Anti-CD70 nanobody and its applications

Abstract

The present invention relates to an anti-CD70 nanobody and its application. The present invention provides a CD70 binding molecule comprising an anti-CD70 nanobody or an antigen-binding fragment thereof, wherein the complementarity determining region (CDR) of the anti-CD70 nanobody comprises CDR1, CDR2 and CDR3. The present invention further provides a chimeric antigen receptor comprising the CD70 binding molecule and a cell expressing the chimeric antigen receptor. The antibody and cell described herein have excellent safety and therapeutic effect of targeting CD70.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This disclosure claims priority to a Chinese patent application bearing application number "CN202210059899.9" and entitled "Anti-CD70 Nanobody and Its Application," filed with the China National Intellectual Property Office on January 19, 2022, the entire contents of which are incorporated herein by reference.

[0002] (Technical field) The present invention relates to the technical field of biological immunotherapy, in particular to anti-CD70 nanobodies and their applications. [Background technology]

[0003] CD70 is a member of the tumor necrosis factor (TNF) superfamily. It is a type II transmembrane glycoprotein in normal tissues, and is only briefly expressed on activated T cells, B cells, and mature dendritic cells, and its receptor is CD27. Studies have shown that CD70 is highly expressed on multiple types of tumor cells under pathological conditions. Currently, immunotherapeutic agents targeting CD70 have been applied in preclinical research.

[0004] Studies have shown that CD70 is abnormally upregulated in several types of malignant tumors, including renal cell carcinoma, acute myeloid leukemia, non-Hodgkin's lymphoma, multiple myeloma, mantle cell lymphoma, diffuse large cell lymphoma, follicular lymphoma, pancreatic cancer, breast cancer, glioblastoma, and other tumors, and is a promising target for tumor immunotherapy.

[0005] Chimeric antigen receptor-T cells (CAR-T) are a new type of immunotherapy targeting specific antibodies on the surface of tumor cells. Currently, many researchers are developing CAR-T cells to treat solid tumors.

[0006] Antibodies, as an important component of CAR, play a crucial role in the specific and highly efficient killing effect of CAR-T and the reduction of toxicity. Therefore, antibodies that can bind to target antigens with high specificity and low immunogenicity are key to developing CAR-T products. Highly specific and high affinity VHH antibodies that can be generated in alpacas can recognize antigen epitopes that cannot be recognized by conventional scFv antibodies due to their own characteristics, and since the alpaca VHH genes have relatively high homology with human VH genes, they have relatively low immunogenicity and can be used for humanization. There have been no reports of CAR-T cells containing anti-CD70 nanobodies in this field. Summary of the Invention

[0007] The present invention provides a CD70 binding molecule comprising an anti-CD70 Nanobody or an antigen-binding fragment thereof, wherein the complementarity determining regions (CDRs) of said anti-CD70 Nanobody comprise CDR1, CDR2 and CDR3, in which CDR1 comprises a sequence as set forth in any one of SEQ ID NOs: 1-7, CDR2 comprises a sequence as set forth in any one of SEQ ID NOs: 8-13, and CDR3 comprises a sequence as set forth in any one of SEQ ID NOs: 14-20.

[0008] In one or more embodiments, the heavy chain variable region sequence of the anti-CD70 Nanobody is depicted in any one of SEQ ID NOs:21-28.

[0009] In one or more embodiments, FR1 of the anti-CD70 Nanobody may be selected from FR1 of the VHH depicted in any one of SEQ ID NOs:21-28, FR2 may be selected from FR2 of the VHH depicted in any one of SEQ ID NOs:21-28, FR3 may be selected from FR3 of the VHH depicted in any one of SEQ ID NOs:21-28, and FR4 may be selected from FR4 of the VHH depicted in any one of SEQ ID NOs:21-28.

[0010] In one or more embodiments, the CD70 binding molecule is a monovalent or multivalent nanobody or single domain antibody comprising one, two or more anti-CD70 nanobodies or antigen-binding fragments thereof, or a multispecific nanobody or single domain antibody.

[0011] In one or more embodiments, the multivalent or multispecific binding molecule is linked to multiple anti-CD70 Nanobodies or antigen-binding fragments thereof by a connexon, the connexon consisting of 1-15 amino acids selected from G and S.

[0012] In one or more embodiments, the Nanobody is a camelid heavy chain antibody or a cartilaginous fish heavy chain antibody.

[0013] In one or more embodiments, the Nanobody further comprises a heavy chain constant region.

[0014] In one or more embodiments, the heavy chain constant region is a camelid heavy chain antibody constant region and comprises a CH2 and a CH3. In one or more embodiments, the CH2 and CH3 are the CH2 and CH3 of human IgG Fc, such as the CH2 and CH3 of IgG1. Preferably, the heavy chain constant region is set forth in SEQ ID NO:37.

