Preparation of a bispecific t-cell engager molecule and uses thereof

Abstract

The present application provides a kind of bispecific T cell engager molecule. Specifically, the present application provides a kind of bispecific cell engager molecule, which includes the first binding domain of targeting uPAR, connecting section and the second binding domain of anti-human CD3 molecule. The first binding domain is combined with uPAR positive target cells (tumor cells, senescent cells and other target cells), and the second binding domain is combined with T lymphocyte, thereby effectively connecting T cell with target cell, targeting T cell and killing target cell, with the potential application in antitumor, anti-aging and other drug preparation.

Description

Technical Field

[0001] This invention relates to the field of biomedicine. Specifically, this invention relates to the preparation and application of bispecific T-cell junction molecules. Background Technology

[0002] Cytotoxic T lymphocytes (CTLs) play a crucial role in the immune response during cancer treatment. Tumor cells can create an immunosuppressive environment through their own secreted cytokines or interactions with the tumor microenvironment, leading to CTL dysfunction within the tumor microenvironment. One strategy for modulating immune cells is to utilize bispecific cell engagers to activate and kill target cells, such as tumor cells. Bispecific T-cell engagers (BiTEs), named for their ability to specifically target antigens expressed by T cells at one end, guide T cells to kill target cells. This process is accompanied by the formation of a transient cytolytic synapse between the T cell and the target tumor cell, followed by T cell proliferation and activation leading to tumor cell lysis. Bispecific cell engager molecules consist of two protein or polypeptide sequences (antibodies are most common) linked together, each binding to a different target protein. As large protein molecules, the size of bispecific T-cell engager molecules is crucial; smaller molecules offer significant advantages in terms of production economy, molecular stability, in vivo half-life, and tissue penetration. The application of smaller nanobodies in bispecific cell engager molecules is increasingly evident.

[0003] Urokinase-type plasminogen activator receptor (uPAR) is a GPI-anchored cell membrane receptor composed of three homologous domains (DI, DII, DIII). Its main function is to activate the proteolytic activity of urokinase (uPA) and regulate the degradation of extracellular matrix (ECM) components. Both uPA proenzyme and uPA can bind to uPAR. A portion of uPAR is cleaved by proteases upon binding to its ligand, producing soluble uPAR (suPAR). Extensive literature demonstrates the importance of uPAR in the progression of most tumors, overexpressing it in most tumor cells and tumor stroma, including solid tumors such as breast cancer, colorectal cancer, prostate cancer, pancreatic cancer, ovarian cancer, lung cancer, and glioma, as well as hematologic malignancies such as acute leukemia and myeloma. However, low uPAR expression is detected only in sternal epithelium, monocytes, macrophages, and neutrophil subsets in normal tissues. Studies have shown that (1) elevated uPAR expression is associated with adverse outcomes (invasion, metastasis, and recurrence) in patients with different types of cancer; (2) uPAR is closely related to tumor metabolism; (3) uPAR expression is associated with RAS gene mutations in patients with non-small cell lung cancer (NSCLC) and colorectal cancer (CRC); (4) uPAR is a key player in regulating the transition between tumor cell dormancy and proliferation; (5) uPAR is associated with multidrug resistance (MDR) of tumor cells; and (6) uPAR is associated with tumor angiogenesis.

[0004] Aging is a complex process influenced by both genetic and environmental factors, closely related to the permanent, progressive decline of physiological cells, and is one of the reasons for the increasingly serious aging of the population. Aging can significantly increase the body's morbidity, namely age-related diseases such as diabetes, Alzheimer's disease, cardiovascular disease, and neurodegenerative diseases. Individual aging is related to, but entirely different from, senescent cells. Senescent cells are cells in a stable state of cell cycle arrest and secrete cytokines that regulate the tissue microenvironment. Physiologically, senescent cells are a mechanism for inhibiting tumors, preventing the expansion of precancerous cells, and playing a beneficial role in wound healing responses. As individuals age, senescent cells accumulate in tissues. Although the proportion of senescent cells in the tissues of elderly individuals is small, their accumulation can promote aging, create an inflammatory environment, and lead to chronic tissue damage, such as liver and lung fibrosis, atherosclerosis, diabetes, and osteoarthritis. Eliminating senescent cells in elderly individuals can improve these pathological symptoms and even promote longevity. Existing studies have confirmed that uPAR is an important surface marker of cellular senescence, and that senescent cells can secrete soluble uPAR (suPAR) as part of the senescence-associated secretory phenotype (SASP).

[0005] Given the important role of uPAR in the development and progression of tumors and aging, a large number of studies have explored the role of uPAR in the diagnosis and prognosis of tumors, aging, and aging-related diseases; as well as the role of uPAR as a therapeutic target for malignant tumors, aging, and aging-related diseases, exploring the role of targeting uPAR in related treatments.

[0006] uPA protein contains an N-terminal ATF (Amino-Terminal Fragment) and a C-terminal catalytic domain. The ATF domain can be further divided into two smaller structural units: a smaller GFD (Growth Factor-like Domain) and a larger Kringle domain. Figure 1 While the GFD domain is essential for uPA binding to uPAR, existing studies have primarily used ATF to investigate uPA-uPAR binding, and to construct bispecific antibodies targeting EGFR and uPAR using the ATF domain and EGF (epidermal growth factor), and to construct uPAR-targeting CAR-T cells using the ATF domain. The Kringle domain in ATF may bind to other proteins, thus affecting the target specificity of bispecific antibodies and potentially causing side effects. Molecular size is a crucial consideration for protein-peptide drugs in terms of cost and stability; whether smaller GFD domains can be used to construct bispecific T-cell conjugates to guide T cells to attack uPAR-positive target cells remains unclear.

[0007] Therefore, there is a need in this field to develop a bispecific T-cell binder molecule that targets uPAR. Summary of the Invention

[0008] The purpose of this invention is to provide a bispecific cell binder molecule that targets GFD (N-terminal domain of uPA) binding proteins.

[0009] In a first aspect of the invention, a cell agonist molecule is provided, comprising:

[0010] (a) A first binding domain having a structure derived from a GFD domain of uPA, and the first binding domain specifically binds to a GFD-binding protein.

[0011] (b) A second binding domain that specifically binds to CD3 molecules.

[0012] In another preferred embodiment, the cell entangler molecule is a bispecific cell entangler molecule.

[0013] In another preferred embodiment, the cell binder molecule specifically connects the GFD-binding protein and the CD3 molecule.

[0014] In another preferred embodiment, the GFD-binding protein refers to a protein capable of binding the GFD domain of uPA, preferably uPAR.

[0015] In another preferred embodiment, the amino acid sequence of the first binding domain is selected from the group consisting of:

[0016] (i) the amino acid sequence as shown in SEQ ID NO.2; and

[0017] (ii) Based on the sequence shown in SEQ ID NO.2, one or more amino acid residues are replaced, deleted, altered, or inserted, or 1 to 10 amino acid residues are added to its N-terminus or C-terminus, preferably 1 to 5 amino acid residues, more preferably 1 to 3 amino acid residues, thereby obtaining an amino acid sequence; and the obtained amino acid sequence has ≥85% (preferably ≥90%, more preferably ≥95%, for example ≥96%, ≥97%, ≥98%, or ≥99%) sequence identity with the sequence shown in SEQ ID NO.2; and the obtained amino acid sequence has the same or similar function as the sequence shown in (i).

[0018] In another preferred embodiment, the second binding domain specifically binds to human CD3.

[0019] In another preferred embodiment, the second binding domain has a structure selected from the group consisting of: single-domain antibody (sdAb), single-chain antibody (scFv), antigen-binding fragment (Fab fragment), ligand, or a polymer thereof, or a combination thereof.

[0020] In another preferred embodiment, the second binding domain has a peptide derived from an anti-human CD3 antibody.

[0021] In another preferred embodiment, the second binding structural domain includes a VH segment having a complementarity determination region (CDR) as follows:

[0022] VH-CDR1, as shown in SEQ ID NO.5,

[0023] VH-CDR2 shown in SEQ ID NO.6, and

[0024] VH-CDR3 as shown in SEQ ID NO.7; and / or

[0025] The second binding structural domain includes a VL segment, which has a complement determination region (CDR) as follows:

[0026] VL-CDR1 shown in SEQ ID NO.8,

[0027] VL-CDR2 shown in SEQ ID NO.9, and

[0028] VL-CDR3 as shown in SEQ ID NO.10;

[0029] Furthermore, any amino acid sequence in the aforementioned CDR sequence may also include a derivative sequence that has been optionally added, deleted, modified, and / or substituted with at least one amino acid, such that the derivative antibody composed of the heavy chain and light chain containing the derived CDR sequence can retain CD3 binding affinity.

[0030] In another preferred embodiment, the VH segment has the amino acid sequence shown in SEQ ID NO.4, or has an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology or sequence identity with the amino acid sequence shown in SEQ ID NO.4; and / or

[0031] The VL segment has the amino acid sequence shown in SEQ ID NO.9, or has an amino acid sequence that has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence homology or sequence identity with the amino acid sequence shown in SEQ ID NO.9.

[0032] In another preferred embodiment, the second structural domain is a single-chain antibody.

[0033] In another preferred embodiment, the first and / or second domains are monovalent or polyvalent (e.g., divalent).

[0034] In another preferred embodiment, the multivalence refers to a plurality of first structural domains and / or second structural domains connected in series.

[0035] In another preferred embodiment, the cell agonist molecule is a single-chain polypeptide.

[0036] In another preferred embodiment, the cell conjugate molecule has a structure selected from formula (I) or (II) from its N-terminus to its C-terminus:

[0037] S-D1-L1-D2-T (I);

[0038] S-D2-L1-D1-T (II),

[0039] In the formula,

[0040] Each "-" independently represents a linking peptide or peptide bond;

[0041] S is either a signal peptide sequence or a no-signal peptide sequence;

[0042] D1 is the first associative structural domain;

[0043] L1 is either absent or the first linker peptide;

[0044] D2 is the second associative domain;

[0045] T is an unlabeled or unmarked protein.

[0046] In another preferred embodiment, S is a signal peptide derived from the mammalian CD8.

[0047] In another preferred embodiment, the amino acid sequence of S is shown in SEQ ID NO.18.

[0048] In another preferred embodiment, the marker protein T is selected from: His tag, GGGS sequence, and FLAG tag.

