CD20-targeting single-domain antibody, car-t cell, and use thereof

Abstract

Provided are a CD20-targeting single-domain antibody, a CAR-T cell, and uses thereof. Specifically, provided are a CD20-targeting single-domain antibody having good specificity and affinity, a CAR-T cell constructed on the basis of the single-domain antibody, and uses of the antibody and the CAR-T cell for treating B-cell tumors and autoimmune diseases.

Description

CD20-targeting single-domain antibodies, CAR-T cells and their applications Technical Field

[0001] This application relates to the biomedical field, specifically to single-domain antibodies targeting CD20, CAR-T cells, and their applications. Background Technology

[0002] Lymphoma is a malignant tumor originating in lymph nodes and extranodal lymphoid tissue, accounting for 3%-4% of all malignant tumors in my country, and is the leading cause of hematologic malignancy. The two most common subtypes of B-cell lymphoma are diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL). Diffuse large B-cell lymphoma (DLBCL) is the most common aggressive B-cell non-Hodgkin's lymphoma (B-NHL), accounting for approximately 30% of newly diagnosed NHL cases. Currently, standard first-line therapy (R-CHOP) is used, but 10% of newly treated DLBCL cases remain refractory, and 40% of those who respond to initial treatment relapse after 2-5 years. Autologous hematopoietic stem cell transplantation (ASCT) after achieving complete remission (CR) or partial remission (PR) following chemotherapy is currently the best treatment option for relapsed / refractory DLBCL, but the 2-year disease-free survival rate after transplantation is still less than 15%. Follicular lymphoma (FL) is the most common indolent B-NHL, accounting for approximately 22% of newly diagnosed NHL cases. Although it progresses slowly, it is generally considered incurable, with a median survival of about 10 years. Within 10 years, about 15-28% of FL will transform into aggressive lymphoma.

[0003] Currently, CAR-T therapy for B-cell lymphoma is primarily based on the CD19 target. Four CD19-CAR-T therapies are already on the market. The main indications for these four CD19-CAR-T therapies are B-cell lymphoma (DLBCL) and follicular lymphoma (FL), with an objective response rate (ORR) of approximately 70%-80% and a complete remission (CR) of 50-60%. Although CD19 CAR-T therapy has achieved higher remission rates than standard therapy for these two major types of B-NHL, follow-up studies have shown that some patients relapse due to antigen escape, i.e., mutations or deletions of the CD19 protein expressed on the surface of tumor cells causing CAR-T cell therapy to fail, accounting for approximately 10%-20%. Therefore, for these relapsed and refractory patients, there is an urgent need to explore CAR-T cell immunotherapy with new targets.

[0004] CD20, a specific biomarker molecule on the surface of B lymphocytes, is expressed from early pre-B cells to mature B cells. In recent years, CD20-targeted drugs have made significant progress in the treatment of cancer and autoimmune diseases (AIDs). Anti-CD20 monoclonal antibodies (such as rituximab and oxutuzumab) are widely used clinically, with rituximab becoming the standard treatment for non-Hodgkin's lymphoma (NHL) and chronic lymphocytic leukemia (CLL). In addition, CD20 monoclonal antibodies have been approved for diseases such as rheumatoid arthritis (RA), multiple sclerosis (MS), and ANCA-associated vasculitis (AAV), and have shown significant efficacy in indications such as refractory systemic lupus erythematosus (SLE) and neuromyelitis optica spectrum disorder (NMOSD). CD20 / CD3 dual antibodies (such as Glofitamab and Mosunetuzumab) have shown high response rates in relapsed / refractory follicular lymphoma and diffuse large B-cell lymphoma (DLBCL), and several drugs have been approved for marketing. In addition, several drugs related to CD20 target ADC and CAR-T therapy are under development. The above data confirms the great potential of CD20 targeted therapy in the fields of oncology and autoimmune diseases. In the future, it is necessary to further optimize precision treatment strategies and explore new drug combinations to maximize clinical benefits.

[0005] Therefore, there is a need in the field to develop single-domain antibodies and chimeric antigen receptor drugs that can exert clinically effective cytotoxic, cytosolic, or immunosuppressive effects on CD20-expressing cells and have no adverse effects on CD20-non-expressing cells. Summary of the Invention

[0006] The purpose of this invention is to provide single-domain antibodies targeting CD20, CAR-T cells, and their applications.

[0007] In a first aspect of the invention, an antibody or antigen-binding fragment thereof targeting CD20 is provided, wherein the heavy chain variable region of the antibody includes HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 are selected from one combination of the following:

[0008] (1) HCDR1 with the amino acid sequence GRTFSSYN (SEQ ID NO:1);

[0009] The amino acid sequence is ISWSGGSP (SEQ ID NO:2) of HCDR2;

[0010] The amino acid sequence is HCDR3 of AAPLTYGSNWLADY (SEQ ID NO:3);

[0011] (2) HCDR1 with the amino acid sequence GRTFSYDT (SEQ ID NO:5);

[0012] The amino acid sequence is ISSSGGFT (SEQ ID NO:6) of HCDR2;

[0013] The amino acid sequence is AADRLQLYMTTTPHY (SEQ ID NO:7) of HCDR3;

[0014] (3) HCDR1 with the amino acid sequence GRTFSSYT (SEQ ID NO:9);

[0015] The amino acid sequence is VSWSGGTT (SEQ ID NO:10) of HCDR2;

[0016] The amino acid sequence is AADRVVYMTTTPQY (SEQ ID NO:11) of HCDR3;

[0017] The antibody also includes variants of the CDR sequence combinations shown in any one of (1)-(3) above, wherein the variants have at least 90% sequence identity with the CDR sequences shown in any one of (1)-(3), or contain at least one and no more than 10, or no more than 5, 4, 3, 2 or 1 amino acid changes on the CDR sequence, and are able to retain CD20 binding affinity.

[0018] In another preferred embodiment, the antibody is a single-domain antibody.

[0019] In another preferred embodiment, the single-domain antibody further includes a frame region (FR).

[0020] In another preferred embodiment, the frame region FR is of human, mouse, rabbit, or camel origin.

[0021] In another preferred embodiment, the frame region FR includes a human-derived FR region, a mouse-derived FR region, or a camel-derived FR region.

[0022] In another preferred embodiment, the antibody is a camel-derived antibody or a humanized antibody.

[0023] In another preferred embodiment, the amino acid sequence of the antibody is as shown in SEQ ID NO:4, or has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it.

[0024] In another preferred embodiment, the amino acid sequence of the antibody is as shown in SEQ ID NO:8, or has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it.

[0025] In another preferred embodiment, the amino acid sequence of the antibody is as shown in SEQ ID NO:12, or has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it.

[0026] In another preferred embodiment, the amino acid sequence of the antibody is as shown in any one of SEQ ID NO: 29, 31, 33, 35, 37, 39, or has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it.

[0027] In another preferred embodiment, the antibody targets the free CD20 antigen protein.

[0028] In another preferred embodiment, the antibody targets the CD20 antigen protein located on the cell membrane surface.

[0029] In a second aspect of the invention, a chimeric antigen receptor (CAR) is provided, wherein the antigen-binding domain of the chimeric antigen receptor comprises an antibody or antigen-binding fragment thereof targeting CD20 as described in the first aspect of the invention.

[0030] In another preferred embodiment, the antigen-binding fragment is a single-chain antibody or a heavy chain variable region of a single-domain antibody.

[0031] In another preferred embodiment, the amino acid sequence of the antigen-binding fragment is as shown in SEQ ID NO:4, or has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it.

[0032] In another preferred embodiment, the amino acid sequence of the antigen-binding fragment is as shown in SEQ ID NO:8, or has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it.

[0033] In another preferred embodiment, the amino acid sequence of the antigen-binding fragment is as shown in SEQ ID NO:12, or has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it.

[0034] In another preferred embodiment, the amino acid sequence of the antigen-binding fragment is as shown in any one of SEQ ID NO: 29, 31, 33, 35, 37, 39, or has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it.

[0035] In another preferred embodiment, the antigen-binding domain of the chimeric antigen receptor comprises one or more of the antigen-binding fragments described above.

[0036] In another preferred embodiment, the structure of the chimeric antigen receptor is shown in Formula I:

[0037] LVH-TM-C-CD3ζ (I)

[0038] In the formula,

[0039] Each "-" independently represents a linking peptide or peptide bond;

[0040] L represents the signal peptide sequence;

[0041] V represents the antigen-binding domain;

[0042] H represents the hinge area;

[0043] TM represents a transmembrane domain;

[0044] C is a co-stimulatory signaling molecule;

[0045] CD3ζ is a cytoplasmic signal transduction sequence derived from CD3ζ.

[0046] In another preferred embodiment, L is a signal peptide of a protein selected from the group consisting of CD8, CD28, GM-CSF, CD4, CD137, or a combination thereof.

[0047] In another preferred embodiment, L is a signal peptide derived from CD8.

[0048] In another preferred embodiment, the amino acid sequence of the signal peptide is shown in SEQ ID NO:16.

[0049] In another preferred embodiment, H is a hinge region derived from a protein selected from the group consisting of Fc, CD8, CD28, CD137, or a combination thereof.

[0050] In another preferred embodiment, H is a hinge region derived from IgG4.

[0051] In another preferred embodiment, the amino acid sequence of the hinge region is as shown in SEQ ID NO:17.

[0052] In another preferred embodiment, the TM is a transmembrane region of a protein selected from the group consisting of: CD8, ICOS, CD28, CD3epsilon, CD45, CD4, CD5, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a combination thereof.

[0053] In another preferred embodiment, the TM is a transmembrane region derived from CD8a.

[0054] In another preferred embodiment, the amino acid sequence of the transmembrane region is as shown in SEQ ID NO:18.

[0055] In another preferred embodiment, C is a co-stimulatory signaling molecule selected from the group consisting of: ICOS, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD1, Dap10, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), NKG2D, GITR, TLR2, or combinations thereof.

[0056] In another preferred embodiment, C is a co-stimulatory signaling molecule derived from 4-1BB.

[0057] In another preferred embodiment, the amino acid sequence of the co-stimulatory signaling molecule is shown in SEQ ID NO:19.

[0058] In another preferred embodiment, the amino acid sequence of the cytoplasmic signal transduction sequence is as shown in SEQ ID NO:20.

[0059] In another preferred embodiment, the CAR (preferably C-terminus) further includes a cell suicide element.

[0060] In another preferred embodiment, the CAR is connected to the cell suicide element via a self-cleaving element.

[0061] In another preferred embodiment, the cell suicide element is connected to the CD3ζ of the CAR via T2A or P2A.

[0062] In another preferred embodiment, the amino acid sequence of the T2A element is shown in SEQ ID NO:21.

[0063] In another preferred embodiment, the cell suicide element is a truncated EGFR (tEGFR).

[0064] In another preferred embodiment, the tEGFR amino acid sequence is shown in SEQ ID NO:22.

[0065] In another preferred embodiment, the amino acid sequence of the CAR is as shown in any one of SEQ ID NO: 23, 25, 27, or has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it.

[0066] In a third aspect of the invention, a recombinant protein is provided, said recombinant protein having:

[0067] (i) an antibody or antigen-binding fragment thereof targeting CD20 as described in the first aspect of the invention; and

[0068] (ii) Optional polypeptide molecules or fragments with therapeutic functions; and / or

[0069] (iii) Optional functional domains that enhance the physicochemical properties or drug-likeness of proteins.

[0070] In a fourth aspect of the invention, a nucleic acid molecule is provided, the nucleic acid molecule encoding a polypeptide selected from the group consisting of:

[0071] (1) An antibody or antigen-binding fragment thereof targeting CD20 as described in the first aspect of the present invention;

[0072] (2) The chimeric antigen receptor as described in the second aspect of the present invention; or

[0073] (3) The recombinant protein as described in the third aspect of the present invention.

[0074] In another preferred embodiment, the polynucleotide includes RNA, DNA, or cDNA.

[0075] In another preferred embodiment, the nucleotide sequence of the nucleic acid molecule encoding the antibody targeting CD20 or its antigen-binding fragment is as shown in any one of SEQ ID NO:13-15.

[0076] In another preferred embodiment, the nucleotide sequence of the nucleic acid molecule encoding the chimeric antigen receptor is shown in any one of SEQ ID NO: 24, 26, 28.