[0015] In one or more embodiments, the heavy chain constant region is a chondrichthyan heavy chain antibody constant region and includes CH1, CH2, CH3, CH4 and CH5.

[0016] In one or more embodiments, the CD70 binding molecule according to any one of the embodiments of the invention is a chimeric antibody or a fully human antibody, preferably a fully human antibody.

[0017] Another aspect of the present invention provides a chimeric antigen receptor comprising any signal peptide sequence, a CD70 binding molecule according to any one of the embodiments herein, a hinge region, a transmembrane region, and an intracellular region.

[0018] In one or more embodiments, the intracellular region comprises an intracellular costimulatory domain and / or an intracellular signaling domain.

[0019] In one or more embodiments, the chimeric antigen receptor comprises, from N-terminus to C-terminus, a signal peptide, a CD70 binding molecule according to any one of the embodiments herein, a hinge region, a transmembrane region, an intracellular costimulatory domain, and an intracellular signaling domain, in that order.

[0020] The present invention relates to a method for the preparation of a medicament comprising the following sequence: (1) a coding sequence for a CD70 binding molecule or a chimeric antigen receptor according to any one of the embodiments herein; (2) The complementary sequence of (1), (3) a 5-50 bp fragment of any one of the sequences of (1) or (2) is further provided.

[0021] In one or more embodiments, the fragment is a primer.

[0022] The present invention further provides a nucleic acid construct comprising a nucleic acid molecule according to the present invention.

[0023] In one or more embodiments, the nucleic acid construct is a cloning vector, an expression vector, or an integrating vector.

[0024] The present invention relates to (1) expressing and / or secreting a CD70 binding molecule or a chimeric antigen receptor according to any one of the embodiments herein; (2) comprising a nucleic acid molecule according to the present specification; and / or (3) comprising a nucleic acid construct according to the present specification.

[0025] In one or more embodiments, the host cell is an immune effector cell, preferably a T cell.

[0026] The invention further provides a method for generating a CD70 binding molecule according to any one of the embodiments herein, comprising culturing a host cell according to the present specification under conditions suitable for the generation of a CD70 binding molecule (e.g. a Nanobody or antigen-binding fragment thereof, a monovalent or multivalent Nanobody or single domain antibody, or a multispecific Nanobody or single domain antibody), and optionally purifying said CD70 binding molecule from the culture.

[0027] The present invention further provides a pharmaceutical composition comprising a CD70 binding molecule, a nucleic acid molecule, a nucleic acid construct or a host cell according to any one of the embodiments herein, and a pharma- ceutically acceptable adjuvant.

[0028] In one or more embodiments, the pharmaceutical composition is used to treat a disease or condition associated with CD70 expression.

[0029] The present invention further provides the application of the CD70 binding molecule, chimeric antigen receptor, nucleic acid molecule, nucleic acid construct or host cell according to any one of the embodiments herein in the production of activated immune cells (e.g., T cells).

[0030] The present invention further provides an application of the CD70 binding molecule, chimeric antigen receptor, nucleic acid molecule, nucleic acid construct or host cell according to any one of the embodiments herein in the manufacture of a medicament for the prevention or treatment of a disease or condition related to CD70 expression.

[0031] In one or more embodiments, the disease or condition is one or more selected from renal cell carcinoma, acute myeloid leukemia, non-Hodgkin's lymphoma, multiple myeloma, mantle cell lymphoma, diffuse large cell lymphoma, follicular lymphoma, pancreatic cancer, breast cancer, and glioblastoma.

[0032] The invention further provides a method for treating or preventing a disease or condition related to CD70 expression, said method comprising administering to a patient in need thereof a therapeutically effective amount of a CD70 binding molecule or a host cell according to any one of the embodiments of the invention, or a pharmaceutical composition according to any one of the embodiments of the invention.

[0033] The present invention further provides a reagent kit for detecting CD70, for example, for use in evaluating the efficacy of drug therapy or in diagnosing cancer, the reagent kit comprising a CD70-binding molecule, a nucleic acid molecule, a nucleic acid construct, or a host cell according to any one of the embodiments herein.

[0034] In one or more embodiments, the reagent kit further comprises a reagent for detecting binding between CD70 and the CD70 binding molecule, for example, by enzyme-linked immunosorbent assay.

[0035] In one or more embodiments, the reagent for detecting binding is a detectable label, such as biotin, that can be attached to the CD70 binding molecule, either attached to the CD70 binding molecule or separately present in the reagent kit.

