Antibody targeting human CD7 or antigen-binding fragment thereof and use thereof

Abstract

An antibody targeting human CD7 or an antigen fragment thereof and the use thereof. The antibody and CD7-CAR based on the antibody fragment have a very strong affinity for a CD7 antigen molecule, and a blocking molecule containing the antibody can almost completely block the expression of the CD7 molecule on the cell surface without affecting the normal amplification of T cells, thereby effectively avoiding the fratricide killing of CD7-CAR-T cells. The CD7-CAR-T cells, which are constructed on the basis of a single domain antibody targeting human CD7, have a strong killing effect with regard to target cells.

Description

Antibodies or antigen-binding fragments targeting human CD7 and their applications

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to Chinese patent application No. 202310906380.4, filed on July 21, 2023, entitled “Antibodies or antigen-binding fragments thereof targeting human CD7 and their applications”, and incorporates its entire contents into this application by reference. Technical Field

[0003] The present invention belongs to the field of tumor immunotherapy and molecular immunology, and specifically relates to a single-domain antibody targeting human CD7 and a CD7-CAR-T cell containing the single-domain antibody, as well as a preparation method and application thereof. Background Art

[0004] Tumor immunotherapy has become one of the most important means of tumor treatment. Only after identifying cancer cells can the immune system effectively attack cancer cells. Canceration is the result of normal somatic cells losing their normal cell regulatory functions and gene mutations accumulating to a certain extent. However, cancer cells can disguise themselves as normal somatic cells and cleverly avoid the attack of the immune system.

[0005] Tumor-associated antigens (TAAs) are self-antigens expressed by tumor cells. They are partially present in normal host cells, primarily through gene amplification or post-translational modification, but tend to be highly or specifically expressed in tumor cells. Currently, many tumor-associated and tumor-specific antigens have been discovered, and targeted therapy targeting TAAs is an important approach to cancer treatment. Numerous cancer immunotherapy drugs that rely on this mechanism are already on the market and have demonstrated excellent clinical efficacy.

[0006] Generally speaking, antibody-mediated TAA recognition can cleverly guide NK cells or T cells to target tumor cells with high TAA expression. Antibodies targeting TAAs (e.g., rituximab and trastuzumab) can not only directly kill tumor cells through ADCC, but can also serve as diagnostic markers or innovatively increase the targeting of traditional cancer therapies.

[0007] Adoptive immunotherapy is a transformative treatment approach in hematology. It involves genetically engineering T cells to express synthetic chimeric antigen receptors (CARs). CARs are engineered receptors that redirect immune cells to target cancer cells. CAR-T cells have achieved impressive results in patients with hematological malignancies, with six FDA-approved CAR-T cells for the treatment of relapsed or refractory B-cell malignancies: Abecma, Breyanzi, Carvykti, Kymriah, Tecartus, and Yescarta. The components of these CARs (extracellular antigen-binding domain, hinge and transmembrane regions, costimulatory domains, and activation domains) are systematically engineered to optimize cell activation and enhance persistence. Using different domains or making minor modifications to the domain sequences can significantly alter the efficacy of CAR-T cells. Despite the success of CAR-T in the treatment of hematological malignancies to date, many patients experience CAR-T cell-related toxicities and relapse from CAR-T cell therapy, potentially related to the CAR binding epitopes, highlighting the need to develop 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 from responding to their surrounding environment. While FDA-approved CAR-T cells target two B-cell antigens, CD19 and B-cell maturation antigen (BCMA), other CAR-Ts targeting B-cell malignancies, other hematologic cancers, and solid tumors are rapidly emerging. Compared to B-cell malignancies, patients with T-cell malignancies often achieve limited clinical responses with first-line cancer treatments (including chemotherapy), resulting in a poor prognosis. Given the clinical success of CAR-T therapy in B-cell malignancies, there is hope that CAR-T therapy will also improve clinical outcomes for patients with T-cell malignancies. However, CAR-T therapy for T-cell malignancies remains challenging. The first challenge is the lack of T-cell tumor-specific CAR-T target antigens. Furthermore, fratricidal killing, T-cell aplasia, and contamination of autologous CAR-T products with malignant T cells during production are among the most significant challenges facing CAR-T therapy for T-cell malignancies, all of which require further investigation.

[0008] CD7 is a transmembrane glycoprotein of the immunoglobulin superfamily. It is expressed as a homodimer on the cell surface of most mature peripheral blood T cells, all thymocytes, and natural killer (NK) cells. It is also expressed on myeloid cell lines and T / B cells that give rise to immature progenitor cells. CD7 is one of the primary markers of T cell maturation. Therefore, it is used as a diagnostic marker for detecting primary T cell cancers. CD7 is highly expressed in acute T-cell leukemia (T-ALL) and subsets of peripheral T-cell lymphomas. Normal CD7 expression is primarily restricted to T cells and natural killer (NK) cells, thereby reducing the risk of off-target organ toxicity. Due to its internalization effect, CD7 has been used as a target for immunotoxin antibodies in patients with T-cell malignancies in some clinical studies. Although there have been no serious, permanent adverse reactions associated with CD7 antibodies, tumor responses have been limited. Therefore, the efficacy of CD7-directed cytotoxicity could be enhanced by replacing monoclonal antibodies with autologous CAR-T cells, potentially improving the 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. CD7-edited T cells expressing a CD7-specific CAR for the therapy of T-cell malignancies. Blood. 2017;130(3):285–296; Sempowski GD, Lee DM, Kaufman RE, Haynes BF. Structure and function of 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).

[0009] Compared to CAR-T therapy for B-ALL, the application of CD7-CAR-T cells for the targeted treatment of T-cell acute lymphoblastic leukemia and CD7-positive lymphomas still faces significant technical challenges. This is because both normal effector T cells and T-cell tumors express the CD7 antigen, which can lead to a "fratricidal" effect of CD7-CAR-T cells. Existing technologies for the in vitro preparation of CD7-CAR-T cells often have a "suicide" effect and are difficult to successfully prepare in vitro. Therefore, it is necessary to develop a new CD7 antibody or its antigen fragment to block CD7 expression on T cells and express it on CD7-CAR-T cells, so that the CD7-CAR-T preparation process will not commit suicide, but can be expanded normally in vitro and have lethality to CD7-positive target cells.

[0010] Summary of the Invention

[0011] In order to solve the technical problems in the prior art, the inventors have conducted in-depth research, prepared and selected a unique single-domain antibody sequence against human CD7. The CDRs of the positive single-domain antibody were grafted into the natural human germline sequence using CDR grafting technology. After combinatorial design, a humanized antibody heavy chain variable region DNA fragment was obtained as the antigen recognition element for the CD7-CAR-T construction of the present invention. At the same time, a CD7 vector that effectively blocks the expression of CD7 molecules on the surface of T cells was constructed, thereby eliminating the "cannibalism" effect of CD7-CAR-T cells, so as to facilitate the in vitro preparation and production of CD7-CAR-T cells. The CD7-CAR-T cells constructed by the single-domain antibody targeting human CD7 provided by the present invention have a better in vitro killing effect on the target cell Jurkat / Luc than the positive reference VHH6 CAR-T cells. Specifically, the present invention includes the following contents.

[0012] In a first aspect, the present invention provides an antibody or antigen-binding fragment thereof that specifically binds to a CD7 antigen, comprising a heavy chain variable region, wherein the heavy chain variable region comprises a complementarity determining region 1 (CDR1), a complementarity determining region 2 (CDR2), and a complementarity determining region 3 (CDR3), wherein:

[0013] CDR1 comprises the sequence set forth in SEQ ID NO: 18, CDR2 comprises the sequence set forth in SEQ ID NO: 19, and CDR3 comprises the sequence set forth in SEQ ID NO: 20;

[0014] CDR1 comprises the sequence set forth in SEQ ID NO:42, CDR2 comprises the sequence set forth in SEQ ID NO:43, and CDR3 comprises the sequence set forth in SEQ ID NO:44;

[0015] CDR1 comprises the sequence set forth in SEQ ID NO: 2, CDR2 comprises the sequence set forth in SEQ ID NO: 3, and CDR3 comprises the sequence set forth in SEQ ID NO: 4;

[0016] CDR1 comprises the sequence shown in SEQ ID NO: 6, CDR2 comprises the sequence shown in SEQ ID NO: 7, and CDR3 comprises the sequence shown in SEQ ID NO: 8;

[0017] CDR1 comprises the sequence set forth in SEQ ID NO: 10, CDR2 comprises the sequence set forth in SEQ ID NO: 11, and CDR3 comprises the sequence set forth in SEQ ID NO: 12;

[0018] CDR1 comprises the sequence set forth in SEQ ID NO: 14, CDR2 comprises the sequence set forth in SEQ ID NO: 15, and CDR3 comprises the sequence set forth in SEQ ID NO: 16;

[0019] CDR1 comprises the sequence set forth in SEQ ID NO: 22, CDR2 comprises the sequence set forth in SEQ ID NO: 23, and CDR3 comprises the sequence set forth in SEQ ID NO: 24;

[0020] CDR1 comprises the sequence set forth in SEQ ID NO: 26, CDR2 comprises the sequence set forth in SEQ ID NO: 27, and CDR3 comprises the sequence set forth in SEQ ID NO: 28;

[0021] CDR1 comprises the sequence set forth in SEQ ID NO:30, CDR2 comprises the sequence set forth in SEQ ID NO:31, and CDR3 comprises the sequence set forth in SEQ ID NO:32;

[0022] CDR1 comprises the sequence set forth in SEQ ID NO:34, CDR2 comprises the sequence set forth in SEQ ID NO:35, and CDR3 comprises the sequence set forth in SEQ ID NO:36;

[0023] CDR1 comprises the sequence shown in SEQ ID NO: 38, CDR2 comprises the sequence shown in SEQ ID NO: 39, and CDR3 comprises the sequence shown in SEQ ID NO: 40; or

[0024] CDR1 comprises the sequence set forth in SEQ ID NO:46, CDR2 comprises the sequence set forth in SEQ ID NO:47, and CDR3 comprises the sequence set forth in SEQ ID NO:48;

[0025] Wherein, said CDR1, CDR2 and CDR3 respectively contain substitutions, insertions or deletions of up to 3 amino acids.

