Title of Invention: Antibody or antigen-binding fragment thereof targeting human-derived CD7 and use thereof

Abstract

This invention provides an antibody or antigen fragment thereof that targets human-derived CD7 and its use. CD7-CARs based on this antibody and antibody fragment have very high affinity for the CD7 antigen molecule, and inhibitory molecules containing this antibody can almost completely inhibit the expression of the CD7 molecule on the cell surface without affecting the normal amplification of T cells, thus effectively avoiding sibling killing of CD7-CAR-T cells. CD7-CAR-T cells constructed using a single-domain antibody that targets human-derived CD7 have a high killing effect on target cells.

Description

Cross-reference of related applications

[0001] This application claims priority to Chinese Patent Application No. 202310906380.4, filed on 21 July 2023, titled "Antibodies or Antigen-Binding Fragments of the Same Targeting Human CD7 and Use thereof," the entire contents of which are incorporated herein by reference. [Technical Field]

[0002] This invention belongs to the fields of tumor immunotherapy and molecular immunology, and specifically relates to a single-domain antibody targeting human-derived CD7, CD7-CAR-T cells containing the single-domain antibody, a method for producing the same, and its use. [Background technology]

[0003] Tumor immunotherapy has become one of the most important methods of treating cancer. The immune system can only effectively attack cancer cells after it has recognized them. Cancer is the result of normal somatic cells losing their normal cellular control functions and accumulating a certain amount of genetic mutations, and cancer cells can cleverly evade the immune system's attack by disguising themselves as normal somatic cells.

[0004] Tumor-associated antigens (TAAs) refer to autoantigens expressed in tumor cells. Some TAAs are present in normal host cells and are mainly derived from gene amplification or post-translational modification, but they tend to be highly expressed or specifically expressed within tumor cells. Currently, many tumor-associated antigens and tumor-specific antigens have been discovered, and targeted therapy targeting TAAs is an important approach to cancer treatment. Many cancer immunotherapy drugs that rely on this mechanism are already commercially available and have excellent clinical efficacy.

[0005] Generally, antibody-mediated TAA recognition can cleverly guide NK cells or T cells to target corresponding TAA-highly expressing tumor cells. Antibodies targeting TAA (e.g., rituximab and trastuzumab) can not only directly kill tumor cells through ADCC activity, but can also function as diagnostic markers and drastically enhance the targeting capabilities of conventional cancer therapies.

[0006] Adoptive immunotherapy is a revolutionary therapeutic approach in hematology that uses genetic techniques to express synthetic chimeric antigen receptors (CARs) within T cells. CARs are genetically modified receptors that redirect immune cells to targeted cancer cells. CAR-T cells have shown remarkable results in patients with hematological malignancies, and the FDA has approved six types of CAR-T cells—Abecma, Breyanzi, Carvykti, Kymriah, Tecartus, and Yescarta—for the treatment of relapsed or refractory B-cell malignancies. The components of these CARs (extracellular antigen-binding domain, hinge, transmembrane domain, costimulatory domain, and activating domain) are systematically designed to optimize cell activation and enhance cell persistence. Using different domains or slightly altering the domain sequences can significantly change the effects of CAR-T cells. To date, CAR-T cells have been successful in treating hematological malignancies, but many patients experience CAR-T cell-related toxicity and relapse after CAR-T cell therapy, which may be related to the epitopes to which CARs bind, suggesting the need for the development of antibodies that bind to multiple epitopes. Next-generation CAR-T cell therapies aim to reduce toxicity and prevent relapse by targeting antigens, modulating the assembly or activation of CAR components, preventing anti-CAR immunity, and / or protecting cells to respond to their surrounding environment. FDA-approved CAR-T cells target two B-cell antigens, CD19 and B-cell maturation antigen (BCMA), but other CAR-T cells targeting B-cell malignancies, other hematological cancers, and solid tumors are rapidly emerging. Compared to B-cell malignancies, using first-line cancer treatments (including chemotherapy) on patients with T-cell malignancies results in limited clinical response and a poor prognosis for these patients. Given the clinical success of CAR-T therapy in treating B-cell malignancies, it is expected that CAR-T therapy will also improve clinical outcomes for patients with T-cell malignancies. However, the treatment of T-cell tumors using CAR-T therapy still faces challenges.The first challenge is the lack of T-cell tumor-specific antigens targeted by CAR-Ts. Furthermore, sibling killing, poor T-cell regeneration, and contamination of products by malignant T cells during autologous CAR-T generation are the most significant challenges in treating T-cell malignancies with CAR-Ts, all of which require further detailed research.

[0007] CD7 is a transmembrane glycoprotein belonging to the immunoglobulin superfamily. It is present on the surface of most mature peripheral blood T cells, all thymocytes, and natural killer (NK) cells in the form of homodimers, and is also expressed on myeloid cells and T / B cells that produce immature progenitor cells. CD7 is one of the major markers of T cell maturation. Therefore, CD7 is used as a diagnostic marker for the detection of primary T-cell cancer. CD7 is highly expressed in T-cell acute leukemia (T-ALL) and subsets of peripheral T-cell lymphoma. Normal expression of CD7 is primarily limited to T cells and natural killer (NK) cells, thus reducing the risk of off-target organ damage. Due to its internalization potential, CD7 has served as a target for immunotoxin antibodies in patients with T-cell malignancies in some clinical trials, with no severe, persistent adverse events associated with CD7 antibodies, although the response to tumors is limited. Therefore, replacing monoclonal antibodies with autologous CAR-T cells can enhance the cytotoxic effect against CD7, thereby improving the therapeutic efficacy of CD7-targeted therapies in patients with T-cell malignancies. (Gomes-Silva D, Srinivasan M, Sharma S, Lee CM, Wagner DL, Davis TH, et al. the CD7 molecule.Crit Rev Immunol.1999;19(4):331-348;Safarzadeh Kozani P, Safarzadeh Kozani P, Rahbarizadeh F.CAR-T cell therapy in T-cell malignancies: Is success a low-hanging fruit?Stem Cell Res Ther.2021 Oct 7;12(1):527).

[0008] Compared to the treatment of B-ALL using CAR-T cells, targeted therapy of T-cell acute lymphoblastic leukemia and CD7-positive lymphoma using CD7-CAR-T cells still faces significant technical challenges. This is because both normal effector T cells and T-cell tumors express the CD7 antigen, causing "sibling killing" of CD7-CAR-T cells. Conventional in vitro production of CD7-CAR-T cells is difficult to succeed because of the presence of "sibling killing." Therefore, it is necessary to develop a new CD7 antibody or its antigen-binding fragment that inhibits CD7 expression on T cells and is expressed on CD7-CAR-T cells, so that CD7-CAR-T cells do not undergo sibling killing during production, can be amplified normally in vitro, and exert a killing effect on CD7-positive target cells. [Overview of the project]

[0009] To solve the technical problems of the prior art, the inventors have diligently researched and produced and selected a unique single-domain antibody sequence for anti-human-derived CD7. Using CDR transplantation technology, CDRs of the positive single-domain antibody are transplanted into a natural human germline sequence, and after designing the combination, a heavy chain variable region DNA fragment of the humanized antibody is obtained. This is used as an antigen recognition element for constructing the CD7-CAR-T cells of the present invention. Furthermore, by constructing a CD7 vector that effectively inhibits the expression of the CD7 molecule on the T cell surface, the "fraternal killing" effect of CD7-CAR-T cells is eliminated, facilitating the in vitro production and manufacturing of CD7-CAR-T cells. CD7-CAR-T cells constructed using the single-domain antibody targeting human-derived CD7 according to the present invention exhibit superior in vitro killing efficacy against target cells Jurkat / Luc compared to the positive control VHH6 CAR-T cells. Specifically, the present invention includes the following:

[0010] In a first aspect, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to the CD7 antigen, wherein the antibody or antigen-binding fragment comprises a heavy chain variable region including complementarity-determining region 1 (CDR1), complementarity-determining region 2 (CDR2), and complementarity-determining region 3 (CDR3). CDR1 contains the sequence shown in SEQ ID NO: 18, CDR2 contains the sequence shown in SEQ ID NO: 19, and CDR3 contains the sequence shown in SEQ ID NO: 20. CDR1 contains the sequence shown in SEQ ID NO: 42, CDR2 contains the sequence shown in SEQ ID NO: 43, and CDR3 contains the sequence shown in SEQ ID NO: 44. CDR1 contains the sequence shown in SEQ ID NO: 2, CDR2 contains the sequence shown in SEQ ID NO: 3, and CDR3 contains the sequence shown in SEQ ID NO: 4. CDR1 contains the sequence shown in SEQ ID NO: 6, CDR2 contains the sequence shown in SEQ ID NO: 7, and CDR3 contains the sequence shown in SEQ ID NO: 8. CDR1 contains the sequence shown in SEQ ID NO: 10, CDR2 contains the sequence shown in SEQ ID NO: 11, and CDR3 contains the sequence shown in SEQ ID NO: 12. CDR1 contains the sequence shown in SEQ ID NO: 14, CDR2 contains the sequence shown in SEQ ID NO: 15, and CDR3 contains the sequence shown in SEQ ID NO: 16. CDR1 contains the sequence shown in SEQ ID NO: 22, CDR2 contains the sequence shown in SEQ ID NO: 23, and CDR3 contains the sequence shown in SEQ ID NO: 24. CDR1 contains the sequence shown in SEQ ID NO: 26, CDR2 contains the sequence shown in SEQ ID NO: 27, and CDR3 contains the sequence shown in SEQ ID NO: 28. CDR1 contains the sequence shown in SEQ ID NO: 30, CDR2 contains the sequence shown in SEQ ID NO: 31, and CDR3 contains the sequence shown in SEQ ID NO: 32. CDR1 contains the sequence shown in SEQ ID NO: 34, CDR2 contains the sequence shown in SEQ ID NO: 35, and CDR3 contains the sequence shown in SEQ ID NO: 36. CDR1 contains the sequence shown in SEQ ID NO: 38, CDR2 contains the sequence shown in SEQ ID NO: 39, CDR3 contains the sequence shown in SEQ ID NO: 40, or CDR1 contains the sequence shown in SEQ ID NO: 46, CDR2 contains the sequence shown in SEQ ID NO: 47, and CDR3 contains the sequence shown in SEQ ID NO: 48. Each of the aforementioned CDR1, CDR2, and CDR3 allows for a maximum of three amino acid substitutions, insertions, or deletions.

[0011] In some embodiments, the heavy chain variable region includes CDR1, CDR2, and CDR3. CDR1 contains the sequence shown in SEQ ID NO: 18, CDR2 contains the sequence shown in SEQ ID NO: 19, and CDR3 contains the sequence shown in SEQ ID NO: 20. CDR1 contains the sequence shown in SEQ ID NO: 42, CDR2 contains the sequence shown in SEQ ID NO: 43, and CDR3 contains the sequence shown in SEQ ID NO: 44. CDR1 contains the sequence shown in SEQ ID NO: 2, CDR2 contains the sequence shown in SEQ ID NO: 3, and CDR3 contains the sequence shown in SEQ ID NO: 4. CDR1 contains the sequence shown in SEQ ID NO: 6, CDR2 contains the sequence shown in SEQ ID NO: 7, and CDR3 contains the sequence shown in SEQ ID NO: 8. CDR1 contains the sequence shown in SEQ ID NO: 10, CDR2 contains the sequence shown in SEQ ID NO: 11, and CDR3 contains the sequence shown in SEQ ID NO: 12. CDR1 contains the sequence shown in SEQ ID NO: 14, CDR2 contains the sequence shown in SEQ ID NO: 15, and CDR3 contains the sequence shown in SEQ ID NO: 16. CDR1 contains the sequence shown in SEQ ID NO: 22, CDR2 contains the sequence shown in SEQ ID NO: 23, and CDR3 contains the sequence shown in SEQ ID NO: 24. CDR1 contains the sequence shown in SEQ ID NO: 26, CDR2 contains the sequence shown in SEQ ID NO: 27, and CDR3 contains the sequence shown in SEQ ID NO: 28. CDR1 contains the sequence shown in SEQ ID NO: 30, CDR2 contains the sequence shown in SEQ ID NO: 31, and CDR3 contains the sequence shown in SEQ ID NO: 32. CDR1 contains the sequence shown in SEQ ID NO: 34, CDR2 contains the sequence shown in SEQ ID NO: 35, and CDR3 contains the sequence shown in SEQ ID NO: 36. CDR1 contains the sequence shown in SEQ ID NO: 38, CDR2 contains the sequence shown in SEQ ID NO: 39, CDR3 contains the sequence shown in SEQ ID NO: 40, or CDR1 contains the sequence shown in SEQ ID NO: 46, CDR2 contains the sequence shown in SEQ ID NO: 47, and CDR3 contains the sequence shown in SEQ ID NO: 48.