[0036] The present invention further provides a non-diagnostic method for detecting the presence of CD70 in a sample, comprising incubating a CD70 binding molecule according to any one of the embodiments herein with the sample and detecting binding of CD70 to said CD70 binding molecule to confirm the presence of CD70 in the sample, said detection being by enzyme-linked immunosorbent assay.

[0037] The present invention further provides an application of the CD70 binding molecule according to any one of the embodiments herein in the manufacture of a reagent kit for use in detecting CD70 in a sample, evaluating the efficacy of drug therapy, or diagnosing cancer.

[0038] The present invention has the following advantages:

[0039] The present invention provides a novel mini-antibody that specifically recognizes CD70 and CAR-modified cells containing said antibody, which have excellent safety and therapeutic effects targeting CD70, and therefore offer a new treatment or improvement route for diseases related to CD70 expression.

[0040] In order to further describe the technical solutions of the embodiments of the present invention in detail, the following briefly introduces the drawings used in the embodiments. It should be understood that the following drawings are only for presenting some embodiments of the present invention, and are not intended to limit the scope, and those skilled in the art can obtain other related drawings based on these drawings without making innovative efforts. [Brief description of the drawings]

[0041] [Figure 1] FIG. 1 shows the results of SDS-PAGE electrophoresis of the recombinant human CD70-huFc protein in Example 1. [Diagram 2] FIG. 2 shows the experimental results of measuring the binding curve between recombinant human CD70-huFc protein and recombinant human CD27 protein in Example 1. [Diagram 3] FIG. 3 shows the results of DNA electrophoresis after PCR amplification of the alpaca VH-CH2 gene in Example 2. [Figure 4] FIG. 4 shows the results of DNA electrophoresis after PCR amplification of the alpaca VHH gene in Example 2. [Diagram 5] FIG. 5 shows the results of DNA electrophoresis in the experiment for detecting VHH / pcomb3X binding efficiency in Example 2. [Figure 6] FIG. 6 shows the results of SDS-PAGE electrophoresis of the recombinant VHH-huFc antibody protein in Example 3. [Figure 7] FIG. 7 shows the results of measuring the binding curve of recombinant VHH-huFc antibody / CD70-huFc recombinant protein in Example 4. [Figure 8] FIG. 8 shows the experimental results of measuring the affinity of the recombinant VHH-huFc antibody in Example 5. [Figure 9] FIG. 9 shows the experimental results of blocking CD27 / CD70 binding by recombinant VHH-huFc in Example 6. [Figure 10] FIG. 10 is a schematic diagram of the CAR structure in Example 7. [Figure 11] Figure 11 shows the experimental results of infection efficiency of each clone CD70 CAR-T cell in Example 8. [Figure 12] Figure 12 shows the experimental results of CD107a activation of different clone CD70 CAR-T cells in Example 9. [Figure 13] Figure 13 shows the experimental results of IFN-γ release when different clone CD70 CAR-T cells were co-incubated with target cells in Example 9. [Figure 14] Figure 14 shows the experimental results of IL-2 release when different clone CD70 CAR-T cells and target cells were co-incubated in Example 9. REST is the NT cell control group. [Figure 15] Figure 15 shows the results of a target cell killing experiment using different clone CD70 CAR-T cells in Example 9. [Figure 16] Figure 16 shows the experimental results of infection efficiency of different clone CD70 CAR-T cells in Example 10. [Figure 17] Figure 17 shows the experimental results of CD107a activation of different clone CD70 CAR-T cells in Example 11. [Figure 18] Figure 18 shows the results of a target cell killing experiment using different clone CD70 CAR-T cells in Example 9. [Figure 19] Figure 19 shows the tumor volume monitoring results of the in vivo efficacy test of different clone CD70 CAR-T cells in Example 11. [Figure 20] Figure 20 shows the T cell survival results of the in vivo efficacy test of different clone CD70 CAR-T cells in Example 11. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0042] As a result of intensive research and repeated selection, the inventors have discovered an anti-CD70 nanobody and an antigen-binding fragment thereof that can specifically recognize CD70, bind to CD70 with high affinity, and block the binding between CD70 and CD27, while also having excellent functional activity.

[0043] Specifically, the present invention involves immunizing alpacas with CD70 protein to obtain a high-quality single-domain antibody gene library. The antibody gene library is then screened by phage display technology to obtain CD70-specific single-domain antibody genes. The genes are then introduced into mammalian cells to obtain antibody strains that can be expressed highly efficiently in mammalian cells and have high specificity. Furthermore, nanobodies with high affinity, high specificity, and high functional activity are identified by methods such as ELISA, biomolecular interaction analysis, and blocking tests. The antibodies or antigen-binding fragments thereof have excellent safety and targeting properties and can specifically bind to the extracellular domain of human CD70.