[0077] In a fifth aspect of the invention, a carrier is provided, said carrier containing nucleic acid molecules as described in the fourth aspect of the invention.

[0078] In another preferred embodiment, the vector is selected from the group consisting of DNA, RNA, plasmids, lentiviral vectors, adenovirus vectors, retroviral vectors, transposons, or combinations thereof.

[0079] In another preferred embodiment, the vector is a lentiviral vector.

[0080] In a sixth aspect of the invention, a host cell is provided, said host cell containing a vector as described in the fifth aspect of the invention, or a chromosome in which exogenous nucleic acid molecules as described in the fourth aspect of the invention are integrated.

[0081] In a seventh aspect of the invention, an engineered immune cell is provided, said immune cell expressing a chimeric antigen receptor as described in the second aspect of the invention.

[0082] In another preferred embodiment, the immune cells are T cells or NK cells.

[0083] In an eighth aspect of the invention, an immunoconjugate is provided, the immunoconjugate comprising:

[0084] (a) An antibody portion, said antibody portion being an antibody or antigen-binding fragment thereof targeting CD20 as described in the first aspect of the invention; and

[0085] (b) A conjugation portion conjugated to the antibody portion, the conjugation portion being selected from the group consisting of: detectable markers, drugs, toxins, cytokines, enzymes, or combinations thereof.

[0086] In another preferred embodiment, the immunoconjugate is a single-domain antibody-drug conjugate.

[0087] In another preferred embodiment, the antibody portion is coupled to the coupling portion via a chemical bond or a linker.

[0088] In another preferred embodiment, the coupling portion is a chemical marker and a biological marker.

[0089] In another preferred embodiment, the chemical label is an isotope, an immunotoxin, and / or a chemical drug.

[0090] In another preferred embodiment, the biomarker is biotin, avidin, or an enzyme label.

[0091] In another preferred embodiment, the coupling portion is a drug or toxin.

[0092] In another preferred embodiment, the drug is a cytotoxic drug.

[0093] In another preferred embodiment, the cytotoxic drug is selected from the group consisting of: anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, or combinations thereof.

[0094] In another preferred embodiment, the coupling portion is a detectable marker.

[0095] 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, single-domain magnetic particles, prodrug-activating enzymes (e.g., DT-cardiacinase (DTD) or biphenyl hydrolase-like protein (BPHL)), chemotherapeutic agents (e.g., cisplatin), or any form of single-domain particle, etc.

[0096] In another preferred embodiment, the immunoconjugate contains: a multivalent (e.g., bivalent) antibody or antigen-binding fragment thereof targeting CD20 as described in the first aspect of the invention.

[0097] In another preferred embodiment, the term "multivalent" means that the amino acid sequence of the immunoconjugate contains a plurality of repeating antibodies or antigen-binding fragments thereof targeting CD20 as described in the first aspect of the invention.

[0098] In another preferred embodiment, the detection is either in vivo or in vitro.

[0099] In another preferred embodiment, the immunoconjugate is used for the diagnosis and / or treatment of tumors expressing the CD20 protein.

[0100] In another preferred embodiment, the immunoconjugate has the following molecular formula:

[0101] in:

[0102] nAb is a single-domain antibody targeting CD20, an antibody targeting CD20, or a multispecific antibody.

[0103] LU stands for connector (also known as a connector).

[0104] D is a drug;

[0105] p is a value selected from 1 to 10.

[0106] In a ninth aspect of the invention, a pharmaceutical composition or formulation is provided, comprising:

[0107] (i) an antibody targeting CD20 or an antigen-binding fragment thereof as described in the first aspect of the present invention, a chimeric antigen receptor as described in the second aspect of the present invention, a recombinant protein as described in the third aspect of the present invention, engineered immune cells as described in the seventh aspect of the present invention, or an immunoconjugate as described in the eighth aspect of the present invention; and

[0108] (ii) Pharmaceutically acceptable carriers.

[0109] In another preferred embodiment, the pharmaceutical composition further comprises other bioactive substances, such as drugs for treating tumors.

[0110] In another preferred embodiment, the method of administration of the pharmaceutical composition is selected from the group consisting of: subcutaneous injection, intradermal injection, intramuscular injection, intravenous injection, intraperitoneal injection, microneedle injection, oral administration, or oral / nasal spray and nebulized inhalation.

[0111] In another preferred embodiment, the dosage form of the pharmaceutical composition is selected from the group consisting of liquid, solid, or gel.

[0112] In another preferred embodiment, the formulation is a liquid formulation.

[0113] In another preferred embodiment, the dosage form of the preparation is an injection.

[0114] In a tenth aspect of the invention, there is provided a use of an active ingredient selected from the group consisting of: antibodies targeting CD20 as described in the first aspect of the invention, or antigen-binding fragments thereof, chimeric antigen receptors as described in the second aspect of the invention, recombinant proteins as described in the third aspect of the invention, engineered immune cells as described in the seventh aspect of the invention, or immunoconjugates as described in the eighth aspect of the invention, or combinations thereof, wherein the active ingredient is used for (a) preparing reagents, detection plates, or kits for detecting CD20; and / or (b) preparing medicaments for the prevention and / or treatment of CD20-related diseases or conditions.

[0115] In another preferred embodiment, the disease or symptom is selected from: cancer, autoimmune diseases.

[0116] In another preferred embodiment, the disease is a CD20-overexpressing cancer.

[0117] In another preferred embodiment, the cancers include leukemia, lymphoma, myeloma, breast cancer (such as triple-negative breast cancer), lung cancer (such as non-small cell lung cancer), pancreatic cancer, malignant glioma, stomach cancer, liver cancer, esophageal cancer, kidney cancer, colorectal cancer, bladder cancer, prostate cancer, endometrial cancer, ovarian cancer, cervical cancer, and angiosarcoma.

[0118] In another preferred embodiment, the cancer is a hematologic malignancy.

[0119] In another preferred embodiment, the cancer includes B-cell lymphoma.

[0120] In another preferred embodiment, the cancer includes multiple myeloma (MM).

[0121] In another preferred embodiment, the hematologic malignancy includes follicular lymphoma, diffuse large B-cell lymphoma (DLBCL), non-Hodgkin lymphoma (NHL), mantle cell lymphoma, marginal zone B-cell lymphoma, and / or B-cell chronic lymphocytic leukemia (CLL).

[0122] In another preferred embodiment, the autoimmune disease includes rheumatoid arthritis (RA), multiple sclerosis (MS), myasthenia gravis, ANCA-associated vasculitis (AAV), systemic lupus erythematosus (SLE), lupus nephritis, nephrotic syndrome, thrombocytopenia, graft-versus-host disease, and / or neuromyelitis optica spectrum disorder (NMOSD).

[0123] In an eleventh aspect of the present invention, an antibody or antigen-binding fragment thereof is provided that competitively binds to the same epitope as an antibody or antigen-binding fragment thereof as described in the first aspect of the present invention.

[0124] In a twelfth aspect of the invention, a kit for detecting CD20 protein in a sample is provided, the kit comprising an antibody or antigen-binding fragment thereof targeting CD20 as described in the first aspect of the invention, or an immunoconjugate as described in the eighth aspect of the invention.

[0125] In a thirteenth aspect of the present invention, a method for in vitro detection of CD20 in a sample is provided, the method comprising the steps of:

[0126] (1) In vitro, the sample is contacted with a single-domain antibody targeting CD20 as described in the first aspect of the present invention, or an immunoconjugate as described in the eighth aspect of the present invention;

[0127] (2) Detect whether an antigen-antibody complex is formed, where the formation of a complex indicates the presence of CD20 in the sample.

[0128] In another preferred embodiment, the detection includes diagnostic or non-diagnostic methods.

[0129] In a fourteenth aspect of the present invention, a method for preparing a recombinant polypeptide is provided, the method comprising:

[0130] (a) Culture host cells as described in the sixth aspect of the invention under suitable expression conditions; and

[0131] (b) Isolate a recombinant polypeptide from the culture, said recombinant polypeptide being a single-domain antibody targeting CD20 as described in the first aspect of the invention, or a recombinant protein as described in the third aspect of the invention.

[0132] In a fifteenth aspect of the invention, a method for treating CD20-related diseases is provided, the method comprising: administering to a desired subject a single-domain antibody targeting CD20 as described in a first aspect of the invention, a recombinant protein as described in a third aspect of the invention, engineered immune cells as described in a seventh aspect of the invention, an immunoconjugate as described in an eighth aspect of the invention, or a pharmaceutical composition or formulation as described in a ninth aspect of the invention, or a combination thereof.

[0133] In another preferred embodiment, the disease or condition is selected from: cancer, autoimmune diseases.

[0134] In another preferred embodiment, the disease is a CD20-overexpressing cancer.

[0135] In another preferred embodiment, the cancer is a hematologic malignancy.

[0136] In another preferred embodiment, the cancer includes B-cell lymphoma.

[0137] In another preferred embodiment, the cancer includes multiple myeloma (MM).

[0138] In another preferred embodiment, the hematologic malignancy includes follicular lymphoma, diffuse large B-cell lymphoma (DLBCL), non-Hodgkin lymphoma (NHL), mantle cell lymphoma, marginal zone B-cell lymphoma, and / or B-cell chronic lymphocytic leukemia (CLL).

[0139] In another preferred embodiment, the autoimmune disease includes rheumatoid arthritis (RA), multiple sclerosis (MS), myasthenia gravis, ANCA-associated vasculitis (AAV), systemic lupus erythematosus (SLE), lupus nephritis, nephrotic syndrome, thrombocytopenia, graft-versus-host disease, and / or neuromyelitis optica spectrum disorder (NMOSD).

[0140] In another preferred embodiment, the method further includes administering other drugs or treatments to the subject in need for combined treatment.

[0141] In another preferred embodiment, the other drugs or treatment methods include: antitumor immunotherapy drugs, tumor-targeted drugs, tumor chemotherapy drugs, and tumor radiotherapy.

[0142] In a sixteenth aspect of the invention, a vector is provided that carries the encoding gene of the chimeric antigen receptor described in the second aspect of the invention, and the vector further comprises a targeting element that targets an immune cell surface antigen.

[0143] In another preferred embodiment, the vector is a viral vector selected from the group consisting of lentiviral vectors, adenovirus vectors, retroviral vectors, or combinations thereof.

[0144] In another preferred embodiment, the vector is a lentiviral vector.

[0145] In another preferred embodiment, the targeting element targets CD7 and / or CD3; preferably, the targeting element comprises an anti-CD7 antibody and / or an anti-CD3 antibody.

[0146] In a seventeenth aspect of the invention, a method for generating engineered immune cells is provided, the method comprising: contacting the immune cells with a carrier as described in a sixteenth aspect of the invention.

[0147] In another preferred embodiment, the immune cells are cells in vitro or cells in vivo.

[0148] In another preferred embodiment, the immune cells include T cells and NK cells.

[0149] In another preferred embodiment, the immune cell is a T cell.

[0150] In an eighteenth aspect of the invention, the use of the carrier as described in the sixteenth aspect of the invention is provided for the preparation of a medicament for the prevention and / or treatment of CD20-related diseases or conditions.

[0151] In another preferred embodiment, the drug is used to generate chimeric antigen receptor-immune cells in vitro or in vivo.

[0152] In another preferred embodiment, the immune cells are cells in vitro or cells in vivo.

[0153] In another preferred embodiment, the immune cells include T cells and NK cells.

[0154] In another preferred embodiment, the immune cell is a T cell.

[0155] In a nineteenth aspect of the invention, a method for treating CD20-related diseases is provided, the method comprising: administering a carrier as described in a sixteenth aspect of the invention to a desired subject.

[0156] In another preferred embodiment, the disease is defined as described in the fifteenth aspect of the invention.

[0157] 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

[0158] The following figures are used to illustrate specific embodiments of the present invention and are not intended to limit the scope of the invention as defined by the claims.

[0159] Figure 1 shows the general workflow of the present invention for screening specific antibodies targeting CD20 from a phage antibody library.

[0160] Figure 2 shows the flow cytometry results of partial phage monoclonal binding to CHO-K1-CD20 and CHO-K1 cells.

[0161] Figure 3 shows the flow cytometry results of the selected phage monoclonal antibodies binding to various CD20 positive and negative cell lines.