[0012] In some embodiments, the sequences of the CDR regions are a group consisting of CDR1 containing the sequence shown in SEQ ID NO: 18, CDR2 containing the sequence shown in SEQ ID NO: 19, and CDR3 containing the sequence shown in SEQ ID NO: 20; a group consisting of CDR1 containing the sequence shown in SEQ ID NO: 42, CDR2 containing the sequence shown in SEQ ID NO: 43, and CDR3 containing the sequence shown in SEQ ID NO: 44; selected from any one of the groups consisting of CDR1 containing the sequence shown in SEQ ID NO: 14, CDR2 containing the sequence shown in SEQ ID NO: 15, and CDR3 containing the sequence shown in SEQ ID NO: 16.

[0013] In some embodiments, the heavy chain variable region contains a framework region (FR) of an alpaca antibody or a human antibody.

[0014] In some embodiments, the antibody or its antigen-binding fragment is selected from the group consisting of a full antibody, Fab, Fab’, (Fab’)2, Fv fragment, scFv, di-scFv, and single domain antibody (sdAb). In one specific embodiment, the antibody or its antigen-binding fragment is a single domain antibody.

[0015] In some embodiments, the antibody or its antigen-binding fragment is a chimeric antibody, an Fc fusion antibody, or a humanized antibody. In some embodiments, the antibody or its antigen-binding fragment is a humanized antibody. In one specific embodiment, the antibody or its antigen-binding fragment is a humanized sdAb.

[0016] In some embodiments, the antibody or its antigen-binding fragment contains a mutant amino acid sequence having at least 80% identity with the amino acid sequence shown in any one of SEQ ID NOs: 17, 41, 1, 5, 9, 13, 21, 25, 29, 33, 37, 45, and 49-51.

[0017] In a second aspect, the present invention provides a CD7 inhibitory molecule comprising the antibody or antigen-binding fragment thereof described in the first aspect and an endoplasmic reticulum localization domain, which is capable of reducing the expression of CD7 molecules on the cell surface.

[0018] In some embodiments, the CD7 inhibitory molecule comprises one or more identical single-domain antibodies linked by a linker. In some embodiments, the linker sequence is the sequence shown in SEQ ID NO: 64.

[0019] In one specific embodiment, the CD7 inhibitory molecule comprises two identical single-domain antibodies linked by a linker.

[0020] In some embodiments, the sequence of the endoplasmic reticulum localization domain includes the sequence shown in Sequence ID No. 63.

[0021] In some embodiments, the CD7 inhibitory molecule further comprises a signal peptide sequence. In some embodiments, the signal peptide sequence comprises the sequence shown in SEQ ID NO: 53.

[0022] In some embodiments, the amino acid sequence of the CD7 inhibitory molecule includes the sequence shown in SEQ ID NO: 52, or a variant sequence having at least 80% identity thereto.

[0023] In a third aspect, the present invention provides a chimeric antigen receptor (CAR), which is a chimeric antigen receptor. 1) An antigen-binding domain that recognizes the CD7 antigen, comprising the antibody or antigen-binding fragment thereof described in the first embodiment, 2) Transmembrane domain, 3) Includes an intracellular signaling domain.

[0024] In some embodiments, the chimeric antigen receptor includes a hinge domain between the antigen-binding domain and the transmembrane domain, wherein the hinge domain comprises one or more proteins selected from the group consisting of CD28, IgG1, IgG4, IgD, 4-1BB, CD4, CD27, CD7, CD8 alpha, PD-1, ICOS, OX40, NKG2D, NKG2C, FcεRIγ, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT.

[0025] In some embodiments, the hinge domain includes a hinge domain derived from CD28. In one specific embodiment, the sequence of the hinge domain includes the sequence shown in Sequence ID No. 55.

[0026] In some embodiments, the transmembrane domain of the chimeric antigen receptor includes a transmembrane domain derived from one or more proteins selected from the group consisting of CD8, CD28, 4-1BB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3e, CD3, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FcεRIγ, BTLA, CD30, GITR, HVEM, DAP10, CD2, NKG2C, LIGHT, DAP12, CD40L, TIM1, CD226, DR3, CD45, CD80, CD86, CD9, CD16, CD22, CD33, CD37, CD64, CD134, CD137, CD154, and SLAM.

[0027] In some embodiments, the transmembrane domain includes a transmembrane domain derived from CD28. In some embodiments, the transmembrane domain includes a transmembrane domain derived from CD8. In one specific embodiment, the sequence of the transmembrane region includes the sequence shown in Sequence ID No. 56.

[0028] In some embodiments, the intracellular signaling domain of the chimeric antigen receptor includes an intracellular signaling domain derived from one or more proteins selected from the group consisting of CD8, CD3ζ, CD3δ, CD3γ, CD3ε, FcγRI-γ, FcγRIII-γ, FcεRIβ, FcεRIγ, DAP10, DAP12, CD32, CD79a, CD79b, CD28, CD3C, CD4, b2c, CD137(4-1BB), ICOS, CD27, CD28δ, CD80, NKp30, and OX40.

[0029] In some embodiments, the intracellular signaling domain includes CD3ζ. In one specific embodiment, the signaling domain includes the sequence shown in Sequence ID No. 58.

[0030] In some embodiments, the chimeric antigen receptor further comprises an intracellular costimulatory signaling domain.

[0031] The intracellular costimulatory signaling domain comprises an intracellular costimulatory signaling domain derived from one or more proteins selected from the group consisting of CD28, CD137, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FcεRIγ, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, the ligand for CD83, CD40, and MyD88.

[0032] In some embodiments, the intracellular co-stimulatory signaling domain includes an intracellular region sequence derived from CD28. In some embodiments, the intracellular co-stimulatory signaling domain includes an intracellular region sequence derived from 4-1BB. In one specific embodiment, the intracellular co-stimulatory signaling domain includes the sequence shown in SEQ ID NO: 57.

[0033] In some embodiments, the structure of the chimeric antigen receptor comprises multiple domains, for example, a signal peptide-VHH-hinge domain-transmembrane region-intracellular costimulatory signaling domain-intracellular signal transduction domain, each domain independently linked by a linker or peptide bond. The VHH may be one or more VHHs linked by a linker.

[0034] In some embodiments, the signal peptide sequence is a signal peptide of a protein selected from the group consisting of CD8, CD28, GM-CSF, CD4, CD137, or a combination thereof. In one specific embodiment, the signal peptide sequence includes the sequence shown in SEQ ID NO: 53.

[0035] In one specific embodiment, the chimeric antigen receptor comprises the amino acid sequence shown in any one of SEQ ID NOs. 59 to 61.

[0036] In a fourth aspect, the present application provides an isolated nucleic acid molecule which encodes an antibody or antigen-binding fragment thereof as described in the first aspect of the present invention, or an inhibitory molecule as described in the second aspect, or a chimeric antigen receptor as described in the third aspect.

[0037] In a fifth aspect, the present application provides a vector comprising the nucleic acid molecule described in the fourth aspect.

[0038] In some embodiments, the vector is DNA, RNA, plasmid, lentiviral vector, adenovirus vector, AAV, retroviral vector, transposon, or a combination thereof.

[0039] In some embodiments, the vector is a lentiviral vector.

[0040] In the sixth aspect, the present application provides a host cell comprising the vector described in the fifth aspect.

[0041] In the seventh aspect, the present application provides genetically modified immune cells comprising an antibody or antigen-binding fragment thereof as described in the first aspect of the present invention, or a CD7 inhibitory molecule as described in the second aspect, or a chimeric antigen receptor as described in the third aspect.

[0042] In some embodiments, the immune cells are at least one selected from leukocytes, monocytes, macrophages, dendritic cells, mast cells, neutrophils, basophils, eosinophils, alpha-beta T cells, gamma-delta T cells, natural killer (NK) cells, natural killer T (NKT) cells, B cells, innate lymphocytes (ILCs), cytokine-induced killer (CIK) cells, cytotoxic T lymphocytes (CTLs), lymphokine-activated killer (LAK) cells, T lymphocytes, peripheral blood mononuclear cells, and hematopoietic stem cells.

[0043] In the eighth aspect, the present application provides a pharmaceutical composition comprising an antibody or antigen-binding fragment thereof as described in the first aspect of the present invention, or an inhibitory molecule as described in the second aspect, or a chimeric antigen receptor as described in the third aspect, or a genetically modified immune cell as described in the seventh aspect, and / or a pharmaceutically acceptable carrier.

[0044] In the ninth aspect, the present application provides the use of an antibody or antigen-binding fragment thereof as described in the first aspect, or an inhibitory molecule as described in the second aspect, or a genetically modified immune cell as described in the seventh aspect, or a composition as described in the eighth aspect, in the treatment of tumors or cancer.

[0045] In some embodiments, the cancer or tumor is a cancer or tumor associated with CD7 expression.

[0046] In some embodiments, the cancer or tumor is a hematological malignancy.

[0047] In some embodiments, the hematological malignancies are T-cell-related tumors, including leukemia, lymphoma, and myeloma.

[0048] In the tenth aspect, the present application provides a method for producing genetically modified immune cells, the method for producing said cells is Step (1) prepares immune cells containing the inhibitory molecule described in the second embodiment that expresses a chimeric antigen receptor, The present invention is characterized by comprising step (2) adding a nucleic acid molecule that expresses the chimeric antigen receptor described in the third embodiment to the cells obtained in step (1).

