essentially of" and "consisting of" are sub-concepts of "containing", "having" or "including".

[0051] Terminology

[0052] As used herein, the term "specifically binds" means that an antibody or polypeptide can preferentially bind to a binding partner, for example, DLL3, in a competitive binding assay, for example, as assessed using a recombinant form of the protein, an epitope therein, or a native protein present on the surface of an isolated target cell. Competitive binding assay methods and other methods for determining specific binding are further described below and are well known in the art.

[0053] As used herein, the term "affinity" means the strength of binding of an antibody or protein to an epitope. The affinity of an antibody is given by the dissociation constant, KD, defined as [Ab] x [Ag] / [Ab-Ag], where [Ab-Ag] is the molar concentration of antibody-antigen complex, [Ab] is the molar concentration of unbound antibody, and [Ag] is the molar concentration of unbound antigen. The affinity constant, KA, is defined by 1 / KD. Preferred methods for determining the affinity of a protein can be found in Harlow et al., "Antibodies: A Laboratory Manual", Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1988); Coligan et al., eds., "Current Protocols in Immunology", Greene Publishing Assoc. and Wiley Interscience, New York, (1992, 1993); and Muller, Meth. Enzymol., Vol. 92: pp. 589-601 (1983). A preferred standard method for determining the affinity of a protein is screening using surface plasmon resonance (SPR) (such as by analysis with a BIAcore™ SPR analytical instrument).

[0054] As used herein, the term "identity" or "identical" when used in the context of the relationship between the sequences of two or more polypeptides refers to the degree of sequence relatedness between polypeptides, as determined by the number of matches between the chains of two or more amino acid residues. The "identity" measure is the percentage of identical matches between the smaller of the two or more sequences with a gap alignment, if any, resolved by a particular mathematical model or computer program (i.e., "algorithm"). Identity of related polypeptides can be readily calculated by known methods. Such methods include, but are not limited to, those described in: "Computational Molecular Biology," Lesk, A. M., ed., Oxford University Press, New York, 1988; "Biocomputing: Informatics and Genome Projects," Smith, D. W., ed., Academic Press, New York, 1993; "Computer Analysis of Sequence Data, Part 1," Griffin, A. M. and Griffin, H. G., eds., Humana Press, New Jersey, 1994; "Sequence Analysis in Molecular Biology," von Heinje, G., Academic Press, 1987; "Sequence Analysis Primer," Gribskov, M. and Devereux, J., eds., M. Stockton Press, New York, 1991 ; and Carillo et al., SIAM J. Applied Math., Vol. 48, pp. 1073-1082 (1988).

[0055] The preferred method for determining identity is designed to give the maximum match between sequences tested. Methods for determining identity are described in publicly available computer programs. Preferred computer program methods for determining identity between two sequences include the GCG suite of programs, including GAP (Devereux et al., Nucl. Acid. Res., vol. 12, p. 387 (1984)); Genetics Computer Group, University of Wisconsin, Madison, Wis., BLASTP, BLASTN, and FASTA (Altschul et al., J. Mol. Biol., vol. 215, pp. 403-410 (1990)). The BLASTX program is publicly available from the National Center for Biotechnology Information (NCBI) and other sources ("BLAST Manual," Altschul et al., NCB / NLM / NIH Bethesda, Md., 20894; Altschul et al., supra). The well-known Smith Waterman algorithm can also be used to determine identity.

[0056] As used herein, "NK cells" refers to a subpopulation of lymphocytes involved in the innate immunity. NK cells can be identified by certain characteristics and biological properties, such as expression of specific surface antigens of human NK cells (including CD56 and / or NKp46), absence of alpha / beta or gamma / delta TCR complex on the cell surface, ability to bind and kill cells that are unable to express "self" MHC / HLA antigens through an activated specific cytolytic mechanism, ability to kill tumor cells or other diseased cells that express ligands for NK activating receptors, and ability to release protein molecules (called cytokines) that stimulate or suppress immune responses. Any of these characteristics and activities can be used to identify NK cells using methods well known in the art. Any subpopulation of NK cells is also encompassed by the term NK cells. In the context herein, "active" NK cells refer to biologically active NK cells, including NK cells that have the ability to lyse target cells or enhance the immune function of other cells. NK cells can be obtained by various techniques known in the art, such as isolation from a blood sample, apheresis, tissue or cell collection, and the like. Useful protocols involving NK cells can be found in "Natural Killer Cells Protocols" (edited by Campbell KS and Colonna M). Humana Press, pp. 219-238 (2000).

[0057] As used herein, the "SpyCatcher-SpyTag" protein ligation system was established by British scientist Mark Howarth in 2012, which is derived from the CnaB2 domain of the Streptococcus pyogenes fibronectin-binding protein FbaB (Zakeri, Fierer et al. 2012). CnaB2 is a split protein that can be split into a 138-amino acid protein SpyCatcher and a 13-amino acid polypeptide SpyTag by structural biology and engineering methods. When they meet, they can be covalently bound to form a complete protein through the formation of an amide bond between the aspartic acid of SpyTag and the lysine of SpyCatcher within a few minutes. The SpyCatcher-SpyTag protein ligation system has the advantages of simple reaction conditions, high specificity, high efficiency, large yield, and stable reaction products compared with traditional methods such as chemical coupling and enzyme coupling.

[0058] As used herein, a "chimeric antigen receptor (CAR)" is a protein that specifically recognizes a target antigen, such as a target antigen on a cancer cell. When bound to a target antigen, a CAR can activate an immune cell to attack and destroy cells (e.g., cancer cells) bearing that antigen. A CAR can also incorporate co-stimulatory or signaling domains to increase its potency.