[0049] In another preferred embodiment, the amino acid sequence of T is shown in SEQ ID NO.17.

[0050] In another preferred embodiment, the amino acid sequence of L1 is shown in SEQ ID NO.16.

[0051] In another preferred embodiment, the D2 has a VH-L2-VL or VL-L2-VH structure from the N-terminus to the C-terminus, wherein VH is the VH segment, VL is the VL segment, and L2 is either absent or a second linker peptide.

[0052] In another preferred embodiment, the amino acid sequence of L2 is shown in SEQ ID NO.13.

[0053] In another preferred embodiment, the amino acid sequence of the cell conjugate molecule is selected from:

[0054] (i) The amino acid sequence as shown in SEQ ID NO.20;

[0055] (ii) Based on the sequence shown in SEQ ID NO. 20, one or more amino acid residues are replaced, deleted, altered, or inserted, or 1 to 30 amino acid residues, preferably 1 to 10 amino acid residues, more preferably 1 to 5 amino acid residues are added to its N-terminus or C-terminus, thereby obtaining an amino acid sequence; and the obtained amino acid sequence has ≥85% (preferably ≥90%, more preferably ≥95%, for example ≥96%, ≥97%, ≥98%, or ≥99%) sequence identity with the sequence shown in SEQ ID NO. 14; and the obtained amino acid sequence has the same or similar function as the sequence shown in (i).

[0056] In a second aspect of the invention, a recombinant protein is provided, said recombinant protein comprising the cell agonist molecule as described in the first aspect of the invention.

[0057] In another preferred embodiment, the recombinant protein (or polypeptide) includes a fusion protein.

[0058] In another preferred embodiment, the recombinant protein is a monomer, dimer, or polymer.

[0059] In another preferred embodiment, the recombinant protein is a fusion protein.

[0060] In another preferred embodiment, the fusion protein is bispecific or multispecific (e.g., trispecific).

[0061] In another preferred embodiment, the multispecific antibody is not only able to bind to uPAR and CD3 simultaneously, but also specifically binds to additional target antigens.

[0062] In another preferred embodiment, the additional target antigen includes tumor antigens, preferably pancreatic cancer-associated antigens.

[0063] In another preferred embodiment, the additional target antigen includes immune cell surface antigens, preferably including: CD2, CD4, CD8, CD11, CD18, or combinations thereof.

[0064] In a third aspect of the invention, a polynucleotide is provided, said polynucleotide encoding a polypeptide selected from the group consisting of:

[0065] (1) A cell-connector molecule as described in the first aspect of the present invention; or

[0066] (2) The recombinant protein as described in the second aspect of the present invention.

[0067] In another preferred embodiment, the sequence of the polynucleotide is shown as positions 1-999 of SEQ ID NO.19.

[0068] In a fourth aspect of the invention, a carrier is provided, the carrier containing the polynucleotide as described in the third aspect of the invention.

[0069] In another preferred embodiment, the vector includes, but is not limited to, bacterial plasmids, bacteriophages, yeast plasmids, plant cell viruses, mammalian cell viruses such as adenoviruses, retroviruses, or other vectors.

[0070] In another preferred embodiment, the carrier includes, but is not limited to, pCDH, pTOMO, pGEM, pELNS, pMSGV, or combinations thereof.

[0071] In a fifth aspect of the invention, an engineered host cell is provided, the host cell containing a vector as described in the fourth aspect of the invention or a genome in which polynucleotides as described in the third aspect of the invention are integrated.

[0072] In another preferred embodiment, the host cell is a T cell, an NK cell, or a combination thereof.

[0073] In another preferred embodiment, the host cell is an in vivo cell or an in vitro cultured cell that can be transplanted into the body.

[0074] In another preferred embodiment, the in vitro cultured cells that can be transplanted into the body are selected from blood cells.

[0075] In another preferred embodiment, the host cell is a T cell.

[0076] In another preferred embodiment, the host cell is derived from a human or a non-human mammal (such as a mouse).

[0077] In a sixth aspect of the invention, a coupling is provided, the coupling comprising:

[0078] (a) a cell-connector molecule as described in the first aspect of the invention; and

[0079] (b) A coupling portion coupled to the cell conjugate, the coupling portion being selected from the group consisting of detectable markers, drugs, or combinations thereof.

[0080] In another preferred embodiment, the detectable marker includes a radionuclide.

[0081] In another preferred embodiment, the drug includes toxins, cytokines, and enzymes.

[0082] In another preferred embodiment, the conjugate is selected from: fluorescent or luminescent markers, radiolabels, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes, radionuclides, biotoxins, cytokines (such as IL-2), antibodies, antibody Fc fragments, antibody scFv fragments, gold nanoparticles / nanorobars, viral particles, liposomes, magnetic nanoparticles, prodrug-activating enzymes (e.g., DT-cardiacinase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (e.g., cisplatin), or any form of nanoparticles.

[0083] In another preferred embodiment, the antibody portion is coupled to the coupling portion via a chemical bond or a linker.

[0084] In another preferred embodiment, the immunoconjugate contains: a multivalent (e.g., bivalent) cell conjugate molecule as described in the first aspect of the invention.

[0085] In another preferred embodiment, the polyvalent meaning refers to the inclusion of multiple repeating cell conjugate molecules as described in the first aspect of the invention in the amino acid sequence of the immunoconjugate.

[0086] In a seventh aspect of the invention, there is provided a use of an active ingredient selected from the group consisting of cell-conjugating molecules as described in the first aspect of the invention, recombinant proteins as described in the second aspect of the invention, conjugates as described in the sixth aspect of the invention, or combinations thereof, wherein the active ingredient is used to prepare diagnostic reagents, detection plates, or kits for detecting uPAR and / or CD3.

[0087] In another preferred embodiment, the reagent, detection plate, or kit is used to detect diseases associated with abnormal uPAR and / or CD3 expression or function.

[0088] In another preferred embodiment, the reagent, detection plate, or kit is used to predict the risk and / or prognosis of tumors and senescent cells.

[0089] In another preferred embodiment, the reagent is prepared as one or more reagents selected from the group consisting of isotope tracers, contrast agents, flow cytometry reagents, cell immunofluorescence reagents, magnetic nanoparticles, and imaging agents.

[0090] In another preferred embodiment, the reagent, test plate, or kit is used to screen for drugs to treat uPAR-positive tumors.

[0091] In another preferred embodiment, the reagent, detection plate, or kit is used to screen for diseases caused by uPAR-positive senescent cells.

[0092] In an eighth aspect of the invention, a pharmaceutical composition is provided, the pharmaceutical composition comprising:

[0093] (i) an active ingredient selected from the group consisting of: T-cell connector molecules as described in the first aspect of the invention, recombinant proteins as described in the second aspect of the invention, immune cells as described in the fifth aspect of the invention, antibody conjugates as described in the sixth aspect of the invention, or combinations thereof; and

[0094] (ii) One or more pharmaceutically acceptable carriers, diluents, fillers, binders, excipients, or combinations thereof.

[0095] In another preferred embodiment, the pharmaceutical composition is a liquid formulation.

[0096] In another preferred embodiment, the pharmaceutical composition is an injection.

[0097] In another preferred embodiment, the pharmaceutical composition comprises 0.01 to 99.99% of a cell conjugate molecule as described in the first aspect of the invention, a recombinant protein as described in the second aspect of the invention, a host cell as described in the fifth aspect of the invention, an antibody conjugate as described in the sixth aspect of the invention, or a combination thereof, and 0.01 to 99.99% of a carrier, wherein the percentage is a percentage by mass of the pharmaceutical composition.

[0098] In another preferred embodiment, the concentration of the engineered immune cells in the formulation is 1 × 10⁻⁶. 3 -1×10 8 Cells / ml, preferably 1×10⁻⁶ 4 -1×10 7 Cells / ml

[0099] In another preferred embodiment, the pharmaceutical composition is used for the prevention and / or treatment of diseases associated with abnormal uPAR and / or CD3 expression or function.

[0100] In a ninth aspect of the invention, a method for detecting uPAR and / or CD3 in a sample is provided, the method comprising the steps of:

[0101] (1) Contact the sample with the cell conjugate molecule as described in the first aspect of the present invention;

[0102] (2) Detect whether a complex is formed, where the formation of a complex indicates the presence of uPAR and / or CD3 in the sample.

[0103] In another preferred embodiment, the detection is for in vitro, non-therapeutic, and non-diagnostic purposes.

[0104] In a tenth aspect of the invention, a composition for in vitro detection of uPAR and / or CD3 in a sample is provided, comprising, as in the first aspect of the invention, a cell binder molecule, as in the second aspect of the invention, a conjugate, as in the sixth aspect of the invention, a host cell, as in the fifth aspect of the invention, or a combination thereof as an active ingredient.

[0105] In an eleventh aspect of the present invention, a detection plate is provided, the detection plate comprising: a substrate (support plate) and a test strip, the test strip containing a cell conjugate molecule as described in the first aspect of the present invention, a recombinant protein as described in the second aspect of the present invention, a conjugate as described in the sixth aspect of the present invention, a host cell as described in the fifth aspect of the present invention, or a combination thereof.

[0106] In a twelfth aspect of the invention, a kit is provided, the kit comprising:

[0107] (1) A first container containing cell-connector molecules as described in the first aspect of the invention; and / or

[0108] (2) A second container containing a secondary antibody against a cell conjugate as described in the first aspect of the present invention;

[0109] Alternatively, the kit may contain a detection plate as described in the eleventh aspect of the present invention.

[0110] In a thirteenth aspect of the present invention, a method for preparing a recombinant polypeptide is provided, the method comprising:

[0111] (a) Culture the host cells as described in the fifth aspect of the present invention under suitable expression conditions;

[0112] (b) Isolate a recombinant polypeptide from the culture, said recombinant polypeptide being a cell conjugate molecule as described in the first aspect of the invention or a recombinant protein as described in the second aspect of the invention.

[0113] In a fourteenth aspect of the invention, the use of a cell-conjugating molecule as described in the first aspect of the invention, or a recombinant protein as described in the second aspect of the invention, or an antibody-drug conjugate as described in the sixth aspect of the invention, or a host cell as described in the fifth aspect of the invention, and / or a pharmaceutical composition as described in the eighth aspect of the invention, in the preparation of a medicament for treating diseases associated with abnormal expression or function of uPAR and / or CD3.