[0049] The beneficial effects of the present invention are as follows:

[0050] The antibody or its antigen-binding fragment according to the present invention exhibits very strong affinity for the CD7 antigen molecule. Furthermore, the inhibitory molecule of the present invention can almost completely inhibit the expression of the CD7 molecule on the cell surface after transfection without affecting the normal amplification of T cells, thereby effectively avoiding sibling killing of CD7-CAR-T cells. CD7-CAR-T cells constructed using the humanized CD7 nanobody sequence of the present invention can further reduce the in vivo immunogenicity of CD7-CAR-T cells constructed using alpaca-derived CD7 antibody sequences, thereby improving clinical therapeutic efficacy. In addition, CD7-CAR-T cells constructed using the human-derived CD7-targeting single-domain antibody according to the present invention exhibit superior target cell killing efficacy compared to the positive control VHH6 CAR-T cells, providing CAR-T cells beneficial for clinical application in cell therapy. [Brief explanation of the drawing]

[0051] [Figure 1] The results of detecting the binding of the antibody described in this application to human-derived CD7 protein are shown. [Figure 2] The results of detecting the binding of the antibody described in this application to monkey-derived CD7 protein are shown. [Figure 3] The results of detecting the binding of the antibody described in this application to mouse-derived CD7 protein are shown. [Figure 4] The FACS detection results for the binding of the antibody described in this application to cells expressing human-derived CD7 protein are shown. [Figure 5] The results of detecting the affinity of the humanized antibodies described in this application to human-derived CD7 protein are shown, with A to G being the detection signals for the affinity of the three antibodies and their corresponding humanized antibodies to human-derived CD7 protein. [Figure 6] The results of detecting the affinity of the humanized antibodies described in this application to monkey-derived CD7 protein are shown, with A to G being the detection signals for the affinity of each of the three humanized antibodies to monkey-derived CD7 protein. [Figure 7] The schematic diagrams of CD7 inhibitory molecules in some examples of this application are shown. [Figure 8] Figures A-F show the inhibitory effect of the CD7 inhibitory molecules described in this application on CD7 expression in T cells, and the detection results of inhibition of CD7 expression on the cell surface by three different CD7 inhibitory molecules under different viral titer conditions. [Figure 9] The AHP19485-VHH1 molecule demonstrates an inhibitory effect on CD7 expression in T cells. [Figure 10] This application outlines the design concept for in vitro activity experiments of CD7-CAR-T cells. [Figure 11] The schematic diagrams of the CAR in several embodiments of this application are shown. [Figure 12] The AHP19485-VHH1 molecule demonstrates an inhibitory effect on CD7 expression in T cells. [Figure 13] This document shows the detection results for the positive rate of CD7 expression in CD7-CAR-T cells constructed in this application. [Figure 14] This application demonstrates the in vitro killing effect of CD7-CAR-T cells against target cells. Figure A shows the killing effect of CAR-T cells with different CD7 antibody molecules against Jurkat target cells, and Figure B shows the killing effect of CAR-T cells with different CD7 antibody molecules against CCRF-CEM target cells. [Modes for carrying out the invention]

[0052] The technical means of the present invention will be described in more detail below with reference to specific examples. It should be understood that the following examples are for illustrative purposes only and should not be interpreted as limiting the scope of protection of the present invention. All technologies realized based on the above-described aspects of the present invention are included within the scope of protection of the present invention.

[0053] Unless otherwise stated, the raw materials and reagents used in the following examples are either commercially available or can be manufactured by known methods. The experimental procedures in the following examples are not described in specific conditions, but are generally carried out according to the general conditions described in Sambrook et al., "Molecular Cloning: A Laboratory Manual" (New York: Cold Spring Harbor Laboratory Press, 1989), or the conditions recommended by the manufacturer.

[0054] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art. Only preferred methods and materials are described in this invention, Methods and materials similar to or equivalent to those described herein may also be used for implementation or testing. All references referred to herein are incorporated by reference to disclose and explain methods and / or materials related to such references. In the event of any conflict with any incorporated reference, the provisions of this specification shall prevail.

[0055] Definition and explanation As used herein, the term “antibody” is used in its broadest sense to refer to a protein or polypeptide, including immunoglobulins or other types of molecules, that contain one or more antigen-binding domains that specifically bind to an antigen, and exhibit binding specificity to a particular antigen. Specific examples of antibodies include complete antibodies (e.g., typical tetrachain antibody molecules), single-chain antibodies, single-domain antibodies, and multispecific antibodies. A typical antibody molecule is usually a tetramer composed of two identical heavy chains and two identical light chains linked to each other by disulfide bonds. Based on differences in the degree of conservation of their amino acid sequences, the heavy and light chains are divided into a variable region (V) located at the amino terminus and a constant region (C) located at the carboxyl terminus. The variable region is used to recognize and bind to the antigen, while the constant region (e.g., the Fc fragment) is used to initiate downstream effects such as antibody-dependent cell-mediated cytotoxicity (ADCC). The variable regions of the heavy and light chains each contain three local regions with higher degrees of variation in amino acid composition and sequence order. These three regions are important sites for antibody-antigen binding and are therefore also called complementarity-determining regions (CDRs). The amino acid sequences of CDRs can be readily determined using numbering schemes known in this art, such as Kabat, Chothia, IMGT, AbM, or Contact.

[0056] Antibodies are primarily classified into five types—IgA, IgD, IgE, IgG, and IgM—based on the amino acid sequence of the constant region of the heavy chain. These antibody types are further divided into subclasses such as IgG1, IgG2a, IgG2b, and IgG3, depending on the size of the hinge domain, the position of interchain disulfide bonds, and molecular weight. The light chain is divided into two types, κ and λ, depending on the amino acid composition and sequence order of the constant region of the antibody's light chain. The subunit structures and three-dimensional structures of different classes of immunoglobulins are known in this field.

[0057] The heavy chain variable region and light chain variable region of an antibody typically contain three complementarity-determining regions (CDRs) and four framework regions (FRs). The complementarity-determining regions are linked by the framework regions, and when an antibody is recognized, the FR molecules bend, bringing the CDR molecules closer together. Since the complementarity-determining regions are the sites that bind to the antigen on the antibody or antigen-binding fragment, the sequence of the complementarity-determining regions determines the specificity of the antibody. As understood in this field, an antibody is a glycoprotein or its antigen-binding portion containing at least two heavy (H) chains and two light (L) chains linked to each other by disulfide bonds. The heavy chain contains a heavy chain variable region (VH) and a heavy chain constant region (CH). The light chain contains a light chain variable region (VL) and a light chain constant region (CL). The variable regions of the heavy and light chains contain framework regions (FRs) and complementarity-determining regions (CDRs). The four FRs are relatively conservative, but the CDR regions (CDR1, CDR2, and CDR3) include hypervariable regions.

[0058] In this application, the term "CDR" usually refers to the complementarity-determining region, which primarily plays a role in binding to the antigen's epitope. The heavy chain CDRs are usually called CDR1, CDR2, and CDR3, numbered sequentially from the N-terminus. The three CDR fragments and the framework region constitute the heavy chain variable region, i.e., the VHH region. Numerous rules exist in this art for defining the CDR region, such as the Chothia rule and the Kabat rule. In this application, the CDRs are divided according to the Kabat definition rule.

[0059] In this specification, “antigen-binding fragment” is a polypeptide fragment that includes a portion of a complete antibody, for example, the antigen-binding region or variable region of a complete antibody, and has the property of specifically targeting CD7, such as Fab, Fab', Fv fragment, F(ab')2, scFv, di-scFv and / or sdAb. Preferably, the antigen-binding fragment includes at least one CDR of the heavy chain variable region and / or light chain variable region of the antibody. More preferably, the antigen-binding fragment may include CDR1-3 of the heavy chain variable region and / or CDR1-3 of the light chain variable region. Antigen-binding fragments can be produced by various techniques, including, but are not limited to, techniques for hydrolyzing and digesting the protein of a complete antibody, or techniques for expressing the antigen-binding fragment in a host cell. Examples of antigen-binding proteins include, but are not limited to, antibodies, antigen-binding fragments, immune complexes, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs, or fusion proteins, as long as they exhibit the desired antigen-binding activity.

[0060] The present invention provides an antibody or antigen-binding fragment thereof that targets CD7, which is excellent in safety and targeting ability, can specifically bind to the extracellular domain of human CD7, and by using a vector containing the coding sequence of the antibody or antigen-binding fragment to introduce immune cells, immune cells with clear killing ability against tumor cells expressing CD7 can be obtained. Since these immune cells can be used to treat or improve diseases related to CD7 expression, this invention lays the foundation for the treatment of CD7-positive tumors.

[0061] In some embodiments, the antibody targeting CD7 includes a heavy chain variable region comprising CDR1, CDR2, and CDR3, where CDR1 comprises the sequence shown in SEQ ID NO: 18, CDR2 comprises the sequence shown in SEQ ID NO: 19, and CDR3 comprises the sequence shown in SEQ ID NO: 20. CDR1 contains the sequence shown in SEQ ID NO: 42, CDR2 contains the sequence shown in SEQ ID NO: 43, and CDR3 contains the sequence shown in SEQ ID NO: 44. CDR1 contains the sequence shown in SEQ ID NO: 2, CDR2 contains the sequence shown in SEQ ID NO: 3, and CDR3 contains the sequence shown in SEQ ID NO: 4. CDR1 contains the sequence shown in SEQ ID NO: 6, CDR2 contains the sequence shown in SEQ ID NO: 7, and CDR3 contains the sequence shown in SEQ ID NO: 8. CDR1 contains the sequence shown in SEQ ID NO: 10, CDR2 contains the sequence shown in SEQ ID NO: 11, and CDR3 contains the sequence shown in SEQ ID NO: 12. CDR1 contains the sequence shown in SEQ ID NO: 14, CDR2 contains the sequence shown in SEQ ID NO: 15, and CDR3 contains the sequence shown in SEQ ID NO: 16. CDR1 contains the sequence shown in SEQ ID NO: 22, CDR2 contains the sequence shown in SEQ ID NO: 23, and CDR3 contains the sequence shown in SEQ ID NO: 24. CDR1 contains the sequence shown in SEQ ID NO: 26, CDR2 contains the sequence shown in SEQ ID NO: 27, and CDR3 contains the sequence shown in SEQ ID NO: 28. CDR1 contains the sequence shown in SEQ ID NO: 30, CDR2 contains the sequence shown in SEQ ID NO: 31, and CDR3 contains the sequence shown in SEQ ID NO: 32. CDR1 contains the sequence shown in SEQ ID NO: 34, CDR2 contains the sequence shown in SEQ ID NO: 35, and CDR3 contains the sequence shown in SEQ ID NO: 36. CDR1 contains the sequence shown in SEQ ID NO: 38, CDR2 contains the sequence shown in SEQ ID NO: 39, CDR3 contains the sequence shown in SEQ ID NO: 40, or CDR1 contains the sequence shown in SEQ ID NO: 46, CDR2 contains the sequence shown in SEQ ID NO: 47, and CDR3 contains the sequence shown in SEQ ID NO: 48. Each of the aforementioned CDR1, CDR2, and CDR3 allows for a maximum of three amino acid substitutions, insertions, or deletions.

[0062] In some embodiments, CDR1, CDR2, and CDR3 each allow for a maximum of one or two amino acid substitutions, insertions, or deletions.

[0063] In some embodiments, the antibody or its antigen-binding fragment comprises an amino acid sequence represented by any one of SEQ ID NOs: 13, 17, 41, and 49-51, or a mutant amino acid sequence that allows for up to five amino acid substitutions, insertions, or deletions in its framework region. In some embodiments, the antibody or its antigen-binding fragment comprises a mutant amino acid sequence that allows for up to five, four, three, two, or one amino acid substitutions, insertions, or deletions in the framework region of the amino acid sequence represented by any one of SEQ ID NOs: 17, 41, 1, 5, 9, 13, 21, 25, 29, 33, 37, 45, and 49-51. In some embodiments, the antibody or its antigen-binding fragment comprises a mutant amino acid sequence that allows for up to five, four, three, two, or one conservative amino acid substitutions in the framework region of the amino acid sequence represented by any one of SEQ ID NOs: 13, 17, 41, and 49-51. In some embodiments, the antibody or its antigen-binding fragment includes a variant sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity with the amino acid sequence shown in any one of SEQ ID NOs: 17, 41, 1, 5, 9, 13, 21, 25, 29, 33, 37, 45, and 49-51.

[0064] In this application, the terms "VHH" and "single-domain antibody (sdAb)" typically refer to an antibody whose target-binding activity is located within a single domain, also known as a nanobody. In this application, the single-domain antibody may be an alpaca single-domain antibody. It is different from, for example, antibodies and single-chain antibodies (antibodies and single-chain antibodies generally provide antigen-binding activity through both heavy-chain and light-chain variable regions). In some examples, the antibody or its antigen-binding fragment described in this application is an sdAb.