[0059] The chimeric antigen receptors disclosed herein comprise an extracellular domain, a transmembrane domain, and an intracellular domain, wherein the extracellular domain comprises a DLL3 antigen binding domain that specifically binds to DLL3. In some embodiments, a DLL3-specific CAR comprises, from 5' to 3', the following elements: a signal sequence, a DLL3 antigen binding domain, a hinge and transmembrane region, and one or more contiguous signaling domains.

[0060] As used herein, an "antigen binding domain" refers to any polypeptide that binds to a specified target antigen, such as a DLL3 protein or fragment thereof (interchangeably referred to herein as a "DLL3 antigen," a "DLL3 target antigen," or a "DLL3 target"). In some embodiments, the antigen binding domain binds to a DLL3 antigen on a tumor cell. In some embodiments, the antigen binding domain binds to a DLL3 antigen on a cell involved in a hyperproliferative disease.

[0061] As used herein, the term "transmembrane domain" has the ability to: (a) be expressed on the surface of an immune cell, such as, but not limited to, a lymphocyte, such as a T helper (Th) cell, a cytotoxic T (Tc) cell, a T regulatory (Treg) cell, or a natural killer (NK) cell, and / or (b) interact with an extracellular antigen-binding domain and an intracellular signaling domain to direct a cellular response of the immune cell to a target cell.

[0062] The transmembrane domain can be derived from a natural or synthetic source. If derived from a natural source, the domain can be derived from any membrane-bound or transmembrane protein.

[0063] The transmembrane region used specifically in the present disclosure can be derived from (comprising or corresponding to) CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, Programmed Death 1 (PD-1), Inducible T-cell Costimulator (ICOS), Lymphocyte Function-Associated Antigen-1 (LFA-1, CD1-1a / CD18), CD3y, CD35, CD3s, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig a (CD79a), DAP-10, Fcy Receptors, Class 1 MHC molecules, TNF Receptor proteins, Immunoglobulin proteins, Cytokine receptors, Integrins, Signaling Lymphocyte Activation Molecules (SLAM proteins), Activating NK cell receptors, BTLA, Toll ligand receptors, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8a, CD8b, IL-2Rb, IL-2Ry, IL-7Ra, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD19a, a ligand that specifically binds with CD83, or any combination thereof.

[0064] As used herein, the term "intracellular (cytoplasmic) domain" can activate at least one normal effector function of the immune cell comprising the CAR, such as signal 1 / activation and / or signal 2 / co-stimulation. For example, an effector function of a T cell can refer to cytolytic activity or helper activity, including secretion of cytokines.

[0065] It will be appreciated that suitable (e.g., activating) intracellular domains include, but are not limited to, signaling domains derived from (or corresponding to) CD3 zeta, CD28, OX-40, 4-1BB / CD137, CD2, CD7, CD27, CD30, CD40, Programmed Death 1 (PD-1), Inducible T-cell Costimulator (ICOS), Lymphocyte Function-Associated Antigen-1 (LFA-1, CD1-1a / CD18), CD3 gamma, CD3 delta, CD3 epsilon, CD247, CD276 (B7-H3), LIGHT, (TNFSF14), NKG2C, Ig alpha (CD79a), DAP-10, Fc gamma receptor, MHC class 1 molecule, TNF receptor proteins, immunoglobulin proteins, cytokine receptors, integrins, signaling lymphocyte activation molecules (SLAM proteins), activating NK cell receptors, BTLA, Toll ligand receptors, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8 alpha, CD8 beta, IL-2R beta, IL-2R gamma, IL-7R alpha, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD11d, ITGAE, CD103, ITGAL, CD11a, LFA-1, ITGAM, CD11b, ITGAX, CD11c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD19a, a ligand that specifically binds with CD83, or any combination thereof.

[0066] The intracellular domain of the CARs disclosed herein can incorporate, in addition to the activating domain described above, a costimulatory signaling domain (interchangeably referred to herein as a costimulatory molecule) to increase its potency. The costimulatory domain can provide a signal in addition to the primary signal provided by the activating molecule described herein.

[0067] It will be appreciated that suitable costimulatory domains within the scope of the present disclosure can be derived from (or correspond to) CD28, OX40, 4-1BB / CD137, CD2, CD3 (a, b, d, e, g, z), CD4, CD5, CD7, CD9, CD16, CD22, CD27, CD30, CD33, CD37, CD40, CD45, CD64, CD80, CD86, CD134, CD137, CD154, PD-1, ICOS, lymphocyte function-associated antigen-1 (LFA-1 (CD11a / CD18), CD247, CD276 (B7-H3), LIGHT (tumor necrosis factor superfamily member 14; TNFSF14), NKG2C, Ig a (CD79a), DAP-10, Fcy receptors, MHC class I molecules, TNFRs, integrins, signaling lymphocytic activation molecules, BTLA, Toll ligand receptors, ICAM-1, B7-H3, CDS, ICAM-1, GITR, BAFFR, LIGHT, HVEM (LIGHTR), KIRDS2, SLAMF7, NKp80 (KLRF1), NKp44, NKp30, NKp46, CD19, CD4, CD8a, CD8b, IL-2Rb, IL-2Ry, IL-7Ra, ITGA4, VLA1, CD49a, ITGA4, IA4, CD49D, ITGA6, VLA-6, CD49f, ITGAD, CD1-1d, ITGAE, CD103, ITGAL, CD1-1a, LFA-1, ITGAM, CD1-1b, ITGAX, CD1-1c, ITGB1, CD29, ITGB2, CD18, LFA-1, ITGB7, NKG2D, TNFR2, TRANCE / RANKL, DNAM1 (CD226), SLAMF4 (CD244, 2B4), CD84, CD96 (Tactile), CEACAM1, CRTAM, Ly9 (CD229), CD160 (BY55), PSGL1, CD100 (SEMA4D), CD69, SLAMF6 (NTB-A, Ly108), SLAM (SLAMF1, CD150, IPO-3), BLAME (SLAMF8), SELPLG (CD162), LTBR, LAT, GADS, SLP-76, PAG / Cbp, CD19a, CD83 ligand, or fragments or combinations thereof, for example.