[0162] Figure 4 shows the results of enzyme-linked immunosorbent assay (ELISA) analysis of the screened phage monoclonal antibodies against various CD20 antigen proteins and unrelated antigens. Negative control is the negative control phage antibody clone; anti-M13 HRP Ab is the negative antibody control with only the first antibody added; ofatumumab / anti-human IgG HRP Ab is the positive control antibody; anti-human IgG HRP Ab is the negative antibody control with only the second antibody added; and anti-his-HRP Ab is the positive antibody control for detecting the antigen tag. In Figure 4, the bar charts for each test antibody and control group, from left to right, represent the test results for proteins SA+Acro Human-CD20-his-Bio, Acro Human CD20, KACTUS human CD20 Loop, KACTUS human CD20 VLP, KACTUS VLP control, and SA, respectively.

[0163] Figure 5 shows the flow cytometry results of the antibodies expressed as IgG by the selected #1-3 antibody clones binding to various CD20 positive and negative cell lines.

[0164] Figure 6 shows the results of human-monkey cross-activity analysis of the antibodies expressed in IgG form by the selected #1-3 antibody clones using enzyme-linked immunosorbent assay (ELISA). The bar charts for each test antibody and control group, from left to right, represent the test results with proteins SA+Acro Human-CD20-his-Bio, Acro cyno CD20, and SA, respectively.

[0165] Figure 7 shows the FACS binding results of antibody clone 13, which is expressed in IgG form, with the Raji tumor cell line, which naturally expresses CD20 antigen.

[0166] Figure 8 shows the FACS binding results of antibodies expressed in IgG form by antibody clones 10 and 20 with Raji, a tumor cell line naturally expressing the CD20 antigen.

[0167] Figure 9 shows the expression of CAR molecules on the surface of CAR-T cells from different clones.

[0168] Figure 10 shows the degranulation results of CAR-T cells of different clones under stimulation by tumor cells from different tissue sources.

[0169] Figure 11 shows the killing effect of different clones of CAR-T cells on different target cells Raji-luc (Figure 11A) and JeKo-1-luc (Figure 11B).

[0170] Figures 12-15 show the antitumor efficacy of CAR-T cells corresponding to different clones in JeKo-1-luc tumor-bearing NPG mice. Figure 12A shows the tumor burden in mice after different CAR-T infusions, and Figures 12B and 12C show the quantitative results of Figure 12A.

[0171] Figure 13 shows the changes in the ratio of T cells to lymphocytes in mouse peripheral blood after different CAR-T cell infusions.

[0172] Figure 14 shows the survival rate of mice after infusion of different CAR-T cells.

[0173] Figure 15 shows the changes in mouse body weight after infusion of different CAR-T cells.

[0174] Figure 16 shows the killing effect of different clones of CAR-T cells on various target cells, namely Raji-luc (Figure 16A), JeKo-1-luc (Figure 16B), and RPMI-8226-luc (Figure 16C).

[0175] Figures 17-18 show the antitumor efficacy of CAR-T cells corresponding to different clones in JeKo-1-luc tumor-bearing NPG mice. Figure 17A shows the tumor burden in mice after different CAR-T cell infusions, Figure 17B shows the quantitative results of Figure 17A, and Figure 18 shows the survival rate of mice after different CAR-T cell infusions.

[0176] Figure 19 shows the dynamic changes in tumor volume after administration of a lentiviral vector containing the CAR-encoding gene.

[0177] Figure 20 shows the changes in the percentage of CAR-T levels in peripheral blood of mice after administration of a lentiviral vector containing the CAR-encoding gene. Detailed Implementation

[0178] Through extensive and in-depth research, the inventors have developed for the first time a single-domain antibody targeting CD20, CAR-T cells, and their applications. The inventors screened CD20-specific antibodies using an antibody library and evaluated the specificity of these antibodies at the phage level using ELISA and FACS experiments. Ultimately, several nanobody clones with good specificity were obtained.

[0179] Furthermore, this invention designs and prepares CAR-T cells based on antibody sequences. Experimental results show that the VHH10 and VHH20 CAR-T cells of this invention have good tumor-suppressing effects both in vitro and in vivo, and are superior to CAR-T cells designed based on Ofatumumab. This invention provides a novel immunotherapy for the treatment of B-cell lymphoma, overcoming the limitation of CD19-negative relapse after CD19 CAR-T therapy, while CD20 CAR-T has the potential to expand indications and improve efficacy. Based on this, this invention was completed.

[0180] the term

[0181] 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.

[0182] As used herein, when referring to a specific enumerated value, the term “about” means that the value can vary by no more than 1% from the enumerated values. 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.).

[0183] As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include “consisting of” and “substantially consisting of”.

[0184] As used herein, the term "pharmaceutically acceptable carrier" refers to a substance that is suitable for use in humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a reasonable benefit / risk ratio.

[0185] As used herein, the term "therapeutic effective amount" refers to an amount that is functional or active in humans and / or animals and is acceptable to humans and / or animals. Those skilled in the art will understand that the "therapeutic effective amount" can vary depending on the form of the pharmaceutical composition, the route of administration, the excipients used, the severity of the disease, and whether it is used in combination with other drugs.

[0186] Antibody

[0187] 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.

[0188] As used herein, the terms "single-domain antibody (VHH)" and "nanobody" have the same meaning: the variable region of the heavy chain of a monoclonal antibody. A single-domain antibody (VHH) consisting of only one variable region of the heavy chain is constructed; it is the smallest antigen-binding fragment with complete function. Typically, antibodies lacking both the light chain and the constant region 1 (CH1) of the heavy chain are first obtained, and then the variable region of the antibody heavy chain is cloned to construct a single-domain antibody (VHH) consisting of only one variable region of the heavy chain.

[0189] As used herein, the term "heavy chain antibody" refers to an antibody containing only the heavy chain. Some antibodies found in the blood of camels are "heavy chain antibodies" lacking the light chain. The heavy chain antibody of this invention comprises a heavy chain variable region (VHH) and heavy chain constant regions CH2 and CH3. The heavy chain antibody of this invention can be a naturally occurring antibody from an animal (e.g., of camel origin) lacking both the light chain and heavy chain constant region 1 (CH1); or it can be a recombinant antibody obtained by recombination of the single-domain antibody (VHH) of this invention with the heavy chain constant region.

[0190] 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.

[0191] As used herein, when referring to amino acid or nucleotide sequences, the term "sequence identity" (also known as "sequence uniformity") refers to the degree of similarity between two amino acid or nucleotide sequences (e.g., a query sequence and a reference sequence), typically expressed as a percentage. Generally, sequence alignment is performed and gaps (if any) are introduced before calculating the percentage of similarity between two amino acid or nucleotide sequences. If, at a certain alignment position, the amino acid residues or bases in the two sequences are the same, the two sequences are considered to be identical or matched at that position; if the amino acid residues or bases in the two sequences are different, they are considered to be inconsistent or mismatched at that position. In some algorithms, sequence identity is obtained by dividing the number of matched positions by the total number of positions in the alignment window. In other algorithms, the number of gaps and / or gap lengths are also taken into account. For the purposes of this invention, the publicly available alignment software BLAST (available at ncbi.nlm.nih.gov) can be used to obtain optimal sequence alignment and calculate the sequence identity between two amino acid or nucleotide sequences using default settings. In some embodiments, the “at least 90% sequence identity” of the present invention includes, but is not limited to, at least 95%, at least 98%, at least 99%, or even 100% sequence identity.

[0192] In some embodiments, the antibody provided by the present invention further includes an amino acid sequence that has at least 90% sequence identity (e.g., at least 95%, at least 98%, at least 99%, or even 100% sequence identity) with the sequence shown in SEQ ID NO:4, 8, 12, 29, 31, 33, 35, 37, or 39.

[0193] Those skilled in the art will understand that, based on the specific sequences provided herein, variants of the CD20-targeting antibodies provided herein can be obtained by substituting, deleting, or adding a few amino acids and verifying or screening the binding ability or biological activity of the resulting products with the corresponding antigen CD20. These variants should also be included within the scope of this invention. For example, the antibodies or antigen-binding fragments of this invention may have at least one and no more than 10, or no more than 5, 4, 3, 2, or 1 amino acid change in their full-length or CDR sequences.

[0194] Those skilled in the art will also understand that, based on the specific heavy chain variable region sequence provided herein, a light chain variable region that matches the heavy chain variable region and maintains CD20 binding ability can be obtained by using CD20 as an antigen to screen antibody light chain libraries (such as human phage light chain libraries). Anti-CD20 antibody molecules obtained in this manner are also included within the scope of this invention.

[0195] In some embodiments, the antigen-binding molecule of the present invention may further comprise post-translational modifications. Examples of post-translational protein modifications include phosphorylation, acetylation, methylation, ADP-riboylation, ubiquitination, glycosylation, carbonylation, ubiquitin-like formation, biotinylation, or the addition of a polypeptide side chain or hydrophobic group. Thus, the modified soluble polypeptide may comprise non-amino acid components, such as lipids, polysaccharides, or monosaccharides, as well as phosphates. A preferred form of glycosylation is sialylation modification, which binds one or more sialic acid groups to the polypeptide. Sialic acid groups improve protein solubility and serum half-life, while also reducing the protein's potential immunogenicity. See Raju et al. Biochemistry. 2001 31; 40(30):8868-76.

[0196] An epitope is a portion of a molecule that is bound to an antigen-binding protein (e.g., an antibody). An epitope can comprise a non-adjacent portion of the molecule (e.g., in a polypeptide, amino acid residues that are not adjacent on the major sequence of the polypeptide but are close enough to each other in the trivalent and tetravalent structures of the polypeptide to be bound to an antigen-binding protein).

[0197] As used in this article, the terms "heavy chain variable region" and "V" are used interchangeably. H "They can be used interchangeably."

[0198] As used in this article, the terms “variable region” and “complementarity determining region (CDR)” are used interchangeably.

[0199] In a preferred embodiment of the present invention, the heavy chain variable region of the antibody includes three complementarity-determining regions CDR1, CDR2, and CDR3.

[0200] In a preferred embodiment of the present invention, the heavy chain of the antibody includes the aforementioned heavy chain variable region and heavy chain constant region.

[0201] In this invention, the terms "antibody of the invention," "protein of the invention," or "peptide of the invention" are used interchangeably and all refer to peptides that specifically bind to the CD20 protein, such as proteins or peptides having a heavy chain variable region. They may or may not contain an initiating methionine.

[0202] The present invention also provides other proteins or fusion expression products having the antibodies of the present invention. Specifically, the present invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a heavy chain containing a variable region, provided that the variable region is the same as or has at least 90% homology with the heavy chain variable region of the antibody of the present invention, preferably at least 95% homology.

[0203] The term "antibody or antigen-binding fragment of the present invention" refers to a polypeptide having CD20 protein-binding activity and including the aforementioned CDR region. This term also includes variants of polypeptides containing the aforementioned CDR region that have the same function as the antibodies of the present invention. These variants include (but are not limited to): deletions, insertions, and / or substitutions of one or more amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10), and the addition of one or more amino acids (typically less than 20, preferably less than 10, more preferably less than 5) to the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids of similar or comparable properties generally does not alter the function of the protein. Similarly, the addition of one or more amino acids to the C-terminus and / or N-terminus generally does not alter the function of the protein. This term also includes active fragments and active derivatives of the antibodies of the present invention.

[0204] The variant forms of the polypeptide include: homologous sequences, conserved variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of the antibody of the present invention under high or low severity conditions, and polypeptides or proteins obtained using antiserum against the antibody of the present invention.

[0205] The present invention also provides other polypeptides, such as fusion proteins comprising a single-domain antibody or a fragment thereof. In addition to nearly full-length polypeptides, the present invention also includes fragments of the single-domain antibody of the present invention. Typically, the fragment has at least about 50 consecutive amino acids of the antibody of the present invention, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids.

[0206] In this invention, "a conserved variant of the antibody of the present invention" refers to a polypeptide 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 antibody of the present invention. These conserved variant polypeptides are preferably generated by amino acid substitutions according to Table A.

[0207] Table A

[0208] The present invention also provides a polynucleotide molecule encoding the above-described antibody or a fragment thereof or a fusion protein thereof. The polynucleotide of the present invention may be in DNA or RNA form. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand.

[0209] 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.

[0210] 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.

[0211] 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 polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0212] The full-length nucleotide sequence or fragments of the antibody 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, the coding sequence of the heavy chain and an expression tag (such as 6His) can be fused together to form a fusion protein.