[0065] In this application, the term “Fc region (fragment crystallizable region)” is also called the constant region and usually refers to the region of an antibody corresponding to the antigen-binding region of the antibody. The Fc region generally maintains a constant state on its own and does not play a role in binding to the antigen, but can bind to the Fc receptor and complement to perform the biological function of the antibody. The Fc region in this application may be the Fc of a human antibody. In some examples, the Fc region may include hinges, CH2, and CH3. If the Fc region includes hinges, it can mediate dimerization between two Fc-containing polypeptides. The Fc fragment may be derived from IgG, IgM, IgD, IgE, or IgA. In some examples, the Fc region is derived from IgG1, IgG2, IgG3, or IgG4. The “Fc fragment” includes mutant Fc fragments derived from natural Fc fragments that have been modified but still retain their effector function. A "mutant Fc fragment" includes an amino acid sequence in which at least one amino acid of the amino acid sequence of a native Fc fragment is modified. In some examples, the mutant Fc fragment has at least one amino acid substitution compared to the parent Fc fragment (native Fc fragment), for example, about 1 to 10 amino acids are substituted, preferably about 1 to 5 amino acids. In some examples, the Fc region of the mutant Fc fragment has at least about 80% sequence identity, at least about 90% sequence identity, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% sequence identity with the parent Fc fragment. The effector function of the "Fc fragment" may include binding to the Fc receptor, C1q binding and complement-dependent cell-mediated cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), and mediating phagocytosis. In some embodiments, the antibody or antigen-binding fragment described in this application includes an Fc region such as a fused scFv, HcAb, or complete antibody.

[0066] For antibodies or their antigen-binding fragments, "binding," "targeting," or "specifically binding" means that the molecule (e.g., the antibody or its antigen-binding fragment) has a higher binding affinity to another molecule (e.g., an antigen) than to other molecules simultaneously present in the environment. A molecule can bind to, target, or specifically bind to one or more molecules; for example, a bispecific antibody has a higher binding affinity to two different antigens than other molecules. The binding affinity of an antibody to an antigen can be measured by parameters such as the EC50 value or KD value of the antibody's binding to the antigen.

[0067] EC 50 (Concentration for 50% of maximal effect) refers to the concentration that produces 50% of the maximum effect. In enzyme-linked immunosorbent assay (ELISA), when EC50 is used to represent the ability of an antibody molecule to bind to a corresponding antigen, it refers to the concentration of the antibody molecule that produces half of the maximum detection signal (such as colorimetric or fluorescence intensity). A lower EC50 value indicates a higher binding affinity to the antigen. In some embodiments, the EC50 value of the binding of the antibody or its antigen-binding fragment to human-derived CD7 molecules is less than 1 nM.

[0068] The KD value may also be used to measure the binding affinity of an antibody to its antigen. The KD value is the equilibrium dissociation constant between an antibody and its antigen, i.e., the koff / kon ratio. Therefore, the lower the KD value (the lower the concentration), the higher the affinity of the antibody. In some embodiments of this application, according to Biacore detection, the antibody or its antigen-binding fragment has a KD value of less than 1 nM for binding to the CD7 molecule. In some embodiments, according to Biacore detection, the antibody or its antigen-binding fragment has a KD value of less than 0.1 nM for binding to the CD7 molecule. In some embodiments, according to Biacore detection, the antibody or its antigen-binding fragment is humanized VHH, and the humanized VHH has a KD value of less than 1 nM, more preferably less than 0.1 nM, for binding to the CD7 molecule.

[0069] In this application, the term "inhibition" typically refers to an effect on the expression of the CD7 molecule on the cell surface, for example, a decrease in the expression of the CD7 molecule on the cell surface. In some embodiments, the inhibitory molecules described in this application have the effect of inhibiting the expression of the CD7 molecule on the surface of T cells, and in some embodiments, the effectiveness of said inhibitory effect is 70%, 80%, 90%, or 100%. In some embodiments, said inhibitory effect is achieved by introducing the nucleic acid encoding the inhibitory molecule into immune cells. In some embodiments, said inhibitory effect is achieved by introducing the inhibitory molecule into immune cells. In some embodiments, said inhibitory effect reaches 100% on day 5, day 6, day 7, day 8, or day 9 after the inhibitory molecule or the nucleic acid encoding said inhibitory molecule is introduced into the cells. In some embodiments, said inhibitory effect can last for 10 days, 15 days, 20 days, or longer.

[0070] In some embodiments, the amino acid sequence of the CD7 inhibitory molecule includes a mutant sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% identity with the sequence shown in SEQ ID NO: 52.

[0071] In this application, the term "transmembrane domain" usually refers to a sequence within a cell surface protein that spans the cell membrane, and may include a hydrophobic alpha-helix. Transmembrane domains can ligate to intracellular signaling domains to play a role in signal transduction, and in this application, transmembrane domains may originate from any type I, type II, or type III transmembrane protein.

[0072] In this application, the term “Chimeric Antigen Receptor (CAR)” typically refers to a fusion protein comprising an extracellular domain capable of binding to an antigen and at least one intracellular domain. A CAR is a core component of a chimeric antigen receptor T cell (CAR-T) and may include an antigen (e.g., tumor-specific antigen and / or tumor-associated antigen) binding domain, a transmembrane domain, a costimulatory domain, and an intracellular signaling domain. A CAR is a genetically modified receptor that allows any specific receptor to be transplanted into immune effector cells, particularly T cells. A CAR allows for the transplantation of scFv or VHH fragments of monoclonal antibodies that specifically recognize tumor antigens into T cells or NK cells. A nucleic acid encoding a CAR can be introduced into T cells, NK cells, or NKT cells using a retroviral vector or the like. In this way, a large number of cancer-specific T cells, NK cells, or NKT cells can be generated for use in adoptive cell transplantation. In this application, the CAR can be combined with a T cell receptor activating intracellular domain based on the antigen (e.g., BCMA) specificity of the antibody. T cells genetically modified to express CARs can specifically recognize and eliminate malignant cells that express target antigens.For explanations of CAR and CAR-T cells, please refer to, for example, Sadelain M, Brentjens R, Rivi ere I. The basic principles of chimeric antigen receptor design. Cancer Discov. 2013;3(4):388-398; Turtle CJ, Hudecek M, Jensen MC, Riddell SR. Engineered T cells for anti-cancer therapy. Curr Opin Immunol. 2012;24(5):633-639; Dotti G, Gottschalk S, Savoldo B, Brenner MK. Design and development of therapies using chimeric antigen receptor-expressing T cells. Immunol Rev 2014;257(1):107-126; and WO2013154760, WO2016014789.

[0073] In this application, the term "co-stimulatory domain" typically refers to an intracellular domain capable of providing immune costimulatory molecules, which are cell surface molecules necessary for an effective response of lymphocytes to antigens.

[0074] In this application, the term "hinge domain" usually refers to the connecting region between an extracellular domain (e.g., a BCMA-targeting portion) and a transmembrane domain. In this application, the CAR may include one or more hinge domains between the BCMA-targeting portion and the transmembrane domain.

[0075] In this application, the term “signaling domain” typically refers to an intracellularly located domain capable of transmitting signals. In this application, the intracellular signaling domain is capable of transmitting signals within a cell. Typically, a signaling domain is any consecutive amino acid sequence that directs a protein to find a target. In some cases, the signaling domain may originate from CD3ζ. CD3ζ can form the T cell receptor-CD3 complex together with the T cell receptor subunits, CD3-gamma, CD3-delta, and CD3-epsilon. CD3ζ contains three ITAM motifs, the ITAM sequence mediating intracellular signal activation of the TCR. The chain is a substrate for receptor-activating protein tyrosine kinase, and when the TCR receptor binds to the polypeptide MHC complex, the chain is rapidly tyrosine-phosphorylated and participates in the transmission of lymphocyte-activating signals. Therefore, CD3ζ plays a crucial role in antigen recognition and TCR signaling.

[0076] In this application, the “vector” typically refers to a nucleic acid molecule capable of self-replication within a suitable host and is used to transfer an inserted nucleic acid molecule into and / or between host cells. The vectors include vectors primarily used for inserting DNA or RNA into cells, vectors primarily used for replicating DNA or RNA, and vectors primarily used for transcription and / or translational expression of DNA or RNA. The vectors further include vectors having a plurality of the above-described functions. The vectors may also be polynucleotides that can be transcribed and translated into polypeptides when introduced into a suitable host cell. Typically, by culturing a suitable host cell containing the vector, the vector can produce the desired expression product. In addition to the transgene insertion sequence and backbone, the vector may include additional characteristics such as promoters, genetic markers, antibiotic resistance, reporter genes, targeting sequences, and protein purification tags. Vectors called expression vectors (expression constructs) can be used to express a transgene in target cells and typically have a regulatory sequence. The vectors described in this application may be expression vectors and include viral vectors (lentiviral vectors and / or retroviral vectors), phage vectors, phagemids, cosmids, artificial chromosomes such as yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), or P1-derived artificial chromosomes (PACs), and / or plasmids.

[0077] In the present invention, "genetically modified immune cells" are immune cells that have been artificially genetically modified to perform immune effector functions. In some embodiments, the genetically modified immune cells express at least FcγRIII and perform ADCC effector functions. Examples of ADCC-mediated immune effector cells include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, neutrophils, and eosinophilic granulocytes. Preferably, the immune cells are at least one selected from immune cells cultured and differentiated from pluripotent stem cells or embryonic stem cells, T lymphocytes, NK cells, peripheral blood mononuclear cells (PBMCs), and hematopoietic stem cells.

[0078] More preferably, the immune cells are T lymphocytes (same as T cells). In some embodiments, the T cells may be CD4+ / CD8-, CD4- / CD8+, CD4+ / CD8+, CD4- / CD8-, or a combination thereof. In some embodiments, the T cells express a chimeric antigen receptor and, upon binding to target cells, produce IL-2, IFN, and / or TNF. In some embodiments, the CD8+ T cells express a chimeric antigen receptor and, upon binding to target cells, lyse antigen-specific target cells.

[0079] The present invention provides a method for producing genetically modified immune cells, comprising introducing an isolated nucleic acid or vector described in the present invention into immune cells. Preferably, the present invention produces genetically modified immune effector cells by introducing a chimeric antigen receptor into immune effector cells (e.g., T cells). In some embodiments, the production method first involves introducing a nucleic acid encoding the inhibitory molecule into the immune cells to be genetically modified to inhibit the expression of the CD7 molecule, and then, on day 5, 6, 7, 8, or 9 after introduction, transfecting the immune cells with a nucleic acid encoding the CD7-CAR molecule to express the CD7-CAR molecule on the surface of the immune cells.

[0080] Methods for introducing nucleic acids or vectors into mammalian cells are known in this art, and such vectors can be introduced into immunoeffector cells by physical, chemical, or biological methods. Physical methods for introducing vectors into immunoeffector cells include calcium phosphate precipitation, liposome transfection, particle impact, microinjection, and electroporation. Chemical means for introducing nucleic acids or vectors into immunoeffector cells include colloidal dispersions such as polymer complexes, nanocapsules, microspheres, beads, and lipid-based systems (including oil-in-water emulsions, micelles, mixed micelles, and liposomes). An exemplary colloidal system used as an in vitro delivery medium is liposomes (e.g., artificial membrane vesicles). Biological methods for introducing nucleic acids or vectors into immunoeffector cells include the use of DNA vectors and RNA vectors. Viral vectors have become the most widely used method for inserting genes into mammalian cells, such as human cells. In some embodiments, after introducing nucleic acids or vectors, the introduced or transfected immunoeffector cells are propagated in vitro.

[0081] In some embodiments, the manufacturing method further comprises evaluating or screening introduced or transfected immune cells to select genetically modified immune cells. In some embodiments, the CAR molecule further comprises a tag sequence for screening cells. In some embodiments, the tag sequence is a flag sequence, and preferably, the tag sequence is the sequence shown in SEQ ID NO: 54.