[0026] In certain embodiments, the heavy chain variable region comprises CDR1, CDR2, and CDR3, wherein:

[0027] CDR1 comprises the sequence set forth in SEQ ID NO: 18, CDR2 comprises the sequence set forth in SEQ ID NO: 19, and CDR3 comprises the sequence set forth in SEQ ID NO: 20;

[0028] CDR1 comprises the sequence set forth in SEQ ID NO:42, CDR2 comprises the sequence set forth in SEQ ID NO:43, and CDR3 comprises the sequence set forth in SEQ ID NO:44;

[0029] CDR1 comprises the sequence set forth in SEQ ID NO: 2, CDR2 comprises the sequence set forth in SEQ ID NO: 3, and CDR3 comprises the sequence set forth in SEQ ID NO: 4;

[0030] CDR1 comprises the sequence set forth in SEQ ID NO:6, CDR2 comprises the sequence set forth in SEQ ID NO:7, and CDR3 comprises the sequence set forth in SEQ ID NO:8;

[0031] CDR1 comprises the sequence set forth in SEQ ID NO: 10, CDR2 comprises the sequence set forth in SEQ ID NO: 11, and CDR3 comprises the sequence set forth in SEQ ID NO: 12;

[0032] CDR1 comprises the sequence set forth in SEQ ID NO: 14, CDR2 comprises the sequence set forth in SEQ ID NO: 15, and CDR3 comprises the sequence set forth in SEQ ID NO: 16;

[0033] CDR1 comprises the sequence set forth in SEQ ID NO: 22, CDR2 comprises the sequence set forth in SEQ ID NO: 23, and CDR3 comprises the sequence set forth in SEQ ID NO: 24;

[0034] CDR1 comprises the sequence set forth in SEQ ID NO: 26, CDR2 comprises the sequence set forth in SEQ ID NO: 27, and CDR3 comprises the sequence set forth in SEQ ID NO: 28;

[0035] CDR1 comprises the sequence set forth in SEQ ID NO:30, CDR2 comprises the sequence set forth in SEQ ID NO:31, and CDR3 comprises the sequence set forth in SEQ ID NO:32;

[0036] CDR1 comprises the sequence set forth in SEQ ID NO:34, CDR2 comprises the sequence set forth in SEQ ID NO:35, and CDR3 comprises the sequence set forth in SEQ ID NO:36;

[0037] CDR1 comprises the sequence shown in SEQ ID NO: 38, CDR2 comprises the sequence shown in SEQ ID NO: 39, and CDR3 comprises the sequence shown in SEQ ID NO: 40; or

[0038] CDR1 comprises the sequence shown in SEQ ID NO:46, CDR2 comprises the sequence shown in SEQ ID NO:47, and CDR3 comprises the sequence shown in SEQ ID NO:48.

[0039] In certain embodiments, the CDR region sequence is selected from any one of the following groups:

[0040] CDR1 comprises the sequence set forth in SEQ ID NO: 18, CDR2 comprises the sequence set forth in SEQ ID NO: 19, and CDR3 comprises the sequence set forth in SEQ ID NO: 20;

[0041] CDR1 comprises the sequence set forth in SEQ ID NO:42, CDR2 comprises the sequence set forth in SEQ ID NO:43, and CDR3 comprises the sequence set forth in SEQ ID NO:44;

[0042] CDR1 comprises the sequence shown in SEQ ID NO: 14, CDR2 comprises the sequence shown in SEQ ID NO: 15, and CDR3 comprises the sequence shown in SEQ ID NO: 16.

[0043] In certain embodiments, the heavy chain variable region comprises a framework region (FR) of an alpaca antibody or a human antibody.

[0044] In certain embodiments, the antibody or antigen-binding fragment thereof is selected from the group consisting of a whole antibody, Fab, Fab', (Fab')2, Fv fragment, scFv, di-scFv and a single domain antibody (sdab). In a specific embodiment, the antibody or antigen-binding fragment thereof is a single domain antibody.

[0045] In certain embodiments, the antibody or its antigen-binding fragment is a chimeric antibody Fc fusion antibody or a humanized antibody. In certain embodiments, the antibody or its antigen-binding fragment is a humanized antibody. In a specific embodiment, the antibody or its antigen-binding fragment is a humanized sdab.

[0046] In certain embodiments, the antibody or antigen-binding fragment thereof comprises a mutant amino acid sequence that is at least 80% identical to 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.

[0047] In a second aspect, the present invention provides a CD7 blocking molecule comprising the antibody or antigen-binding fragment thereof described in the first aspect and an endoplasmic reticulum localization domain, wherein the blocking molecule can reduce the expression of CD7 molecules on the cell surface.

[0048] In certain embodiments, the CD7 blocking molecule comprises one or more identical single-domain antibodies, wherein the single-domain antibodies are connected by a linker. In certain embodiments, the linker sequence is the sequence shown in SEQ ID NO: 64.

[0049] In a specific embodiment, the CD7 blocking molecule comprises two identical single domain antibodies, and the single domain antibodies are connected by a linker.

[0050] In certain embodiments, the endoplasmic reticulum localization domain sequence comprises the sequence shown in SEQ ID NO:63.

[0051] In certain embodiments, the CD7 blocking molecule further comprises a signal peptide sequence. In certain embodiments, the signal peptide sequence comprises the sequence shown in SEQ ID NO:53.

[0052] In certain embodiments, the amino acid sequence of the CD7 blocking molecule comprises the sequence shown in SEQ ID NO: 52 or a mutant sequence thereof having at least 80% identity thereto.

[0053] In a third aspect, the present invention provides a chimeric antigen receptor (CAR), comprising:

[0054] 1) an antigen-binding domain that recognizes the CD7 antigen, wherein the antigen-binding domain comprises the antibody or antigen-binding fragment thereof according to the first aspect;

[0055] 2) a transmembrane domain; and

[0056] 3) Intracellular signaling domain.

[0057] In certain embodiments, the chimeric antigen receptor includes a hinge region between the antigen binding domain and the transmembrane domain, wherein the hinge region comprises a hinge region derived from one or more proteins selected from the group consisting of CD28, IgG1, IgG4, IgD, 4-IBB, CD4, CD27, CD7, CD8 alpha, PD-1, ICOS, OX40, NKG2D, NKG2C, Fc e RI y, BTLA, GITR, DAP10, CD40L, TIM1, CD226, SLAM, CD30, and LIGHT.

[0058] In certain embodiments, the hinge region comprises a hinge region derived from CD28. In a specific embodiment, the hinge region sequence comprises the sequence shown in SEQ ID NO:55.

[0059] In certain embodiments, the transmembrane domain of the chimeric antigen receptor comprises a transmembrane domain derived from one or more proteins selected from the group consisting of CD8, CD28, 4-IBB, CD4, CD27, CD7, PD-1, TRAC, TRBC, CD3e, CD3, CTLA-4, LAG-3, CD5, ICOS, OX40, NKG2D, 2B4, CD244, FceRly, 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.

[0060] In certain embodiments, the transmembrane domain comprises a transmembrane domain derived from CD28. In certain embodiments, the transmembrane domain comprises a transmembrane domain derived from CD8. In a specific embodiment, the transmembrane region sequence comprises the sequence shown in SEQ ID NO:56.

[0061] In certain embodiments, the intracellular signaling domain of the chimeric antigen receptor comprises 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, OX40, or any combination thereof.

[0062] In certain embodiments, the intracellular signal transduction domain comprises CD3ζ. In a specific embodiment, the signal transduction domain comprises the sequence shown in SEQ ID NO:58.

[0063] In certain embodiments, the chimeric antigen receptor further comprises an intracellular co-stimulatory signaling domain.

[0064] The intracellular co-stimulatory signaling domain comprises an intracellular co-stimulatory signaling domain derived from one or more proteins selected from the following group: CD28, CD137, CD27, CD2, CD7, CD8, OX40, CD226, DR3, SLAM, CDS, ICAM-1, NKG2D, NKG2C, B7-H3, 2B4, FceRly, BTLA, GITR, HVEM, DAP10, DAP12, CD30, CD40, CD40L, TIM1, PD-1, LFA-1, LIGHT, JAML, CD244, CD100, ICOS, a ligand of CD83, CD40 and MyD88.

[0065] In certain embodiments, the intracellular costimulatory signal domain comprises a sequence derived from the intracellular region of CD28. In certain embodiments, the intracellular costimulatory signal domain comprises a sequence derived from the intracellular region of 4-1BB. In a specific embodiment, the intracellular costimulatory signal domain comprises a sequence shown in SEQ ID NO:57.

[0066] In certain embodiments, the structure of the chimeric antigen receptor comprises multiple domains, such as: signal peptide-VHH-hinge region-transmembrane region-intracellular co-stimulatory signaling domain-intracellular signal transduction domain, wherein each domain is independently connected by a linker or peptide bond. The VHH can be one or more VHHs connected by a linker.

[0067] In certain embodiments, the signal peptide sequence is selected from the group consisting of signal peptides of proteins including CD8, CD28, GM-CSF, CD4, CD137, or a combination thereof. In a specific embodiment, the signal peptide sequence comprises the sequence shown in SEQ ID NO: 53.

[0068] In certain embodiments, the chimeric antigen receptor comprises the amino acid sequence shown in any one of SEQ ID NOs: 59-61.

[0069] In a fourth aspect, the present application provides an isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to the first aspect of the present invention, or the blocking molecule according to the second aspect, or the chimeric antigen receptor according to the third aspect.

[0070] In a fifth aspect, the present application provides a vector comprising the nucleic acid molecule according to the fourth aspect.

[0071] In certain embodiments, the vector is DNA, RNA, a plasmid, a lentiviral vector, an adenoviral vector, AAV, a retroviral vector, a transposon, or a combination thereof.

[0072] In certain embodiments, the vector is a lentiviral vector.

[0073] In a sixth aspect, the present application provides a host cell comprising the vector according to the fifth aspect.