[0213] Chimeric antigen receptor (CAR)

[0214] The chimeric antigen receptor (CAR) of this invention comprises an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain includes a target-specific binding element (also referred to as an antigen-binding domain). The intracellular domain includes a co-stimulatory signaling region and a ζ-chain portion. The co-stimulatory signaling region refers to a portion of the intracellular domain containing a co-stimulatory molecule. The co-stimulatory molecule is a cell surface molecule required for an effective lymphocyte response to an antigen, rather than an antigen receptor or its ligands.

[0215] A linker may be incorporated between the extracellular and transmembrane domains of the CAR, or between the cytoplasmic and transmembrane domains of the CAR. As used herein, the term "linker" generally refers to any oligopeptide or polypeptide that functions to link the transmembrane domain to the extracellular or cytoplasmic domain of the polypeptide chain. Linkers may comprise 0-300 amino acids, preferably 2 to 100 amino acids, and most preferably 3 to 50 amino acids.

[0216] In a preferred embodiment of the present invention, the extracellular domain of the CAR provided by the present invention includes an antigen-binding domain targeting CD20. When expressed in T cells, the CAR of the present invention is capable of antigen recognition based on antigen-binding specificity. When it binds to its associated antigen, it affects tumor cells, causing them to stop growing, be induced to die, or otherwise be affected, leading to a reduction or elimination of the patient's tumor burden. The antigen-binding domain is preferably fused with an intracellular domain derived from one or more of the co-stimulatory molecule and the ζ chain. Preferably, the antigen-binding domain is fused with an intracellular domain combining a 4-1BB signaling domain and a CD3ζ signaling domain.

[0217] As used in this article, "antigen-binding domain" refers to the Fab fragment, Fab' fragment, F(ab')2 fragment, single Fv fragment, or heavy chain variable region of a single-domain antibody that has antigen-binding activity.

[0218] carrier

[0219] The present invention includes a DNA construct comprising a CAR sequence, wherein the sequence comprises a nucleic acid sequence of an antigen-binding domain operatively linked to a signal transduction domain.

[0220] The nucleic acid sequence encoding the desired molecule can be obtained using recombination methods known in the art, such as, for example, by screening a library from a cell expressing the gene, by obtaining the gene from a vector known to contain the gene, or by directly isolating the gene from cells and tissues containing the gene using standard techniques. Optionally, the gene of interest can be synthesized.

[0221] The present invention also provides vectors in which the DNA of the present invention is inserted. Vectors derived from retroviruses, such as lentiviruses, are suitable tools for achieving long-term gene transfer because they allow for long-term, stable integration of transgenes and their proliferation in daughter cells.

[0222] In short, cloning typically involves operatively linking a nucleic acid encoding a CAR polypeptide or a portion thereof to a promoter and incorporating the construct into an expression vector to express the natural or synthetic nucleic acid encoding the CAR. This vector is suitable for replication and integration into eukaryotic cells. A typical cloning vector contains transcription and translation terminators, an initial sequence, and a promoter that can be used to regulate the expression of the desired nucleic acid sequence.

[0223] This nucleic acid can be cloned into many types of vectors. For example, it can be cloned into vectors including, but not limited to, plasmids, phage particles, phage derivatives, animal viruses, and granules. Specific vectors of interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0224] Furthermore, expression vectors can be provided to cells in the form of viral vectors. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Typically, a suitable vector contains at least one origin of replication functioning in an organism, a promoter sequence, a convenient restriction enzyme site, and one or more optional markers.

[0225] Many virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Selected genes can be inserted into vectors and packaged into retroviral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to target cells in vivo or in vitro. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In one embodiment, lentiviral vectors are used.

[0226] Additional promoter elements, such as enhancers, can regulate the frequency of transcription initiation. These are typically located in a 30–110 bp region upstream of the start site, although recent studies have shown that many promoters also contain functional elements downstream of the start site. The spacing between promoter elements is often flexible to maintain promoter function when an element is inverted or moved relative to another. In the thymidine kinase (TK) promoter, the spacing between promoter elements can be increased to 50 bp before activity begins to decline. Depending on the promoter, individual elements can function cooperatively or independently to initiate transcription.

[0227] An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strongly constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operatively linked thereto. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including but not limited to the early promoter of simian virus 40 (SV40), mouse mammary cancer virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Russ's sarcoma virus promoter, and human gene promoters, such as, but not limited to, actin promoter, myosin promoter, heme promoter, and creatine kinase promoter. Furthermore, the invention should not be limited to the application of constitutive promoters. Inducible promoters are also considered as part of the invention. The use of inducible promoters provides a molecular switch that can turn on the expression of a polynucleotide sequence operatively linked to the inducible promoter when such expression is desired, or turn off expression when expression is undesirable. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0228] To assess the expression of CAR peptides or portions thereof, the expression vector introduced into cells may also contain either or both of an optional marker gene or a reporter gene to facilitate the identification and selection of expressing cells from a population of cells seeking transfection or infection via a viral vector. Alternatively, the optional marker may be carried on a separate DNA segment and used in co-transfection procedures. Both the optional marker and the reporter gene may be flanked by appropriate regulatory sequences to enable expression in host cells. Useful optional markers include, for example, antibiotic resistance genes such as neo.

[0229] Reporter genes are used to identify potentially transfected cells and to evaluate the functionality of regulatory sequences. Typically, a reporter gene is a gene that is either absent from or expressed by the recipient organism or tissue, and that encodes a polypeptide whose expression is clearly indicated by readily detectable properties such as enzyme activity. The expression of the reporter gene is determined at an appropriate time after DNA has been introduced into the recipient cells. Suitable reporter genes may include those encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secretory alkaline phosphatase, or green fluorescent protein genes (e.g., Ui-Tei et al., 2000 FEBS Letters 479:79-82). Suitable expression systems are well-known and can be prepared using known techniques or are commercially available. Typically, a construct with at least five flanking regions that exhibits the highest level of reporter gene expression is identified as a promoter. Such promoter regions can be ligated into reporter genes and used to evaluate the ability of reagents to regulate promoter-driven transcription.

[0230] Methods for introducing genes into cells and expressing genes into cells are known in the art. Within the scope of expression vectors, the vector can be readily introduced into host cells, such as mammalian, bacterial, yeast, or insect cells, by any method in the art. For example, expression vectors can be transferred into host cells by physical, chemical, or biological means.

[0231] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, lipid transfection, particle bombardment, microinjection, electroporation, and so on. Methods for producing cells comprising vectors and / or exogenous nucleic acids are well known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). Calcium phosphate transfection is a preferred method for introducing polynucleotides into host cells.

[0232] Biological approaches to introducing polynucleotides of interest into host cells include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian cells, such as human cells. Other viral vectors may be derived from lentiviruses, poxviruses, herpes simplex virus I, adenoviruses, and adeno-associated viruses, among others.

[0233] Chemical means of introducing polynucleotides into host cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, and beads; and lipid-based systems, including oil-in-water emulsions, micelles, mixed micelles, and liposomes. An exemplary colloidal system used as a delivery vehicle in both in vitro and in vivo is the liposome (e.g., an artificial membrane capsule).

[0234] In the case of using a non-viral delivery system, an exemplary delivery tool is a liposome. Consider using a lipid formulation to introduce nucleic acid into host cells (in vitro, ex vivo, or in vivo). Alternatively, the nucleic acid may be associated with a lipid. Lipid-associated nucleic acid can be encapsulated within the aqueous interior of a liposome, dispersed within the lipid bilayer of the liposome, attached to the liposome via a linker molecule associated with both the liposome and the oligonucleotide, trapped within the liposome, complexed with the liposome, dispersed in a solution containing lipids, mixed with lipids, conjugated with lipids, contained in lipids as a suspension, contained in or complexed with micelles, or otherwise associated with lipids. The lipids, lipid / DNA, or lipid / expression vector associated with the composition are not limited to any specific structure in solution. For example, they may be present in a bilayer structure, as micelles, or have a “collapsed” structure. They may also be simply dispersed in solution, possibly forming aggregates of varying sizes or shapes. Lipids are fatty substances and can be naturally occurring or synthetic lipids. For example, lipids include fat droplets, which occur naturally in the cytoplasm and in compounds containing long-chain aliphatic hydrocarbons and their derivatives such as fatty acids, alcohols, amines, amino alcohols and aldehydes.

[0235] In a preferred embodiment of the present invention, the vector is a lentiviral vector. The inventors have confirmed that the CAR constructed using this lentiviral vector exhibits high transfection efficiency for T cells and high reproducibility.

[0236] In a preferred embodiment of the present invention, the vector further includes a signal peptide sequence. Preferably, the signal peptide sequence is linked upstream of the nucleic acid sequence of the antigen-tuberculous domain.

[0237] Antibody preparation

[0238] The DNA sequences of the antibodies or fragments thereof of this invention can be obtained using conventional techniques, such as PCR amplification or genomic library screening. Furthermore, the coding sequences of the light and heavy chains can be fused together to form single-chain antibodies.

[0239] 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.

[0240] 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.

[0241] Currently, the DNA sequence encoding the antibody (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) and cells known in the art. Furthermore, mutations can be introduced into the protein sequence of the present invention through chemical synthesis.

[0242] 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.

[0243] 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 animal cells include (but are not limited to): CHO-S and HEK-293 cells.

[0244] Typically, host cells transformed with the antibody are cultured under conditions suitable for antibody expression according to the present invention. The antibody of the present invention is 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, which are well known to those skilled in the art.

[0245] The obtained monoclonal antibodies can be identified using conventional methods. For example, the binding specificity of monoclonal antibodies can be determined by immunoprecipitation or in vitro binding assays (such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA)). The binding affinity of monoclonal antibodies can be determined, for example, by the Scatchard analysis described by Munson et al., Anal. Biochem., 107:220 (1980).

[0246] The antibodies of this invention can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be separated and purified using various separation methods utilizing their 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, permeation, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

[0247] Immunoconjugates

[0248] The present invention also provides immunoconjugates (ADCs) based on the antibodies of the present invention, preferably nanobody-drug conjugates (NDCs).

[0249] Typically, the antibody-drug conjugate comprises an antibody and an effector molecule, wherein the antibody is conjugated to the effector molecule, preferably chemically conjugated. The effector molecule is preferably a drug with therapeutic activity. Furthermore, the effector molecule may be one or more of a toxic protein, a chemotherapeutic agent, a small molecule drug, or a radionuclide.

[0250] The antibody and the effector molecule of this invention can be coupled via a coupling agent. Examples of the coupling agent include any one or more of non-selective coupling agents, carboxyl-based coupling agents, peptide chains, and disulfide bonds. The non-selective coupling agent refers to a compound that covalently links the effector molecule and the antibody, 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).

[0251] Certain residues on antibodies (such as Cys or Lys) are used to link to a variety of functional groups, including imaging reagents (e.g., chromophores and fluorophores), diagnostic reagents (e.g., MRI contrast agents and radioisotopes), stabilizers (e.g., ethylene glycol polymers), and therapeutic agents. Antibodies can be conjugated to functional agents to form antibody-functional agent conjugates. Functional agents (e.g., drugs, detection reagents, stabilizers) are conjugated (covalently linked) to antibodies. Functional agents can be directly attached to antibodies or indirectly through linkers.

[0252] Single-domain antibodies can be conjugated to drugs to form antibody-drug conjugates (NDCs). Typically, an NDC contains a linker between the drug and the antibody. 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 antibody. 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 under conditions where the antibody is hydrolyzed by proteases.

[0253] Prior to attachment to the antibody, the linker has a reactive group capable of reacting with certain amino acid residues, and the attachment is achieved through the reactive group. Thiol-specific reactive groups are preferred and include, for example, maleimide compounds, haloamides (e.g., iodinated, brominated, or chlorinated); haloesters (e.g., iodinated, brominated, or chlorinated); halomethyl ketones (e.g., iodinated, brominated, or chlorinated); benzyl halides (e.g., iodinated, brominated, or chlorinated); vinyl sulfones; pyridyl disulfides; mercury derivatives such as 3,6-di-(mercurymethyl)dioxane, with the counter ion being acetate, chloride, or nitrate; and polymethylene dimethyl sulfide thiosulfonate. The linker may include, for example, a maleimide attached to the antibody via a thiosuccinimide.