[0114] In another preferred embodiment, the abnormal expression of uPAR and / or CD3 refers to the overexpression of uPAR and CD3.

[0115] In another preferred embodiment, the overexpression refers to the ratio of the expression level of uPAR and / or CD3 (F1) to the expression level of normal cell tissue (F0) (i.e., F1 / F0) being ≥1.5, preferably ≥2, and more preferably ≥2.5.

[0116] In another preferred embodiment, the drug is used to prevent and / or treat tumor occurrence, growth and / or metastasis.

[0117] In another preferred embodiment, the GFD-binding protein includes (but is not limited to) uPAR.

[0118] In another preferred embodiment, the diseases associated with uPAR overexpression include: tumors, aging, aging-related diseases, or combinations thereof.

[0119] In another preferred embodiment, the disease is a malignant tumor that overexpresses uPAR (i.e., is uPAR positive).

[0120] In another preferred embodiment, the tumor includes hematologic malignancies and solid tumors.

[0121] In another preferred embodiment, the hematologic malignancy is acute myeloid leukemia (AML).

[0122] In another preferred embodiment, the solid tumor is selected from the group consisting of: breast cancer, gastric cancer, hepatobiliary cancer, colorectal cancer, bladder cancer, non-small cell lung cancer, ovarian cancer and esophageal cancer, glioma, lung cancer, pancreatic cancer, prostate cancer, etc., or combinations thereof.

[0123] In another preferred embodiment, the drug is used to inhibit uPAR-positive cells, preferably including: human pancreatic cancer cell line ASPC1, human pancreatic cancer cell line BXPC3, human lung cancer cell line A549, human lung cancer cell line H1299, or a combination thereof.

[0124] In another preferred embodiment, the drug is used to: kill tumor cells, clear senescent cells, or a combination thereof.

[0125] In a fifteenth aspect of the invention, a method for treating a disease associated with abnormal expression or function of uPAR and CD3 is provided, comprising administering to a subject in need an effective amount of a cell-conjugating molecule as described in a first aspect of the invention, or a recombinant protein as described in a second aspect of the invention, or a host cell as described in a fifth aspect of the invention, or an antibody-drug conjugate as described in a sixth aspect of the invention, or a pharmaceutical composition as described in an eighth aspect of the invention, or a combination thereof.

[0126] In another preferred embodiment, the diseases associated with abnormal uPAR expression or function include: tumors, aging, aging-related diseases, or combinations thereof.

[0127] In another preferred embodiment, the age-related diseases include: type II diabetes, autoimmune diseases, fatty liver, cirrhosis, liver fibrosis, pulmonary fibrosis, osteoarthritis, infections, and cardiovascular and cerebrovascular diseases.

[0128] In another preferred embodiment, the disease associated with abnormal uPAR expression or function includes tumors, preferably including pancreatic cancer.

[0129] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0130] The following figures are used to illustrate specific embodiments of the invention and are not intended to limit the scope of the invention as defined by the claims.

[0131] Figure 1The structural diagram of uPA is shown (Kumar AA, Buckley BJ, Ranson M. The Urokinase Plasminogen Activation System in Pancreatic Cancer: Prospective Diagnostic and Therapeutic Targets. Biomolecules. 2022;12(2):152. Published 2022Jan 18. doi:10.3390 / biom12020152).

[0132] Figure 2 The diagram shows the VH and VL chains of OKT3 as described in Journal of Biochemistry, 1996, 120:657-662. The VH amino acid sequence positions 1-19 are the signal peptide, and the VL amino acid sequence positions 1-23 are the signal peptide.

[0133] Figure 3 (A) shows schematic diagrams of the control CD19-BiTE and GFD-BiTE, SMB-BiTE, and Clone20-BiTE sequences, where CD8 SP is the CD8 signal peptide (1-21AA), GFD is the GFD domain of the uPAR ligand uPA (22-69AA), Linker is the linker sequence (70-84AA), OKT3 is the CD3 antibody scFv fragment (85-327AA), and His-tag is the HIS marker protein (328-333AAA).

[0134] Figure 3 (B) shows a schematic diagram of the GFD-BiTE plasmid structure, where copGFP is a fluorescent label used to detect the infection efficiency of BiTE.

[0135] Figure 3 (C) shows a schematic diagram of HindIII restriction enzyme digestion identification of the control CD19-BiTE and GFD-BiTE, SMB-BiTE, and Clone20-BiTE plasmid vectors.

[0136] Figure 4 The results show the plasmid transfection with HEK-293T;

[0137] Figure 5 The results show the infection of T cells with lentiviruses packaged with control CD19-BiTE and GFD-BiTE, SMB-BiTE, and Clone20-BiTE.

[0138] Figure 6This diagram illustrates the proliferation and viability of T cells after lentiviral infection.

[0139] Figure 7 The diagram shows the killing ability of control CD19-BiTE, GFD-BiTE, SMB-BiTE, and Clone20-BiTE on target cells.

[0140] Figure 8 The diagram shows the ability of control CD19-BiTE, GFD-BiTE, SMB-BiTE, and Clone20-BiTE to secrete TNF-α.

[0141] Figure 9 The study demonstrated the killing effect of control CD19-BiTE and GFD-BiTE proteins on tumor cells.

[0142] Figure 10 The diagram shows the killing effect of control CD19-BiTE and GFD-BiTE proteins on senescent cells and the release of cytokine IFN-γ. Detailed Implementation

[0143] Through extensive and in-depth research and numerous screenings, the inventors have, for the first time, developed a bispecific cell-binding molecule based on uPAR and its application. Experimental results show that the BiTE targeting the uPAR receptor of this invention exhibits significant cytotoxic effects on target cells and specific anti-tumor cell effects. Based on this, the present invention was completed.

[0144] The bispecific cell binder molecule provided by this invention consists of three parts: a first binding domain (ligand segment) targeting tumor cell surface antigen receptors, a linker segment, and a second binding domain (antibody segment) against human CD3 molecules. The ligand segment connects to tumor cells, while the anti-CD3 antibody segment connects to T lymphocytes, thereby effectively chemotactically attracting effector immune cells to the tumor site, enabling the body to exert anti-tumor effects more effectively.

[0145] the term

[0146] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Before describing the invention, it should be understood that the invention is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary. It should also be understood that the terminology used herein is intended only to describe particular embodiments and is not intended to be restrictive; the scope of the invention will be limited only by the appended claims.

[0147] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. As used herein, when referring to a specifically enumerated numerical value, the term “about” means that the value can vary from the enumerated value by no more than 1%. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0148] The amino acid three-letter codes and single-letter codes used in this invention are as described in J. biol. chem, 243, p3558 (1968).

[0149] As used herein, the term "treatment" refers to the administration of an oral or topical therapeutic agent to a patient, comprising the BiTE targeting the uPAR receptor of the present invention, T cells expressing the BiTE of the present invention, or combinations thereof, wherein the patient has one or more disease symptoms, and the therapeutic agent is known to have a therapeutic effect on these symptoms. Typically, the patient is given an amount of the therapeutic agent that effectively relieves one or more disease symptoms (therapeutic effective amount).

[0150] As used herein, the terms “optional” or “optionally” mean that the events or conditions described below may occur but are not required to occur. For example, “optionally containing 1-3 antibody heavy chain variable regions” means that the antibody heavy chain variable regions of a particular sequence may be present but are not required to be present, and may be 1, 2 or 3.

[0151] The term "sequence identity" as used in this invention refers to the degree of identity between two nucleic acid or two amino acid sequences when optimally aligned and compared with appropriate mutations such as substitutions, insertions, or deletions. The sequence identity between the sequences described in this invention and sequences exhibiting identity with them can be at least 85%, 90%, or 95%, preferably at least 95%. Non-limiting embodiments include 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%.

[0152] plasminogen activating system

[0153] The plasminogen activator (PA) system is an extracellular proteolytic enzyme system involved in various physiological and pathophysiological processes. The primary physiological function of the PA system is the conversion of inactive plasminogen into plasmin, mediated by two types of PA: tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). The PA system was initially thought to play a role in the dissolution of fibrous clots. However, subsequent studies have shown that the PA system has additional functions in other biological processes, such as embryogenesis, angiogenesis, cell migration, wound healing, inflammatory responses, and apoptotic cell death. In cancer, the PA system plays a dominant role in tumor growth, angiogenesis, tumor cell invasion, migration, and metastasis.

[0154] uPAR and Tumors: Nearly 90% of human cancer-related deaths are caused by the metastasis and spread of tumor cells. One of the key events behind metastasis is the proteolysis and degradation of the extracellular matrix (ECM) to facilitate tumor cell invasion, migration, and homing to distant organs. Although several protease systems are involved in this process, substantial evidence suggests that the uPA-uPAR system is a central player mediating proteolysis during cancer invasion and metastasis. The function of the uPA-uPAR system extends beyond proteolysis, playing a broader role in multiple stages of cancer development from tumorigenesis to metastasis.

[0155] Urokinase-type plasminogen activator receptor (uPAR) is a GPI-anchored cell membrane receptor composed of three homologous domains (DI, DII, DIII). Its primary function is to focus on the hydrolytic activity of urokinase (uPA) proteins on the cell surface, which are responsible for the degradation of extracellular matrix (ECM) components. Extensive literature demonstrates the importance of uPAR in the progression of most tumors, showing overexpression in the vast majority of tumor cells and tumor stroma, including solid tumors such as breast cancer, colorectal cancer, prostate cancer, pancreatic cancer, ovarian cancer, lung cancer, and glioma, as well as hematologic malignancies such as acute leukemia and myeloma. However, low uPAR expression is detected only in sternal epithelium, monocytes, macrophages, and neutrophil subsets in normal tissues. Studies have shown that (1) elevated uPAR expression is associated with adverse outcomes (invasion, metastasis, and recurrence) in patients with different types of cancer; (2) uPAR is closely related to tumor metabolism; (3) uPAR expression is associated with RAS mutations in patients with non-small cell lung cancer (NSCLC) and colorectal cancer (CRC); (4) uPAR is a key participant in regulating the transition between dormancy and proliferation in single-cell tumors; (5) uPAR is associated with multidrug resistance (MDR) in tumor cells; and (6) uPAR is associated with tumor angiogenesis. Given the important role of uPAR in tumorigenesis and development, a large number of studies have been conducted on: (1) exploring the role of uPAR in tumor diagnosis and prognosis; and (2) using uPAR as a therapeutic target for malignant tumors to explore the role of targeting uPAR in tumor treatment.