[0068] It will be appreciated that other costimulatory molecules or fragments thereof not listed above are also within the scope of the present disclosure.

[0069] As used herein, an “immune cell” is a T cell (e.g., inflammatory T lymphocyte, cytotoxic T lymphocyte, regulatory T lymphocyte (Treg), helper T lymphocyte, tumor infiltrating lymphocyte (TIL)), natural killer T cell (NKT), TCR-expressing cell, dendritic cell, killer dendritic cell, mast cell, or B cell. In some embodiments, the cell can be derived from a group consisting of CD4 + T lymphocytes and CD8 + T lymphocytes. In some exemplary embodiments, the engineered immune cell is a T cell. In some exemplary embodiments, the engineered immune cell is a gd T cell. In some exemplary embodiments, the engineered immune cell is a macrophage. In some exemplary embodiments, the engineered immune cell is a natural killer (NK) cell.

[0070] As used herein, the term “proliferative disease” includes neoplasm, solid tumor, hematoma, malignant ascites or malignant pleural effusion, refers to a disease, disorder or condition in which cells exhibit or have exhibited relatively abnormal, uncontrolled and / or autonomous growth, such that they exhibit or have exhibited an abnormally elevated proliferation rate and / or an abnormal growth phenotype; wherein the solid tumor can be benign or malignant, primary or metastatic, the malignant solid tumor can be a carcinoma or a sarcoma, e.g., an epithelial cell carcinoma, an endothelial cell carcinoma, a squamous cell carcinoma, a teratoma, a lung tumor, a papillomavirus-induced carcinoma, an adenocarcinoma, a carcinoma, a melanoma, an angiosarcoma, a neuroblastoma, a metastatic lung carcinoma, a non-small cell lung carcinoma, a small cell lung carcinoma, a breast carcinoma, a Merkel cell carcinoma, an ovarian carcinoma, a renal cell carcinoma, a metastatic renal carcinoma, a head and neck carcinoma, a bladder carcinoma, a non-muscular invasive bladder carcinoma; the hematoma can be selected from a leukemia, a lymphoma, a multiple myeloma, e.g., a B-cell lymphoma, a T-cell lymphoma, a cutaneous T-cell lymphoma, a T-cell large granular lymphocyte leukemia.

[0071] The above cancers are all within the scope of the present application.

[0072] As used herein, the term “solid tumor” refers to an abnormal mass of tissue that includes cancer cells. In various embodiments, e.g., as set forth herein, a solid tumor is or includes an abnormal mass of tissue that does not include a cyst or a fluid region. In some embodiments, e.g., as set forth herein, a solid tumor can be benign; in some embodiments, a solid tumor can be malignant. Examples of solid tumors include carcinomas, lymphomas, and sarcomas. In some embodiments, e.g., as set forth herein, a solid tumor can be or include an adrenal, biliary, bladder, bone, brain, breast, cervical, colon, endometrial, esophageal, eye, gallbladder, gastrointestinal, kidney, laryngeal, liver, lung, nasal, nasopharyngeal, oral, ovarian, penile, pituitary, prostate, retinal, salivary gland, skin, small intestine, stomach, testicular, thymic, thyroid, uterine, vaginal, and / or vulvar tumor.

[0073] The above-mentioned solid tumors are all within the scope of the present application.

[0074] As used herein, the term "autoimmune disease" refers to a predominantly defensive pathological reaction of the body to various physical, chemical, biological, and the like, harmful stimuli, resulting in a disease. In various embodiments, for example, as set forth herein, autoimmune diseases include rheumatoid arthritis, ankylosing spondylitis, systemic lupus erythematosus, cutaneous lupus erythematosus, lupus nephritis, IgA nephropathy, Sjogren's syndrome, polymyositis, dermatomyositis, scleroderma, psoriasis, plaque psoriasis, alopecia areata, multiple sclerosis, amyotrophic lateral sclerosis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, graft-versus-host disease, organ transplant rejection, autoimmune hepatitis, type I diabetes, autoimmune vasculitis, eczema, or asthma.

[0075] As used herein, the term "pharmaceutical composition" refers to a composition comprising extracellular vesicles formulated together with one or more pharmaceutically acceptable carriers.

[0076] The formulation of the pharmaceutical composition can be adjusted according to the application. In particular, the pharmaceutical composition can be formulated using methods known in the art in order to provide rapid, continuous, or delayed release of the active ingredient after administration to a mammal. For example, the formulation can be any one selected from the group consisting of plasters, granules, lotions, liniments, lemonades, aromatic waters, powders, syrups, eye ointments, liquids and solutions, aerosols, sprays, extracts, elixirs, ointments, fluid extracts, emulsions, suspensions, decoctions, infusions, eye drops, tablets, suppositories, injections, alcohol, capsules, creams, lozenges, tinctures, pastes, pills, and soft or hard gelatin capsules.