[0254] The drug can be any cytotoxic, cell growth-inhibiting, or immunosuppressive drug. In one embodiment, the linker connects the antibody 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 linked to the linker, the drug has a reactive group before being linked to the antibody.

[0255] 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 tubulin 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.

[0256] In this invention, the drug-linker can be used to form NDC in a simple step. In other embodiments, bifunctional linker compounds can be used to form NDC 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 NDC.

[0257] 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.

[0258] The present invention also provides a method for preparing NDC, which may further include: binding an antibody to a drug-adaptor compound under conditions sufficient to form an antibody-drug conjugate (NDC).

[0259] In some embodiments, the method of the present invention includes binding an antibody to a bifunctional adapter compound under conditions sufficient to form an antibody-adaptor conjugate. In these embodiments, the method of the present invention further includes binding the antibody-adaptor conjugate to a drug moiety under conditions sufficient to covalently link a drug moiety to the antibody via the adapter.

[0260] In some embodiments, the structure of the immunoconjugate, preferably a single-domain antibody-drug conjugate NDC, is shown in the following molecular formula:

[0261] in:

[0262] nAb refers to the aforementioned single-domain antibody targeting CD20, or an antibody targeting CD20 or a multispecific antibody.

[0263] LU stands for connector / connector;

[0264] D is a drug;

[0265] Furthermore, the subscript p is selected from values ​​from 1 to 10.

[0266] application

[0267] This invention also provides uses for the antibodies of this invention, such as in the preparation of diagnostic agents or in the preparation of drugs for the prevention and / or treatment of CD20-related diseases. These CD20-related diseases include tumorigenesis, growth and / or metastasis, tumor drug resistance-related diseases, autoimmune diseases, inflammation, metabolic-related diseases, etc.

[0268] The uses of the antibodies or ADCs of this invention include (but are not limited to):

[0269] (i) Diagnosis, prevention and / or treatment of tumor occurrence, growth and / or metastasis, especially tumors with high CD20 expression, including (but not limited to): lymphoma, multiple myeloma;

[0270] (ii) Diagnosis, prevention and / or treatment of autoimmune diseases.

[0271] Pharmaceutical Composition

[0272] The present invention also provides a composition. In a preferred embodiment, the composition is a pharmaceutical composition comprising the aforementioned antibody or its active fragment or its fusion protein or its NDC or corresponding CAR-T cells, and a pharmaceutically acceptable carrier. Typically, these substances are formulated in a non-toxic, inert, and pharmaceutically acceptable aqueous carrier medium. The formulated pharmaceutical composition can be administered via conventional routes, including (but not limited to): intratumoral, intraperitoneal, intravenous, or local administration.

[0273] The antibody described in this invention can also be expressed in cells by a nucleotide sequence for cell therapy, such as for chimeric antigen receptor T-cell immunotherapy (CAR-T).

[0274] The pharmaceutical compositions of the present invention can be directly used to bind to CD20 protein molecules, and therefore can be used for the prevention and treatment of diseases such as tumors. Furthermore, other therapeutic agents can be used simultaneously.

[0275] 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 above-described monoclonal antibody (or conjugate thereof) of the present invention, and a pharmaceutically acceptable carrier or excipient. Such carriers include (but are not limited to): saline, buffer, 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 by conventional methods using physiological saline or an aqueous solution containing glucose and other excipients. Pharmaceutical compositions such as injections and solutions are preferably 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.

[0276] When using a pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to mammals. This safe and effective amount is typically at least about 10 micrograms per kilogram of body weight, and in most cases does not exceed about 50 milligrams per kilogram of body weight. Preferably, the dose is about 10 micrograms per kilogram of body weight to about 20 milligrams per kilogram of body weight. Of course, the specific dosage should also take into account 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.

[0277] For NDCs, because the single-domain antibody-drug conjugates provided by this invention can target specific cell populations and bind to cell surface-specific proteins (antigens), thereby releasing the drug into the cell in its active form through conjugate endocytosis or drug infiltration, the single-domain antibody-drug conjugates of this invention can be used to treat target diseases. The aforementioned antibody-drug conjugates can be administered to subjects (e.g., humans) in therapeutically effective amounts via appropriate routes. Subjects requiring treatment may be patients at risk or suspected of having a condition related to the activity or expression level of a specific antigen. Such patients can be identified through routine physical examinations.

[0278] When treated with the single-domain antibody-drug conjugate of the present invention, delivery can be performed using methods conventional in the art. For example, it can be introduced into cells using liposomes, hydrogels, cyclodextrins, biodegradable nanocapsules, or bioadhesive microspheres. Alternatively, the nucleic acid or carrier can be delivered locally by direct injection or by using an infusion pump.

[0279] Antibody screening methods

[0280] This invention utilizes an alpaca immunization and phage display technology platform to screen antibodies against CD20 target antigens, obtaining CD20 nanobodies. CD20 is a four-transmembrane protein, and the conformation of recombinantly expressed CD20 protein may differ from that of the natively expressed CD20 protein on cells. Therefore, this study employed an alternating protein / cell line panning method to enrich phage antibodies that can simultaneously bind to both recombinantly expressed CD20 protein and CHO-K1-CD20 cells, and screened for nanobodies that specifically bind to CD20 antigens on the cell membrane surface.

[0281] This study used phage display technology to screen for specific antibodies targeting CD20, and evaluated the specificity of these antibodies at the phage level using ELISA and FACS experiments. Ultimately, several anti-CD20 nanobody clones with good specificity were obtained.

[0282] Specifically, this study used two antibody libraries and, through alternating panning of recombinant CD20 protein and CHO-K1-CD20 cells, selected a total of 133 monoclonal antibodies for initial screening using flow cytometry (FACS). Of these, 54 clones specifically bound to CD20-positive cells (CHO-K1-CD20) but not to CD20-negative cells (CHO-K1). Sequencing yielded three different monoclonal sequences. These three antibodies were then identified by flow cytometry (FACS) with various CD20-positive (CHO-K1-CD20, Raji) and CD20-negative cell lines (CHO-K1, Jurkat), and by enzyme-linked immunosorbent assay (ELISA) with CD20 proteins from different species (human-CD20-his-Bio, Cyno-CD20-his) and unrelated proteins (KACTUS-VLP, SA). The three clones exhibited good binding affinity and specificity on multiple cell lines and various protein antigens. Obtaining these clones laid the foundation for the subsequent development of CD20 CAR-T products or antibody drugs. The overall project flow is shown in Figure 1.

[0283] CAR-T screening methods

[0284] Based on screened antibody sequences, this invention designed a CAR molecular vector targeting CD20 and obtained CD20 CAR-T cells via lentiviral transduction into T cells. CD107a degranulation assays showed that these CD20 CAR-T cells were effectively activated upon stimulation by CD20-positive target cells Raji and JeKo-1, while no significant CD107a release was observed upon stimulation by negative target cells NCI-H929, indicating that these CD20 CAR-T cells specifically bind to CD20. In vitro cell killing assays showed that these CAR-T cells exhibited strong in vitro killing activity against Raji and JeKo-1. Further in vivo efficacy evaluation of these CAR-T cells in mice showed that VHH10 and VHH20 CAR-T cells had better tumor-suppressing effects in vivo, and were superior to Ofa (Ofatumumab). VHH10H1-VHH10H6 were obtained by humanizing VHH10, and the efficacy of the humanized CAR-T was further evaluated in vitro and in vivo. The results showed that VHH10H1 had a good in vivo tumor-suppressing effect, comparable to OFA, but weaker than wild-type VHH10 CAR-T.

[0285] The main advantages of this invention include:

[0286] 1) The anti-CD20 antibody provided by this invention is a single-domain antibody. Compared with traditional antibodies, single-domain antibodies have the characteristics and advantages of small size, strong penetrability, high stability, low immunogenicity, high affinity, and ease of modification.

[0287] 2) This invention provides single-domain antibodies with strong affinity and good specificity, and further verifies the function of CAR-T cells constructed based on the single-domain antibodies VHH10 and VHH20 of this invention. The CAR-T cells of this invention exhibit the best in vivo anti-tumor efficacy and good safety in mice, which is beneficial for clinical drug development.

[0288] 3) The high-affinity specific antibody provided by this invention can also be used in the development of detection reagents.

[0289] 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.

[0290] Example 1. Alpaca Immunization

[0291] Healthy adult alpacas were used to immunize themselves by injection after mixing CD20 antigen or CD20 overexpressing cell line CHO-K1-CD20 with adjuvant. The immunization schedule is shown in Table 1. On the 7th day after the fifth booster immunization, peripheral blood of alpacas was collected to construct a phage display library.

[0292] Table 1 Alpaca Immunization Schedule

[0293] Example 2. Construction of a phage antibody library

[0294] Alpaca were immunized with CD20-VLP protein and / or CD20-overexpressing cells CHO-K1-CD20 to obtain peripheral blood. RNA was extracted and reverse transcribed into cDNA. The heavy chain variable region was amplified, the target fragment was recovered, digested with restriction endonucleases, and ligated into a library construction vector to construct a phage antibody library.

[0295] Example 3. Enrichment of specific antibody clones targeting the CD20 protein from a phage antibody library by affinity panning.

[0296] Appropriate negative and positive panning strategies were employed to enrich the desired specific antibody clones from the phage antibody library.

[0297] 3.1 CD20 protein selection

[0298] Multiple rounds of panning were performed using CD20-His as the positive selection protein to obtain a phage pool enriched with target antibody clones. The experimental procedures are briefly described below:

[0299] 1) Block the SA magnetic beads with blocking solution for 2 hours, and then bind the target antigen (CD20-His) to the blocked SA magnetic beads;

[0300] 2) Add phage library (containing 5x10) 12 (One phage particle) and a clean SA magnetic bead are incubated together to eliminate phage antibody clones that do not specifically bind to the SA magnetic beads;

[0301] 3) After incubation, the supernatant was transferred to SA magnetic beads bound to the target antigen and incubated for a longer period to allow the phage to bind to the target antigen.

[0302] 4) Wash the magnetic beads with detergent to remove unbound phages;

[0303] 5) Elute the positive phages from the target antigen with elution buffer, and then neutralize them with neutralization solution;

[0304] 6) Reinfect the host bacteriophage XL1-blue with the eluted phage to amplify the recovered phage. Reserve a small amount of sample for serial dilution, infect the host bacteriophage, plate with Amp antibody-resistant plates, and calculate the number of recovered phages.

[0305] 7) Repeat steps 1) to 6) until a significant increase in phage recovery rate (number of eluted phages / number of introduced phages) is observed.

[0306] The enriched phage pool can be used for subsequent monoclonal selection and FACS screening.

[0307] 3.2 CHO-K1-CD20 / CHO-K1 cell panning

[0308] Multiple rounds of panning were performed using CD20-negative CHO-K1 cells as negative panning cells and CD20-positive CHO-K1-CD20 cells as positive panning cells to obtain a phage pool enriched with the target antibody clone.

[0309] The simplified experimental steps are as follows:

[0310] 1) The phage pool enriched with specific clones after protein panning (containing 5 x 10^6 phages) 11 (one phage particle) and 1x10 7 Mix the negatively panned cells with CHO-K1 and incubate at room temperature for 2 hours using a rotary mixer. This allows the antibody clones that bind to the negatively panned cell lines to fully bind to these cells.

[0311] 2) Centrifuge at 1500 rpm for 5 minutes to pellet the cells. Transfer the supernatant to a new tube and mix with 1x10⁻⁶ cells. 7 Mix the CHO-K1-CD20 cells (CD20 positive cells) thoroughly and incubate at room temperature for 2 hours using a rotary mixer.

[0312] 3) Wash the cells 6 times with PBS, discarding the supernatant each time, resuspend, and centrifuge at 1500 rpm for 5 minutes to remove unbound phages;

[0313] 4) Elute the positive phages from the target antigen with elution buffer, and then neutralize them with neutralization solution;

[0314] 5) Reinfect the host bacteria with the eluted phage to amplify the recovered phage. Reserve a small amount of sample for serial dilution, infect the host bacteria with the diluted sample, plate with Amp antibody-resistant plates, and calculate the number of recovered phages.

[0315] 6) Repeat steps 1) to 5), usually requiring two rounds of screening, until a significant increase in phage recovery rate (number of eluted phages / number of phages added) is observed.

[0316] A well-enriched phage pool can then be used for the next step of single-clone selection and FACS screening.