[0156] uPAR and Aging and Related Diseases: Aging is a complex process influenced by both genetic and environmental factors, closely related to permanent, progressive physiological cellular decline, and is one of the reasons for the increasingly serious aging of the population. Aging can significantly increase the body's morbidity, namely aging-related diseases, such as diabetes, Alzheimer's disease, cardiovascular disease, and neurodegenerative diseases.

[0157] Cellular senescence is characterized by stable cell cycle arrest and senescence-related secretory programs that regulate the tissue microenvironment. Physiologically, senescence is a tumor-suppressing mechanism that can prevent the expansion of precancerous cells and plays a beneficial role in wound healing responses. Pathologically, the abnormal accumulation of senescent cells creates an inflammatory environment, leading to chronic tissue damage and diseases such as liver and lung fibrosis, atherosclerosis, diabetes, and osteoarthritis. Eliminating senescent cells from damaged tissues can improve these pathological symptoms and even promote longevity.

[0158] Previous studies have confirmed that uPAR is an important surface marker of senescent cells. A portion of uPAR is cleaved by proteases upon binding to its ligand, producing soluble uPAR (suPAR). Senescent cells secrete suPAR as part of the senescence-associated secretory phenotype (SASP).

[0159] Therefore, the bispecific cell binder molecule targeting uPAR of the present invention has important value for the treatment of solid tumors, hematologic malignancies, aging, aging-related diseases or other diseases related to abnormal uPAR expression.

[0160] uPA

[0161] Urokinase-type plasminogen activator (urokinase, uPA) is a key serine protease involved in the conversion of inactive plasminogen into active plasmin, which in turn plays a role in a series of events in the plasminogen cascade. It was first discovered in urine in 1947 by MacFarlane and Pilling, who reported a novel, unnamed protein with fibrinolytic activity. Five years later, Sobel and colleagues named this unnamed protein "urokinase." Further research has shown that urokinase is also present in plasma and many tissues of the extracorporeal membrane (ECM).

[0162] pro-uPA (411 amino acids) is the precursor structure of uPA. Pro-uPA consists of three domains: a growth factor-like domain (GFD) homologous to epidermal growth factor (EGF), a kringle domain (KD), and a serine protease domain. GFD (1-49 amino acids) and KD (50-131 amino acids) are located at the N-terminus, while the serine protease domain (159-411 amino acids) is located at the C-terminus. Between the N- and C-termini is a linker region (132-158 amino acids). When pro-uPA needs to be secreted, the peptide bond between Lys158 and IIe159 in the linker region is cleaved to produce a disulfide-linked double-stranded form of uPA. After another round of proteolytic cleavage at the peptide bond between Lys135 and Lys136, the double-stranded form of uPA can be further cleaved into two parts: (1) a low molecular weight form of uPA with a serine protease domain and catalytic activity, and (2) an inactive amino-terminal fragment (ATF) composed of GFD and KD. Studies have shown that the GFD domain is the key region for uPA to specifically bind to uPAR, and it has been confirmed that the ATF fragment, the double-stranded form of uPA, and pro-uPA can all bind specifically to uPAR with almost the same affinity.

[0163] uPA's specific binding to uPAR depends on its GFD domain. This invention uses the GFD domain as the first molecular binding domain of a bispecific cell binder molecule to recognize target (uPAR) positive cells, thereby exerting its effect.

[0164] Antibody

[0165] The second binding domain of the cell-binding molecule of the present invention specifically binds to CD3. This second binding domain may be an anti-CD3 antibody or an antigen-binding fragment of an antibody.

[0166] As used herein, the terms "antibody" or "immunoglobulin" refer to isotetraglycoproteins of approximately 150,000 Daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by a covalent disulfide bond, although the number of disulfide bonds between heavy chains varies among different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other; the constant regions of the light chains are opposite the first constant region of the heavy chains, and the variable regions of the light chains are opposite the variable regions of the heavy chains. Specific amino acid residues form interfaces between the variable regions of the light and heavy chains.

[0167] As used herein, the term "variable" refers to the fact that certain portions of the variable region of an antibody differ sequentially, contributing to the binding and specificity of various specific antibodies to their specific antigens. However, variability is not uniformly distributed throughout the entire variable region of an antibody. It is concentrated in three segments within the variable regions of the light and heavy chains, known as complementarity-determining regions (CDRs) or hypervariable regions. The more conserved portions of the variable region are called framework regions (FRs). The variable regions of the native heavy and light chains each contain four FRs, which are generally β-sheet configurations linked by three CDRs forming a linking loop, and in some cases, partially β-sheet structures. The CDRs in each chain are tightly packed together by the FR regions and, together with the CDRs of the other chain, form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)). Constant regions do not directly participate in antibody-antigen binding, but they exhibit different effector functions, such as participating in antibody-dependent cytotoxicity.

[0168] The term "antibody fragment" or "antigen-binding fragment" is used to refer to a portion of an antibody, such as F(ab')2, F(ab)2, Fab', Fab, Fv, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv), fragments containing VL or VH domains, fragments generated from Fab expression libraries, and anti-id antibodies. Regardless of structure, antibody fragments bind to the same antigen recognized by the intact antibody. The term "antibody fragment" includes DART and biantibodies. The term "antibody fragment" also includes any synthetic or genetically engineered protein containing a variable region of an immunoglobulin that functions like an antibody by forming a complex with a specific antigen. A "single-chain variable region" or "scFv" refers to a fusion protein of the variable region of the heavy chain (VH) and light chain (VL) of an immunoglobulin. In some aspects, the domain of said region is linked to a short linker peptide of 10 to about 25 amino acids. The linker can be enriched with glycine for flexibility and serine or threonine for solubility, and can connect to the N-terminus of VH or the C-terminus of VL, or vice versa. Despite the removal of the constant region and the introduction of the linker, this protein retains the specificity of the original immunoglobulin. Regarding IgG, the standard immunoglobulin molecule contains two identical light chain polypeptides with a molecular weight of approximately 23,000 Daltons and two identical heavy chain polypeptides with molecular weights of 53,000–70,000. The four chains are typically linked by disulfide bonds in a “Y” configuration, with the light chain linking to the heavy chain from the “Y” and extending through the variable region.

[0169] As described above, the variable region allows antibodies to selectively recognize and specifically bind to epitopes on antigens. That is, the VL and VH domains of the antibody, or a subset of the antibody's complementarity-determining regions (CDRs), combine to form a variable region defining a three-dimensional antigen-binding site. This quaternary antibody structure forms the antigen-binding site located at the ends of each arm of each Y-configuration. More specifically, this antigen-binding site is defined by three CDRs (i.e., HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, and LCDR3) on each of the VH and VL chains. In some cases, such as certain immunoglobulin molecules derived from camel species or engineered based on camel immunoglobulins, or immunoglobulin molecules that consist only of a heavy chain lacking a light chain or only of a light chain lacking a heavy chain, the immunoglobulin molecule may be composed of a heavy chain lacking only a light chain.

[0170] As used herein, antibodies, antibody fragments, or antibody domains also include “variants” thereof, which are defined as antibodies, antibody fragments, or antibody domains that: (1) have at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the original antibody, antibody fragment, or antibody domain; and (2) specifically bind to the same target that specifically binds to the original antibody, antibody fragment, or antibody domain. It should be understood that when sequence identity is expressed in the form of “at least x% identical” or “at least x% identity,” such embodiments include any and all numerical percentages equal to or higher than the lower limit. Furthermore, it should be understood that the presence of an amino acid sequence in this application should be interpreted as additional disclosure or inclusion of an amino acid sequence having 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% identity with that amino acid sequence.

[0171] The antibody contained in the cell binder molecule of this invention can be an immunologically active antibody fragment, such as a Fab or (Fab')2 fragment; an antibody heavy chain; or an antibody light chain. The antibody used in this invention is preferably in the form of a single-chain antibody (scFv), which contains variable regions of the antibody heavy chain and light chain, but no constant regions, and is the smallest antibody fragment with all antigen-binding sites. Generally, Fv antibodies also contain a polypeptide linker between VH and VL domains and are capable of forming the structure required for antigen binding.

[0172] The term "epitope" or "antigenic determinant" refers to a site on an antigen where an immunoglobulin or antibody specifically binds. An epitope typically comprises at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive or discontinuous amino acids in a unique spatial conformation. An epitope can be a discontinuous three-dimensional spatial site on the antigen, recognized by the antibody or antigen-binding fragment of the present invention.

[0173] The terms "specific binding," "selective binding," "selective binding," and "specific binding" refer to the binding of an antibody to a pre-defined epitope on an antigen. Typically, antibodies bind at a concentration of approximately less than 10... -7 M, for example, approximately less than 10 -8 M, 10 -9 M or l0 -10 M or lower affinity (KD) binding.

[0174] The antibodies used in this invention include not only complete antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Therefore, the antibodies used in this invention also include antibody fragments, derivatives, and analogs.

[0175] In this invention, the antibody can be monospecific, bispecific, trispecific, or more multiple specific.

[0176] As used in this article, the terms “heavy chain variable region” and “VH” are used interchangeably.

[0177] As used in this article, the terms “variable region” and “complementarity determining region (CDR)” are used interchangeably.

[0178] The term "CDR" refers to one of the six hypervariable regions within the variable domain of an antibody that primarily facilitate antigen binding. One of the most commonly used definitions of these six CDRs is provided by Kabat EA et al., (1991) Sequences of proteins of immune interest. NIH Publication 91-3242.

[0179] In a preferred embodiment of the present invention, the bispecific cell binder molecule is a single-chain polypeptide comprising an anti-CD3 single-chain antibody segment, a linker peptide, and a Pep42 ligand segment, wherein the anti-CD3 single-chain antibody is a conventional single-chain antibody in the art, comprising a heavy chain variable region and a light chain variable region.

[0180] In this invention, the animal is preferably a mammal, such as a mouse.

[0181] Meanwhile, those skilled in the art should understand that although the first binding domain of BiTE in this invention is preferably a ligand of uPAR, the first binding domain can also be selected as a specific anti-uPAR antibody, as long as it can achieve the cell binding effect of this invention.