[0074] In a seventh aspect, the present application provides an engineered immune cell comprising the antibody or antigen-binding fragment thereof according to the first aspect of the present invention, or the CD7 blocking molecule according to the second aspect, or the chimeric antigen receptor according to the third aspect.

[0075] In certain embodiments, the immune cells are selected from at least one of leukocytes, monocytes, macrophages, dendritic cells, mast cells, neutrophils, basophils, eosinophils, αβT cells, γδT cells, natural killer (NK) cells, natural killer T (NKT) cells, B cells, innate lymphoid cells (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.

[0076] In an eighth aspect, the present application provides a pharmaceutical composition comprising: the antibody or antigen-binding fragment thereof according to the first aspect of the present invention, or the blocking molecule according to the second aspect, or the chimeric antigen receptor according to the third aspect, or the engineered immune cell according to the seventh aspect, and / or a pharmaceutically acceptable carrier.

[0077] In the ninth aspect, the present application provides an antibody or antigen-binding fragment thereof according to the first aspect, or the blocking molecule according to the second aspect, or the engineered immune cell according to the seventh aspect, or the composition according to the eighth aspect for use in the treatment of tumors or cancer.

[0078] In certain embodiments, the cancer or tumor is a cancer or tumor associated with CD7 expression.

[0079] In certain embodiments, the cancer or tumor is a hematological malignancy.

[0080] In certain embodiments, the hematological malignancy is a T-cell related tumor, including leukemia, lymphoma, and myeloma.

[0081] In a tenth aspect, the present application provides a method for preparing engineered immune cells, characterized in that it comprises the following steps:

[0082] (1) providing a cell to be expressed in a chimeric antigen receptor, wherein the immune cell comprises the blocking molecule described in the second aspect; and

[0083] (2) Adding a nucleic acid molecule to the cells obtained in step 1, wherein the nucleic acid molecule expresses the chimeric antigen receptor described in the third aspect.

[0084] Beneficial effects of the present invention:

[0085] The antibodies or antigen-binding fragments thereof provided by the present invention have extremely strong affinity for CD7 antigen molecules. At the same time, the blocking molecules of the present invention can almost completely block the expression of CD7 molecules on the cell surface after transfection without affecting the normal proliferation of T cells, thereby effectively avoiding the cannibalism of CD7-CAR-T cells. The CD7-CAR-T cells constructed with humanized CD7 nano antibody sequences of the present invention can further reduce the immunogenicity of CD7-CAR-T cells constructed with CD7 antibody sequences of ovine origin in vivo, thereby improving the clinical treatment effect. In addition, the CD7-CAR-T cells constructed with single-domain antibodies targeting human CD7 provided by the present invention have a strong killing effect on target cells, which is better than the positive reference VHH6 CAR-T cells, and provides beneficial CAR-T cells for clinical application of cell therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0086] Figure 1 shows the binding test results of the antibodies described in this application and human CD7 protein.

[0087] FIG2 shows the binding test results of the antibodies described in the present application and monkey CD7 protein.

[0088] FIG3 shows the binding test results of the antibodies described in this application and mouse CD7 protein.

[0089] FIG4 shows the FACS detection results of the binding of the antibodies described in the present application to cells expressing human CD7 protein.

[0090] Figure 5 shows the affinity test results of the humanized antibody and human CD7 protein described in this application.

[0091] Shown are the affinity detection signals of three antibodies and their corresponding humanized antibodies to human CD7 protein.

[0092] Figure 6 shows the affinity test results of the humanized antibody described in this application and the monkey CD7 protein.

[0093] Shown are the affinity detection signals of three humanized antibodies to monkey-derived CD7 protein.

[0094] Figure 7 shows a schematic diagram of the structure of CD7 blocking molecules in some embodiments of the present application.

[0095] Figure 8 shows the blocking effect of the CD7 blocking molecules described in this application on T cell CD7 expression. Figures AF show the blocking test results of three different CD7 blocking molecules on cell surface CD7 expression under different virus titers.

[0096] Figure 9 shows the effect of AHP19485-VHH1 molecule in blocking CD7 expression on T cells.

[0097] Figure 10 shows the design ideas for the in vitro activity experiment of CD7 CAR-T cells in this application.

[0098] Figure 11 shows a schematic diagram of the CAR structure in some embodiments of the present application.

[0099] FIG. 12 shows the blocking effect of AHP19485-VHH1 on CD7 expression on T cells.

[0100] Figure 13 shows the results of the positive rate detection of CD7 expression on the CD7 CAR-T cells constructed in this application.

[0101] Figure 14 shows the in vitro cytotoxicity of CD7 CAR-T cells constructed in this application. Panel A shows the cytotoxicity of CAR-T cells carrying different CD7 antibody molecules against Jurkat target cells. Panel B shows the cytotoxicity of CAR-T cells carrying different CD7 antibody molecules against CCRF-CEM target cells. DETAILED DESCRIPTION

[0102] The technical solutions of the present invention will be described in further detail below with reference to specific embodiments. It should be understood that the following embodiments are merely illustrative and explanations of the present invention and should not be construed as limiting the scope of protection of the present invention. All technologies implemented based on the above content of the present invention are encompassed within the scope of protection that the present invention is intended to protect.

[0103] Unless otherwise noted, the raw materials and reagents used in the following examples are commercially available or can be prepared by known methods. Experimental procedures in the following examples, where specific conditions are not specified, were generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer.

[0104] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0105] Definition and explanation:

[0106] As used herein, the term "antibody" is used in its broadest sense and includes immunoglobulins or other types of molecules comprising one or more antigen-binding domains that specifically bind to an antigen, and is a protein or polypeptide that exhibits binding specificity to a specific antigen. Specific examples of antibodies may include complete antibodies (e.g., classic four-chain antibody molecules), single-chain antibodies, single-domain antibodies, multispecific antibodies, and the like. Classical antibody molecules are typically tetramers composed of two identical heavy chains and two identical light chains interconnected by disulfide bonds. Based on the conservative differences in the amino acid sequences, the heavy chain and light chain are divided into a variable region (V) at the amino terminus and a constant region (C) at the carboxyl terminus. The variable region is used to recognize and bind to the antigen, and the constant region (e.g., Fc fragment) is used to initiate downstream effects, such as antibody-dependent cellular cytotoxicity (ADCC). Within the variable regions of the heavy and light chains, there are three local regions, each with a higher degree of variation in amino acid composition and arrangement order, which are key positions for antibody-antigen binding and are therefore also referred to as complementary determining regions (CDRs). The amino acid sequence of a CDR can be readily determined using art-recognized numbering schemes, such as Kabat, Chothia, IMGT, AbM, or Contact.

[0107] Based on the amino acid sequence of the constant region of its heavy chains, antibodies can be divided into five major classes: IgA, IgD, IgE, IgG, and IgM. These antibody classes are further divided into subclasses based on the size of the hinge region, the location of interchain disulfide bonds, and molecular weight, such as IgG1, IgG2a, IgG2b, and IgG3. Based on the amino acid composition and arrangement of the constant region of its light chains, light chains can be divided into two types: kappa and lambda. The subunit structures and three-dimensional conformations of the different classes of immunoglobulins are well known in the art.

[0108] The heavy and light chain variable regions of an antibody typically include three complementary determining regions (CDRs) and four framework regions (FRs). The CDRs are connected by the framework regions. During antibody recognition, the FR molecules curl to bring the CDR molecules closer together. The CDRs are the binding sites between the antibody or antigen-binding fragment and the antigen. Therefore, the sequence of the CDRs determines the specificity of the antibody. As understood in the art, an antibody is a glycoprotein or antigen-binding portion thereof comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. The heavy chain comprises a heavy chain variable region (VH) and a heavy chain constant region (CH). The light chain comprises a light chain variable region (VL) and a light chain constant region (CL). The variable regions of the heavy and light chains comprise framework regions (FRs) and complementary determining regions (CDRs). The four FRs are relatively conserved, while the CDR regions (CDR1, CDR2, and CDR3) comprise hypervariable regions.

[0109] In this application, the term "CDR" generally refers to the complementary determining region, which is primarily responsible for binding to an antigenic epitope. The CDRs of the heavy chain are generally referred to as CDR1, CDR2, and CDR3, and are numbered sequentially starting from the N-terminus. The three CDR segments and the framework region together constitute the heavy chain variable region, i.e., the VHH segment. There are various rules for defining CDR regions in the art, such as the Chothia rule, the Kabat rule, etc. In this application, the CDRs can be divided according to the Kabat definition rule.

[0110] The "antigen-binding fragment" herein refers to a polypeptide fragment that contains a portion of an intact antibody, such as the antigen-binding region or variable region of an intact antibody, and has the ability to specifically target CD7, such as Fab, Fab', Fv fragment, F(ab')2, scFv, di-scFv and / or sdAb, etc. Preferably, it contains at least one CDR of the heavy chain variable region and / or light chain variable region of the antibody; further preferably, it may contain CDR1-3 of the heavy chain variable region and / or CDR1-3 of the light chain variable region. Antigen-binding fragments can be prepared by a variety of techniques, including but not limited to proteolytic digestion of intact antibodies, or expression by host cells containing antigen-binding fragments. Examples of antigen-binding proteins include but are not limited to antibodies, antigen-binding fragments, immunoconjugates, multispecific antibodies (e.g., bispecific antibodies), antibody fragments, antibody derivatives, antibody analogs or fusion proteins, etc., as long as they exhibit the desired antigen-binding activity.

[0111] The present invention provides the above-mentioned CD7-targeting antibody or antigen-binding fragment thereof, which has good safety and targeting properties, can specifically bind to the extracellular domain of human CD7, and a vector containing the coding sequence of the antibody or antigen-binding fragment thereof is used to infect immune cells, thereby obtaining immune cells that have a significant killing ability against CD7-expressing tumor cells. The immune cells can be used to treat or improve diseases related to CD7 expression, thereby laying the foundation for the treatment of CD7-positive tumors.