[0317] Main materials and reagents:

[0318] Alpaca immune antibody library;

[0319] Biotinylated Human CD20 / MS4A1 Full Length Protein,His,Avitag TM (Detergent)(SPR verified),ACRObiosystem,CD0-H82E5

[0320] BeaverBeads TM Streptavidin, Beaver Biology, 22307-10;

[0321] Blocking solution: PBS + 3% BSA

[0322] Rinse solution: PBS + 0.1% Tween 20

[0323] Elution buffer: 0.2M Glycine, pH 2.2

[0324] Neutralization solution: 1M Tris, pH 9.1

[0325] Experimental results:

[0326] Using different antibody libraries, three rounds of protein panning were conducted, and a significant increase in recovery rate was observed in each panning (Table 2), demonstrating that antibody clones were effectively enriched.

[0327] Table 2 Results of Protein / Cell Panning Experiment

[0328] As can be seen, after two rounds of panning, different antibody libraries were enriched. In terms of recovery rate, all three libraries were enriched and can be used for the next step of selecting monoclonal antibodies.

[0329] Example 4. Screening of specific clones from an enriched phage pool using flow cytometry (FACS).

[0330] Objective and Principle: The phage pool enriched through affinity panning contains phage antibodies of various properties: specific clones, non-specific clones, and negative clones. To obtain specific clones, single clones are isolated, packaged into monoclonal phages, and a large number of single clones are initially screened using flow cytometry (FACS) to select single clones that specifically bind to the CD20-positive cell line CHO-K1-CD20. Specific single clones are then further analyzed using DNA sequencing to determine the unique antibody sequence they contain.

[0331] In the initial FACS screening, the CD20-positive cell line CHO-K1-CD20 and the CD20-negative cell line CHO-K1 were used. Cells that only bind to CHO-K1-CD20 and not to CHO-K1 were identified as specific clones. Through FACS screening, candidate antibodies that can specifically recognize native CD20 molecules on the cell surface can be obtained for further screening.

[0332] Brief steps of FACS initial screening experiment:

[0333] 1) Culture and package monoclonal phages using deep-well 96-well plates;

[0334] 2) CHO-K1-CD20 and CHO-K1 cells were washed twice with PBS and resuspended in PBS to a density of 1x10⁻⁶ cells / mL. 7 / mL concentration, dispensed into 50μL portions into 96-well deep-well plates;

[0335] 3) Add 50 μL of packaged monoclonal phage to each well, mix well, and incubate at 4°C for 2 h;

[0336] 4) Wash twice with 200 μL PBS;

[0337] 5) Add 100 μL of mouse anti M13 primary antibody diluted 1:2000 per well, mix well by pipetting, and incubate at room temperature for 45 min.

[0338] 6) Wash twice with 200 μL PBS;

[0339] 7) Add 100 μL of Fluorescein (FITC) Affini Pure Goat Anti-Mouse IgG (H+L) diluted 1:300 to each well, mix well by pipetting, and incubate at room temperature for 45 min.

[0340] 8) Wash twice with 200 μL PBS; finally, resuspend the cells in 200 μL PBS;

[0341] 9) Detect the fluorescence intensity of the FITC channel of the sample using a flow cytometer and analyze the results.

[0342] Main materials and reagents:

[0343] Helper phage KO7, Thermo / Invitrogen, 18311019

[0344] Anti-M13 Bacteriophage Coat Protein g8p antibody,abcam,ab9225

[0345] Fluorescein(FITC)AffiniPure Goat Anti-Mouse IgG(H+L),Jackson ImmunoReseach,115-095-003

[0346] Experimental results:

[0347] Single clones were randomly selected from the enriched phage antibody pool, packaged into phages, and their binding to CD20-overexpressing cell lines CHO-K1-CD20 and CD20-negative cell lines CHO-K1 was detected using phage FACS to identify CD20-specific phage antibody clones. The initial FACS screening results are shown in Figure 2. Clones A1 and A3 bound to CHO-K1-CD20 cells but not to CHO-K1 cells, indicating they were specific clones; the other clones were non-specific binding clones (binding to both cell types).

[0348] A total of 54 FACS-positive and highly specific clones were obtained through initial screening using FACS. These 54 positive and highly specific clones were then sequenced, yielding three different monoclonal sequences. The binding specificity of these three monoclonal sequences was further tested using FACS identification on multiple cell lines and ELISA identification on multiple antigens.

[0349] Example 5. Identification of monoclonal specificity at the phage level using multiple cell lines via FACS

[0350] Experimental Objective and Principle: Antibodies used for treatment must possess excellent target specificity, binding only to the target antigen and excluding any irrelevant antigens. Furthermore, different cell lines may exhibit differences in the amino acid sequence of the same antigen (isomers or mutants) or bind different ligands, necessitating an examination of whether the antibody can bind to cells positive for various target proteins. To further analyze the specificity and universality of these monoclonal antibodies and identify the best candidate clones, flow cytometry was used to further evaluate the specificity of the initial screening clones. In this experiment, multiple CD20-positive and CD20-negative cell lines were reacted with these monoclonal phage antibodies to analyze whether these clones could bind to the CD20 antigen on different cell lines and whether they exhibited any non-specific binding to other cell lines that do not express CD20. This experiment yielded several clones with excellent specificity.

[0351] Experimental method: Same as FACS initial screening;

[0352] Main samples and reagents:

[0353] CHO-K1-CD20 cell line, CD20 positive cell line;

[0354] CHO-K1 cell line, CD20 negative cell line;

[0355] Raji cell line, CD20 positive cell line;

[0356] Jurkat cell line, CD20 negative cell line;

[0357] The remaining reagents are the same as those used for the FACS initial screening.

[0358] Experimental results:

[0359] The specific clones obtained in Example 4 were identified by flow cytometry in a wider range of cell lines. The results, as shown in Figure 3, indicate that Clone10, Clone13, and Clone20 bound to the CD20-highly expressing cell line CHO-K1-CD20 and the CD20-positive tumor cell line Raji, but not to the two CD20-negative cell lines CHO-K1 and Jurkat, demonstrating good specificity.

[0360] Example 6. Identification of monoclonal specificity at the phage level using antigens with different structures via ELISA.

[0361] Experimental Objective and Principle: Antibodies used for treatment must possess excellent target specificity, binding only to the target antigen and excluding any unrelated antigens. To further analyze the binding specificity of these monoclonal antibodies and their binding to antigens with different structures, enzyme-linked immunosorbent assay (ELISA) was used to further evaluate the specificity of the initial screening clones. In this experiment, CD20 antigens with different structures and other unrelated antigens reacted with these monoclonal phage antibodies to analyze whether these clones could bind to CD20 antigens with different structures and whether they exhibited any non-specific binding to other unrelated CD20 antigens.

[0362] Experimental methods:

[0363] Brief steps of the ELISA experiment:

[0364] 1) Single-tube culture and packaging of monoclonal phages;

[0365] 2) Dilute Strepavidin to 2 μg / mL with PBS and add 100 μL / well to a high-binding microplate. Incubate at room temperature for 2 hours.

[0366] 3) Discard the coating solution, add 250 μL of blocking solution to each well, and seal overnight at 4°C;

[0367] 4) Wash the plate twice with 250μL of rinsing solution;

[0368] 5) Dilute the biotin-tagged target protein and control protein to 2 μg / mL with PBS, add 100 μg / well to a microplate pre-coated with Strepavidin, and bind at room temperature for 1 h.

[0369] 6) Wash the plate twice with 250μL of rinsing solution;

[0370] 7) Add 100 μL of the phage supernatant cultured in step 1) to the well coated with the target antigen, and allow it to bind at room temperature for 2 hours;

[0371] 8) Wash the plate 4 times with 250μL of rinsing solution;

[0372] 9) Add 100 μL of Anti-M13 Antibody (HRP) diluted 1:10000 to each well and incubate at room temperature for 45 min;

[0373] 10) Wash the plate 6 times with 250μL of rinsing solution;

[0374] 11) Add 100 μL of TMB substrate and develop for 5 to 10 minutes;

[0375] 12) Add 100 μL of 2M H2SO4 to stop the reaction and read the results on the microplate reader.

[0376] Main samples and reagents:

[0377] The remaining reagents are the same as those used for the initial ELISA screening.

[0378] Experimental results:

[0379] Antibodies used for treatment must possess very high target specificity. To further analyze the specificity of these monoclonal antibodies, multiple clones obtained in Example 4 were identified using enzyme-linked immunosorbent assay (ELISA) on various antigens. The results are shown in Figure 4. The negative control phage antibody clone did not bind to either the target antigen or the control antigen; the anti-M13 phage HRP Ab was a negative antibody control with only the second antibody added; the anti-human IgG HRP Ab was a negative control with only the second antibody added to the ofatumumab control antibody; neither of these bound to either the target antigen or the control antigen; and the anti-his HRP Ab was a positive control antibody for detecting the antigen tag. This indicates that the coated antigen has bound to the ELISA plate. Clone10 clone binds strongly to the KACTUS human CD20 VLP antigen, weakly to the Biotinylated Acro human CD20 and Acro human CD20 antigens, and does not bind to the KACTUS human CD20 Loop antigen. The binding behavior is similar to that of the control antibody ofatumumab, indicating that Clone10's binding epitope differs from the other clones and is not located on the large loop; the binding epitope may be due to spatial conformation. Clone13 and Clone20 clones bind to all four CD20 target antigens, with binding behavior similar to Rituximab, indicating that their binding epitopes are on the large loop and are Type I antibodies. Furthermore, the three screened antibody clones do not bind to the two unrelated antigens, indicating that these three clones are antibodies that specifically bind to CD20 target antigens with good specificity.

[0380] Example 7. Identification of monoclonal specificity at the protein level using multiple cell lines via FACS

[0381] To further analyze whether these monoclonal antibodies retained their original binding ability and specificity after being expressed as IgG antibodies in a eukaryotic system, the three clones obtained in Example 4 were expressed via CHOS, and the antibodies purified from Protein A to IgG form were identified by flow cytometry (FACS). The results, shown in Figure 5, indicate that Ofatumumab served as a positive control antibody for CD20. Clone10, Clone13, and Clone20 bound to the CD20-highly expressed cell line CHO-K1-CD20 and the CD20-positive tumor cell line Raji, but not to either of the two CD20-negative cell lines, CHO-K1 and Jurkat, demonstrating good specificity.

[0382] Example 8. Study on cross-activity of antibodies between humans and monkeys

[0383] To determine the cross-binding activity of CD20 antibodies against monkeys, an enzyme-linked immunosorbent assay (ELISA) was used to detect the strength of antibody binding to monkey CD20 antigen. Monoclonal antibodies were constructed from the three obtained clones and prepared using CHOS expression for the study of cross-species activity. Different species CD20 antigens (Acro Human CD20 bio, Cyno-human CD20) were diluted to 2 μg / mL with PBS, and 100 μL / well was coated onto 96-well ELISA plates and incubated overnight at 4°C. Blocking was performed with 3% BSA at 37°C for 2 hours. Antibodies were diluted to 1 μg / mL with PBS, and 100 μL / well was incubated at 37°C for 2 hours. HRP-labeled anti-human IgG antibody (HRP Donkey anti-human IgG, 410902, Biolegend) was diluted 1:2000, 100 μL / well, and incubated at 37°C for 1 hour. After washing six times with 0.01% PBST, add 100 μL / well of substrate TMB, react in the dark for 5 minutes, and terminate the reaction with 100 μL / well of 2M H2SO4. Detect A450 using a microplate reader.

[0384] The test results are shown in Figure 6. All three screened clone antibodies were able to specifically bind to human-monkey antigens, indicating that they all have monkey cross-activity.

[0385] Example 9. Detection of monoclonal affinity by FACS

[0386] The affinity between CD20 antibody molecules and antigens may have a significant impact on the killing effect and duration of CAR-T or antibody drugs in patients. To determine this important property, the half-maximal effective concentration (Ec50) of antibody molecules was analyzed using the FACS binding method, providing important information for the research and development process.