[0082] In this application, the term “treatment” typically means (i) preventing a disease, disorder and / or symptom in a patient who is susceptible to such disease, disorder and / or symptom but has not yet been diagnosed with such disease, (ii) suppressing such disease, disorder or symptom, i.e., inhibiting its progression, and (iii) alleviating such disease, disorder or condition, i.e., reducing the disease, disorder and / or condition and / or symptoms associated with such disease, disorder and / or condition.

[0083] In this specification, “CD7 expression-related cancer” refers to a disease directly or indirectly caused by abnormal CD7 expression, and usually refers to a disease caused by CD7 overexpression. Preferably, the cancer or tumor is a hematological malignancy. More preferably, the hematological malignancy is a T-cell related tumor, including leukemia, lymphoma, and myeloma.

[0084] In this application, the term “pharmaceutically acceptable carrier” typically refers to one or more non-toxic substances that do not interfere with the efficacy of the biological activity of the active ingredient. Such formulations may generally include salts, buffers, preservatives, compatible carriers, and any other therapeutic agents. Such pharmaceutically acceptable formulations may further include compatible solid or liquid fillers, diluents, or encapsulating materials suitable for administration to humans. Other conceivable carriers, excipients, and / or additives that may be used in the formulations described herein include flavorings, antimicrobial agents, sweeteners, antioxidants, antistatic agents, lipids, protein excipients (such as serum albumin, gelatin, and casein), and salt-forming counterions (such as sodium).

[0085] In this application, the term "and / or" should be understood to mean either one of the options or both of the options.

[0086] In this application, the term "includes" generally means including the features explicitly specified, but not excluding other elements.

[0087] In this application, the term "about" usually refers to a variation within a range of 0.5% to 10% above or below a specified value, for example, a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value.

[0088] Unless otherwise defined or clearly indicated by the context, all technical and scientific terms in this disclosure have the same meanings as those commonly understood by those skilled in the art.

[0089] Example 1: Discovery of a human-derived CD7 single-domain antibody-positive cloned molecule 1) Animal immunity and construction of a phage library As the antigen, we used the recombinant human CD7 extracellular domain protein CD7-His (CD7-H52H7, ACROBiosystems, created by fusion with a His tag, referencing the amino acid sequence from positions 26 to 180 of P09564-1). Alpaca JSR49 was subcutaneously immunized using a 1:1 emulsion containing 0.5 mg of CD7-His fusion protein in Freund's complete adjuvant. Subsequently, alpaca was boosted every two weeks by subcutaneous injection of a 1:1 emulsion containing 0.25 mg of CD7-His in Freund's incomplete adjuvant (Sigma-Aldrich) up to five times. Alpaca serum titers were detected using both whole IgG antibodies and single-domain antibodies. Goat anti-Llama IgG(H+L) Secondary Antibody [HRP] (Anorogen, NB7242) and MonoRab® Rabbit Anti-Camelid VHH Cocktail [HRP] (GenScript, A02016) were used as secondary detection antibodies. After the fourth immunization, the serum titers of the single-domain antibodies met the library construction requirements (library construction criteria: OD450nm response value of approximately 1.0 at a dilution ratio of 1:8000). To enhance library diversity, peripheral blood mononuclear cells (PBMCs) isolated from blood collections after the third and fourth immunizations were combined and used for subsequent phage library construction.

[0090] PBMC lysate samples from the third and fourth immunizations were mixed in equal proportions, and total RNA samples were extracted according to the standard RNA extraction procedure. Qualitative and quantitative analysis of the extracted RNA was performed by gel electrophoresis and OD260 / OD280. RNA was reverse transcribed into cDNA according to the Primescript® 1st Strand cDNA synthesis kit manual. The cDNA was used as a template for the first round of PCR, separating the conventional IgG1 sample from the single-domain antibody sample. The first round of PCR products recovered by gel electrophoresis were used as a template to separate the VHH fragment by PCR. The VHH fragment and the single-domain antibody phage display vector plasmid (Nanjing Probio) were digested with Sfi I endonuclease, the VHH fragment was fused to a linearization vector using T4 ligase, the ligated product was electroporated, and the plate (ampicillin-containing agar plate) was incubated overnight. The library volume was determined by counting bacteria on a plate, and the library quality was evaluated by selecting a single clone and sequencing it. The library volume and quality are shown in Table 1.

[0091] [Table 1]

[0092] 2) Screening of lead antibody molecules that specifically target human-derived CD7 using an alpaca immunotherapy library Using the aforementioned alpaca immunotherapy library, phage library eluates were obtained by performing two cell pannings with a positive Jurkat cell line and a negative Jurkat / KO CD7 (human-derived CD7 display gene knockout) cell line (Nanjing Probio). The neutralized phage panning eluates were added to the prepared TG1 bacterial suspension, mixed uniformly, and allowed to stand at 37°C for 45 minutes to infect the TG1 host bacteria. After sufficient infection, the bacterial suspension was diluted with a gradient, spread onto agar plates with relevant resistance, inverted, and cultured overnight at 37°C. A sterile 96-well deep-well plate was prepared with 0.5 ml of 2YT medium (containing 0.2% w / v glucose and 0.1 mg / ml ampicillin), and monoclonal colonies cultured on the plate were separated into the well plate using a sterile pipette tip. These were cultured overnight (16-18 hours) at 37°C with shaking at 220 rpm. 0.05-0.1 ml of a single-clonal bacterial suspension cultured overnight was transferred to a newly prepared sterile deep-well plate (containing 0.5 ml of 2YT medium with a final concentration of 0.1 mg / ml ampicillin), and the cells were cultured until the OD value reached approximately 0.6-0.8 (under OD600 detection conditions). Helper phages were added, and the mixture was shaken to ensure homogeneity. The mixture was then allowed to stand at 37°C for 45 minutes to infect the cells. Subsequently, 0.5 ml of 2YT medium (containing 0.1 mg / ml ampicillin and kanamycin with a final concentration of 0.05 mg / ml after addition) was added, and the cells were cultured overnight (16-18 hours) at 25°C with shaking at 220 rpm. The overnight-expressed bacterial suspension was centrifuged at 4000 rpm for 10 minutes to obtain the phage display supernatant. This supernatant was used for ELISA binding detection of the human-derived CD7-His protein, which is the antigen protein of the single-clonal antigen after panning, and for FACS binding detection of the cell antigen, to obtain a positive single-domain antibody clone.

[0093] ELISA binding detection method: The binding ability of phage display antibodies in the supernatant to human-derived CD7-related proteins was evaluated by indirect ELISA. ELISA plates were coated with 100 μl / well of CBS containing 1 μg / ml recombinant human-derived CD7-His protein and left overnight at 4°C. The plates were washed with PBS-T (0.05% Tween) and blocked with 300 μl / well of PBS containing 3% skim milk at 37°C for 1 hour. The blocking solution was then discarded, and 50 μl of phage expression supernatant and 50 μl of 0.1% PBS-T were added to each plate and incubated at room temperature for 2 hours. The plates were washed three times with PBS-T and incubated with 100 μl / well of horseradish peroxidase-conjugated goat anti-M13 phage antibody (Sino-Biological) at room temperature for 45 minutes. The plate was washed six times with PBS-T, TMB colorimetric solution (GenScript) was added, and the plate was incubated at room temperature in the dark for 10-15 minutes. The reaction was stopped by adding 50 μl of 1 M HCl stop solution (Sigma). The plate was read at 450 nm using a microplate reader.

[0094] FACS detection method: The binding ability of antibodies in the supernatant to human-derived CD7 antigen expressed on the surface of Jurkat cell membranes was evaluated using a FACS binding assay. Jurkat cells expressing human-derived CD7 for detection and negative Jurkat / KO CD7 (human-derived CD7 display knocked out) cell lines were collected and washed three times with PBS. 2.5 × 10⁶ cells were placed in a 96-well plate. 5 Each detection cell, 100 μl of the supernatant, and 3.5 μg / ml of biotin-conjugated anti-phage antibody were added and incubated at 4°C for 1 hour. The cells were then washed three times with PBS, 100 μl of iFluor-conjugated streptavidin protein (Jackson ImmunoResearch, 016-600-084) was added, and the cells were incubated at 4°C for 45 minutes. Finally, the cells were washed three times with PBS, and the signal was read using FACS Calibur (BD). Example 2: Sequencing of the variable region of positive clones and recombinant production of single-domain antibodies

[0095] Based on the detection results of ELISA and FACS, positive clones were selected. The original colonies corresponding to the positive clones were taken and cultured in bacterial suspension, expression plasmids were extracted, and PCR and Sanger sequencing of single clones were performed to obtain 12 positive clones. The correspondence between the clones and their sequence numbers is shown in Table 2.

[0096] [Table 2]

[0097] DNA fragments containing codon-optimized heavy chain variable regions were synthesized and inserted into a pcDNA3.4-Fc(HuIgG1) expression vector to form an expression plasmid.

[0098] The above plasmid was transfected into HEK293-6E cells, which were cultured in a shaking flask at 37°C for 10 days. The supernatant was collected and used for antibody purification. Before purification, pyrogenic substances were removed from the tubes and Protein A column with 0.2 M NaOH. The column was re-equilibriumated with a buffer containing 0.05 M Tris and 1.5 M NaCl (pH=8.0). The collected cell culture supernatant was diluted 1:1 with twice the volume of the above buffer, filtered, and sterilized. The filtered supernatant was incubated with the Protein A column at room temperature for 2 hours. After washing the column with one-fold volume of the above buffer, IgG was eluted with sterile 0.1 M sodium citrate (pH3.5). The eluate was collected and neutralized with 1 / 9 volume of sterile 1 M Tris-HCl (pH=9.0). Under sterile conditions, the buffer of the product was replaced with PBS (pH7.4) to remove all elution buffer and concentrate the sample. After concentration, the antibody was quantified at OD280nm using an extinction coefficient Ec(0.1%) of 1.43.

[0099] Purified antibodies were analyzed by SDS-PAGE using a 10% precast gel (GenScript) in a BioRad electrophoresis system. The gel was stained with Estain2.0 (GenScript), and the stained bands were compared with a Protein Ladder (GenScript) to estimate molecular size and purity.

[0100] Example 3: ELISA-based detection of purified antibodies against human, monkey, and mouse-derived CD7 recombinant proteins. The binding ability of the purified antibody from Example 2 to recombinant proteins associated with human CD7, monkey CD7 (Sino-Biological, 90993-H08H), and mouse CD7 (ACROBiosystems, CD7-M52H3) was evaluated by indirect ELISA. ELISA plates were coated with 100 μl / well each of CBS containing 1 μg / ml human CD7 recombinant protein, monkey CD7 recombinant protein, or mouse CD7 recombinant protein, and left overnight at 4°C. The plates were washed with PBS-T (0.05% Tween) and blocked at 37°C for 1 hour with 300 μl / well of PBS containing 3% skim milk. The blocking solution was then discarded, and 100 μl of 8 μg / ml (approximately 100 nM) purified antibody was added to the first well and diluted with a 3-fold gradient to create a total of 11 test concentration gradients. These were then incubated at room temperature for 1 hour. The plates were washed three times with PBS-T and incubated with 100 μl / well of horseradish peroxidase-conjugated mouse anti-human IgG Fc fragment (GenScript) at 37°C for 0.5 hours. The plates were washed five times with PBS-T, TMB chromogenic solution (GenScript) was added, and incubated at room temperature for 15 minutes under light shielding. The reaction was stopped by adding 50 μl of 1 M HCl stop solution (Sigma). The plates were read at 450 nm using a microplate reader. The ELISA experimental results for the binding of 12 positive recombinant antibody clones to CD7 antigen proteins from different species are shown in Figures 1-3, along with the specific EC values ​​for each antibody. 50The results are shown in Table 3 below. Compared to the positive control antibody PA3-17 VHH6-Fc(HuIgG1), these detected antibodies have equivalent binding ability to human-derived CD7 antigen protein and do not exhibit cross-reactivity with monkey-derived or mouse-derived CD7.