[0112] In certain embodiments, the antibody targeting CD7 comprises a heavy chain variable region comprising CDR1, CDR2, and CDR3, wherein CDR1 comprises the sequence of SEQ ID NO: 18, CDR2 comprises the sequence of SEQ ID NO: 19, and CDR3 comprises the sequence of SEQ ID NO: 20;

[0113] CDR1 comprises the sequence set forth in SEQ ID NO:42, CDR2 comprises the sequence set forth in SEQ ID NO:43, and CDR3 comprises the sequence set forth in SEQ ID NO:44;

[0114] CDR1 comprises the sequence set forth in SEQ ID NO: 2, CDR2 comprises the sequence set forth in SEQ ID NO: 3, and CDR3 comprises the sequence set forth in SEQ ID NO: 4;

[0115] CDR1 comprises the sequence set forth in SEQ ID NO:6, CDR2 comprises the sequence set forth in SEQ ID NO:7, and CDR3 comprises the sequence set forth in SEQ ID NO:8;

[0116] CDR1 comprises the sequence set forth in SEQ ID NO: 10, CDR2 comprises the sequence set forth in SEQ ID NO: 11, and CDR3 comprises the sequence set forth in SEQ ID NO: 12;

[0117] CDR1 comprises the sequence set forth in SEQ ID NO: 14, CDR2 comprises the sequence set forth in SEQ ID NO: 15, and CDR3 comprises the sequence set forth in SEQ ID NO: 16;

[0118] CDR1 comprises the sequence set forth in SEQ ID NO: 22, CDR2 comprises the sequence set forth in SEQ ID NO: 23, and CDR3 comprises the sequence set forth in SEQ ID NO: 24;

[0119] CDR1 comprises the sequence set forth in SEQ ID NO: 26, CDR2 comprises the sequence set forth in SEQ ID NO: 27, and CDR3 comprises the sequence set forth in SEQ ID NO: 28;

[0120] CDR1 comprises the sequence set forth in SEQ ID NO:30, CDR2 comprises the sequence set forth in SEQ ID NO:31, and CDR3 comprises the sequence set forth in SEQ ID NO:32;

[0121] CDR1 comprises the sequence set forth in SEQ ID NO:34, CDR2 comprises the sequence set forth in SEQ ID NO:35, and CDR3 comprises the sequence set forth in SEQ ID NO:36;

[0122] CDR1 comprises the sequence shown in SEQ ID NO: 38, CDR2 comprises the sequence shown in SEQ ID NO: 39, and CDR3 comprises the sequence shown in SEQ ID NO: 40; or

[0123] CDR1 comprises the sequence set forth in SEQ ID NO:46, CDR2 comprises the sequence set forth in SEQ ID NO:47, and CDR3 comprises the sequence set forth in SEQ ID NO:48;

[0124] Wherein, said CDR1, CDR2 and CDR3 respectively contain substitutions, insertions or deletions of up to 3 amino acids.

[0125] In certain embodiments, the CDR1, CDR2 and CDR3 each contain at most 1 or 2 amino acid substitutions, insertions or deletions.

[0126] In certain embodiments, the antibody or antigen-binding fragment thereof comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 13, 17, 41, and 49-51, or an amino acid sequence as set forth in any one of SEQ ID NOs: 13, 17, 41, and 49-51, or a mutant amino acid sequence thereof comprising up to 5 amino acid substitutions, insertions, or deletions in the framework region. In certain embodiments, the antibody or antigen-binding fragment thereof comprises a mutant amino acid sequence as set forth in any one of SEQ ID NOs: 17, 41, 1, 5, 9, 13, 21, 25, 29, 33, 37, 45, and 49-51, comprising up to 5, 4, 3, 2, or 1 amino acid substitutions, insertions, or deletions in the framework region. In certain embodiments, the antibody or antigen-binding fragment thereof comprises a substitution of up to 5, 4, 3, 2, or 1 conservative amino acids in the framework region compared to the amino acid sequence as set forth in any one of SEQ ID NOs: 13, 17, 41, and 49-51. In some embodiments, the antibody or antigen-binding fragment thereof comprises a mutant sequence having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to 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.

[0127] In this application, the terms "VHH" and "single-domain antibody (sdAb)" generally refer to antibodies whose target binding activity is located within a single domain, also known as nanobodies. In this application, the single-domain antibody can be a single-domain antibody of an alpaca. Unlike, for example, antibodies and single-chain antibodies (antibodies and single-chain antibodies generally contribute antigen binding activity to both the heavy chain variable domain and the light chain variable domain). In certain embodiments, the antibody or antigen-binding fragment thereof described in this application is an sdAb.

[0128] In the present application, the term "Fc region (fragment crystallizable region)", also known as the constant region, generally refers to the region of an antibody corresponding to the antigen binding region of an antibody. The Fc region can generally remain constant by itself and is not responsible for binding to the antigen, but can bind to Fc receptors and complement to exert the biological function of the antibody. The Fc region of the present application may be the Fc of a human antibody. In some instances, the Fc region may include a hinge, CH2, and CH3. When the Fc region includes a hinge, it can mediate dimerization between two Fc-containing polypeptides. The Fc fragment may be from IgG, IgM, IgD, IgE, or IgA. In some instances, the Fc region is from IgG1, IgG2, IgG3, or IgG4. "Fc fragment" also includes variant Fc fragments derived from natural Fc fragments that have been modified but still retain their effector functions. "Variant Fc fragment" comprises an amino acid sequence having at least one amino acid change in the amino acid sequence of a natural Fc fragment. In some instances, the variant Fc fragment has at least one amino acid substitution compared to the parent Fc fragment (native Fc fragment), for example, about 1 to about 10 amino acids are substituted in the parent Fc fragment, and preferably about 1 to about 5 amino acid substitutions. In some instances, the variant Fc fragment Fc region 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 functions of the "Fc fragment" may include binding to Fc receptors, Clq binding and complement dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), mediating phagocytosis, and the like. In certain embodiments, the antibodies or antigen-binding fragments thereof described herein include an Fc region, such as a fused scFv, HcAb, or a whole antibody.

[0129] For antibodies or antigen-binding fragments thereof, "binding," "directed against," or "specifically binds" means that one molecule (e.g., an antibody or antigen-binding fragment thereof) has a higher binding affinity for another molecule (e.g., an antigen) relative to other molecules present in the environment. A molecule can bind to, be directed against, or specifically bind to more than one molecule. For example, a bispecific antibody can have a higher binding affinity for two different antigens relative to other molecules. The binding affinity of an antibody for an antigen can be measured by a number of parameters, such as the EC50 value or KD value for antibody-antigen binding.

[0130] EC 50 Concentration for 50% of maximal effect refers to the concentration that elicits 50% of the maximum effect. In enzyme-linked immunosorbent assays (ELISAs), when used to indicate the binding ability of an antibody molecule to its corresponding antigen, it refers to the concentration of the antibody molecule that produces half the maximum detection signal (e.g., colorimetric or fluorescence intensity). The lower the EC50 value, the greater the binding affinity for the antigen. In certain embodiments, the EC50 value for binding of the antibody or antigen-binding fragment thereof to the human CD7 molecule is less than 1 nM.

[0131] KD value can also be used to measure the binding affinity between an antibody and its antigen. KD value is the equilibrium dissociation constant between an antibody and its antigen, i.e., the ratio of koff / kon. Therefore, the lower the KD value (the lower the concentration), the higher the affinity of the antibody. In some embodiments of the present application, after Biacore detection, the KD value of the binding of the antibody or its antigen-binding fragment to the CD7 molecule is less than 1nM. In some embodiments, after Biacore detection, the KD value of the binding of the antibody or its antigen-binding fragment to the CD7 molecule is less than 0.1nM. In some embodiments, after Biacore detection, the antibody or its antigen-binding fragment is a humanized VHH, and the KD value of the binding of the humanized VHH to the CD7 molecule is less than 1nM, more preferably, less than 0.1nM.

[0132] In this application, the term "blocking" generally refers to that the expression of CD7 molecules on the cell surface is affected, for example, the expression of CD7 molecules on the cell surface is downregulated. In some embodiments, the blocking molecules described in the present application have a blocking effect on the expression of CD7 molecules on the surface of T cells; in some embodiments, the efficiency of the blocking effect is 70%, 80%, 90% or 100%. In some embodiments, the blocking effect is achieved by introducing a nucleic acid encoding a blocking molecule into an immune cell. In some embodiments, the blocking effect is achieved by introducing a blocking molecule into an immune cell. In some embodiments, the blocking effect reaches 100% after the 5th, 6th, 7th, 8th or 9th day after the blocking molecule or the nucleic acid encoding the blocking molecule is introduced into the cell. In some embodiments, the blocking effect can last for 10 days, 15 days, 20 days and longer.

[0133] In certain embodiments, the amino acid sequence of the CD7 blocking molecule comprises a mutant sequence having 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to the sequence shown in SEQ ID NO: 52. In certain embodiments, the amino acid sequence of the CD7 blocking molecule comprises the sequence shown in SEQ ID NO: 52.

[0134] In this application, the term "transmembrane domain" generally refers to a sequence in a cell surface protein that spans the cell membrane and may include a hydrophobic alpha helix. The transmembrane domain may be connected to an intracellular signal transduction domain to play a role in signal transmission. In this application, the transmembrane domain can be derived from any type I, type II, or type III transmembrane protein.

[0135] In the present application, the term "chimeric antigen receptor (CAR)" generally refers to a fusion protein comprising an extracellular domain capable of binding to an antigen and at least one intracellular domain. CAR is a core component of a chimeric antigen receptor T cell (CAR-T), which may include an antigen (e.g., tumor-specific antigen and / or tumor-associated antigen) binding domain, a transmembrane domain, a co-stimulatory domain, and an intracellular signaling domain. CAR is an engineered receptor that can implant any specific receptor into immune effector cells, especially T cells. In CAR, scFv fragments or VHH fragments of monoclonal antibodies that specifically recognize tumor antigens can be implanted into T cells or NK cells. CAR encoding nucleic acids can be introduced into T cells, NK cells, or NK T cells using, for example, a retroviral vector. In this way, a large number of cancer-specific T cells, NK cells, or NKT cells can be generated for adoptive cell transfer. In the present application, the CAR can be combined with the T cell receptor activation intracellular domain based on the antigen (e.g., BCMA) specificity of the antibody. Genetically modified T cells expressing CAR can specifically recognize and eliminate malignant cells expressing target antigens. For descriptions of CAR and CAR-T cells, see, for example, Sadelain M, Brentjens R, Riviere 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 WO2013 154760, WO2016014789.