[0182] Bispecific cell-connector molecules

[0183] As used herein, the terms “bispecific cell binder molecule,” “bispecific cell binder,” “cell binder,” “bispecific antibody,” and “BiTE” are used interchangeably and all refer to the cell binder molecule provided in the first aspect of the present invention that is capable of simultaneously binding uPAR and CD3.

[0184] Bispecific cell conjugate molecules are composed of two protein or polypeptide sequences (antibodies being the most common) that bind to different target proteins. The function of the BiTE of this invention is determined by the specific gene sequences of the uPA ligand segment and the CD3 antibody segment. The BiTE of this invention can simultaneously bind uPAR and CD3, connecting to tumor cells via the ligand segment and T lymphocytes via the anti-CD3 antibody segment, thereby effectively connecting effector immune cells to tumor cells and exerting a more effective anti-tumor effect. Using the VL, VH segment genes or complementarity-determining region (CDR) genes of this invention, different forms of genetically engineered cell conjugate molecules can be modified and produced in any expression system utilizing prokaryotic and eukaryotic cells.

[0185] As used herein, the term "bispecific" refers to a molecule comprising at least two binding domains with different binding specificities. Each binding domain is capable of specifically binding to a target molecule. In some embodiments, a bispecific cell binder is a polymer molecule having two or more peptides. In some embodiments, the binding domain comprises a single-domain antibody that specifically binds to a target protein, an antigen-binding fragment of an antibody, a single-chain variable fragment, or a variable region, or a CDR, or a combination thereof. In some embodiments, the binding domain comprises a ligand or a fragment thereof that specifically binds to a target protein. In some embodiments, the binding domain comprises a combination of the above structures.

[0186] At least two targeting domains of the cell-connector molecule of the present invention are optionally linked by a linker peptide. Preferred linker peptide sequences are shown in SEQ ID NO.16, but are not limited thereto.

[0187] In this invention, the BiTE of this invention also includes its conserved variants, which refer to polypeptides formed by replacing up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids with amino acids of similar or analogous properties compared to the amino acid sequence of the BiTE of this invention. These conserved variant polypeptides are preferably generated by amino acid substitutions according to Table 1.

[0188] Table 1

[0189]

[0190]

[0191] Furthermore, the amino acid sequence also includes a sequence formed by adding, deleting, modifying and / or substituting at least one amino acid sequence, preferably with at least 80% homology or sequence identity, more preferably at least 85%, more preferably at least 90%, and most preferably at least 95% amino acid sequence.

[0192] Methods for determining sequence homology or identity known to those skilled in the art include, but are not limited to: Computational Molecular Biology, Lesk, AM, ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, DW, ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, AM and Griffin, HG, eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., Stockton Press, New York, 1991; and Carillo, H. and Lipman, D., SIAM J. Applied. Math., 48:1073 (1988). Preferred methods for determining identity aim to achieve the largest possible match between the tested sequences. Methods for determining identity are compiled into publicly available computer programs. Preferred computer program methods for determining identity between two sequences include, but are not limited to: the GCG package (Devereux, J. et al., 1984), BLASTP, BLASTN, and FASTA (Altschul, S., F. et al., 1990). The BLASTX program is publicly available from NCBI and other sources (BLAST manual, Altschul, S. et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S. et al., 1990). The well-known Smith-Waterman algorithm can also be used for identity determination.

[0193] In the above-described content of the present invention, the number of added, deleted, modified and / or substituted amino acids is preferably no more than 40% of the total number of amino acids in the initial amino acid sequence, more preferably no more than 35%, more preferably 1-33%, more preferably 5-30%, more preferably 10-25%, and more preferably 15-20%.

[0194] In the above-described content of the present invention, more preferably, the number of added, deleted, modified and / or substituted amino acids can be 1-7, more preferably 1-5, more preferably 1-3, and more preferably 1-2.

[0195] Recombinant protein

[0196] The present invention also provides a recombinant protein comprising the cell agonist molecule of the present invention.

[0197] The recombinant protein of the present invention may include a monomer, dimer, or polymer of the cell conjugate molecule of the present invention; or a multispecific (e.g., trispecific) cell conjugate.

[0198] The recombinant protein is prepared using conventional methods in the art. Preferably, the preparation method involves isolating the protein from a recombinant expression transformant or obtaining it through artificial synthesis of the protein sequence. The preferred method for isolating the protein from the recombinant expression transformant is as follows: cloning a nucleic acid molecule encoding the protein and carrying a point mutation into a recombinant vector; transforming the obtained recombinant vector into a transformant to obtain a recombinant expression transformant; and culturing the obtained recombinant expression transformant to isolate and purify the recombinant protein.

[0199] Nucleic acid

[0200] The present invention also provides a polynucleotide molecule encoding the aforementioned cell binder molecule. The polynucleotide of the present invention can be in DNA or RNA form. The DNA form includes cDNA, genomic DNA, or synthetically produced DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand. The coding region sequence encoding the mature polypeptide can be identical to or a degenerate variant of the coding region sequence of the cell binder of the present invention. As used herein, "degenerate variant" refers to a nucleic acid sequence encoding a sequence having the same amino acid sequence as the polypeptide of the present invention, but with a different coding region sequence.

[0201] The polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence that encodes only the mature polypeptide; a coding sequence of the mature polypeptide and various additional coding sequences; a coding sequence of the mature polypeptide (and optional additional coding sequences) and a non-coding sequence.

[0202] The term "polynucleotide encoding a polypeptide" can refer to a polynucleotide that includes the polypeptide, or it can also include additional coding and / or non-coding sequences.

[0203] The present invention also relates to polynucleotides that hybridize with the above-described sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that hybridize with the polynucleotides described herein under stringent conditions. In the present invention, “stringent conditions” means: (1) hybridization and elution at lower ionic strength and higher temperatures, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with a denaturing agent, such as 50% (v / v) formamide, 0.1% fetal bovine serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization only occurs when the identity between the two sequences is at least 90%, preferably at least 95%. Furthermore, the polypeptides encoded by the hybridizable polynucleotides have the same biological functions and activities as the mature polypeptides shown in SEQ ID NO. 4 and SEQ ID NO. 9.

[0204] The full-length nucleotide sequence or fragments thereof of the cell conjugate molecule of the present invention can generally be obtained by PCR amplification, recombinant methods, or artificial synthesis. One feasible method is to synthesize the relevant sequence artificially, especially when the fragment length is short. Typically, long fragments can be obtained by first synthesizing multiple small fragments and then ligating them. Furthermore, its coding sequence and expression tag (such as 6His) can be fused together to form a fusion protein.

[0205] Vector and host cell

[0206] The present invention also provides a recombinant expression vector containing the nucleic acid.

[0207] The recombinant expression vector described herein can be obtained by conventional methods in the art, namely, by linking the nucleic acid molecule described in this invention to various expression vectors. The expression vector can be any conventional vector in the art, as long as it can accommodate the aforementioned nucleic acid molecule. Preferably, the vector includes various plasmids, granules, bacteriophages, or viral vectors, etc.

[0208] The present invention also provides a recombinant expression transformant comprising the above-described recombinant expression vector.

[0209] The recombinant expression transformant is prepared using conventional methods in the art, preferably by transforming the recombinant expression vector into host cells. The host cells can be any type of host cell commonly used in the art, as long as they allow the recombinant expression vector to replicate stably and effectively express the carried nucleic acid. Preferably, the host cells are E. coli TG1 or E. coli BL21 cells, or HEK-293T or CHO cells. Transforming the aforementioned recombinant expression plasmid into host cells yields the preferred recombinant expression transformant of this invention. The transformation method is a conventional method in the art, preferably a chemical transformation, heat shock, or electroporation.

[0210] In a preferred embodiment of the present invention, the carriers that can be selected include: pCDH, pTOMO, pGEM, pELNS, pMSGV, or combinations thereof.

[0211] In a preferred embodiment of the present invention, the host cells that can be selected include: T cells, NK cells, or combinations thereof.

[0212] Preparation of cell conjugate molecules

[0213] The preferred method for preparing the DNA sequence of the cell-connector molecule or its fragment of the present invention involves fusing the coding sequences of the ligand segment and the antibody segment together to form a single-chain polypeptide. Alternatively, conventional techniques, such as PCR amplification or genomic library screening, can be used.

[0214] Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transferring it into cells, and then isolating the sequence from the proliferated host cells using conventional methods.

[0215] In addition, sequences can be synthesized artificially, especially when the fragment length is short. Typically, long sequences can be obtained by first synthesizing multiple small fragments and then joining them.

[0216] Currently, the DNA sequence encoding the cell conjugate (or a fragment thereof, or a derivative thereof) of the present invention can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors, etc.) and cells known in the art. Furthermore, mutations can be introduced into the protein sequence of the present invention through chemical synthesis.

[0217] The present invention also relates to vectors comprising the aforementioned suitable DNA sequences and suitable promoters or control sequences. These vectors can be used to transform suitable host cells to enable them to express proteins.

[0218] The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Preferred cells include (but are not limited to): T cells.

[0219] Typically, host cells transformed with this invention are cultured under conditions suitable for the expression of the cell conjugate molecules of this invention. The cell conjugate molecules of this invention are then purified using conventional immunoglobulin purification steps, such as protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, ion exchange chromatography, hydrophobic chromatography, molecular sieve chromatography, or affinity chromatography—common separation and purification methods well-known to those skilled in the art—to obtain the cell conjugate molecules of this invention.

[0220] The resulting cell conjugates can be identified using conventional methods. For example, their binding specificity can be determined by immunoprecipitation or in vitro binding assays (such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA)). Their binding affinity can be determined, for example, by the Scatchard analysis described by Munson et al., Anal. Biochem., 107:220 (1980).

[0221] The cell conjugate of the present invention can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant protein can be separated and purified by various separation methods utilizing its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeabilization, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques and combinations thereof.