[0077] As used herein, the term "pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient" refers to any and all solvents, dispersion media, preservatives, antioxidants, coatings, isotonic and absorption delaying agents, surfactants, fillers, disintegrants, binders, diluents, lubricants, glidants, pH adjusting agents, buffers, enhancers, humectants, solubilizers, surfactants, antioxidants, and the like, which are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. The composition can comprise other active compounds that provide supplementary, additional, or enhanced therapeutic functions. Solid carriers or excipients, such as lactose, starch, or talc, or liquid carriers, such as water, fatty oils, or liquid paraffin. Other examples of the carriers include culture media, such as DMEM or RPMI; and cryogenic storage media, including components that scavenge free radicals, provide pH buffering, osmotic / osmotic support, energy substrates, and ion concentrations that can balance intracellular states at low temperatures; and mixtures of organic solvents with water.

[0078] The pharmaceutical composition is formulated for administration by an administration route selected from the group consisting of intranasal, intralesional, intrathecal, intravenous, intramuscular, subcutaneous, sublingual, oral, and intracerebral administration routes. According to one embodiment, the pharmaceutical composition is formulated for intranasal administration. According to some embodiments, such pharmaceutical composition is a liquid solution, a nasal drop, a spray, a metered spray form. According to other embodiments, the pharmaceutical composition is formulated as a preparation for injection, for example, intralesional, intrathecal or intravenous injection. According to such embodiment, the pharmaceutical composition is in the form of a sterile injectable solution.

[0079] Any known method can be used to administer the pharmaceutical composition of the present application. The term "administering" or "administering" a substance, compound, or agent to a subject can be performed using one of a variety of methods known to those of skill in the art. For example, the compound or agent can be administered intranasally (e.g., by inhalation), intrathecally (into the spinal canal or subarachnoid space), intraarterially, intradermally, intramuscularly, intraperitoneally, intravenously, subcutaneously, ocularly, sublingually, orally (by ingestion), intracerebrally, and transdermally (by absorption, e.g., through a skin catheter). The compound or agent can also be suitably introduced by a rechargeable or biodegradable polymeric device or other device (e.g., patches and pumps or formulations) that provides extended, slowed, or controlled release of the compound, agent. Administration can also be performed, for example, once, multiple times, and / or over one or more extended periods.

[0080] As used herein, the term "therapeutically effective amount" means an amount that is sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level includes the type of subject and severity, age, gender, drug activity, sensitivity to drugs, time of administration, route of administration, and excretion rate, duration of treatment, factors including concomitant drugs, and other factors well known in the medical field.

[0081] As used herein, the term "treatment" of a condition or patient refers to taking steps to obtain a beneficial or desired result, including clinical results. Beneficial or desired clinical results include, but are not limited to, or eliminate, substantially inhibit, slow or reverse the progression of a disease, condition or disorder, substantially improve or alleviate the clinical or aesthetic symptoms of some condition, substantially prevent the clinical or aesthetic symptoms of a disease, condition or disorder, and avoid deleterious or unpleasant symptoms. Treatment also refers to one or more of the following: (a) reducing the severity of the disorder; (b) limiting the development of symptoms characteristic of the disorder being treated; (c) limiting the worsening of symptoms characteristic of the disorder being treated; (d) limiting the recurrence of the disorder in patients who previously had the disorder; and / or (e) limiting the recurrence of symptoms in patients who were previously asymptomatic for the disorder.

[0082] The experimental methods used in the following examples are conventional methods, and the reagents, methods and equipment used are conventional reagents, methods and equipment in the technical field unless otherwise specified.

[0083] Example 1: Bio-Layer Interferometry (BLI) assay for DLL3-targeting binding proteins to detect the affinity to DLL3 molecules

[0084] The DLL3-targeting binding proteins #1, #2 and #3 were synthesized using an E. coli expression system, i.e. the DLL3-targeting binding protein sequence with a 6xHis tag was inserted into a pET-30a(+) vector, transformed into host bacteria BL21(DE3) for expression, and subjected to affinity purification using a nickel column and endotoxin removal using a Kimson endotoxin removal kit to <0.1 EU / μg.

[0085] DLL3-targeting binding protein #1 (SEQ ID NO. 1 with a histidine tag at the N-terminus)

[0086] DLL3-targeting binding protein #2 (SEQ ID NO. 2 with a histidine tag at the N-terminus)

[0087] DLL3-targeting binding protein #3 (SEQ ID NO. 3 with a histidine tag at the N-terminus)

[0088] The specific amino acid sequences of SEQ ID NO. 1, SEQ ID NO. 2 and SEQ ID NO. 3 are as follows:

[0089] SEQ ID NO. 1:

[0090] DLGKKLLEAASAGQDDEVRILLKAGADVNALDNFGQTPLHLAAKVGHLEIVEVLLKYGADVNALDWAGVTPLHLAALDGHLEIVEVLLKYGADVNAFDVMGATPLHLAAIAGHLEIVEVLLKYGADVNAQDKSGKTPADLAADACHEDIAEVLQKAA

[0091] SEQ ID NO. 2:

[0092] DLGKKLLEAASAGQDDEVRILLKAGADVNACAGDTPLHLAAEVGHLEIVEVLLKYGADVNAADWFGVTPLHLAAAWGHLEIVEVLLKYGADVNARDDKGRTPLHLAAVFGHLEIVEVLLKYGADVN A QDKSGKTPAYLAADAGHEDIAEVLQKAA

[0093] SEQ ID NO. 3:

[0094] DLGKKLLEAARAGQDDEVRILLKAGADVNAYDFLGFTPLHLAASYGHLEIVEVLLKYGADVNASDNNGSTPLHLAAAWGHLEIVEVLLKYGADVNAMDAHGWTPLHLAAIDGHLEIVEVLLKYGADVNAQDKSGKTPADLAADAGHEDIAEVLQKAA