[0136] In this application, the term "costimulatory domain" generally refers to an intracellular domain that can provide immune co-stimulatory molecules, which are cell surface molecules required for lymphocytes to effectively respond to antigens.

[0137] In this application, the term "hinge region" generally refers to the connecting region between the extracellular domain (e.g., BCMA targeting moiety) and the transmembrane region. In this application, the CAR may comprise one or more hinge regions between the BCMA targeting moiety and the transmembrane domain.

[0138] In this application, the term "signaling domain" generally refers to a domain located inside a cell that can transduce signals. In this application, the intracellular signaling domain can transmit signals into the cell. Generally, a signaling domain is any continuous amino acid sequence used to guide a protein to find a target. In some cases, the signaling domain can be derived from CD3ζ. CD3ζ can form a T cell receptor-CD3 complex with T cell receptor subunits and CD3-gamma, -delta, and -epsilon. CD3ζ contains three ITAM motifs, and the ITAM sequence mediates intracellular signal activation of the TCR. The chain is a receptor-activated protein tyrosine kinase substrate. When the TCR receptor binds to the polypeptide MHC complex, the chain can quickly undergo tyrosine phosphorylation and participate in the transduction of lymphocyte activation signals. Therefore, CD3ζ plays a key role in antigen recognition and TCR signal transduction.

[0139] In this application, the "vector" generally refers to a nucleic acid molecule capable of self-replication in a suitable host, for transferring the inserted nucleic acid molecule into and / or between host cells. The vector may include a vector primarily used to insert DNA or RNA into a cell, a vector primarily used to replicate DNA or RNA, and a vector primarily used for expression of the transcription and / or translation of DNA or RNA. The vector also includes vectors with a variety of the above-mentioned functions. The vector may be a polynucleotide that can be transcribed and translated into a polypeptide when introduced into a suitable host cell. Typically, the vector can produce a desired expression product by culturing a suitable host cell containing the vector. The vector may encompass additional features other than the transgenic insertion sequence and the backbone: promoters, genetic markers, antibiotic resistance, reporter genes, targeting sequences, protein purification tags. A vector called an expression vector (expression construct) can be used to express a transgene in a target cell and typically has a control sequence. The vector described in the present application can be an expression vector, which can include a viral vector (lentiviral vector and / or retroviral vector), a phage vector, a phagemid, a cosmid, a cosmid, an artificial chromosome such as a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC) or a P1-derived artificial chromosome (PAC) and / or a plasmid.

[0140] In the present invention, "engineered immune cells" are immune cells that can perform immune effector functions after artificial modification. In some embodiments, the engineered immune cells express at least FcγRIII and perform ADCC effector functions. Examples of immune effector cells that mediate ADCC include peripheral blood mononuclear cells (PBMC), natural killer (NK) cells, monocytes, cytotoxic T cells, neutrophils and eosinophils. Preferably, the immune cells are selected from at least one of: immune cells differentiated by culture of pluripotent stem cells or embryonic stem cells, T lymphocytes, NK cells, peripheral blood mononuclear cells (PBMC) and hematopoietic stem cells.

[0141] More preferably, the immune cell is a T lymphocyte (same as T cell). In some embodiments, the T cell can be CD4+ / CD8-, CD4- / CD8+, CD4+ / CD8+, CD4- / CD8- or a combination thereof. In some embodiments, the T cell produces IL-2, IFN and / or TNF when expressing a chimeric antigen receptor and binding to a target cell. In some embodiments, the CD8+ T cell lyses antigen-specific target cells when expressing a chimeric antigen receptor and binding to a target cell.

[0142] The present invention provides a preparation method of the engineered immune cell, which includes using the isolated nucleic acid of the present invention or the vector infected immune cell. Preferably, the present invention prepares genetically modified immune effector cells by introducing chimeric antigen receptors into immune effector cells (such as T cells). In some embodiments, the preparation method first imports the nucleic acid encoding the blocking molecule into immune cells to be modified to block CD7 molecule expression, and on the 5th day, 6th day, 7th day, 8th day or 9th day after importing, the nucleic acid encoding the CD7-CAR molecules is transfected to express CD7-CAR molecules on the surface of immune cells.

[0143] It should be noted that the method for introducing nucleic acid or vector into mammalian cells is known in the art, and the vector can be transferred to immune effector cells by physical, chemical or biological methods. The physical method for introducing vector into immune effector cells includes calcium phosphate precipitation, liposome transfection, particle bombardment, microinjection, electroporation, etc. The chemical means for introducing nucleic acid or vector into immune effector cells include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads and lipid-based systems (including oil-in-water emulsions, micelles, mixed micelles and liposomes). The exemplary colloidal system used as an in vitro delivery vehicle is liposomes (such as artificial membrane vesicles). The biological method for introducing nucleic acid or vector into immune effector cells includes the use of DNA and RNA vectors. Viral vectors have become the most widely used method for inserting genes into mammals, such as human cells. In some embodiments, transduced or transfected immune effector cells are propagated in vitro after introducing nucleic acid or vector.

[0144] In some embodiments, the preparation further includes further evaluating or screening the transduced or transfected immune cells to select engineered immune cells. In some embodiments, the CAR molecule also includes a tag sequence for screening cells. In some embodiments, the tag sequence is a flag sequence, preferably, the tag sequence is a sequence shown in SEQ ID NO:54.

[0145] As used herein, the term "treat," ...

[0146] As used herein, "cancer associated with CD7 expression" refers to a disease directly or indirectly caused by abnormal CD7 expression, and typically refers to a disease caused by overexpression of CD7. Preferably, the cancer or tumor is a hematological malignancy. Further preferably, the hematological malignancy is a T-cell-related tumor, which includes leukemia, lymphoma, and myeloma.

[0147] In this application, the term "pharmaceutically acceptable carrier" generally refers to one or more non-toxic materials that do not interfere with the effectiveness of the biological activity of the active ingredient. Such preparations can conventionally contain salts, buffers, preservatives, compatible carriers and optionally other therapeutic agents. Such pharmaceutically acceptable preparations can also contain compatible solid or liquid fillers, diluents or encapsulating materials suitable for administration to people. Other envisioned carriers, excipients, and / or additives that can be used in the formulations described herein include, for example, flavorings, antimicrobials, sweeteners, antioxidants, antistatic agents, lipids, protein excipients (such as serum albumin, gelatin, casein), salt-forming counterions (such as sodium), etc.

[0148] In this application, the term "and / or" should be understood to mean either one of the alternatives or both of the alternatives.

[0149] In this application, the term "comprising" generally means including the features specifically stated, but not excluding other elements.

[0150] In this application, the term "about" generally refers to a variation within a range of 0.5%-10% above or below the 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 the specified value.

[0151] Unless defined otherwise or clearly indicated by the context, all technical and scientific terms in this disclosure have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0152] Example 1. Discovery of human CD7 single-domain antibody positive clone molecules

[0153] 1) Animal immunization and phage library construction

[0154] The antigen used was a recombinant protein CD7-His, representing the extracellular domain of human CD7 (sequence reference P09564-1, amino acid sequence 26-180, fused to a His protein tag, Biopsies, CD7-H52H7). JSR49 alpacas were subcutaneously immunized with a 1:1 emulsion of Freund's complete adjuvant containing 0.5 mg of CD7-His fusion protein. Subsequently, the alpacas were boosted with a 1:1 emulsion of Freund's incomplete adjuvant (Sigma-Aldrich) containing 0.25 mg of CD7-His subcutaneously up to five times every two weeks. Alpaca serum titers were tested for both full IgG antibodies and single-domain antibodies using Goat anti-Llama IgG (H+L) Secondary Antibody [HRP] (Anolon, NB7242) and MonoRab, respectively. TM Rabbit Anti-Camelid VHH Cocktail [HRP] (GenScript A02016) was used as the secondary antibody for detection. The serum titer against the single-domain antibody reached the library construction requirement after four immunizations (the library construction standard is: when the dilution factor is 1:8000, its OD450nm response value is approximately 1.0). To increase the diversity of the library, peripheral blood mononuclear cells (PBMCs) isolated from the third and fourth immunizations were selected and used in combination for subsequent phage library construction.

[0155] 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 protocol. The extracted RNA was qualitatively and quantitatively analyzed by gel electrophoresis and the OD260 / OD280 method. RNA was reverse transcribed into cDNA according to the instruction manual of the Primescript™ 1st Strand cDNA Synthesis Kit. The cDNA was used as a template for a single-round PCR to separate traditional IgG1 and single-domain antibody samples. The PCR product recovered from the first round of gel electrophoresis was used as a template for PCR to isolate the VHH fragment. The VHH fragment and the single-domain antibody phage display vector plasmid (Nanjing Pengbo) were digested with Sfi I endonuclease, and the VHH fragment was fused to the linearized vector using T4 ligase. The ligation product was electroporated and plated (ampicillin-containing agar plates) for overnight incubation. The plate was counted to determine the library capacity, and single clones were selected for sequencing to assess library quality. The library capacity and quality are shown in Table 1.

[0156] Table 1. Capacity and quality of alpaca immune library

[0157] 2) Using the alpaca immune library to screen lead antibody molecules specifically targeting human CD7

[0158] Two rounds of cell panning were performed using the alpaca immune library described above and the positive Jurkat cell line and the negative Jurkat / KO CD7 (gene knockout human CD7 display) cell line (Nanjing Pengbo) to obtain the phage library eluate. The neutralized phage panning eluate was added to the prepared TG1 bacterial solution, mixed well, and infected at 37°C for 45 minutes. After sufficient infection, the bacterial solution was gradiently diluted and spread on an agar plate with relevant resistance, inverted and cultured at 37°C overnight. Prepare a 96-well sterile deep-well plate filled with 0.5ml 2YT medium (with 0.2% w / v glucose and 0.1mg / ml ampicillin antibiotics), pick the monoclonal colonies cultured in the plate with a sterile pipette tip and culture them in the well plate, and shake at 37°C 220rpm overnight (16-18 hours). Take 0.05-0.1ml of overnight cultured monoclonal bacterial solution and transfer it to a freshly prepared sterile deep-well plate (containing 0.5ml of 2YT medium containing a final concentration of 0.1mg / ml ampicillin). Cultivate to an OD value of approximately 0.6-0.8 (under OD600 detection conditions). Add helper phage and shake to mix well. After infecting at 37°C for 45 minutes, add 0.5ml of 2YT medium (containing 0.1mg / ml ampicillin and kanamycin with a final concentration of 0.05mg / ml after addition) and culture overnight at 25°C with shaking at 220rpm (16-18 hours). Centrifuge the overnight expression bacterial solution at 4000rpm for 10 minutes to obtain the phage display expression supernatant for subsequent testing of the selected monoclonal antibody and the antigen protein human CD7-His protein ELISA binding test and FACS binding test for cellular antigens to obtain positive single-domain antibody clones.