[0222] The following are two conventional methods for preparing bispecific cell-binding molecules in this field;

[0223] 1. First, a bispecific antibody gene was prepared using PCR. This gene was then cloned into the expression vector pRB199 and transformed into *E. coli* strain BL21(λDE3) to prepare inclusion bodies. Subsequently, the inclusion bodies were denatured by adding 6M guanidine-HCl and DET (dithioerythritol), followed by a 100-fold dilution with renaturation buffer. The mixture was rapidly mixed at 4°C and incubated at 4°C for 72 h to allow the protein to refold. After renaturation, the protein was dialyzed against 0.1M Tris and 0.5M NaCl at a 1:10 ratio, repeated three times, and then filtered (0.2 μm). Metal ion affinity chromatography was then performed. Finally, the protein was purified using a rapid protein liquid chromatography system (BioLogic DuoFlow 10 System; Bio-Rad) using a histidine-tagged fusion protein purification column. The protein was eluted with an imidazole stepwise gradient at a flow rate of 1 mL / min. The product was subjected to column chromatography (Sartorius Stedim Biotech) to remove proteins with a molecular weight greater than 10,000, dialyzed with PBS, and filtered for sterilization. After determining the concentration, silver staining identification was performed using SDS / PAGE (see PNAS, 2013, 110(1):270-275).

[0224] 2. CHO cells were infected with lentivirus containing bispecific antibodies. After culturing for 72 hours post-infection, fluorescent expression was observed in the CHO cells. Successfully infected cell lines were expanded. CHO cells stably expressing bispecific antibodies continued to secrete the protein. Cell supernatant was collected for protein purification and concentration. Subsequently, purification was performed using a rapid protein liquid chromatography system (BioLogic DuoFlow 10 System; Bio-Rad), employing a histidine-tagged fusion protein purification column. The sample was passed through a nickel column with five times its volume of equilibration buffer at a flow rate of 0.5-1 ml / min. After equilibration, the sample was passed through the nickel column again at a flow rate of 0.5 ml / min. The nickel column was washed with five times its volume of equilibration buffer to remove background proteins until the eluent absorbance at 280 nm was 0. The target protein was eluted with imidazole at a flow rate of 0.5 ml / min. The protein was then concentrated using ultrafiltration and the salt solution was replaced. After the concentration was determined, Western blot was used for identification (see Oncoimmunology, 2015, 4(4):e989776).

[0225] Those skilled in the art can make conventional selections or equivalent modifications to the above methods to prepare or produce the bispecific cell-binding molecule of the present invention.

[0226] Antibody-drug conjugates (ADCs)

[0227] The term "antibody-drug conjugate (ADC)" as used in this invention refers to the conjugate formed by the cell binder molecule and the effector molecule of this invention.

[0228] Typically, the antibody-drug conjugate includes the cell-conjugating molecule and an effector molecule, the cell-conjugating molecule being conjugated to the effector molecule, preferably chemically conjugated. The effector molecule is preferably a drug with therapeutic activity. Furthermore, the effector molecule can be one or more of a toxic protein, a chemotherapeutic agent, a small molecule drug, or a radionuclide.

[0229] The cell binder molecule and the effector molecule of the present invention can be coupled via a coupling agent. Examples of the coupling agent can be any one or more of non-selective coupling agents, carboxyl-based coupling agents, peptide chains, and disulfide-based coupling agents. The non-selective coupling agent refers to a compound that covalently links the effector molecule and the cell binder molecule, such as glutaraldehyde. The carboxyl-based coupling agent can be any one or more of maleic aconitine-based coupling agents (e.g., maleic aconitine) and acylhydrazone-based coupling agents (with an acylhydrazone as the coupling site).

[0230] Certain residues on cell binder molecules (such as Cys or Lys) are used to attach to a variety of functional groups, including imaging agents (e.g., chromophores and fluorophores), diagnostic agents (e.g., MRI contrast agents and radioisotopes), stabilizers (e.g., ethylene glycol polymers), and therapeutic agents. Cell binder molecules can be coupled to functional agents to form cell binder molecule-functional agent conjugates. Functional agents (e.g., drugs, diagnostic reagents, stabilizers) are coupled (covalently linked) to cell binder molecules. Functional agents can be attached to cell binder molecules directly or indirectly through linkers.

[0231] Cell-conjugator molecules can be coupled with drugs to form antibody-drug conjugates (ADCs). Typically, an ADC contains a linker between the drug and the cell-conjugator molecule. The linker can be degradable or non-degradable. Degradable linkers are typically readily degraded in intracellular environments, such as at the target site, thereby releasing the drug from the cell-conjugator molecule. Suitable degradable linkers include, for example, enzyme-degradable linkers, including peptide-containing linkers that can be degraded by intracellular proteases (e.g., lysosomal proteases or endosomal proteases), or sugar linkers, such as glucuronidase-containing linkers. Peptide linkers can include, for example, dipeptides, such as valine-citrulline, phenylalanine-lysine, or valine-alanine. Other suitable degradable linkers include, for example, pH-sensitive linkers (e.g., linkers that hydrolyze at pH less than 5.5, such as hydrazone linkers) and linkers that degrade under reducing conditions (e.g., disulfide linkers). Non-degradable linkers typically release the drug when the cell-conjugator molecule is hydrolyzed by proteases.

[0232] The drug can be any cytotoxic, cell growth-inhibiting, or immunosuppressive drug. In one embodiment, a linker connects the cell binder molecule and the drug, and the drug has a functional group that can bond with the linker. For example, the drug may have an amino, carboxyl, thiol, hydroxyl, or ketone group that can bond with the linker. In the case where the drug is directly connected to the linker, the drug has a reactive group before being connected to the cell binder molecule.

[0233] Useful drug classes include, for example, anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemotherapy sensitizers, topoisomerase inhibitors, and vinca alkaloids. Examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and microtubule inhibitors. Typical cytotoxic drugs include, for example, auristatins, camptothecins, duocarmycins, etoposides, maytansines and maytansinoids (e.g., DM1 and DM4), taxanes, benzodiazepines or benzodiazepine-containing drugs (e.g., pyrrolo[1,4]benzodiazepines (PBDs), indolinobenzodiazepines and oxazolidinobenzodiazepines)) and vinca alkaloids.

[0234] In this invention, the drug-linker can be used to form an ADC in a simple step. In other embodiments, bifunctional linker compounds can be used to form an ADC in a two- or multi-step process. For example, cysteine ​​residues react with the reactive portion of the linker in a first step, and in a subsequent step, the functional groups on the linker react with the drug to form an ADC.

[0235] Typically, functional groups on the linker are selected to facilitate specific reaction with suitable reactive groups on the drug moiety. As a non-limiting example, azide-based moieties can be used to specifically react with reactive alkynyl groups on the drug moiety. The drug is covalently bound to the linker via a 1,3-dipolar cycloaddition between the azide and alkynyl groups. Other useful functional groups include, for example, ketones and aldehydes (suitable for reaction with hydrazides and alkoxyamines), phosphine (suitable for reaction with azides); isocyanates and isothiocyanates (suitable for reaction with amines and alcohols); and activated esters, such as N-hydroxysuccinimide esters (suitable for reaction with amines and alcohols). These and other linking strategies, such as those described in Bioconjugation Techniques, Second Edition (Elsevier), are well known to those skilled in the art. Those skilled in the art will understand that for selective reaction between the drug moiety and the linker, when a complementary pair of reactive functional groups is selected, each member of that complementary pair can be used for either the linker or the drug.

[0236] application

[0237] The present invention also provides the use of the bispecific cell conjugate molecules, antibody-drug conjugates (ADCs), recombinant proteins, chimeric antigen receptor (CAR) constructs and / or immune cells of the present invention, for example, for the preparation of diagnostic agents or pharmaceuticals.

[0238] Preferably, the drug is a drug for the prevention and / or treatment of diseases associated with abnormal uPAR expression or function.

[0239] In this invention, the diseases associated with abnormal uPAR expression or function are conventional diseases associated with abnormal uPAR expression or function in the art, including: tumors, aging, aging-related diseases, and combinations thereof.

[0240] In this invention, the cancer referred to is a conventional cancer in the art, including hematologic malignancies and solid tumors.

[0241] Detection uses and kits

[0242] The bispecific cell-interceptor molecule of the present invention or its ADC can be used in detection applications, such as for detecting samples, thereby providing diagnostic information.

[0243] In this invention, the samples used include cells, tissue samples, and biopsy specimens. The term "biopsy" as used in this invention should include all types of biopsies known to those skilled in the art. Therefore, biopsies used in this invention can include, for example, resected tumor samples, tissue samples prepared by endoscopic methods or puncture or needle biopsy of organs.

[0244] The samples used in this invention include fixed or preserved cell or tissue samples.

[0245] The present invention also provides a kit containing the cell conjugate (or a fragment thereof) of the present invention. In a preferred embodiment of the present invention, the kit further includes a container, instructions for use, a buffer, etc. In a preferred embodiment, the cell conjugate of the present invention can be fixed to a detection plate.

[0246] Pharmaceutical Composition

[0247] The present invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition containing the aforementioned cell binder or its active fragment or fusion protein or its ADC or corresponding immune cell, and a pharmaceutically acceptable carrier. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium, wherein the pH is typically about 5-8, preferably about 6-8, although the pH may vary depending on the nature of the formulated substance and the condition to be treated.

[0248] The prepared pharmaceutical composition can be administered via conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or local administration. Typically, the pharmaceutical composition of the present invention is preferably administered by injection or oral administration. Injection administration preferably includes intravenous injection, arterial injection, intramuscular injection, intraperitoneal injection, intradermal injection, or subcutaneous injection. The pharmaceutical composition is in various dosage forms conventional in the art, preferably in solid, semi-solid, or liquid form, and can be an aqueous solution, non-aqueous solution, or suspension, more preferably tablets, capsules, granules, injections, or infusions.

[0249] The cell conjugate described in this invention can also be expressed intracellularly by a nucleotide sequence for cell therapy.

[0250] The pharmaceutical composition described in this invention is a pharmaceutical composition for the prevention and / or treatment of diseases associated with abnormal expression or function of uPAR and / or CD3.

[0251] The pharmaceutical compositions of the present invention contain a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the cell conjugate (or its conjugate) described above, or a safe and effective amount (1 × 10⁻⁶ wt%) of the present invention. 3 -1×10 8 Cells / ml, more optimal 1×10 4 -1×10 7Engineered immune cells (cells / ml) and pharmaceutically acceptable carriers or excipients. Such carriers include (but are not limited to): saline, buffer solutions, glucose, water, glycerol, ethanol, and combinations thereof. The pharmaceutical formulation should be matched to the route of administration. The pharmaceutical compositions of the present invention can be formulated into injectable forms, for example, prepared using conventional methods with physiological saline or aqueous solutions containing glucose and other excipients. Pharmaceutical compositions such as injections and solutions should preferably be manufactured under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 1 microgram / kg body weight to about 5 milligrams / kg body weight per day. Furthermore, the peptides of the present invention can also be used with other therapeutic agents.