[0101] [Table 3]

[0102] Example 4: Binding of monoclonal antibody to Jurkat cell line expressing human-derived CD7. Jurkat cells expressing human-derived CD7 for detection and negative Jurkat cells (Jurkat / KO CD7) in which human-derived CD7 expression was knocked out were collected and washed three times with PBS. 2.5 × 10⁶ cells were placed in a 96-well plate. 5 The cells were mixed with 100 μl of purified antibody (10 μg / ml) and incubated at 4°C for 1 hour. The cells were then washed three times with PBS, and 100 μl of iFluor-conjugated goat anti-human IgG, Fcγ-specific fragment antibody was added and incubated at 4°C for 45 minutes. Finally, the cells were washed three times with PBS, and the signal was read using a FACS BD Calibur. As shown in Figure 4, all antibodies bound to Jurkat cells expressing human-derived CD7 but not to negative cells. This indicates that all 12 purified antibodies (i.e., monoclonal antibodies) specifically bound to the human-derived CD7 cell antigen.

[0103] Example 5: Humanization design and recombinant manufacturing of a single-domain antibody molecule targeting human-derived CD7. The structure of the parent antibody was modeled using computer-aided homology modeling software (MOE). A natural human germline sequence with high homology to the parent sequence was selected. Using CDR transplantation technology, CDRs of the positive monoclonal antibody were transplanted into the natural human germline sequence to obtain a humanized chimeric antibody of the parent antibody, i.e., a humanized VHH antibody. By comparing different amino acid residues of the chimeric antibody and the parent antibody, key points that may affect subsequent affinity were determined based on standard residues, interaction loop regions, core regions, mutation hotspots, etc., and appropriate sites were selected and combinations were designed to obtain the amino acid sequence of the humanized antibody variable region. The amino acid sequence of the humanized antibody was codon-optimized, and a DNA fragment containing the codon-optimized heavy chain variable region was synthesized and inserted into a pcDNA3.4-Fc (human IgG1) expression vector to form an expression plasmid. The associated expression plasmid was prepared using the antibody recombinant expression and purification method of Example 2 to obtain humanized antibodies. The humanized clones and their corresponding sequence numbers are shown in Table 4, and each CDR sequence was defined using the Kabat definition method.

[0104] [Table 4]

[0105] Example 6: Detection of binding of humanized antibodies to human and monkey-derived CD7 proteins using SPR. The affinity of purified antibodies to human and monkey-derived CD7 proteins was measured using a surface plasmon resonance (SPR) biosensor, Biacore T200 (GE Healthcare). Antibodies were immobilized on the sensor tip using the Fc capture method. Human or monkey-derived CD7 proteins were used as the analytes. Dissociation (kd) rate constants and association (ka) rate constants were obtained using Biacore T200 evaluation software. The equilibrium dissociation constant (KD) was calculated based on the ratio of kd to ka. Antibodies were ranked based on the equilibrium dissociation constant, and humanized antibodies that did not experience a decrease in affinity or whose affinity decreased by 3 times or less were selected for subsequent detection. The SPR affinity measurement results are shown in Tables 5-6 below, and the corresponding sensorgrams are shown in Figures 5-6. Affinity measurements by SPR showed that all three groups of antibodies—parental antibodies (AHP19484-VHH, AHP19485-VHH, AHP19509-VHH) and humanized antibodies (AHP19484-VHH3, AHP19485-VHH1, AHP19509-VHH1)—showed affinity equivalent to that of human-derived CD7 antigen protein, and all had an equilibrium dissociation constant (KD) of 10. -10 Within this range, none of them bound to the monkey-derived CD7 protein.

[0106] [Table 5]

[0107] [Table 6]

[0108] Example 7: Searching for the optimal antibody molecule with CD7 inhibitory effect The structures of the CD7 inhibitor molecules are shown in Figure 7. Three CD7 inhibitor molecules with different VHH sequences were prepared, and the VHH amino acid sequences are shown in SEQ ID NOs. 49-51. These inhibitor molecules were subcloned into lentiviral vectors, and CD7 inhibitor molecule viruses were obtained using general lentiviral packaging techniques for preparation for use. The viral titer (TU / ml) was measured using a functional viral titration method based on flow cytometry.

[0109] T cell activation: Frozen PBMCs (Cyagen) were thawed and revived, and the cells were resuspended in AIM V complete medium (ThermoFisher, Cat#31035025) containing 300 IU / ml of rhIL-2, counted, and then activated with T cells in a 1:1 ratio of cells to CD3 / CD28 antibody magnetic beads (GenScript, Cat#L00899-1) (activation time was designated as D0, and the times on day 1 and day 2 of activation were designated as D1 and D2, respectively). The cells and magnetic beads were incubated together for 24 hours to obtain activated T cells.

[0110] Activated T cells were placed in a 24-well plate, and the cell density per well was set to 1 × 10⁶. 6 Each well was individually prepared and a CD7 inhibitory lentivirus (MOI=5, MOI=10) with three different VHH sequences was added to make a total system volume of 400 μl. Simultaneously, 16 μl of the virus transfection accelerator HiTransG P (Genechem, Cat#REVG005) was added. 16 hours after transfection, 1 ml of culture medium was added to each well and the cells were cultured. After amplification and culture for 6 days, CD7 expression on the T cell surface was detected by flow cytometry, using PE anti-human CD7 Antibody (Biolegend, CAT#343106) as the detection antibody.

[0111] The results are shown in Figure 8 (in the figure, AHP19485 corresponds to the AHP19485-VHH1 molecule, AHP19484 corresponds to the AHP19484-VHH3 molecule, and AHP19509 corresponds to the AHP19509-VHH1 molecule). Under experimental conditions of MOI=5 and MOI=10, the inhibitory effect of the AHP19485-VHH1 molecule on CD7 was clearly superior to that of AHP19484-VHH3 and AHP19509-VHH1. Six days after transfection, the inhibitory effect of the AHP19485-VHH1 molecule on CD7 on the T cell surface reached 70%, and thereafter, CD7-inhibiting T cells were constructed by selecting the AHP19485-VHH1 molecule.

[0112] Example 8: Exploration of the duration of inhibition of CD7 molecules on the T cell surface by CD7 inhibitory molecules. The structure of the CD7 inhibitor molecule is shown in Figure 7. The AHP19485-VHH1 molecule was used as the inhibitory antibody sequence, which is the amino acid sequence (SEQ ID NO: 52). CD7 expression on the surface of T cells was continuously monitored for 22 days by flow cytometry (detection antibody: PE anti-human CD7 Antibody, Biolegend, Cat#343106). As shown in the results in Figure 9, the CD7 expression level on T cells was clearly reduced on day 8 after introduction of the CD7 inhibitor molecule, and 90% of CD7 molecule expression was inhibited on day 15. CD7 expression continued to decrease until day 22, approaching 100%.

[0113] Based on these results, we selected day 8 after introduction as the time for detecting CD7 expression when producing CD7-inhibiting T cells.

[0114] Example 9: Construction and production of CD7-CAR-T cells The present invention constructs a second-generation CAR vector targeting human-derived CD7, comprising, in order, two repeating CD7 VHH antibody sequences, a Flag tag as an introduction indicator protein, a CD28 hinge domain, a CD28 transmembrane region, a CD28 intracellular region sequence, and a CD3ζ sequence. The CAR structure is shown in Figure 11. The amino acid sequences of each component are shown in SEQ ID NOs. 53-58.

[0115] To compare the differences in CAR-T functional activity of three VHH molecules, three CD7-CAR-T cells (SEQ ID NOs. 59-61) with different CD7 VHH sequences were constructed by lentiviral transfection, and VHH6 CAR-T cells constructed with VHH6 (Chinese Patent No. CN110760007A) were used as the positive control for the experiment (SEQ ID NO. 62). The specific construction steps are as follows.

[0116] T cell activation: Frozen PBMCs (Cyagen) were thawed and revived, and the cells were resuspended in AIM V complete medium (ThermoFisher, Cat#31035025) containing 300 IU / ml of rhIL-2, counted, and then activated with T cells in a 1:1 ratio of cells to CD3 / CD28 antibody magnetic beads (GenScript, Cat#31035025) (activation time was designated as D0, and the times on day 1 and day 2 of activation were designated as D1 and D2, respectively). The cells and magnetic beads were incubated together for 24 hours to obtain activated T cells.

[0117] Production of CD7-inhibiting T cells: A CD7 inhibitor molecule (amino acid sequence: SEQ ID NO: 52) constructed using the AHP19485-VHH1 molecule was subcloned into a lentiviral vector. The CD7 inhibitor molecule virus was obtained using general lentiviral packaging techniques and prepared for use. Viral titer (TU / ml) was measured by functional viral titration using flow cytometry. Activated T cells were placed in a 24-well plate, and the cell density per well was set to 1 × 10⁶. 6 Each well was individually prepared, and a CD7 inhibitory lentivirus (MOI=10) was added to bring the total volume of the system to 400 μl. Simultaneously, 16 μl of the virus transfection promoter HiTransG P (Genechem, Cat#REVG005) was added. 16 hours after transfection, 1 ml of culture medium was added to each well for amplification culture. On day 8, CD7 expression on the T cell surface was detected by flow cytometry.

[0118] The results are shown in Figure 12. The CD7 positivity rate on the surface of T cells that were not introduced with the CD7 inhibitory virus (corresponding to the pan-T group in the figure) was 93.1%, while the CD7 positivity rate in the introduced group (corresponding to the AHP19485 CD7 Blocking group in the figure) was only 4.22%. CD7 expression was hardly detectable on the surface of T cells, and it was shown that the inhibitory effect on CD7 expression in T cells reached over 90% after 8 days had passed since the introduction of the CD7 inhibitory molecule virus. As a result, it was shown that CD7-inhibited T cells were successfully constructed and can be used for the subsequent production of CD7-CAR-T cells.

[0119] Lentivirus infection of CD7 CARs: On day 9, the manufactured CD7 inhibitory T cells were placed in a 24-well plate, and the cell density per well was set to 1 × 10⁶ 6 Each well was individually prepared, and CD7 CAR lentivirus (MOI=10) was added to bring the total volume of the system to 400 μl. Simultaneously, 16 μl of the virus transfection accelerator HiTransG P (Genechem, Cat#REVG005) was added. 16 hours after transfection, 1 ml of culture medium was added to each well for amplification and culture to obtain CAR-T cells targeting CD7.

[0120] On day 13, the proportion of CD7 CAR cells was detected by flow cytometry. PE-Labeled Human CD7 Protein, His Tag (ACROBiosystems, Cat#CD7-HP2E3) was used as the detection antibody. CD7 Blocking T cells were defined as T cells that inhibited CD7 expression but did not undergo CAR virus introduction, and were used as a negative control. VHH6 CAR cells were used as a positive control, and the CAR positivity rates are shown in Table 7 and Figure 13. In both cases, the CD7 CAR positivity rate exceeded 90%.

[0121] [Table 7]

[0122] Example 10: Experiment on in vitro toxicity of CD7-CAR-T cells against CD7-positive target cells. Target cells Jurkat / Luc (Nanjing Probio) and CCRF-CEM / Luc (Nanjing Probio) were collected, resuspended using complete medium RPMI 1640 (Gibco, Cat#22400-089) + 10% FBS (Gibco, Cat#10091-148), adjusted to a density of 4×10 4 cells / mL, and then 50 μL of the target cell suspension was transferred to a 96-well plate. CD7-CAR-T cells were collected, resuspended using complete medium RPMI 1640 (Gibco, Cat#22400-089) + 10% FBS (Gibco, Cat#10091-148), and based on the CAR positive rates of different transgenes (see Table 7) and different effector / target ratios (E / T), the theoretical densities were 4×10 4 cells / mL (E / T = 1:1), 2×10 5 cells / mL (E / T = 5:1), and 8×10 5 cells / mL (E / T = 20:1), respectively. Then, 50 μL of the CD7-CAR-T cell suspension and 50 μL of 2% Triton X-100 (J&K Scientific, Cat#993361) (as the maximum killing signal value) were transferred to the 96-well plate containing the target cells. The culture plate was transferred to an incubator and incubated at 37°C with 5% CO2 for 24 hours. After incubating for 24 hours, the culture plate was taken out, the luciferase detection reagent of the Bio-Lite Luciferase Assay System, a detection reagent (Novozymes, Cat#DD1201-02), was added, incubated at room temperature for 5 minutes, and the Luminescence signal value was detected using a microplate reader (PHERAstar FSX, BMG).