[0095] 2. Affinity determination with DLL3 protein

[0096] The inter-protein affinity determination was performed using ForteBio Octet system, and the specific process was as follows:

[0097] Using streptavidin (SA) sensor, first, initialization was performed with PBS buffer (containing 0.01% Tween 20), and the sensor was inserted into the well of 96-well plate containing 200 μL PBS buffer (containing 0.01% Tween 20) for 5 minutes. Then, the SA sensor was inserted into the biotinylated DLL3 protein solution with a concentration of 10 μg / mL for protein coupling reaction, and the time was 10 minutes. After the coupling was completed, the sensor was washed with PBS buffer (containing 0.01% Tween 20) for 2 minutes to remove the unbound DLL3 protein. The DLL3 targeted binding protein solution was prepared, and the concentration gradient was set as 1000 nM, 500 nM, 250 nM, 125 nM, 62.5 nM, 31.25 nM, 15.625 nM. In the 96-well plate, 200 μL of DLL3 targeted binding protein solution with different concentrations was added to each well. The sensor coupled with DLL3 protein was inserted into the DLL3 targeted binding protein solution with corresponding concentration in turn, and the real-time signal change of the binding reaction was recorded (the binding time was set as 300 seconds). Subsequently, the sensor was transferred to PBS buffer (containing 0.01% Tween 20), and the dissociation reaction signal was recorded (the dissociation time was set as 300 seconds). The signal curves (nm change) of the binding and dissociation stages were recorded in real time by BLI analyzer. The binding model was used to fit the binding curve and dissociation curve, and the binding rate constant, dissociation rate constant and affinity constant were calculated. The results are shown in Figure 1.

[0098] As Figure 1 A shows, the affinity of DLL3 targeting binding protein #1 is 8.62 x 10 -9 M; Figure 1 B shows, the affinity of DLL3 targeting binding protein #2 is 3.97 x 10 -8 M; Figure 1 C shows, the affinity of DLL3 targeting binding protein #3 is 2.82 x 10 - 8 M.

[0099] In the following examples, the DLL3 targeting binding protein #3 with relatively low affinity is selected as the experimental object.

[0100] Example 2: Using polyacrylamide gel electrophoresis (SDS-PAGE) to detect the purity and molecular weight of DLL3 targeting binding protein

[0101] 1. Electrophoresis

[0102] Take 20 μL of the purified DLL3 targeting binding protein and mix with 5 μL of 5x buffer, and place in a metal bath pot to denature the protein at 100°C for 10 min; configure 15% SDS-PAGE separation gel and 5% concentrated gel; add 5 μL marker to the well, and add 10 μL of the DLL3 targeting binding protein sample; electrophorese at 70V for 30 min, and then adjust the voltage to 120V for electrophoresis for 60 min after the protein has completely entered the separation gel.

[0103] 2. Staining

[0104] After electrophoresis, carefully pry open the clamp, cut off the edge according to the lowermost edge of the marker band, cut along the concentrated gel and separation gel, and carefully take out the gel piece into the staining solution and stain with Coomassie brilliant blue for half an hour.

[0105] 3. Determination

[0106] Take out the gel piece and place it in water to decolorize, take pictures on the machine, and analyze the molecular weight. The results are shown in Figure 2 .

[0107] As Figure 2 shown, the molecular weight of the DLL3 targeting binding protein is about 17 KDa, and the purity is >90%.

[0108] Example 3: Flow cytometry to detect the binding of DLL3 targeting binding protein to DLL3 positive cell lines and DLL3 negative cell lines

[0109] The cell suspensions of DLL3 positive cell SHP-77 and DLL3 negative cell A549 were set as the same type control protein group Ctrl DP group and the DLL3 targeted binding protein group D3DP group respectively, and then 100 μg / mL of the same type control protein (the targeted binding protein for other target points screened) and D3DP (DLL3 targeted binding protein) were added to the cells in the corresponding groups respectively, mixed, and then incubated at 4°C for 1 h; after the incubation, the unbound proteins were removed by washing, and then His Tag flow antibody (Nanjing Kingsriver Biological Technology Co., Ltd., A01802) was added, and incubated at room temperature for 30 min in the dark; after the incubation, the unbound antibody was removed by washing, and finally the proportion and average fluorescence intensity of positive cells were detected by flow cytometry. The results are shown in Figure 3 .

[0110] As shown in Figure 3 , the DLL3 targeted binding protein binds to the DLL3 positive SHP-77 cells, and does not bind to the DLL3 negative A549 cells.

[0111] Example 4: Cell immunofluorescence detection of the binding of DLL3 targeted binding protein to DLL3 positive cell line SHP-77 and DLL3 negative cell line A549

[0112] DLL3 positive cell SHP-77 and DLL3 negative cell A549 were cultured, wherein the DLL3 positive cell SHP-77 was a suspension cell, and the DLL3 negative cell A549 was an adherent cell. First, the DLL3 positive cell SHP-77 was centrifuged and fixed with 4% paraformaldehyde, and then dropped on a high-adhesion glass slide and naturally dried; the DLL3 negative cell A549 was plated on a confocal dish at an appropriate density, and then fixed with 4% paraformaldehyde after adherent growth for 8 hours. Then, both cells were blocked with 1% BSA at room temperature for 1 h, and then incubated with 100 μg / mL of DLL3 targeted binding protein at 4°C overnight; after the incubation, the unbound proteins were removed by washing, and then His Tag fluorescent antibody (Nanjing Kingsriver Biological Technology Co., Ltd., A01802) was added, and incubated at room temperature for 30 min in the dark; after the incubation, the unbound antibody was removed by washing, and finally DAPI staining solution was added to stain the nucleus for 5 min, and then observed under a confocal microscope. The results are shown in Figure 4 .