[0387] Brief steps of the FACS binding experiment:

[0388] 1) Preparation of antibodies at different concentrations: Anti-CD20 IgG was diluted 5-fold from 300 nM to 0.00384 nM using PBS to prepare 8 concentrations for testing the binding ability of antibodies to Raji cells;

[0389] 2) Wash Raji cells twice with PBS and resuspend them in PBS to a concentration of 1x10⁻⁶ cells / mL. 7 / mL concentration, dispensed into 50μL portions into 96-well deep-well plates;

[0390] 3) Add 50 μL of diluted antibody to each well, mix well, and incubate at 4°C for 2 hours;

[0391] 4) Wash twice with 200 μL PBS;

[0392] 5) Add Alexa diluted 1:300 647AffiniPureGoat Anti-Human IgG, 100μL / well, mix well by pipetting and incubate at room temperature for 45min;

[0393] 6) Wash twice with 200 μL PBS; finally, resuspend the cells in 200 μL PBS;

[0394] 7) Analyze samples on a flow cytometer (Alexa) Fluorescence intensity of channel 647;

[0395] 8) Analyze the binding constant using Graphpad Prism software.

[0396] Main samples and reagents:

[0397] Raji cell line, CD20-positive cell line;

[0398] Alexa 647AffiniPure Goat Anti-Human IgG,Fcγfragment specific,Jackson ImmunoReseach,109-605-008

[0399] Experimental results

[0400] Affinity refers to the strength of the binding between a single molecule and its ligand. It is typically assessed and ranked by FACS (Fluorescence Antibody-Coding Acid) detection of the binding ability to positive cell lines. A lower Ec50 value indicates a greater affinity of the antibody for its target. As shown in Tables 3 and 4 and Figures 7 and 8, Clone 10, Clone 13, and Clone 20 can all bind to the CD20-positive Raji cell line.

[0401] Table 3

[0402] Table 4

[0403] Example 10. Determination of affinity for anti-CD20 SdAbs

[0404] (1) Experimental objective and principle:

[0405] The affinity between CD20 SdAb and the antigen can significantly influence the cytotoxic effects and duration of CAR-T or antibody drugs in patients. To determine this important property, this embodiment employed Sartorius's Biomembrane Interference (BLI) technique for measurement. This system utilizes biomembrane interference, a label-free technique that provides high-throughput biomolecular interaction information in real time. The instrument emits white light onto the sensor surface and collects the reflected light. The reflected spectra at different frequencies are affected by the thickness of the biosensor's optical film layer; some frequencies of reflected light form constructive interference (blue), while others experience destructive interference (red). These interferences are detected by the spectrometer, forming an interference spectrum, which is displayed as the phase shift intensity (nm). Therefore, any increase or decrease in the number of molecules bound to the sensor surface will be detected in real time by the spectrometer, and this shift directly reflects the thickness of the biomembrane on the sensor surface. High-quality data on biomolecular interactions can be obtained, enabling the determination of biomolecular interaction dynamics parameters (Kon, Kdis, and KD), providing crucial information for the research and development process.

[0406] (2) The experimental steps are as follows:

[0407] 1) Dilute anti-CD20 IgG (composed by fusing the VHH sequence of CD20 with human IgG4 Fc) to 10 μg / mL with loading buffer (1×PBS, pH 7.4, 0.01% BSA and 0.02% Tween 20) and load it onto the biosensor.

[0408] 2) After the 60s equilibrium phase, the binding kinetics of CD20 antigen (Acrobiosystem, CD0-H52H3) were monitored at various antigen concentrations (100 to 6.25 nM). Binding and dissociation were performed for 30s and 300s respectively at each concentration.

[0409] 3) Regenerate the chip by washing it three times with 10mM Glycine-HCl at pH 1.5.

[0410] 4) The binding constant was analyzed using a 1:1 binding site model (BLI analysis software V11.0).

[0411] (3) Experimental results:

[0412] Affinity refers to the strength of binding between a single molecule and its ligand, and is usually measured and reported using the equilibrium dissociation constant (KD). The equilibrium dissociation constant can be used to assess and rank the strength of the interaction between two molecules. The binding of an antibody to its antigen is a reversible process, and the rate of the binding reaction is directly proportional to the concentration of the reactant. The smaller the KD value, the greater the affinity of the antibody for its target. The affinity test results are shown in Table 5 (Affinity test results of anti-CD20 IgG): Clone 10 IgG, Clone 13 IgG, and Clone 20 IgG can all bind to the CD20 antigen.

[0413] Table 5

[0414] Example 11. Preparation of CAR-T cells and detection of CAR molecule expression

[0415] To verify the function of the candidate clone on CAR-T cells, a CAR vector was constructed based on the candidate antibody sequence. The CAR structure contains a CD8α signal peptide (SP) and a V receptor that binds to the target antigen CD20. HH Alternatively, it can be scFv (VH-linker-VL), the hinge region and transmembrane region between the cell membrane and extracellular binding region (IgG4 hinge-CH2-CH3, CD8αTM), the 4-1BB co-stimulatory molecule, and the CD3ζ intracellular domain, linked with a truncated EGFR molecule (tEGFR) via T2A. This can be used as a safety switch during clinical translation and can also characterize CAR molecule expression. tEGFR is not an essential element of the CAR molecule and does not affect CAR molecule expression or function.

[0416] T cells were activated using CD3 / CD28 Dynabeads (Gibco, 11132D). After 24 hours of activation, CAR molecules were transduced into T cells via lentivirus. The medium was changed 24 hours after viral transduction. CAR molecule expression was detected 7 days after viral transduction. The specific experimental steps are as follows:

[0417] 1) Take 1×10 5 Wash CAR-T or T cells once with PBS, centrifuge at 400g for 5 minutes, and discard the supernatant.

[0418] 2) Resuspend the cell pellet in 50 μL PBS, add 1 μL of Alexa Fluor488-EGFR (Clone AY13, BioLegend, Cat. No. 352908) or Alexa Fluor488-G4S linker (E702V, Cell Signaling, Cat. No. 50515) antibody respectively, and incubate at 4°C in the dark for 15 minutes.

[0419] 3) After incubation, wash twice with PBS, centrifuge at 400g for 5 minutes, and discard the supernatant.

[0420] 4) Resuspend the cell pellet in 100 μL PBS and perform flow cytometry analysis.

[0421] The expression of CAR molecules is shown in Figure 9. 3508 represents the CAR molecule constructed using the positive control of ofatumumab, 3710 is the CAR molecule constructed using Clone10, 3713 is the CAR molecule constructed using Clone13, and 3720 is the CAR molecule constructed using Clone20. CAR conversion of 3508 was characterized using the G4S linker, while CAR conversion of 3710, 3713, and 3720 was indirectly characterized using tEGFR. The main structures of each CAR molecule are as described above.

[0422] The results showed that when mockT staining was negative, all CAR molecules were effectively expressed, with conversion rates all above 40%.

[0423] Example 12. Cytotoxic T cell degranulation evaluation assay (CD107a stimulation assay)

[0424] Experimental principle and purpose:

[0425] CD107a is a marker of intracellular microvesicles. When granzyme-loaded microvesicles fuse with the cell membrane, the amount of CD107a on the cell membrane increases. When monensin (BioLegend) is used to block its reuptake, the intensity of microvesicle release can be quantitatively reflected. When CAR-T cells are stimulated by target antigens on target cells, granzyme release occurs, and the increase in CD107a can be detected by flow cytometry to determine the activation status of T cells.

[0426] Brief steps of the cytotoxic T cell degranulation assay:

[0427] 1) Centrifuge the CAR-T cells to be tested and the target cells separately at 300g for 5 min at room temperature, discard the supernatant, and resuspend them in RPMI-1640 medium + 10% FBS (Gibco, Cat. No. 10099141) to a concentration of 1x10⁻⁶. 6 cells / mL;

[0428] 2) In a 96-well plate, add 100 μL of the CAR-T cells to be tested and 100 μL of Raji-luc (CD20+) or JeKo-1-luc (CD20+) or NCI-H929-luc (CD20-) target cells or 100 μL of buffer and mix well (Figure 10).

[0429] 3) Add 1.5 μL of PE / Cy7 anti-human CD107a antibody (BD, Cat. No. 561348) and 0.2 μL of monensin (BioLegend, Cat. No. 420701) to each well of cells, and then incubate in a 37°C, 5% CO2 cell culture incubator for 4 h;

[0430] 4) After incubation, centrifuge at 300g for 5 minutes at 4℃, discard the supernatant, and wash the cells with 200μL PBS;

[0431] 5) Resuspend the cells in 100 μL PBS and add 1.5 μL of BV421 anti-human CD3 (Clone OKT3, BD, Cat. No. 317344), 1.5 μL of APC anti-human CD8 (Clone SK1, BioLegend, Cat. No. 344721), and Alexa Fluor488-EGFR (Clone AY13, BioLegend, Cat. No. 352908) or Alexa Fluor488-G4Slinker (E702V, Cell Signaling, Cat. No. 50515) antibody respectively. After mixing, incubate at 4°C in the dark for 20 min.

[0432] 6) After incubation, wash the cells twice with 200 μL PBS; finally, resuspend the cells in 100 μL PBS for flow cytometry analysis.

[0433] Experimental results:

[0434] CAR-T cells were obtained via lentiviral transduction and cultured in vitro for 10-14 days for CD107a degranulation. CAR-T cells to be tested, target cells, monensin, and CD107a antibody were co-incubated for 4 hours. After antibody labeling, flow cytometry analysis was performed. In FlowJo software, the lymphocyte phylum (P1) was selected from the scatter plot, and cell debris was removed. Cells in the P1 phylum were further separated into individual dispersed phylum (P2) using FSC-A and FSC-H. CD3-positive cells were further selected from the P2 phylum (P3). CD8-positive cells were further selected from the P3 phylum (P4). Finally, the proportion of CD107a-positive cells among EGFR- or G4S-positive cells (i.e., CAR-positive cells) in the P4 phylum was analyzed.

[0435] Experimental results:

[0436] As shown in Figure 10, the degranulation level of each CD20 CAR-T cell significantly increased only after co-incubation with CD20-positive target cells (Raji or JeKo-1), while no significant degranulation level was observed after co-incubation with CD20-negative target cells (NCI-H929). Among them, the degranulation level of CAR-T cells constructed using VHH10 (Clone10), VHH13 (Clone13), and VHH20 (Clone20) (i.e., 3710, 3713, and 3720) after co-incubation with CD20-positive cells was comparable to that of CAR-T cells constructed using Ofa (Ofatumumab) (3508).

[0437] Example 13. In vitro cell killing experiment

[0438] Experimental objective and principle:

[0439] In vitro cell killing experiments used human Burkitt's lymphoma cells Raji (Figure 11A) and human mantle cell lymphoma cells JeKo-1 (Figure 11B) as CD20-positive target cells to evaluate the antigen-specific killing ability of anti-CD20 CAR-T cells. These target cells were obtained through lentiviral transduction to stably express firefly luciferase; therefore, the luciferase activity in the sample reflects the number of target cells. CAR-T cells and target cells were co-cultured. When target cells were killed by CAR-T cells, luciferase was released and quickly inactivated (firefly luciferase half-life is approximately 0.5 h). If target cells were not killed or inhibited by CAR-T cells, more luciferase would be produced as target cells proliferated and luciferase expression continued. Adding a luciferin substrate would induce fluorescence, and the emitted photons could be detected by a photosensitive element. Therefore, the lower the luciferase activity reading, the more target cells were killed, reflecting a stronger CAR-T killing ability against that target cell.

[0440] Brief steps of in vitro cell killing experiment:

[0441] 1) Based on different effector-to-target ratios, resuspend the required number of CAR-T cells in RPMI-1640 + 10% FBS medium, and add 100 μL of CAR-T cells to each well of a 96-well plate.

[0442] 2) Take the required number of target cells and resuspend them in RPMI-1640 + 10% FBS medium to a concentration of 1 x 10⁻⁶ cells / mL. 5 100 μL of target cells / mL were added to each of the above 96-well plates, mixed with CAR-T cells and co-cultured, and then incubated in a carbon dioxide incubator for 24 h.

[0443] 3) After incubation, the luciferase activity in each well was detected using a luciferase assay kit (Steady-Glo Luciferase Assay System, Promega, Cat. No. E2520).

[0444] Experimental results:

[0445] As shown in Figure 11, all CAR-T cell samples were able to effectively kill CD20-positive target cells, and the killing ability of VHH10, VHH13, and VHH20 CAR-T cells was stronger than that of the control Ofa CAR-T cells.