[0123] The calculation formula for the killing rate: %Cytotoxicity = 100×(1 - (RLU Experimental - RLU Min ) / (RLU UnT - RLU MinBased on the above, the CD7-CAR-T killing rates for different transdermal molecules were calculated. The results are shown in Figure 14, Table 8, and Table 9 (in the figures and tables, AHP19484 corresponds to the AHP19484-VHH3 molecule, AHP19485 corresponds to the AHP19485-VHH1 molecule, and AHP19509 corresponds to the AHP19509-VHH1 molecule). When the target cells were Jurkat / Luc, the CAR-T cells with molecular sequences AHP19484-VHH3, AHP19485-VHH1, and AHP19509-VHH1 showed higher killing efficacy than the positive control VHH6 CAR-T in all three E / T ratios. When the target cells were CCRF-CEM / Luc with E / T ratios of 5:1 and 20:1, and the molecular sequences were AHP19484-VHH3, AHP19485-VHH1, and AHP10509-VHH1, they showed a higher killing effect than the positive control VHH6 CAR-T.

[0124] RLU Experimental :Signal value of CD7-CAR T cells + target cells (Jurkat / Luc or CCRF-CEM / Luc), RLU UnT :Non-transduced T cell + target cell (Jurkat / Luc or CCRF-CEM / Luc) signal value, RLU Min : Signal value of 1% Triton X-100+ target cells (Jurkat / Luc or CCRF-CEM / Luc).

[0125] [Table 8]

[0126] [Table 9]

[0127] Although embodiments of the present invention have been described above, the present invention is not limited to the embodiments described above. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and scope of the present invention should all be within the scope of protection of the present invention.

[0128] The amino acid sequence information referred to in this specification is as follows:

[0129] Amino acid sequence of the variable region of the heavy chain of the single-domain antibody AHP19472: SEQ ID NO: 1 AVQLVDSGGGLVQPGGSLGLSCAASGFTFSGFDMSWYRQAPGKERMLVATITRFGGSSNYEDSVKGRFTISRDNARDTVYLQMNSLKPEDTAMYYCHAEAPAGSLWFGNNYWGQGTQVTVSS

[0130] Amino acid sequence of the CDR1 region of AHP19472: Sequence ID No. 2 GFTFSGFD

[0131] Amino acid sequence of the CDR2 region of AHP19472: Sequence ID No. 3 ITRFGGSS

[0132] Amino acid sequence of the CDR3 region of AHP19472: Sequence ID No. 4 HAEAPAGSLWFGNNY

[0133] Amino acid sequence of the variable region of the heavy chain of the single-domain antibody AHP19481: SEQ ID NO: 5 AVQLVDSGGGLVQPGGSLRLSCAASGFTFSGFAMSWYRQAPGKERELVATISRFGESSNYEDSVKGRFTISRDNAKDTGYLQMNSLKPEDTAVYYCNAEAPAGSLWYGNNYWGQGTQVTVSS

[0134] Amino acid sequence of the CDR1 region of AHP19481: Sequence ID No. 6 GFTFSGFA

[0135] Amino acid sequence of the CDR2 region of AHP19481: Sequence ID No. 7 IRFGESS

[0136] Amino acid sequence of the CDR3 region of AHP19481: Sequence ID No. 8 NAEAPAGSLWYGNNY

[0137] AHP19483 Single-Domain Antibody Heavy Chain Variable Region Amino Acid Sequence: SEQ ID NO: 9 AVQLVDSGGGLVQAGGSLTLSCAASGRTFSRYTMGWFRQAPGKERELVASISWSGGSTDYADSVKDRFTISRDNTKNTVYLQMNGLKPEDTAVYYCNTDRMPYRPVVGGILSWEPYWGQGTQVTVTP

[0138] Amino acid sequence of the CDR1 region of AHP19483: SEQ ID NO: 10 GRTFSRYT

[0139] Amino acid sequence of the CDR2 region of AHP19483: SEQ ID NO: 11 ISWSGGST

[0140] Amino acid sequence of the CDR3 region of AHP19483: SEQ ID NO: 12 NTDRMPYRPVVGGILSWEPY

[0141] AHP19484 Single-Domain Antibody Heavy Chain Variable Region Amino Acid Sequence: SEQ ID NO: 13 QVKLEESGGGLVQPGGSLRLSCVASGSSFHFAFMGWYRQAPGKQRELVADISPGNSTNYADPVKGRFVISRDNAKNTVYLQMNSLKPEDTAVYFCRDLRSWGAWGRGTQVTVSS

[0142] Amino acid sequence of the CDR1 region of AHP19484: SEQ ID NO: 14 GSSFHFAF

[0143] Amino acid sequence of the CDR2 region of AHP19484: SEQ ID NO: 15 ISPGNST

[0144] Amino acid sequence of the CDR3 region of AHP19484: SEQ ID NO: 16 RDLRSWGA

[0145] Amino acid sequence of the variable region of the heavy chain of the single-domain antibody AHP19485: SEQ ID NO: 17 EVQLVESGGGLVQPGGSLRLSCASSERIFSIHAMGWYRQAPGKQRELVASITIGGSTHYADSVKGRFTISRDNAKNTVYLHMNSLKPEDTAVYYCNSMLLAGTVGGSWGQGTQVTVSS

[0146] Amino acid sequence of the CDR1 region of AHP19485: SEQ ID NO: 18 ERIFSIHA

[0147] Amino acid sequence of the CDR2 region of AHP19485: SEQ ID NO: 19 ITIGGST

[0148] Amino acid sequence of the CDR3 region of AHP19485: SEQ ID NO: 20 NSMLLAGTVGGS

[0149] AHP19487 Single-Domain Antibody Heavy Chain Variable Region Amino Acid Sequence: SEQ ID NO: 21 DVQLVESGGGLVQPGGSLRLSCAFPGSAFSFYFMGWYRQAPGKQRELVGDITPGGTANYADSVKGRFTISRDSAKNTVYLQMNSLKPEDTAVYYCNADRRVTIRPDGYWGQGTQVTVSS

[0150] Amino acid sequence of the CDR1 region of AHP19487: SEQ ID NO: 22 GSAFSFYF

[0151] Amino acid sequence of the CDR2 region of AHP19487: SEQ ID NO: 23 ITPGGTA

[0152] Amino acid sequence of the CDR3 region of AHP19487: SEQ ID NO: 24 NADRRVTIRPDGY

[0153] AHP19490 Single-Domain Antibody Heavy Chain Variable Region Amino Acid Sequence: SEQ ID NO: 25 QVQLVESGGGLVQPGGSLRLSCAASGFTFSGFDMSWYRQAPGKERELVATISRFGGSSNYEDSVKGRFTISRDNVKDTVYLQMNSLKPEDTAVYYCYALAPAGSLWHGDNYWGQGTQVTVSS

[0154] Amino acid sequence of the CDR1 region of AHP19490: SEQ ID NO: 26 GFTFSGFD

[0155] Amino acid sequence of the CDR2 region of AHP19490: SEQ ID NO: 27 ISRFGGSS

[0156] Amino acid sequence of the CDR3 region of AHP19490: SEQ ID NO: 28 YALAPAGSLWHGDNY

[0157] AHP19494 Single-Domain Antibody Heavy Chain Variable Region Amino Acid Sequence: SEQ ID NO: 29 QVKLEESGGGLVQAGGSLLSCAAPGRTLSASIKAWFRQAPGKDREFAAAIRWSGDSTYYADSVKGRFTISRDNAKNTVDLQMNSLKPEDTAVYYCNAGGLLSNGYQPGSSWGQGTQVTVSS

[0158] Amino acid sequence of the CDR1 region of AHP19494: SEQ ID NO: 30 GRTLSASI

[0159] Amino acid sequence of the CDR2 region of AHP19494: SEQ ID NO: 31 IRWSGDST

[0160] Amino acid sequence of the CDR3 region of AHP19494: SEQ ID NO: 32 NAGGLLSNGYQPGSS

[0161] AHP19495 Single-Domain Antibody Heavy Chain Variable Region Amino Acid Sequence: SEQ ID NO: 33 AVQLVESGGGLVQPGGSLRLSCAASGFTASRDVMSWYRQPPGKERELVASIFSAGSKETYAESVKGRFTISIDNVKNTVNLQMDNLKPEDTAVYYCHRWPGYWGQGTQVTVSS

[0162] Amino acid sequence of the CDR1 region of AHP19495: SEQ ID NO: 34 GFTASRDV

[0163] Amino acid sequence of the CDR2 region of AHP19495: SEQ ID NO: 35 IFSAGSKE

[0164] Amino acid sequence of the CDR3 region of AHP19495: SEQ ID NO: 36 HRWPGY

[0165] Amino acid sequence of the variable region of the heavy chain of the single-domain antibody AHP19508: SEQ ID NO: 37 AVQLVDSGGGLVQPGGSLRLSCAASGDFARLYEMAWYRQTPGQQREVVAQITVGGSTKYADSVKGRFTISRDSAKNTVYLQMNSLRPEDTAVYYCHAWPALWGQGTQVTVSS

[0166] Amino acid sequence of the CDR1 region of AHP19508: SEQ ID NO: 38 GDFARLYE

[0167] Amino acid sequence of the CDR2 region of AHP19508: SEQ ID NO: 39 ITVGGST

[0168] Amino acid sequence of the CDR3 region of AHP19508: SEQ ID NO: 40 HAWPAL

[0169] Amino acid sequence of the variable region of the heavy chain of the single-domain antibody AHP19509: SEQ ID NO: 41 QVKLEESGGGLVQAGGSLRLLSCAASGGTFNRYTMGWFRQAPGKEREFVAAISWSGGSTDYADSVKGRFTISRDNTKNTVYLQMNSLKPEDAAVYYCDTDRMPYRPVAAGILSWEAANWGQGTQVTVSS

[0170] Amino acid sequence of the CDR1 region of AHP19509: SEQ ID NO: 42 GGTFNRYT

[0171] Amino acid sequence of the CDR2 region of AHP19509: SEQ ID NO: 43 ISWSGGST

[0172] Amino acid sequence of the CDR3 region of AHP19509: SEQ ID NO: 44 DTDRMPYRPVAAGILSWEAN

[0173] Amino acid sequence of the variable region of the heavy chain of the single-domain antibody AHP19511: SEQ ID NO: 45 AVQLVESGGGLVQPGGSLRLSCAASGFTFSNAVIGWYRQAPGKQRELVASITPGGRTTYPDSVKGRFIISRDNAKNTVYLEMNSLKLEDTAVYYCNDFRGLGGYWGQGTQVTVSS

[0174] Amino acid sequence of the CDR1 region of AHP19511: SEQ ID NO: 46 GFTFSNAV

[0175] Amino acid sequence of the CDR2 region of AHP19511: SEQ ID NO: 47 ITPGGRT

[0176] Amino acid sequence of the CDR3 region of AHP19511: SEQ ID NO: 48 NDFRGLGGY