[0159] ELISA Binding Assay: Indirect ELISA was used to assess the binding ability of phage-displayed antibodies in the supernatant to human CD7-related proteins. ELISA plates were coated with 100 μl / well of CBS containing 1 μg / ml of recombinant human CD7-His protein 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% PBST were added to each plate, followed by incubation at room temperature for 2 hours. The plates were washed three times with PBST and incubated with 100 μl / well of horseradish peroxidase-conjugated goat anti-M13 phage antibody (Sino-Bio) at room temperature for 45 minutes. The plates were washed six times with PBST, and TMB color development solution (GenScript) was added and incubated in the dark at room temperature 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.

[0160] FACS assay: FACS binding assay was used to evaluate the ability of antibodies in the supernatant to bind to the human CD7 antigen expressed on the surface of Jurkat cell membranes. Jurkat cells expressing human CD7 and negative cells Jurkat / KO CD7 (knockout human CD7 expression) cell lines were collected for testing and washed three times with PBS. 2.5X10 5 Each test cell was incubated with 100 μl of the supernatant to be tested and 3.5 μg / ml of biotinylated anti-phage antibody at 4°C for 1 hour. The cells were then washed three times with PBS and 100 μl of iFluor-labeled streptavidin (Jackson, 016-600-084) 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 Calibur (BD).

[0161] Example 2. Variable region sequencing of positive clones and recombinant production of single domain antibodies

[0162] Positive clones were selected based on the ELISA and FACS test results. The original colonies corresponding to the positive clones were cultured, and the expression plasmids were extracted, PCR was performed, and single-clone Sanger sequencing was performed. Finally, 12 positive clones were obtained. The corresponding relationship between the clones and their sequence numbers is shown in Table 2.

[0163] Table 2. Positive clones and their corresponding sequence numbers

[0164] A DNA fragment containing the codon-optimized heavy chain variable region was synthesized and inserted into the pcDNA3.4-Fc (HuIgG1) expression vector to form an expression plasmid.

[0165] HEK293-6E cells were transfected with the above plasmids and cultured in shake flasks at 37°C for 10 days. The supernatant was then collected for antibody purification. Prior to purification, the tubing and Protein A column were depyrogenated with 0.2 M NaOH. The column was re-equilibrated with a buffer containing 0.05 M Tris and 1.5 M NaCl (pH 8.0). The harvested cell culture supernatant was then diluted 1:1 with 2× the above buffer and sterilized by filtration. The filtered supernatant and Protein A column were incubated at room temperature for 2 hours. After washing the column with 1× the above buffer, the IgG was eluted with sterile 0.1 M sodium citrate (pH 3.5). The eluate was collected and neutralized with one-ninth volume of sterile 1 M Tris-HCl (pH 9.0). Under sterile conditions, the product buffer was exchanged into PBS (pH 7.4) to remove any elution buffer and the sample was concentrated. After concentration, the antibodies were quantified by OD280nm using an extinction coefficient Ec of 1.43 (0.1%).

[0166] The purified antibodies were analyzed by SDS-PAGE using a BioRad electrophoresis system with a 10% precast gel (GenScript). The gel was stained with Estain 2.0 (GenScript) and the molecular size and purity were estimated by comparing the stained bands with Protein Ladder (GenScript).

[0167] Example 3. ELISA binding detection of purified antibodies to human, monkey and mouse CD7 recombinant proteins

[0168] Indirect ELISA was used to evaluate the binding ability of the purified antibodies of Example 2 to recombinant proteins related to human CD7, monkey CD7 (Yi Qiao Shenzhou, 90993-H08H) and mouse CD7 (Bipusaisi, CD7-M52H3). The ELISA plates were coated overnight at 4 ° C with 1 μg / ml of human CD7 recombinant protein, monkey CD7 recombinant protein or mouse CD7 recombinant protein in 100 μl / well of CBS. The plate was 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 100 μl of purified antibody at 8 μg / ml (about 100nM) was added to the first well and diluted 3 times for a total of 11 test concentration gradients. It was then incubated at room temperature for 1 hour. The plate was washed three times with PBST and incubated with 100 μl / well of horseradish peroxidase-conjugated mouse anti-human IgG Fc fragment (GenScript) at 37°C for 0.5 h. The plate was washed five times with PBST, then TMB color development solution (GenScript) was added and incubated in the dark at room temperature for 15 min. The reaction was terminated by adding 50 μl of 1 M HCl stop solution (Sigma). The plate was read at 450 nm using a microplate reader. The results of the ELISA experiments of 12 positive recombinant antibody clones binding to CD7 antigen proteins related to different species are shown in Figures 1-3. The specific EC values ​​of each antibody are shown in Figures 1-3. 50 As shown in Table 3 below: Compared with the Yang Shen antibody PA3-17 VHH6-Fc (HuIgG1), the binding ability of these tested antibodies to human CD7 antigen protein is comparable to that of the Yang Shen antibody, and there is no species cross-reaction with monkey or mouse CD7.

[0169] Table 3. Purified antibodies and CD7 protein ELISA EC from different species 50 Detection

[0170] Note: NA means the purified antibody does not bind to the test antigen protein.

[0171] Example 4. Binding of monoclonal antibodies to Jurkat cell lines expressing human CD7

[0172] The Jurkat cells expressing human CD7 and Jurkat / KO CD7 cells expressing human CD7 were collected for detection and washed three times with PBS. 2.5X10 5 The cells were washed three times with PBS and 100 μl of 10 μg / ml purified antibody was added and incubated at 4°C for 1 hour. The cells were then washed three times with PBS, and 100 μl of iFluor-labeled goat anti-human IgG and 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 FACS BD Calibur. As shown in Figure 4, all antibodies bound to Jurkat cells expressing human CD7 but not to negative cells, indicating that all 12 purified antibodies (i.e., monoclonal antibodies) specifically bound to human CD7 cell antigens.

[0173] Example 5. Humanized design and recombinant production of human CD7-targeted single-domain antibody molecules

[0174] The parent antibody structure is modeled by computer-assisted homology modeling software (MOE). A human natural germline sequence with high homology to the parent sequence is selected. The CDRs of the positive monoclonal antibody are grafted into the human natural germline sequence using CDR grafting technology to obtain a humanized chimeric antibody of the parent antibody, i.e., a humanized VHH antibody. The different amino acid residues of the chimeric antibody and the parent antibody are compared, and the key points that will affect subsequent affinity are judged based on classic residues, interaction loop regions, core regions, mutation hotspots, etc., and appropriate sites are selected for combinatorial design to obtain the humanized antibody variable region amino acid sequence. The humanized antibody amino acid sequence is codon-optimized, and a DNA fragment containing the codon-optimized heavy chain variable region DNA fragment is synthesized and inserted into the pcDNA3.4-Fc (Human IgG1) expression vector to form an expression plasmid. The relevant expression plasmid is expressed according to the antibody recombinant method of Example 2, and the purification method is implemented to produce humanized antibodies. The humanized clones and their corresponding sequence numbers are shown in Table 4, and each CDR sequence is defined with the Kabat definition scheme.

[0175] Table 4. Humanized antibodies and their corresponding sequence numbers

[0176] Example 6. SPR Binding Detection of Humanized Antibodies to Human and Monkey CD7 Proteins

[0177] The affinity of the purified antibodies to human and monkey CD7 proteins was determined individually using the surface plasmon resonance (SPR) biosensor Biacore T200 (GE Healthcare). The antibodies were immobilized on a sensor chip using an Fc capture assay. Human or monkey CD7 proteins were used as analytes. Dissociation (kd) and association (ka) rate constant data were obtained using Biacore T200 evaluation software. The equilibrium dissociation constant (KD) was calculated from the ratio of kd to ka. Antibodies were ranked by their equilibrium dissociation constant, and humanized antibodies with no or no more than a 3-fold decrease in affinity were selected for subsequent testing. The results of SPR affinity determination are shown in Tables 5-6 below, and the relevant sensorgrams are shown in Figures 5-6. According to SPR affinity determination, the three groups of parents (AHP19484-VHH, AHP19485-VHH, AHP19509-VHH) and humanized antibodies (AHP19484-VHH3, AHP19485-VHH1, AHP19509-VHH1) all have comparable affinity levels with human CD7 antigen protein, and the equilibrium dissociation constants (KD) are all within 10 -10 range, and none of them bound to monkey CD7 protein.

[0178] Table 5. Affinity determination of humanized single domain antibodies and human CD7 protein

[0179] Table 6. Affinity determination of humanized single domain antibodies and monkey CD7 protein

[0180] Example 7. Search for the optimal antibody molecule for CD7 blocking effect

[0181] The structure of the CD7 blocking molecule is shown in Figure 7. Three CD7 blocking molecules with different VHH sequences were prepared, and the VHH amino acid sequences are shown in SEQ ID NOs: 49-51. The above blocking molecules were subcloned into lentiviral vectors. CD7 blocking molecule viruses were obtained by conventional lentiviral packaging technology for use, and the virus titer (TU / ml) was obtained by a virus functional titer detection method based on flow cytometry.