[0113] As shown in Figure 4 , the DLL3 targeted binding protein binds to the DLL3 positive SHP-77 cells, and does not bind to the DLL3 negative A549 cells.

[0114] Example 5: Preparation of DLL3-targeting binding protein-CD16A-targeting binding protein fusion protein and its activation effect on NK cells

[0115] 1. Preparation and purification of DLL3-targeting binding protein-CD16A-targeting binding protein fusion protein

[0116] The nucleic acid sequence encoding DLL3-targeting binding protein-Spycatcher-DLL3-targeting binding protein (hereinafter referred to as "DLL3 end fusion protein") and the nucleic acid sequence encoding CD16A-targeting binding protein-SpyTag-CD16A-targeting binding protein (hereinafter referred to as "CD16A end fusion protein") with a 6xHis tag were inserted into the pET-30a(+) vector, and the host bacteria clear E. coli was transformed for expression, and nickel column was used for affinity purification, and high-purity DLL3 end fusion protein (amino acid sequence as shown in SEQ ID NO. 4) and CD16A end fusion protein (amino acid sequence as shown in SEQ ID NO. 5) were obtained, respectively.

[0117] Then the above obtained DLL3 end fusion protein and CD16A end fusion protein were mixed in a molar ratio of 1:1 and placed in PBS buffer, and incubated at 4°C overnight. Based on the spontaneous formation of covalent hetero-linkage of SpyTag and SpyCatcher modules under mild conditions, finally a stable DLL3xCD16A fusion protein (hereinafter referred to as D316A) can be constructed, and its purity was verified by SDS-PAGE, and the results are shown in Figure 5 .

[0118] DLL3 end fusion protein sequence SEQ ID NO. 4

[0119] DLGKKLLEAARAGQDDEVRILLKAGADVNAYDFLGFTPLHLAASYGHLEIVEVLLKYGADVNASDNNGSTPLHLAAAWGHLEIVEVLLKYGADVNAMDAHGWTPLHLAAIDGHLEIVEVLLKYGADVNAQDKSGKTPADLAADAGHEDIAEVLQKAAGGGGSGGGGSGGGGSGGGGSEDSATHIKFSKRDEDGKELAGATMELRDSSGKTISTWISDGQVKDFYLYPGKYTFVETAAPDGYEVATAITFTVNEQGQVTVNGKATKGDAHIGGGGSGGGGSGGGGSDLGKKLLEAARAGQDDEVRILLKAGADVNAYDFLGFTPLHLAASYGHLEIVEVLLKYGADVNASDNNGSTPLHLAAAWGHLEIVEVLLKYGADVNAMDAHGWTPLHLAAIDGHLEIVEVLLKYGADVNAQDKSGKTPADLAADAGHEDIAEVLQKAA

[0120] CD16A N-terminal fusion protein sequence SEQ ID NO. 5

[0121] DLGKKLLEAARAGQDDEVRILLKAGADVNAHDKQGSTPLHLAAEEGHLEIVEVLLKYGADVNATDVSGWTPLHLAAFYGHLEIVEVLLKYGADVNAHDHLGTTPLHLAAAVGHLEIVEVLLKYGADVNAQDKSGKTPADLAADAGHEDIAEVLQKAAGGGGSGGGGSGGGGSAHIVMVDAYKPTKGGGGSGGGGSGGGGSDLGKKLLEAARAGQDDEVRILLKAGADVNAHDKQGSTPLHLAAEEGHLEIVEVLLKYGADVNATDVSGWTPLHLAAFYGHLEIVEVLLKYGADVNAHDHLGTTPLHLAAAVGHLEIVEVLLKYGADVNAQDKSGKTPADLAADAGHEDIAEVLQKAA

[0122] As shown in Figure 5 lane 1 is CD16A N-terminal fusion protein, lane 2 is DLL3 N-terminal fusion protein, and lane 3 is DLL3xCD16A fusion protein D316A. As shown in Figure 5It can be seen that the DLL3 end fusion protein and the CD16A end fusion protein are mixed and incubated to obtain a high-purity DLL3xCD16A fusion protein D316A.

[0123] 2. Flow cytometry detection of NK cell degranulation and intracellular cytokines

[0124] The NK cells and DLL3-positive cells SHP-77 cells were mixed at an effector-to-target ratio of 5:1 to form an NK+SHP-77 co-incubation system, and then the cells were divided into four groups: an NK group (referring to a group of NK cells alone), an NK+SHP-77 group, an NK+SHP-77+D316A group, and an NK+SHP-77+Ctrl16A group. Then the cells in each group were treated as follows:

[0125] The NK+SHP-77+D316A group: 10 μg / mL of the DLL3-targeted binding protein-CD16A-targeted binding protein fusion protein (D316A) was added to the NK+SHP-77 co-incubation system;

[0126] The NK+SHP-77+Ctrl16A group: 10 μg / mL of a homologous control fusion protein of any target other than DLL3xCD16A fusion protein (Ctrl16A) was added to the NK+SHP-77 co-incubation system;

[0127] The NK+SHP-77 group and the NK group: no protein was added.

[0128] 1) Immediately after the start of co-incubation, fluorescently labeled anti-CD107a antibody (LAMP-1 marker) was added to the cells in each group, and after 1 hour of incubation, GolgiStop (Brefeldin A inhibitor, BD554724) was added, and the incubation was continued until the 6th hour. Then the cells were collected, and surface markers (CD56 + CD3 - NK cell gating) were detected, and the CD107a expression level was detected using flow cytometry to evaluate the degranulation activation state of the NK cells. The results are shown in Figure 6 .