[0177] Amino acid sequence of the variable region of the heavy chain of the single-domain antibody AHP19484-VHH3: SEQ ID NO: 49 QVQLVESGGGLVKPGGSLRLSCAASGSSFHFAFMGWYRQAPGKQRELVADISPGNSTNYADPVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYFCRDLRSWGAWGQGTLVTVSS

[0178] Amino acid sequence of the variable region of the heavy chain of the single-domain antibody AHP19485-VHH1: SEQ ID NO: 50 EVQLVESGGGLVQPGGSLRLSCAASERIFSIHAMGWYRQAPGKQRELVSSITIGGSTHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCNSMLLAGTVGGSWGQGTLVTVSS

[0179] Amino acid sequence of the variable region of the heavy chain of the single-domain antibody AHP19509-VHH1: SEQ ID NO: 51 EVQLVESGGGLVQPGGSLRLSCAASGGTFNRYTMGWFRQAPGKEREFVSAISWSGGSTDYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCDTDRMPYRPVAAGILSWEANWGQGTLVTVSS

[0180] CD7 inhibitor amino acid sequence: SEQ ID NO: 52 MLLLVTSLLLCELPHPAFLLIPEVQLVESGGGLVQPGGSLRLSCAASERIFSIHAMGWYRQAPGKQRELVSSITIGGSTHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCNSMLLAGTVGGSWGQGTLVTVSSGGGS GGGSGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASERIFSIHAMGWYRQAPGKQRELVSSITIGGSTHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCNSMLLAGTVGGSWGQGTLVTVSSEQKLISEEDLKDEL*

[0181] Signal peptide sequence: SEQ ID NO: 53 MLLLVTSLLLCELPHPAFLLIP

[0182] Flag tag array: Array 54 DYKDDDDK

[0183] Hinge domain sequence: Sequence ID 55 IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP

[0184] Transmembrane region sequence: Sequence ID 56 FWVLVVVGGVLACYSLLVTVAFIIFWV

[0185] CD28 sequence: Sequence ID 57 RSKRSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRS

[0186] CD3ζ sequence: Sequence ID 58 RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR

[0187] AHP19484-VHH3 CAR sequence: Sequence ID 59 MLLLVTSLLLCELPHPAFLLIPQVQLVESGGGLVKPGGSLRLSCAASGSSFHFAFMGWYRQAPGKQRELVADISPGNSTNYADPVKGRFTISRDNAKNSLYLQMNSLRAEDTAVYFCRDLRS WGAWGQGTLVTVSSGGGSGGGSGGGSGGGSQVQLVESGGGLVKPGGSLRLSCAASGSSFHFAFMGWYRQAPGKQRELVADISPGNSTNYADPVKGRFTISRDNAKNSLYLQMNSLRAEDTAV YFCRDLRSWGAWGQGTLVTVSSAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQPYA PPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR*

[0188] AHP19485-VHH1 CAR sequence: Sequence ID 60 MLLLVTSLLLCELPHPAFLLIPEVQLVESGGGLVQPGGSLRLSCAASERIFSIHAMGWYRQAPGKQRELVSSITIGGSTHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCNSMLLAG TVGGSWGQGTLVTVSSGGGSGGGSGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASERIFSIHAMGWYRQAPGKQRELVSSITIGGSTHYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAV YYCNSMLLAGTVGGSWGQGTLVTVSSAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRKHYQP YAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR*

[0189] AHP19509-VHH1 CAR sequence: Sequence ID 61 MLLLVTSLLLCELPHPAFLLIPEVQLVESGGGLVQPGGSLRLSCAASGGTFNRYTMGWFRQAPGKEREFVSAISWSGGSTDYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCDTDRMPYRPV AAGILSWEANWGQGTLVTVSSGGGSGGGSGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGGTFNRYTMGWFRQAPGKEREFVSAISWSGGSTDYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTA VYYCDTDRMPYRPVAAGILSWEANWGQGTLVTVSSAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTRK HYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR*

[0190] VHH6 CAR sequence: Sequence ID 62 MLLLVTSLLLCELPHPAFLLIPDVQLQESGGGLVQAGGSLRLSCAVSGYPYSSYCMGWFRQAPGKEREGVAAIDSDGRTRYADSVKGRFTISQDNAKNTLYLQMNRMKPEDTAMYYCAARFGPMGCVDL STLSFGHWGQGTQVTVSITGGGGSGGGGSGGGGSGGGGSDVQLQESGGGLVQAGGSLRLSCAVSGYPYSSYCMGWFRQAPGKEREGVAAIDSDGRTRYADSVKGRFTISQDNAKNTLYLQMNRMKPEDT AMYYCAARFGPMGCVDLSTLSFGHWGQGTQVTVSITAAAIEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSKRSRLLHSDYMNMTPRRPGPTR KHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR*

[0191] endoplasmic reticulum localization domain sequence: Sequence ID 63 EQKLISEEDLKDEL

[0192] Linker array: Sequence ID 64 GGGSGGGSGGGSGGGS

Claims

1. Includes heavy chain variable regions including CDR1, CDR2 and CDR3, CDR1 contains the sequence shown in SEQ ID NO: 18, CDR2 contains the sequence shown in SEQ ID NO: 19, and CDR3 contains the sequence shown in SEQ ID NO:

20. CDR1 contains the sequence shown in SEQ ID NO: 42, CDR2 contains the sequence shown in SEQ ID NO: 43, and CDR3 contains the sequence shown in SEQ ID NO:

44. CDR1 contains the sequence shown in SEQ ID NO: 2, CDR2 contains the sequence shown in SEQ ID NO: 3, and CDR3 contains the sequence shown in SEQ ID NO:

4. CDR1 contains the sequence shown in SEQ ID NO: 6, CDR2 contains the sequence shown in SEQ ID NO: 7, and CDR3 contains the sequence shown in SEQ ID NO:

8. CDR1 contains the sequence shown in SEQ ID NO: 10, CDR2 contains the sequence shown in SEQ ID NO: 11, and CDR3 contains the sequence shown in SEQ ID NO:

12. CDR1 contains the sequence shown in sequence number 14, CDR2 contains the sequence shown in sequence number 15, and CDR3 contains the sequence shown in sequence number 16. CDR1 contains the sequence shown in SEQ ID NO: 22, CDR2 contains the sequence shown in SEQ ID NO: 23, and CDR3 contains the sequence shown in SEQ ID NO:

24. CDR1 contains the sequence shown in SEQ ID NO: 26, CDR2 contains the sequence shown in SEQ ID NO: 27, and CDR3 contains the sequence shown in SEQ ID NO:

28. CDR1 includes the sequence shown in SEQ ID NO: 30, CDR2 includes the sequence shown in SEQ ID NO: 31, and CDR3 includes the sequence shown in SEQ ID NO:

32. CDR1 contains the sequence shown in SEQ ID NO: 34, CDR2 contains the sequence shown in SEQ ID NO: 35, and CDR3 contains the sequence shown in SEQ ID NO:

36. CDR1 contains the sequence shown in SEQ ID NO: 38, CDR2 contains the sequence shown in SEQ ID NO: 39, CDR3 contains the sequence shown in SEQ ID NO: 40, or CDR1 contains the sequence shown in SEQ ID NO: 46, CDR2 contains the sequence shown in SEQ ID NO: 47, and CDR3 contains the sequence shown in SEQ ID NO:

48. An antibody or antigen-binding fragment thereof that specifically binds to human-derived CD7, characterized in that CDR1, CDR2, and CDR3 each allow for up to three amino acid mutations, insertions, or deletions.

2. The arrangement of the CDR region is, A group comprising CDR1 containing the sequence shown in SEQ ID NO: 18, CDR2 containing the sequence shown in SEQ ID NO: 19, and CDR3 containing the sequence shown in SEQ ID NO: 20, A group in which CDR1 contains the sequence shown in SEQ ID NO: 42, CDR2 contains the sequence shown in SEQ ID NO: 43, and CDR3 contains the sequence shown in SEQ ID NO: 44, The antibody or antigen-binding fragment according to claim 1, characterized in that CDR1 is selected from any of the groups including the group including the sequence shown in SEQ ID NO: 14, CDR2 is selected from the group including the sequence shown in SEQ ID NO: 15, and CDR3 is selected from the group including the sequence shown in SEQ ID NO:

16.

3. The antibody or antigen-binding fragment according to claim 1, characterized in that the heavy chain variable region includes a framework region of an alpaca antibody or a human antibody.

4. The antibody or antigen-binding fragment according to claim 1, characterized in that it is selected from the group consisting of a complete antibody, Fab, Fab', Fv fragment, scFv, di-scFv, and a single-domain antibody, and is preferably a single-domain antibody.

5. The antibody or antigen-binding fragment according to claim 1, characterized in that it is a chimeric antibody, an Fc fusion antibody, or a humanized antibody.

6. The antibody or antigen-binding fragment according to claim 5, characterized in that it includes an amino acid sequence shown in any one of SEQ ID NOs: 17, 41, 1, 5, 9, 13, 21, 25, 29, 33, 37, 45 and 49-51, or a mutant amino acid sequence having at least 80% identity thereto.

7. A CD7 inhibitory molecule comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 6 and an endoplasmic reticulum localization domain, characterized in that it is capable of reducing the expression of CD7 molecules on the cell surface.

8. The CD7 inhibitory molecule according to claim 7, characterized by comprising two identical single-domain antibodies linked by a linker.

9. The inhibitory molecule according to claim 7, characterized in that the sequence of the endoplasmic reticulum localization domain includes the sequence shown in Sequence ID No.

63.

10. The CD7 inhibitory molecule according to claim 7, further comprising a signal peptide sequence, preferably the signal peptide sequence comprising the sequence shown in SEQ ID NO:

53.

11. The CD7 inhibitory molecule according to claim 7, characterized in that the amino acid sequence of the CD7 inhibitory molecule includes the sequence shown in SEQ ID NO: 52, or a mutant sequence having at least 80% identity thereto.

12. 1) An antigen-binding domain that recognizes a CD7 antigen, comprising an antibody or antigen-binding fragment thereof as described in any one of claims 1 to 6, 2) Transmembrane domain and 3) A chimeric antigen receptor characterized by comprising an intracellular signaling domain.

13. The chimeric antigen receptor according to claim 12, further comprising an intracellular co-stimulatory signaling domain.

14. The chimeric antigen receptor according to claim 12, characterized in that the sequence includes the amino acid sequence shown in any one of sequence numbers 59 to 61.

15. An isolated nucleic acid molecule encoding an antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, an inhibitory molecule according to any one of claims 7 to 11, or a chimeric antigen receptor according to any one of claims 12 to 14.

16. A vector comprising the nucleic acid molecule described in claim 15.

17. A host cell comprising the vector according to claim 16.

18. Genetically modified immune cells characterized by comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, an inhibitory molecule according to any one of claims 7 to 11, or a chimeric antigen receptor according to any one of claims 12 to 14.

19. The genetically modified immune cell according to claim 18, characterized in that it expresses the chimeric antigen receptor described in any one of claims 12 to 14.

20. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, an inhibitory molecule according to any one of claims 7 to 11, a chimeric antigen receptor according to any one of claims 12 to 14, or a genetically modified immune cell according to claim 18 or 19, and / or a pharmaceutically acceptable carrier.

21. Use of an antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, or an inhibitory molecule according to any one of claims 7 to 11, or a genetically modified immune cell according to claim 18 or 19, or the composition according to claim 20, in the treatment of a tumor or cancer.

22. The use according to claim 21, characterized in that the cancer or tumor is a hematological malignancy, and preferably the hematological malignancy is a T cell-associated tumor.

23. (1) A step of preparing immune cells containing an inhibitory molecule according to any one of claims 7 to 11 that expresses a chimeric antigen receptor, A method for producing genetically modified immune cells, comprising the steps of: (1) adding a nucleic acid molecule encoding a chimeric antigen receptor according to any one of claims 12 to 14 to the cells obtained in step (1); (2)

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