[0252] In a preferred embodiment of the invention, the polypeptide of the invention can be used in combination with other therapeutic agents for treating and / or preventing cancer and / or cancer metastasis.

[0253] In this invention, preferably, the pharmaceutical composition further includes one or more pharmaceutical carriers. The pharmaceutical carrier is a conventional pharmaceutical carrier in the art, and can be any suitable physiologically or pharmaceutically acceptable pharmaceutical excipient. The pharmaceutical excipient is a conventional pharmaceutical excipient in the art, preferably including pharmaceutically acceptable excipients, fillers, or diluents. More preferably, the pharmaceutical composition comprises 0.01–99.99% of the above-mentioned protein and 0.01–99.99% of the pharmaceutical carrier, where the percentage is a percentage by mass of the pharmaceutical composition.

[0254] In this invention, preferably, the dosage of the pharmaceutical composition is an effective amount, which is an amount capable of alleviating or delaying the progression of a disease, degenerative or damaging condition. The effective amount can be determined on an individual basis and will be partly based on considerations of the symptoms to be treated and the desired outcome. Those skilled in the art can determine the effective amount by using the aforementioned factors, such as individual baselines, and by using experiments not exceeding the conventional range.

[0255] When using conjugates, a safe and effective amount is administered to mammals, typically at least about 10 micrograms per kilogram of body weight, and in most cases not exceeding about 50 milligrams per kilogram of body weight. Preferably, this dose is between about 10 micrograms per kilogram of body weight and about 20 milligrams per kilogram of body weight. Of course, the specific dosage should also consider factors such as the route of administration and the patient's health condition, which are all within the scope of a skilled physician's expertise.

[0256] Methods and compositions for detecting uPAR and / or CD3 in samples

[0257] The present invention also provides a method for detecting uPAR and CD3 in a sample (e.g., detecting overexpression of uPAR and CD3, comprising the following steps: the cell conjugate described above is brought into contact with the sample to be tested in vitro, and the method for detecting whether the cell conjugate and the sample to be tested bind to form an antigen-antibody complex and / or a receptor-ligand complex is detected.

[0258] The term "overexpression" is conventional in the art and refers to the overexpression of uPAR and CD3 in RNA or protein in the sample to be tested (due to increased transcription, post-transcriptional processing, translation, post-translational processing, and changes in protein degradation), as well as local overexpression and increased functional activity due to changes in protein transport patterns (increased nuclear localization) (e.g., in the case of increased enzymatic hydrolysis of the substrate).

[0259] In this invention, the detection method for whether or not a complex is formed is a conventional detection method in the art, and preferably a flow cytometry (FACS) assay.

[0260] This invention provides a composition for detecting uPAR and CD3 in a sample, comprising the aforementioned cell conjugate, recombinant protein, antibody conjugate, immune cell, or a combination thereof as an active ingredient. Preferably, it further comprises a compound composed of functional fragments of the aforementioned antibody as an active ingredient.

[0261] Main advantages of the invention

[0262] The bispecific cell-binding molecule constructed in this invention simultaneously targets uPAR tumor antigen and T cells. It can be directly injected into the body, or carried and continuously expressed in vivo by in vivo cells (such as NK cells, T cells, CAR-T cells, etc.), thereby enabling the bispecific cell-binding molecule to exert its cytotoxic effect in vivo while simultaneously co-presenting with a sufficient number of T effector cells, thus optimizing the efficiency of the effector's action. Its main advantages include:

[0263] 1) High targeting: Prepare bispecific cell binder molecules that target uPAR-positive tumors, which can effectively bind to the receptor sites of tumor target cells.

[0264] 2) Small molecular weight: The preferred bispecific cell binder of the present invention has an scFv structure at one end and a structure with only 48 amino acids at the other end. In the blood, it is not only conducive to binding tumor cells and T cell surface antigens, but also has a small total protein molecular weight, which is easy to produce.

[0265] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0266] The sequences involved in the embodiments of the present invention are shown in the table below.

[0267] Table 2 Sequence List

[0268]

[0269]

[0270]

[0271]

[0272] In SEQ ID NO.19, positions 1-999 constitute the nucleotide coding sequence, and the last three bases... TAG To terminate the codon.

[0273] Example 1: Preparation of molecular genes for bispecific cell conjugates

[0274] In this embodiment, the sequence of a bispecific cell-connector molecule was constructed.

[0275] The GFD ligand sequence for binding uPAR used in this embodiment is derived from the uPA molecule (see Kumar AA, et al. Biomolecules. 2022; 12(2):152.).

[0276] The SMB sequence that binds uPAR used in this embodiment is derived from the VTN ligand (see Smith HW, Marshall CJ. Nat Rev Mol Cell Biol. 2010 Jan; 11(1):23-36.; Deng G, et al. J Cell Biol. 1996 Sep; 134(6):1563-71; Huai Q, et al. Nat Struct Mol Biol. 2008 Apr; 15(4):422-3.).

[0277] The specific binding peptide Clone 20 sequence for uPAR used in this embodiment is from the literature (see Goodson RJ, et al. Proc Natl Acad Sci US A. 1994 Jul 19; 91(15):7129-33. doi:10.1073 / pnas.91.15.7129.PMID:8041758; PMCID:PMC44352.).

[0278] The anti-CD19 antibody sequence used in this example is from patent (PCT / EP99 / 02693) and is used as a control.

[0279] The anti-CD3 antibody sequence used in this embodiment is derived from the sequence of the antibody clone OKT3 (see Arakawa F, Kuroki M, Kuwahara M, Senba T, Ozaki H, Matsuoka Y, Misumi Y, Kanda H, Watanabe T. Cloning and sequencing of the VH and V kappa genes of an anti-CD3 monoclonal antibody, and construction of a mouse / human chimeric antibody. J Biochem. 1996 Sep; 120(3):657-62. doi:10.1093 / oxfordjournals.jbchem.a021462.PMID:8902633). The VH and VL chains of OKT3 are as follows: Figure 2 As shown. The VH chain sequence of the anti-CD3 antibody used in this invention is obtained from GenBank BAA11539.1, and the VL chain sequence is obtained from GenBank AAC28463.1.

[0280] The anti-CD3 antibody VH and VL are linked by linker2 (SEQ ID NO.13) to form the VH-linker2-VL single-chain structure of OKT3, which will be referred to as OKT3 in the following text. Its amino acid sequence and nucleotide sequence are shown in Table 2.

[0281] The cell conjugate molecule of the present invention (GFD-linker1-OKT3) is constructed by linking GFD and OKT3 using linker1 (SEQ ID NO.16), and its structure is as follows. Figure 3 As shown in (a), its full-length amino acid sequence and nucleotide sequence are shown in Table 2.

[0282] Example 2 Plasmid Construction

[0283] The plasmid was constructed using the lentiviral vector pCDH-CMV-MCS-EF1-Puro (see Myeloid Leukemia. MolTher, 2016. 24(9): p. 1615-26.), and its map is shown below. Figure 3 As shown in (b). The target gene was synthesized using a direct synthesis method, with EcoRI and SwaI restriction sites and protective bases added to its 3' and 5' ends, respectively. After digestion with EcoRI and SwaI, the gene was ligated into a vector that had also been digested with EcoRI and SwaI. After confirming correct sequencing results, the gene was transformed into competent *E. coli* (Stbl3). All plasmids were extracted using the QIAGEN endotoxin-free large-scale extraction kit and identified by Hind III digestion. The identification results are shown below. Figure 3 As shown in (c).

[0284] Example 3 Virus Packaging

[0285] HEK-293T cells were cultured in 15cm culture dishes for virus packaging. When the HEK-293T cells reached approximately 80%-90% confluence, transfection was performed. A plasmid mixture (core plasmid 20ug, pCMVΔR 8.910ug, PMD2.G 4ug) was prepared in 2ml OPTIMEM solution; 2ml OPTIMEM and 68ul Lipo 8000 were added to another centrifuge tube. After incubation at room temperature for 5 min, the plasmid complex was added to the liposome complex and incubated at room temperature for 20 min. The mixture was then added dropwise to HEK-293T cells and incubated at 37°C for 6 hours. The culture medium was then removed, and preheated complete culture medium was added again. Transfection was observed under a microscope 24 hours after transfection. The results are shown in the figure below. Figure 4 After collecting viral supernatants at 48 and 72 hours, the virus was centrifuged at 3000 rpm for 20 minutes at 4°C. The supernatant was then filtered through a 0.45 μm filter and centrifuged at 25000 rpm at 4°C for 2.5 hours for virus concentration. The concentrated virus was dissolved overnight in 30 μL of viral lysis buffer, and the viral titer was detected by qPCR. The results showed that the viral titer met the requirements.

[0286] Example 4: Preparation of T cells secreting bispecific cell-binding organelle molecules

[0287] Monocytes were isolated from human peripheral blood using Ficool separation medium, and purified CD3+ T cells were obtained using RosetteSep Human T Cell Enrichment Cocktail (Stemcell technologies). T cells were activated with CD3 / CD28 magnetic beads (Life technology), followed by the addition of 200 U / ml IL-2 (PeproTech). After 48 hours of stimulation and culture, viral infection was performed. Lentiviral cells were infected with lentiboost at an MOI of 100 to prepare T cells secreting bispecific single-stranded cell binder molecules. The culture medium was changed 24 hours after infection. The ability to infect T cells was observed 48 hours after lentivirus infection, and T cell proliferation and viability were also measured.

[0288] Results: 48 hours after lentiviral infection, compared with the control group, T cell proliferation and activity (…) Figure 6 No significant changes were observed. This result indicates that infection with a virus expressing the cell-interceptor molecule of this invention has no significant effect on T cell activity.

[0289] Example 5: Construction of target cells carrying luciferase

[0290] The packaging steps for pTomo-CMV-Luciferase-IRES-Puro lentivirus were the same as in Example 1.2. After infecting PANC1, BXPC3, ASPC1, MCF7, MDA-MB-468, and MDA-MB-231 cells with the virus (MOI = 100), cells were screened with Puromycin (1 μg / ml) for 2 weeks, successfully obtaining stable transfected PANC1, BXPC3, ASPC1, MCF7, MDA-MB-468, and MDA-MB-231-luciferase cells.