[0129] 2) GolgiStop (BD554724) and GolgiPlug (BD555029) were simultaneously added to the cells in each group 1 hour after the start of co-incubation, and the incubation was continued until the 6th hour. After the incubation was completed, the cells were subjected to Fixation / Permeabilization fixation and membrane rupture treatment, and then intracellular IFN-γ and TNF-α staining was performed. The intracellular cytokine level was detected using flow cytometry under CD56 + CD3 - NK cell gating. The results are shown in Figure 6 .

[0130] As shown in Figure 6 , compared with the NK+SHP-77+Ctrl16A group, the NK+SHP-77 group and the NK group, the concentration of CD 107a released by degranulation of NK cells and the concentration of IFN-γ and TNF-α in the NK+SHP-77+D316A group were significantly increased, and the difference was statistically significant. The above results show that the DLL3 targeted binding protein-CD16A targeted binding protein fusion protein D316A prepared in this embodiment can effectively promote the degranulation release (CD107a) and cytokine (IFN-γ, TNF-α) secretion of NK cells, thereby further enhancing the activation response of NK cells.

[0131] Example 6: DLL3 targeted binding protein-CD16A targeted binding protein fusion protein promotes the killing effect of NK cells on SHP-77

[0132] The NK cells and the DLL3 positive cells SHP-77 cells were mixed according to different effector to target ratios (E:T ratios), 10:1, 5:1, 2:1, 1:1, respectively, to form the corresponding co-incubation systems. Then different concentrations (50 μg / mL, 10 μg / mL, 1 μg / mL, 0.1 μg / mL, 0 μg / mL) of DLL3 targeted binding protein-CD16A targeted binding protein fusion protein D316A (prepared in Example 5) were added to the corresponding co-incubation systems with different E:T ratios. The incubation system was set up in a 96-well plate, the incubation temperature was 37°C, and the incubation time was 6 hours. After incubation, luciferase activity was determined by adding luciferin potassium salt to reflect the number of residual living cells. The results are shown in Figure 7 .

[0133] As shown in Figure 7 , under different E:T ratios, the experimental group with the addition of fusion protein D316A showed significantly higher SHP-77 tumor cell specific killing rate compared with the control group, and still maintained high killing activity as the E:T ratio decreased. In addition, under different concentrations of D316A, the killing activity on SHP-77 tumor cells showed a concentration-dependent increase, and the fusion protein D316A concentration of 50 μg / mL group reached the maximum killing effect (right panel).

[0134] Example 7: Anti-tumor effect of DLL3 targeted binding protein-CD16A targeted binding protein fusion protein in a mouse small cell lung cancer subcutaneous tumor model

[0135] 1. Construction of a mouse small cell lung cancer subcutaneous tumor model

[0136] Select 6-week-old NCG mice, and inoculate 5×10 6The SHP-77-Luc cells were subcutaneously inoculated in the groin of the mice at a seeding amount of 1 cell / animal, and observed for several days until the tumor grew to 100 mm 3 The tumor-bearing model mice were constructed.

[0137] 2. Grouping and treatment

[0138] The tumor-bearing mice constructed above were randomly divided into three groups: a PBS group, an NK group, and an NK+DLL3-targeting binding protein-CD16A-targeting binding protein fusion protein D316A group, each group having 6 animals. The mice in each group were treated as follows:

[0139] The PBS group: 100 μL of PBS was injected into the tail vein of each mouse once a week for two weeks.

[0140] The NK group: 1×10 7 cells were injected into the tail vein of each mouse once a week for two weeks.

[0141] The NK+D316A group: 1×10 7 cells and 2 mg / kg of the fusion protein D316A (prepared in Example 5) were injected into the tail vein of each mouse once a week for two weeks.

[0142] During the treatment, 50,000 IU of rIL 2 was injected into the peritoneal cavity of each mouse three times a week to maintain the activity of the NK cells.

[0143] 3. Survival time statistics

[0144] The survival state of each mouse in the PBS group, the NK group, and the NK+D316A group was observed and counted, and the observation and counting time was 60 days. The survival curve of the mice was plotted using the Kaplan Meier method, and the difference in the survival time of the mice in each group was statistically compared by log rank (Mantel Cox) test. The results are shown in Figure 8 .

[0145] As shown in Figure 8 , the survival time of the mice in the NK+D316A group was significantly longer than that of the mice in the PBS group and the NK group, which indicated that the prepared DLL3-targeting binding protein-CD16A-targeting binding protein fusion protein D316A effectively inhibited the proliferation and growth of small cell lung cancer and significantly prolonged the survival time of the mice.

[0146] The above merely describes preferred embodiments of the present application, and is not intended to limit the present application in any form. Although the present application has been disclosed with the preferred embodiments as above, it is not intended to limit the present application. Any person skilled in the art can make some changes or modifications to the above disclosed methods and technical contents to make equivalent embodiments with equivalent changes, without departing from the technical solution of the present application. Any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present application, without departing from the technical solution of the present application, still belongs to the scope of the technical solution of the present application.

Claims

1. A DLL3-targeting binding protein, characterized in that, The targeting binding protein is selected from: (1) a targeting binding protein having an amino acid sequence set forth in SEQ ID NO. 1, SEQ ID NO. 2, or SEQ ID NO. 3; or (2) a protein that is homologous to or has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid sequence identity to the amino acid sequence set forth in SEQ ID NO. 1, SEQ ID NO. 2, or SEQ ID NO. 3; or (3) a targeting binding protein derived from (1) or (2) with one or several amino acids substituted, deleted, or added in the amino acid sequence of (1) or (2).