[0446] Example 14. Antitumor efficacy experiment in NPG tumor-bearing mice

[0447] 1x10 6One JeKo-1-luc cell was injected into immunodeficient NPG mice via the tail vein (Beijing Vitonda). Three days after tumor formation, 1x10 cells were reinfused via the tail vein. 6 CAR-T cells were collected, and imaging and blood sampling were performed weekly. The mice were weighed twice a week, and their survival status was observed.

[0448] Experimental results:

[0449] Figures 12-15 show the antitumor efficacy of different CAR-T cells in mice. As shown in Figure 12, after infusion of VHH10 and VHH20 CAR-T cells, tumors were well suppressed, indicating good in vivo tumor-suppressing effects. However, after infusion of Ofa and VHH13 CAR-T cells, the tumor burden continued to increase, indicating poor in vivo tumor-suppressing effects, although the tumor burden was lower than that of the MockT and PBS groups. Correspondingly, T cell proliferation was better in mice in the VHH10 and VHH20 groups, while T cell proliferation was poor in the other groups (Figure 13). Within 7 weeks of CAR-T infusion, mice in the PBS group had a high tumor burden and severe weight loss, and all died by day 40. 40% of mice in the MockT group died, while no mice died in the other CAR-T groups, and their weight remained normal (Figures 14 and 15). These results indicate that VHH10 and VHH20 CAR-T cells have better in vivo tumor-suppressing effects than Ofa CAR-T.

[0450] Example 15. In vitro cell killing experiment

[0451] Experimental objective and principle:

[0452] The in vitro cell-killing ability of the humanized VHH10 clone was evaluated. Human Burkitt's lymphoma cells Raji (Figure 16A), human mantle cell lymphoma cells JeKo-1 (Figure 16B) were used as CD20-positive target cells, and human multiple myeloma cells RPMI-8226 (Figure 16C) were used as negative target cells. All target cells were transduced via lentivirus to obtain target cells stably expressing firefly luciferase.

[0453] Brief steps of in vitro cell killing experiment:

[0454] 1) Based on different effector-to-target ratios, resuspend the required number of CAR-T cells in RPMI-1640 + 10% FBS medium, and add 100 μL of CAR-T cells to each well of a 96-well plate.

[0455] 2) Take the required number of target cells and resuspend them in RPMI-1640 + 10% FBS medium to a concentration of 1 x 10⁻⁶ cells / mL. 5100 μL of target cells / mL were added to each of the above 96-well plates, mixed with CAR-T cells and co-cultured, and then incubated in a carbon dioxide incubator for 24 h.

[0456] 3) After incubation, the luciferase activity in each well was detected using a luciferase assay kit (Steady-Glo Luciferase Assay System, Promega, Cat. No. E2520).

[0457] Experimental results:

[0458] As shown in Figure 16, all CAR-T cell samples were able to effectively kill CD20-positive target cells (Figure 16A-B), and had no or weak killing effect on negative cells (Figure 16C).

[0459] Example 16. Antitumor efficacy experiment in NPG tumor-bearing mice

[0460] 1x10 6 One JeKo-1-luc cell was injected into immunodeficient NPG mice via the tail vein (Beijing Vitonda). Four days after tumor formation, 1x10 cells were reinfused via the tail vein. 6 CAR-T cells were collected, and imaging and blood sampling were performed weekly. The mice were weighed twice a week, and their survival status was observed.

[0461] Experimental results:

[0462] Figures 17-18 show the antitumor efficacy of different CAR-T cells in mice. As shown in Figure 17, after VHH10 CAR-T infusion, the tumor was completely suppressed, indicating a good in vivo tumor-suppressing effect. Ofa and VHH10H1 showed good tumor-suppressing effects within 4 weeks after CAR-T infusion, followed by gradual tumor recurrence. The other CAR-T groups showed poor tumor-suppressing effects, with rapid tumor recurrence after CAR-T infusion, but the tumor burden was lower than that of the MockT and PBS groups (Figures 17A-B). Within 8 weeks of CAR-T infusion, all mice in the PBS and MockT groups died, while no mice died in the other CAR-T groups (Figure 18). These results indicate that VHH10 CAR-T has a good in vivo tumor-suppressing effect, followed by Ofa and VHH10H1, while VHH10H2, VHH10H3, VHH10H4, VHH10H5, and VHH10H6 have some in vivo tumor-suppressing effects.

[0463] Example 17. In vivo CAR-T cell generation and its tumor-suppressing effect

[0464] In this embodiment, using the CAR molecule constructed with VHH10H1 as an example, a lentiviral vector carrying the CAR gene was injected into mice, and the CAR-T cells generated in vivo and their tumor-suppressing activity were detected.

[0465] Experimental methods:

[0466] A plasmid carrying the CAR gene encoding the molecule was mixed with a plasmid for lentiviral packaging and transfected into HEK293T cells to prepare a lentiviral vector. In this embodiment, the lentiviral envelope surface also expressed a targeting molecule containing an anti-CD7 antibody and a T cell activation molecule containing an anti-CD3 antibody, for targeted delivery of the CAR gene encoding the molecule to T cells. The prepared lentiviral vector was named LV0181.

[0467] Severely immunodeficient NOD.Cg-Prkdcscid Il2rgtm1 / Vst(NPG) (Vitonica) mice were inoculated with 5E6 Jeko-1 lymphoma cells in their flanks. Prior to inoculation, Jeko-1 cell suspension was mixed with an equal volume of matrix gel. Seven days after tumor cell inoculation, when the tumor was palpable, each mouse was intraperitoneally injected with 2E7 PBMCs. One day after PBMC injection, the lentiviral vector LV0181 was administered via tail vein injection at doses of 1.53E7 TU (low-dose group), 3.06E7 TU (medium-dose group), and 6.12E7 TU (high-dose group). Control mice received 0.2 mL of the formulation solution via tail vein. Six mice were in each group. Tumor volume was measured twice weekly after administration. On days 10 and 17 post-administration, peripheral blood PBMC reconstitution levels and CAR-T cell levels were monitored to assess the success of PBMC reconstitution and CAR-T cell generation.

[0468] Experimental results:

[0469] As shown in Figure 19, compared with the control group, all LV0181 administration groups showed significant tumor-suppressive activity (excluding two mice from the control group whose PBMC reconstruction failed). Tumor growth slowed down 10 days after administration, and the tumor volume began to shrink after 14 days. By day 21 after administration, 50% of the mice in the medium-dose and high-dose groups had completely disappeared from their tumors.

[0470] As shown in Figure 20, after excluding mice with failed PBMC reconstruction, peripheral blood CAR-T detection showed that CAR-T amplification could be detected in each lentiviral vector administration group on day 10 after administration, and on day 17 after administration, the proportion of peripheral blood CAR-T cells in total T cells reached 20%-45%.

[0471] This application involves the following nucleic acid and amino acid sequences:

[0472] 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. An antibody or antigen-binding fragment thereof targeting CD20, characterized in that, The heavy chain variable region of the antibody includes HCDR1, HCDR2, and HCDR3, wherein HCDR1, HCDR2, and HCDR3 are selected from one of the following combinations: (1) HCDR1 with the amino acid sequence GRTFSSYN (SEQ ID NO:1); The amino acid sequence is ISWSGGSP (SEQ ID NO:2) of HCDR2; The amino acid sequence is HCDR3 of AAPLTYGSNWLADY (SEQ ID NO:3); (2) HCDR1 with the amino acid sequence GRTFSYDT (SEQ ID NO:5); The amino acid sequence is ISSSGGFT (SEQ ID NO:6) of HCDR2; The amino acid sequence is AADRLQLYMTTTPHY (SEQ ID NO:7) of HCDR3; (3) HCDR1 with the amino acid sequence GRTFSSYT (SEQ ID NO:9); The amino acid sequence is VSWSGGTT (SEQ ID NO:10) of HCDR2; The amino acid sequence is AADRVVYMTTTPQY (SEQ ID NO:11) of HCDR3; The antibody also includes variants of the CDR sequence combinations shown in any one of (1)-(3) above, wherein the variants have at least 90% sequence identity with the CDR sequences shown in any one of (1)-(3), or contain at least one and no more than 10, or no more than 5, 4, 3, 2 or 1 amino acid changes on the CDR sequence, and are able to retain CD20 binding affinity.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein, The antibody described is a single-domain antibody.

3. The antibody or antigen-binding fragment thereof of claim 1, wherein The antibody is a camel-derived antibody or a humanized antibody.

4. The antibody or antigen-binding fragment thereof of claim 1, wherein, The amino acid sequence of the antibody is shown in any one of SEQ ID NO:4, 8, 12, or has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it.

5. The antibody or antigen-binding fragment thereof of claim 1, wherein The amino acid sequence of the antibody is shown in any one of SEQ ID NO:29, 31, 33, 35, 37, 39, or has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it.

6. A chimeric antigen receptor (CAR), characterized in that, The antigen-binding domain of the chimeric antigen receptor comprises an antibody or antigen-binding fragment thereof targeting CD20 as described in claim 1.

7. The chimeric antigen receptor of claim 6, wherein, The structure of the chimeric antigen receptor is shown in Formula I: LVH-TM-C-CD3ζ (I) In the formula, Each "-" independently represents a linking peptide or peptide bond; L represents the signal peptide sequence; V represents the antigen-binding domain; H represents the hinge area; TM represents a transmembrane domain; C is a co-stimulatory signaling molecule; CD3ζ is a cytoplasmic signal transduction sequence derived from CD3ζ.

8. The chimeric antigen receptor of claim 6, wherein, The amino acid sequence of the CAR is shown in any one of SEQ ID NO:23, 25, 27, or has at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity with it.

9. A recombinant protein, characterized in that, The recombinant protein has the following characteristics: (i) the antibody targeting CD20 as described in claim 1, or its antigen-binding fragment; and (ii) Optional polypeptide molecules or fragments with therapeutic functions; and / or (iii) Optional functional domains that enhance the physicochemical properties or drug-likeness of proteins.

10. A nucleic acid molecule, characterized in that, The nucleic acid molecule encodes a polypeptide selected from the following group: (1) The antibody or antigen-binding fragment thereof targeting CD20 as described in claim 1; (2) The chimeric antigen receptor as described in claim 6; or (3) The recombinant protein as described in claim 9.

11. A vector, characterized in that, The carrier contains the nucleic acid molecule as described in claim 10.

12. An engineered immune cell, characterized in that, The immune cells express the chimeric antigen receptor as described in claim 6.

13. An immunoconjugate, comprising, The immunoconjugate contains: (a) The antibody portion, wherein the antibody portion is the antibody targeting CD20 as described in claim 1 or its antigen-binding fragment; and (b) A conjugation portion conjugated to the antibody portion, the conjugation portion being selected from the group consisting of: detectable markers, drugs, toxins, cytokines, enzymes, or combinations thereof.

14. A pharmaceutical composition or formulation comprising: (i) the antibody targeting CD20 as claimed in claim 1 or its antigen-binding fragment, the chimeric antigen receptor as claimed in claim 6, the recombinant protein as claimed in claim 9, the vector as claimed in claim 11, the engineered immune cell as claimed in claim 12, or the immunoconjugate as claimed in claim 13; and (ii) Pharmaceutically acceptable carriers.

15. A vector, characterized in that, The vector contains the encoding gene of the chimeric antigen receptor as described in claim 6, and the viral vector further contains a targeting element that targets immune cell surface antigens.

16. A method of generating an engineered immune cell, the method comprising, The method includes contacting immune cells with the carrier of claim 17.

17. Use of an active ingredient, characterized in that The active ingredient is selected from the group consisting of: the antibody targeting CD20 as described in claim 1 or its antigen-binding fragment, the chimeric antigen receptor as described in claim 6, the recombinant protein as described in claim 9, the vector as described in claim 11, the engineered immune cell as described in claim 12, the immunoconjugate as described in claim 13, or the vector as described in claim 15, or combinations thereof, wherein the active ingredient is used for (a) preparing reagents, detection plates or kits for detecting CD20; and / or (b) preparing medicaments for the prevention and / or treatment of CD20-related diseases or conditions.

18. A method for treating CD20-related diseases, characterized in that, The method includes administering to a desired object an antibody targeting CD20 as described in claim 1 or an antigen-binding fragment thereof, a chimeric antigen receptor as described in claim 6, a recombinant protein as described in claim 9, a vector as described in claim 11, engineered immune cells as described in claim 12, or an immunoconjugate as described in claim 13, a pharmaceutical composition or formulation as described in claim 14, or a vector as described in claim 15, or a combination thereof.

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