[0291] Example 6: Molecular Screening for Targeted uPAR Bispecific Cell Engager—Killing Assay

[0292] Forty-eight hours after infection with the above lentiviruses, T cells from each group were compared with PANC1, ASPC1, BXPC3, MCF7, MDA-MB-468, and MDA-MB-231 tumor cells at an effector-target ratio of 10:1 (T cells were effector cells, with a concentration of 2*10). 5 / mL, 100uL per well; target cells, concentration 2*10 4 / mL, 100uL per well, 100uL per well) were incubated for 48 hours (cell culture medium: Advance 1640 medium (Gibco) + 10% fetal bovine serum (Gibco) + 1% penicillin, streptomycin (Gibco)).

[0293] Cell-killing efficacy was assessed using a Promega fluorescence detection kit. Cells were first treated with 30 μL of 1*PLB lysis buffer for 30 minutes. Then, 30 μL of substrate was added to each well, and the cells were immediately analyzed using a BioTek microplate reader. The percentage of cells killed by cytotoxicity was calculated as (1 - fluorescence value of target cells with effector cells / fluorescence value of target cells without effector cells) × 100%.

[0294] Result: As Figure 7 As shown, compared with the control group (NTD, CD19-BiTE), T cells secreting SMB-BiTE and Clone 20-BiTE showed no significant difference in their ability to kill tumor cells. However, the ability of T cells secreting GFD-BiTE bispecific single-chain antibody to specifically kill PANC1, ASPC1, BXPC3, MCF7, MDA-MB-468, and MDA-MB-231 cells was significantly upregulated. The results of this embodiment demonstrate that not all cell conjugate molecules constructed by binding uPAR sequences can achieve the effect of killing tumor cells. The GFD-BiTE of this invention has unexpectedly excellent anti-tumor effects.

[0295] Example 7: TNF-α release assay targeting uPAR bispecific cell binder molecule

[0296] In this embodiment, the release of cytokines was detected when T cells that secrete bispecific cell-interceptor molecules were co-incubated with target cells. The supernatant from co-incubated cells was used in a cell-killing assay for detection.

[0297] The method is as follows: Take the cell supernatant from the co-incubation in Example 6 and detect TNF-α according to the human TNF-alpha double antibody sandwich ELISA detection kit (proteintech).

[0298] The standard was dissolved in Sample Diluent PT6 and serially diluted to 500 pg / ml, 250 pg / ml, 125 pg / ml, 62.5 pg / ml, 31.2 pg / ml, 15.6 pg / ml and 0 pg / ml.

[0299] Add 100 μL of test sample, standard, or Sample Diluent PT6 (as a blank well) to each well, mix well, cover with sealing film, and incubate at 37°C for 120 minutes.

[0300] Then follow these steps in sequence:

[0301] Wash the hole 4 times with 1*Wash Buffer, each time for 1 minute;

[0302] Add 100 μL of 1× detection antibody to each well, cover with sealing film, and incubate at 37°C for 1 hour;

[0303] Repeat step (1).

[0304] Add 100 μL of 1×HRP-labeled secondary antibody to each well and incubate at 37°C for 40 minutes.

[0305] Repeat step (1).

[0306] Add 100 μL of TMB colorimetric solution to each well and incubate at 37°C in the dark for 15 min.

[0307] Add 100ul of stop solution to each well and mix well.

[0308] Absorbance was detected at 450 nm.

[0309] TNF-α secretion status as follows Figure 8 As shown, the results further confirm that not all cell-binding molecules constructed from uPAR sequences can recognize target proteins, activate T cells, and release large amounts of cytokines to exert a killing effect. The GFD-BiTE of the present invention has unexpectedly excellent anti-tumor effects.

[0310] Example 8: Detection of the tumor cell killing ability of the bispecific cell binder molecule GFD-BiTE protein

[0311] After infecting CHO cells with the aforementioned lentivirus, the cell supernatant was collected and concentrated to purify the target protein. The supernatant was then co-incubated with MDA-MB-468, MDA-MB-231, and BXPC3 tumor cells at an effector-to-target ratio of 10:1 (T cells were used as effector cells, with a concentration of 1*10). 4 / mL, 100uL per well; tumor cells are the target cells, concentration 1*10 3 / mL, 100uL per well), and add the corresponding concentration of protein, and incubate for 24 hours (cell culture medium: advance 1640 medium (Gibco) + 10% fetal bovine serum (Gibco) + 1% penicillin, streptomycin (Gibco)).

[0312] Cell-killing efficacy was assessed using a Promega fluorescence detection kit. Cells were first treated with 30 μL of 1*PLB lysis buffer for 30 minutes. Then, 30 μL of substrate was added to each well, and the cells were immediately analyzed using a BioTek microplate reader. The percentage of cells killed by cytotoxicity was calculated as (1 - fluorescence value of target cells with effector cells / fluorescence value of target cells without effector cells) × 100%.

[0313] Result: As Figure 9As shown, compared with the control CD19-BiTE, the GFD-BiTE bispecific single-chain antibody has a good killing effect on tumor cells, with IC50 values ​​all below 0.21 ng / ml.

[0314] Example 9: Cytotoxicity assay of the bispecific cell binder molecule GFD-BiTE on senescent cells.

[0315] HEL1-P16 is a senescent cell line stably transfected with green fluorescent protein (EGFP). HEL1-P16 cells were co-cultured with the GFD-BiTE protein of this invention and controls (NTD (T cells only) and CD19-BiTE protein) at a 2:1 effector-target ratio for 20 h. The killing effect was observed under a microscope, and the number of cells was counted for statistical analysis. Simultaneously, cell supernatant was collected to detect the release of the cytokine IFN-γ. Results are as follows: Figure 10 As shown, compared with the control group, the GFD-BiTE group exhibited a significant killing effect on HEL1-P16 cells. Figure 10 A, B) and higher release of the cytokine IFN-γ (A, B) Figure 10 C).

[0316] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. A cell conjugate molecule, characterized in that, The cell-connector molecule includes: (a) A first binding domain having a structure derived from a GFD domain of uPA, and the first binding domain specifically binds to a GFD-binding protein; the amino acid sequence of the first binding domain is shown in SEQ ID NO:

2. (b) A second binding domain that specifically binds to CD3 molecules; and the second binding domain comprises a VH segment and a VL segment, the VH segment having a complementarity-determining region (CDR) as follows: VH-CDR1 shown in SEQ ID NO: 5, VH-CDR2 shown in SEQ ID NO: 6, and VH-CDR3 shown in SEQ ID NO: 7; and The VL segment has the following complementary determination region (CDR): VL-CDR1 shown in SEQ ID NO: 8, VL-CDR2 shown in SEQ ID NO: 9, and VL-CDR3 as shown in SEQ ID NO: 10; Furthermore, the cell conjugate molecule has a structure selected from the following formula from its N-terminus to its C-terminus: S-D1-L1-D2-T (I); and S-D2-L1-D1-T (II), In the formula, Each "-" independently represents a linking peptide or peptide bond; S is either a signal peptide sequence or a no-signal peptide sequence; D1 is the first associative structural domain; L1 is a linker peptide; D2 is the second associative domain; T is an unlabeled or unmarked protein.

2. The cell-connector molecule as claimed in claim 1, characterized in that, The amino acid sequence of the VH segment is shown in SEQ ID NO: 4; and / or the amino acid sequence of the VL segment is shown in SEQ ID NO:

9.

3. The cell-connector molecule as claimed in claim 1, characterized in that, The amino acid sequence of the cell conjugate molecule is shown in SEQ ID NO:

20.

4. A polynucleotide, characterized in that, The polynucleotide encodes the cell conjugate molecule as described in claim 1.

5. A carrier, characterized in that, The carrier contains the polynucleotide as described in claim 4.

6. An engineered host cell, characterized in that, The host cell contains the vector as described in claim 5 or the genome is integrated with the polynucleotide as described in claim 4.

7. A coupling agent, characterized in that, The coupling contains: (a) The cell conjugate molecule as claimed in claim 1; and (b) A coupling portion coupled to the cell conjugate molecule, wherein the coupling portion is a detectable marker.

8. The use of an active ingredient in the preparation of a diagnostic reagent, test plate, or kit for detecting uPAR and / or CD3, characterized in that, The active ingredient is selected from the group consisting of: the cell conjugate molecule as described in claim 1, the conjugate as described in claim 7, or a combination thereof.

9. A pharmaceutical composition, characterized in that, The pharmaceutical composition contains: (i) an active ingredient selected from the group consisting of: the cell-connector molecule of claim 1, the host cell of claim 6, or combinations thereof; and (ii) One or more pharmaceutically acceptable carriers.

10. A method for detecting uPAR and / or CD3 in a sample in vitro for non-therapeutic and non-diagnostic purposes, characterized in that, The method includes the following steps: (1) Contact the sample with the cell conjugate molecule as described in claim 1; (2) Detect whether a complex is formed, where the formation of a complex indicates the presence of uPAR and / or CD3 in the sample.

11. A composition for in vitro detection of uPAR and / or CD3 in a sample, characterized in that, It includes the cell conjugate molecule as claimed in claim 1, the conjugate as claimed in claim 7, or a combination thereof as an active ingredient.

12. A detection plate, characterized in that, The detection plate comprises a substrate and a test strip, wherein the test strip contains the cell conjugate molecule as described in claim 1, the conjugate as described in claim 7, or a combination thereof.

13. A reagent kit, characterized in that, The kit includes: A first container, the first container containing the cell-connector molecule as described in claim 1; Alternatively, the kit may contain the detection plate as described in claim 12.

14. A method for preparing a recombinant polypeptide, characterized in that, The method includes: (a) Culturing the host cells as described in claim 6 under suitable expression conditions; (b) Isolate a recombinant polypeptide from the culture, said recombinant polypeptide being the cell conjugate molecule as described in claim 1.

15. Use of a cell-connector molecule as claimed in claim 1, a host cell as claimed in claim 6, and / or a pharmaceutical composition as claimed in claim 9 in the preparation of a medicament for treating diseases associated with abnormal expression or function of uPAR and / or CD3; The disease is selected from: tumors, aging, or a combination thereof; The tumor is selected from the group consisting of breast cancer, pancreatic cancer, or a combination thereof.

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