2. The targeted binding protein of claim 1, wherein, The amino acid sequence of SEQ ID NO. 1 at positions 31, 33, 34, 36, 44, 45, 64, 66, 67, 69, 77, 78, 97, 99, 100, 102, 110, and 111 comprises an amino acid change.

3. A fusion protein, characterized in that, The fusion protein comprises: (1) the DLL3 targeting binding protein of claim 1 or 2; (2) a CD16A targeting binding protein; and (3) a bridging intermediate.

4. The fusion protein of claim 3, wherein, The bridging intermediate is SpyCatcher-SpyTag, which comprises a SpyCatcher and a SpyTag; wherein the SpyCatcher is modified to the DLL3 targeting binding protein, the SpyTag is modified to the CD16A targeting binding protein, and the SpyCatcher and SpyTag react via an amide bond to generate the bridging intermediate SpyCatcher-SpyTag coupling the DLL3 targeting binding protein and the CD16A targeting binding protein.

5. The fusion protein according to claim 3 or 4, characterized in that, The fusion protein is formed by the DLL3-end fusion protein having the amino acid sequence set forth in SEQ ID NO. 4 connected to the CD16A-end fusion protein having the amino acid sequence set forth in SEQ ID NO.

5.

6. A chimeric antigen receptor, characterized in that, The chimeric antigen receptor comprises an extracellular domain, a transmembrane domain, and an intracellular domain; wherein the extracellular domain comprises a DLL3 antigen binding domain that specifically binds to DLL3, and wherein the antigen binding domain is selected from the DLL3 targeting binding protein of claim 1 or 2 or the fusion protein of any one of claims 3-5.

7. A nucleic acid molecule, characterized in that, comprises a sequence selected from: (i) encodes the DLL3 targeting binding protein of claim 1 or 2 or the fusion protein of any one of claims 3-5 or the chimeric antigen receptor of claim 6; (ii) a variant having at least 80%, at least 90%, at least 95%, at least 98%, or at least 99% sequence identity to (i); or (iii) a complement of (i) or (ii).

8. A vector, characterized in that, The vector comprises the nucleic acid molecule of claim 7.

9. The carrier of claim 8, wherein, The vector comprises one or a combination of a retroviral vector, a DNA vector, a plasmid, an RNA vector, an adenoviral vector, an adenovirus-associated vector, a lentiviral vector.

10. An engineered immune cell, characterized in that, The engineered immune cell expresses the chimeric antigen receptor of claim 6, the nucleic acid molecule of claim 7, or the vector of claim 8 or 9.

11. The engineered immune cell of claim 10, wherein, The immune cell is one or a combination of a T cell, a tumor infiltrating lymphocyte, an NK cell, a TCR-expressing cell, a dendritic cell, or an NK-T cell.

12. Use of the DLL3-targeting binding protein of claim 1 or 2, the fusion protein of any one of claims 3-5, the chimeric antigen receptor of claim 6, the nucleic acid molecule of claim 7, the vector of claim 8 or 9, or the engineered immune cell of claim 10 or 11 in the manufacture of a medicament for treating a tumor and / or a disease and / or a symptom associated with a tumor.

13. A pharmaceutical composition, characterized by, The pharmaceutical composition comprises: (1) a therapeutically effective amount of the DLL3-targeting binding protein of claim 1 or 2, the fusion protein of any one of claims 3-5, the chimeric antigen receptor of claim 6, the nucleic acid molecule of claim 7, the vector of claim 8 or 9, or the engineered immune cell of claim 10 or 11; (2) a pharmaceutically or immunologically acceptable carrier or excipient.

14. The pharmaceutical composition of claim 13, wherein, The medicament is for treating an autoimmune disease or a proliferative disease; The autoimmune disease comprises rheumatoid arthritis, ankylosing spondylitis, systemic lupus erythematosus, cutaneous lupus erythematosus, lupus nephritis, IgA nephropathy, Sjogren's syndrome, polymyositis, dermatomyositis, scleroderma, psoriasis, plaque psoriasis, alopecia areata, multiple sclerosis, amyotrophic lateral sclerosis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, graft-versus-host disease, organ transplant rejection, autoimmune hepatitis, type I diabetes, autoimmune vasculitis, eczema, or asthma. The proliferative disease comprises a neoplasm, a solid tumor, a hematological tumor, a malignant ascites, or a malignant pleural effusion; wherein the solid tumor can be benign or malignant, primary or metastatic, the malignant solid tumor can be a carcinoma or a sarcoma, for example, an epithelial cell carcinoma, an endothelial cell carcinoma, a squamous cell carcinoma, a teratoma, a lung tumor, a papillomavirus-induced carcinoma, an adenocarcinoma, a carcinoma, a melanoma, an angiosarcoma, a neuroblastoma, a metastatic lung carcinoma, a non-small cell lung carcinoma, a small cell lung carcinoma, a breast carcinoma, a Merkel cell carcinoma, an ovarian carcinoma, a renal cell carcinoma, a metastatic renal carcinoma, a head and neck carcinoma, a bladder carcinoma, a non-muscular invasive bladder carcinoma; the hematological tumor can be selected from a leukemia, a lymphoma, a multiple myeloma, for example, a B-cell lymphoma, a T-cell lymphoma, a cutaneous T-cell lymphoma, a T-cell large granular lymphocyte leukemia.

15. A pharmaceutical preparation, characterized in that, The pharmaceutical preparation comprises the pharmaceutical composition of claim 13 or 14.

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