[0182] T cell activation: After thawing, frozen PBMCs (Sai ​​Li) were resuspended in AIM V complete medium (Thermo Fisher, Cat#31035025) containing 300 IU / ml rhIL-2 and counted. T cells were then activated at a 1:1 ratio of cells to CD3 / CD28 antibody magnetic beads (GenScript, Cat#L00899-1) (the activation time point was designated as D0, and the time points 1 day and 2 days after activation were designated as D1 and D2, respectively). The cells were incubated with the magnetic beads for 24 hours to obtain the activated T cells.

[0183] The activated T cells were plated into 24-well plates and the cell density per well was adjusted to 1×10 6 Three CD7-blocking lentiviruses expressing different VHH sequences were added to each well (MOI = 5, MOI = 10) in a total volume of 400 μl. 16 μl of the viral infection-promoting reagent HiTransGP (Gene Gene, Cat#REVG005) was also added. 16 hours after transfection, 1 ml of culture medium was added to each well for incubation. After 6 days of expansion, flow cytometry was used to detect CD7 expression on the T cell surface. The detection antibody was PE anti-human CD7 Antibody (Biolegend, Cat#343106).

[0184] 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 the experimental conditions of MOI = 5 and MOI = 10, the CD7 blocking effect of the AHP19485-VHH1 molecule was significantly better than that of AHP19484-VHH3 and AHP19509-VHH1. Six days after transfection, the blocking effect of the AHP19485-VHH1 molecule on CD7 on the surface of T cells reached 70%. Subsequently, the AHP19485-VHH1 molecule was selected for the construction of CD7 blocking T cells.

[0185] Example 8. Exploring the blocking time of CD7 molecules on T cell surface by CD7 blocking molecules

[0186] The structure of the CD7 blocking molecule is shown in Figure 7. AHP19485-VHH1 was used as the blocking antibody sequence, with an amino acid sequence (SEQ ID NO: 52). Flow cytometry (detection antibody: PE anti-human CD7 Antibody, Biolegend, Cat# 343106) was used to continuously monitor CD7 expression on the surface of T cells for 22 days. As shown in Figure 9, the results show that on the eighth day after transduction with the CD7 blocking molecule, the level of CD7 expression on T cells was significantly reduced. On the 15th day after transduction, 90% of CD7 molecules were blocked. CD7 expression continued to decrease until the 22nd day, approaching 100%.

[0187] According to the above results, when preparing CD7 blocking T cells, the eighth day after transduction was selected as the time point for CD7 expression detection.

[0188] Example 9. Construction and preparation of CD7 CAR-T cells

[0189] The present invention constructs a second-generation human CD7-targeting CAR vector, which sequentially includes two repeats of the CD7 VHH antibody sequence, a Flag tag serving as a transduction indicator protein, a CD28 hinge region, a CD28 transmembrane region, a CD28 intracellular region sequence, and a CD3ζ sequence. The CAR structure is shown in Figure 11, and the amino acid sequences of each component are shown in SEQ ID NOs: 53-58.

[0190] To compare the differences in the functional activity of the three VHH molecules, CD7-CAR-T cells with three different CD7 VHH sequences (SEQ ID NOs: 59-61) were constructed by lentiviral transfection. VHH6 CAR-T cells constructed with VHH6 (patent number: CN 110760007A) served as a positive control for the experiment (SEQ ID NO: 62). The specific steps for construction are as follows:

[0191] T cell activation: After thawing the frozen PBMCs (Sai ​​Li), the cells were resuspended in AIM V complete medium (Thermo Fisher Scientific Cat#31035025) containing 300 IU / ml rhIL-2 and counted. Then, T cells were activated at a 1:1 ratio of cells to CD3 / CD28 antibody magnetic beads (GenScript Cat#31035025) (the activation time point was marked as D0, and the time points 1 day and 2 days after activation were marked as D1 and D2, respectively). The cells were incubated with the magnetic beads for 24 hours to obtain the activated T cells.

[0192] Preparation of CD7 blocking T cells: Using the CD7 blocking molecule constructed by AHP19485-VHH1 molecule, the amino acid sequence (SEQ ID NO: 52) was subcloned into a lentiviral vector. The CD7 blocking molecule virus was obtained by conventional lentiviral packaging technology and the virus titer (TU / ml) was obtained by flow cytometry-based virus functional titer detection method. The activated T cells were plated into 24-well plates and the cell density per well was adjusted to 1×10 6 16 cells were transfected with CD7-blocking lentivirus (MOI = 10) in a total volume of 400 μl. 16 μl of the viral infection-promoting reagent HiTransGP (Gene Gene, Cat#REVG005) was also added. 16 hours after transfection, 1 ml of culture medium was added to each well for expansion. On day 8, CD7 expression on the T cell surface was assessed by flow cytometry.

[0193] The results are shown in Figure 12. The surface CD7 positivity rate of T cells not transduced with the CD7 blocking virus (corresponding to the pan-T group in the figure) was 93.1%, while the CD7 positivity rate of the transduced group (corresponding to the AHP19485 CD7 Blocking group in the figure) was only 4.22%, indicating that CD7 expression was almost undetectable on the T cell surface. Eight days after CD7 blocking molecule viral transduction, the blocking effect on T cell CD7 expression reached over 90%. These results demonstrate the successful construction of CD7-blocked T cells, which can be used for subsequent CD7 CAR-T cell preparation.

[0194] Lentiviral infection of CD7 CAR: On day 9, the prepared CD7 blocking T cells were plated into 24-well plates, and the cell density per well was adjusted to 1×10 6 16 cells were transfected with CD7 CAR lentivirus (MOI = 10) for a total volume of 400 μl. 16 μl of the viral infection-promoting reagent HiTransG P (Gene Gene, Cat#REVG005) was also added. 16 hours after transfection, 1 ml of culture medium was added to each well for expansion to obtain CD7-targeted CAR-T cells.

[0195] On day 13, the proportion of CD7 CAR cells was detected by flow cytometry. The detection antibody was PE-Labeled Human CD7 Protein, His Tag (Biopsy, Cat#CD7-HP2E3). CD7 Blocking T cells represented T cells that blocked CD7 expression but were not transduced with CAR virus and served as a negative control. VHH6 CAR served as a positive control. The results of the CAR positivity rate are shown in Table 7 and Figure 13. The CD7 CAR positivity rate was greater than 90%.

[0196] Table 7. CD7-CAR-T positive rate test results

[0197] Example 10. In vitro killing experiment of CD7-CAR-T cells on CD7-positive target cells

[0198] Target cells Jurkat / Luc (Nanjing Pengbo) and CCRF-CEM / Luc (Nanjing Pengbo) were collected and resuspended in complete medium RPMI 1640 (Gibco, Cat#22400-089) + 10% FBS (Gibco, Cat#10091-148) and adjusted to a density of 4×10 4cells / mL, and then transfer 50 μL of the target cell suspension to a 96-well plate. CD7-CAR-T cells were collected and resuspended in complete medium RPMI 1640 (Gibco, Cat#22400-089) + 10% FBS (Gibco, Cat#10091-148). The theoretical density was adjusted to 4×10 based on the CAR positive rate of different transduction molecules (see Table 7) and different effector-target ratios (E / T). 4 cells / mL (E / T = 1:1), 2 x 10 5 cells / mL (E / T = 5:1) and 8 x 10 5 cells / mL (E / T=20:1), then transfer 50 μL of CD7-CAR-T cell suspension and 50 μL of 2% Triton X-100 (Beijing Bailingwei Technology Co., Ltd., Cat#993361) (as the maximum killing signal value) to a 96-well plate with target cells, and then transfer the culture plate to an incubator at 37 ° C, 5% CO2 and incubate for 24 hours. After incubation for 24 hours, the culture plate was transferred out and the detection reagent Bio-Lite Luciferase Assay System luciferase detection reagent (Novozyme, Cat#DD1201-02) was added and incubated at room temperature for 5 minutes, and then the luminescence signal value was detected by a microplate reader (PHERAstar FSX, BMG).

[0199] Killing calculation formula % Cytotoxicity = 100*(1-(RLU Experimental–RLU Min ) / (RLU UnT –RLU Min )) The CD7-CAR-T killing value of different transduction molecules was calculated. The results are shown in Figure 14, Tables 8 and 9 (AHP19484 in the figure and table corresponds to the AHP19484-VHH3 molecule, AHP19485 corresponds to the AHP19 485-VHH1 molecule, and AHP19509 corresponds to AHP19509-VHH1). When the target cells are Jurkat / Luc, the molecular sequences AHP19484-VHH3, AHP19485-VHH1, and AHP19509-VHH1 show a stronger killing effect on CAR-T cells than the positive reference VHH6 CAR-T at three E / T ratios. The target cells were CCRF-CEM / Luc, E / T=5:1 and 20:1, and the molecular sequences AHP19484-VHH3, AHP19485-VHH1, and AHP10509-VHH1 showed stronger killing effects than the positive reference VHH6 CAR-T.

[0200] RLU Experimental: signal value of CD7-CAR T cells + target cells (Jurkat / Luc or CCRF-CEM / Luc);

[0201] RLU UnT : Signal value of non-transduced T cells + target cells (Jurkat / Luc or CCRF-CEM / Luc);

[0202] RLU Min : Signal value of 1% TritonX-100+target cells (Jurkat / Luc or CCRF-CEM / Luc).

[0203] Table 8. Killing value of CD7 CAR-T cells against target cells Jurkat / Luc

[0204] Table 9. Killing value of CD7 CAR-T cells against target cells CCRF-CEM / Luc

[0205] The above describes the embodiments of the present invention. However, the present invention is not limited to the above embodiments. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention shall be included in the scope of protection of the present invention.

[0206] The amino acid sequence information mentioned in this article is as follows.

[0207] AHP19472 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 1

[0208] AHP19472 CDR1 region amino acid sequence: SEQ ID NO: 2

[0209] AHP19472 CDR2 region amino acid sequence: SEQ ID NO: 3

[0210] AHP19472 CDR3 region amino acid sequence: SEQ ID NO: 4

[0211] AHP19481 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 5

[0212] AHP19481 CDR1 region amino acid sequence: SEQ ID NO: 6

[0213] AHP19481 CDR2 region amino acid sequence: SEQ ID NO: 7

[0214] AHP19481 CDR3 region amino acid sequence: SEQ ID NO: 8

[0215] AHP19483 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 9

[0216] AHP19483 CDR1 region amino acid sequence: SEQ ID NO: 10

[0217] AHP19483 CDR2 region amino acid sequence: SEQ ID NO: 11

[0218] AHP19483 CDR3 region amino acid sequence: SEQ ID NO: 12

[0219] AHP19484 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 13

[0220] AHP19484 CDR1 region amino acid sequence: SEQ ID NO: 14

[0221] AHP19484 CDR2 region amino acid sequence: SEQ ID NO: 15

[0222] AHP19484 CDR3 region amino acid sequence: SEQ ID NO: 16

[0223] AHP19485 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 17

[0224] AHP19485 CDR1 region amino acid sequence: SEQ ID NO: 18

[0225] AHP19485 CDR2 region amino acid sequence: SEQ ID NO: 19

[0226] AHP19485 CDR3 region amino acid sequence: SEQ ID NO: 20

[0227] AHP19487 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 21

[0228] AHP19487 CDR1 region amino acid sequence: SEQ ID NO: 22

[0229] AHP19487 CDR2 region amino acid sequence: SEQ ID NO: 23

[0230] AHP19487 CDR3 region amino acid sequence: SEQ ID NO: 24

[0231] AHP19490 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 25

[0232] AHP19490 CDR1 region amino acid sequence: SEQ ID NO: 26

[0233] AHP19490 CDR2 region amino acid sequence: SEQ ID NO: 27

[0234] AHP19490 CDR3 region amino acid sequence: SEQ ID NO: 28

[0235] AHP19494 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 29

[0236] AHP19494 CDR1 region amino acid sequence: SEQ ID NO: 30

[0237] AHP19494 CDR2 region amino acid sequence: SEQ ID NO: 31

[0238] AHP19494 CDR3 region amino acid sequence: SEQ ID NO: 32

[0239] AHP19495 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 33

[0240] AHP19495 CDR1 region amino acid sequence: SEQ ID NO: 34

[0241] AHP19495 CDR2 region amino acid sequence: SEQ ID NO: 35

[0242] AHP19495 CDR3 region amino acid sequence: SEQ ID NO: 36

[0243] AHP19508 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 37

[0244] AHP19508 CDR1 region amino acid sequence: SEQ ID NO: 38

[0245] AHP19508 CDR2 region amino acid sequence: SEQ ID NO: 39

[0246] AHP19508 CDR3 region amino acid sequence: SEQ ID NO: 40

[0247] AHP19509 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 41

[0248] AHP19509 CDR1 region amino acid sequence: SEQ ID NO: 42

[0249] AHP19509 CDR2 region amino acid sequence: SEQ ID NO: 43

[0250] AHP19509 CDR3 region amino acid sequence: SEQ ID NO: 44

[0251] AHP19511 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 45

[0252] AHP19511 CDR1 region amino acid sequence: SEQ ID NO: 46

[0253] AHP19511 CDR2 region amino acid sequence: SEQ ID NO: 47

[0254] AHP19511 CDR3 region amino acid sequence: SEQ ID NO: 48

[0255] AHP19484-VHH3 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 49

[0256] AHP19485-VHH1 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 50

[0257] AHP19509-VHH1 single domain antibody heavy chain variable region amino acid sequence: SEQ ID NO: 51

[0258] CD7 blocking amino acid sequence: SEQ ID NO: 52

[0259] Signal peptide sequence: SEQ ID NO: 53

[0260] Flag tag sequence: SEQ ID NO: 54

[0261] Hinge region sequence: SEQ ID NO: 55

[0262] Transmembrane region sequence: SEQ ID NO: 56

[0263] CD28 sequence: SEQ ID NO: 57

[0264] CD3ζ sequence: SEQ ID NO: 58

[0265] AHP19484-VHH3 CAR sequence: SEQ ID NO: 59

[0266] AHP19485-VHH1 CAR sequence: SEQ ID NO: 60

[0267] AHP19509-VHH1 CAR sequence: SEQ ID NO: 61

[0268] VHH6 CAR sequence: SEQ ID NO: 62

[0269] Endoplasmic reticulum localization sequence: SEQ ID NO: 63

[0270] Linker sequence: SEQ ID NO: 64

Claims

1. An antibody or antigen-binding fragment thereof that specifically binds to human CD7, characterized in that: comprising a heavy chain variable region, the heavy chain variable region comprising CDR1, CDR2 and CDR3, wherein, 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 comprises the sequence shown in SEQ ID NO:42, CDR2 comprises the sequence shown in SEQ ID NO:43, and CDR3 comprises the sequence shown in SEQ ID NO:44; CDR1 comprises the sequence shown in SEQ ID NO:2, CDR2 comprises the sequence shown in SEQ ID NO:3, and CDR3 comprises the sequence shown in SEQ ID NO:4; CDR1 comprises the sequence shown in SEQ ID NO:6, CDR2 comprises the sequence shown in SEQ ID NO:7, and CDR3 comprises the sequence shown in SEQ ID NO:8; CDR1 comprises the sequence shown in SEQ ID NO: 10, CDR2 comprises the sequence shown in SEQ ID NO: 11, and CDR3 comprises the sequence shown in SEQ ID NO: 12; CDR1 comprises the sequence shown in SEQ ID NO: 14, CDR2 comprises the sequence shown in SEQ ID NO: 15, and CDR3 comprises the sequence shown in SEQ ID NO: 16; CDR1 comprises the sequence shown in SEQ ID NO:22, CDR2 comprises the sequence shown in SEQ ID NO:23, and CDR3 comprises the sequence shown in SEQ ID NO:24; CDR1 comprises the sequence shown in SEQ ID NO:26, CDR2 comprises the sequence shown in SEQ ID NO:27, and CDR3 comprises the sequence shown in SEQ ID NO:28; CDR1 comprises the sequence shown in SEQ ID NO:30, CDR2 comprises the sequence shown in SEQ ID NO:31, and CDR3 comprises the sequence shown in SEQ ID NO:32; CDR1 comprises the sequence shown in SEQ ID NO:34, CDR2 comprises the sequence shown in SEQ ID NO:35, and CDR3 comprises the sequence shown in SEQ ID NO:36; CDR1 comprises the sequence shown in SEQ ID NO:38, CDR2 comprises the sequence shown in SEQ ID NO:39, and CDR3 comprises the sequence shown in SEQ ID NO:40; or CDR1 comprises the sequence shown in SEQ ID NO:46, CDR2 comprises the sequence shown in SEQ ID NO:47, and CDR3 comprises the sequence shown in SEQ ID NO:48; The CDR1, CDR2 and CDR3 each contain mutations, insertions or deletions of up to 3 amino acids.

2. The antibody or antigen-binding fragment thereof according to claim 1, characterized in that: The CDR region sequence is selected from any of the following groups: 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 comprises the sequence shown in SEQ ID NO:42, CDR2 comprises the sequence shown in SEQ ID NO:43, and CDR3 comprises the sequence shown in SEQ ID NO:44; CDR1 comprises the sequence shown in SEQ ID NO:14, CDR2 comprises the sequence shown in SEQ ID NO:15, and CDR3 comprises 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 The antibody or antigen-binding fragment thereof is selected from the group consisting of whole antibody, Fab, Fab', Fv fragment, scFv, di-scFv and single-domain antibody; preferably, the antibody or antigen-binding fragment thereof is a single-domain antibody.

5. The antibody or antigen-binding fragment according to claim 1, characterized in that The antibody or antigen-binding fragment thereof 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 The antibody or antigen-binding fragment thereof comprises: 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 blocking molecule, characterized in that The invention comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6 and an endoplasmic reticulum localization domain, wherein the blocking molecule can reduce the expression of CD7 molecules on the cell surface.

8. The CD7 blocking molecule according to claim 7, characterized in that The CD7 blocking molecule includes two identical single-domain antibodies, and the single-domain antibodies are connected by a linker.

9. The blocking molecule according to claim 7, characterized in that The sequence of the endoplasmic reticulum localization domain includes the sequence shown in SEQ ID NO:

63.

10. The CD7 blocking molecule according to claim 7, characterized in that It also includes a signal peptide sequence; preferably, the signal peptide sequence includes the sequence shown in SEQ ID NO:

53.

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

12. A chimeric antigen receptor, characterized in that The chimeric antigen receptor comprises: 1) an antigen-binding domain that recognizes the CD7 antigen, wherein the antigen-binding domain comprises the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6; 2) a transmembrane domain; and 3) Intracellular signaling domain.

13. The chimeric antigen receptor according to claim 12, characterized in that It further includes an intracellular co-stimulatory signaling domain.

14. The chimeric antigen receptor according to claim 12, characterized in that The chimeric antigen receptor sequence comprises the amino acid sequence shown in any one of SEQ ID NOs: 59-61.

15. An isolated nucleic acid molecule encoding the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, or the blocking molecule according to any one of claims 7 to 11, or the chimeric antigen receptor according to any one of claims 12 to 14. A vector comprising the nucleic acid molecule according to claim 15 .

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

18. An engineered immune cell, characterized in that: The immune cell contains the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, or the blocking molecule according to any one of claims 7 to 11, or the chimeric antigen receptor according to any one of claims 12 to 14.

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

20. A pharmaceutical composition, characterized in that The composition comprises the antibody or antigen-binding fragment thereof according to any one of claims 1-6, or the blocking molecule according to any one of claims 7-11, or the chimeric antigen receptor according to any one of claims 12-14, or the engineered immune cell according to claim 18 or 19, and / or a pharmaceutically acceptable carrier.

21. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, or the blocking molecule according to any one of claims 7 to 11, or the engineered immune cell according to claim 18 or 19, or the composition according to claim 20 in the treatment of tumors or cancer.

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

23. A method for preparing engineered immune cells, characterized in that: The following steps are involved: (1) providing an immune cell to be expressed with a chimeric antigen receptor, wherein the immune cell contains the blocking molecule according to any one of claims 7 to 11; and (2) Adding a nucleic acid molecule encoding the chimeric antigen receptor according to any one of claims 12 to 14 to the cells obtained in step 1.

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