[0044] The present invention further provides a chimeric antigen receptor (CAR) comprising said nanobody. By infecting immune cells with a vector containing the coding sequence of said CAR, immune effector cells with significant cytotoxicity against tumor cells overexpressing CD70 are obtained. Such immune effector cells can be used to treat or ameliorate diseases related to CD70 expression, thus laying the foundation for the treatment of CD70-positive tumors.

[0045] antibody As used herein, a "CD70 binding molecule" is a protein that specifically binds to CD70, including, but not limited to, an antibody, a heavy chain antibody, a nanobody, or an antigen-binding fragment thereof.

[0046] As used herein, the term "antibody" includes monoclonal antibodies (including full-length antibodies having an immunoglobulin Fc region), antibody compositions with multi-epitope specificity, multispecific antibodies (e.g., bispecific antibodies), diabodies, single-chain molecules, and antibody fragments (particularly antigen-binding fragments, such as Fab, F(ab')2, and Fv). As used herein, "antibody" and "immunoglobulin" can be used interchangeably.

[0047] A conventional "antibody" comprises a basic four-chain antibody unit, which is a heterotetrameric glycoprotein composed of two identical light chains (L) and two identical heavy chains (H). Each heavy chain has a variable domain (VH) at the N-terminus, three (CH1, CH2, and CH3 for α and γ chains) and four (CH1, CH2, CH3, and CH4 for μ and ε isotypes) constant domains (CH), and a hinge region (Hinge) between the CH1 and CH2 domains. Each light chain has a variable domain (VL) at the N-terminus, and a constant domain (CL) at the other end. The pair of VH and VL together form an antigen-binding site. For the structure and properties of various antibodies, see Basic and Clinical Immunology, 8th Edition, Daniel P. Sties, Abba I. Terr, and Tristram G. Parsolw (eds.), Appleton & Lange, Norwalk, CT, 1994, page 71 and chapter 6. Light chains from any vertebrate species can be divided into two distinct classes, called kappa and lambda, depending on the amino acid sequence of their constant domains. The gamma and alpha classes are further divided into subclasses, e.g., IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1, and IgA2, expressed in humans, with relatively minor differences in CH sequence and function.

[0048] A "heavy chain antibody" according to the present specification is an antibody derived from a camelid or elasmobranch organism. Compared to the above-mentioned four-chain antibodies, a heavy chain antibody lacks light and heavy chain constant region 1 (CH1) and contains only two heavy chains consisting of a variable region (VHH) and other constant regions, with the variable region being linked to the constant region by a structure similar to a hinge region structure. Each heavy chain of a camelid heavy chain antibody contains one variable region (VHH) and two constant regions (CH2 and CH3), while each heavy chain of an elasmobranch heavy chain antibody contains one variable region and five constant regions (CH1-CH5). Antigen-binding fragments of heavy chain antibodies include VHH and single-chain heavy chain antibodies. A heavy chain antibody may have the CH2 and CH3 of human IgG Fc by fusion with the constant region of human IgG Fc.

[0049] As used herein, the terms "single domain antibody", "anti-CD70 single domain antibody", "heavy chain variable region domain of a heavy chain antibody", and "VHH" are used interchangeably, and all refer to single domain antibodies that specifically recognize and bind to CD70. A single domain antibody is a variable region of a heavy chain antibody. Typically, a single domain antibody comprises three CDRs and four FRs. Preferably, the single domain antibody of the present invention has a CDR1 as shown in any one of SEQ ID NOs: 1-7, a CDR2 as shown in any one of SEQ ID NOs: 8-13, and a CDR3 as shown in any one of SEQ ID NOs: 14-20. A single domain antibody is the smallest functional antigen-binding fragment. Typically, an antibody that naturally lacks a light chain and a heavy chain constant region 1 (CH1) is obtained, and then the variable region of the antibody heavy chain is cloned to construct a single domain antibody consisting of only one heavy chain variable region.

[0050] As used herein, a "nanobody" refers to an antibody comprising a VHH according to the present specification. It may be any of the heavy chain antibodies described above, multivalent or multispecific antibodies comprising multiple VHHs, and recombinant antibodies obtained by recombining a VHH with an antibody Fc (e.g., CH2 and CH3, or CH2, CH3 and CH4).

[0051] A binding molecule comprising two or more single domain antibodies is a multivalent single domain antibody, and a binding molecule comprising two or more single domain antibodies with different specificities is a multispecific single domain antibody. A multivalent or multispecific single domain antibody is linked to the multiple single domain antibodies by a connexon, which typically consists of 1-15 amino acids selected from G and S.

[0052] In this specification, the terms heavy chain antibody and antibody (conventional four-chain antibody) are used to distinguish different combinations of antibodies. Because the two have structural similarities, the following explanation of the antibody structure applies to both light chain antibodies and heavy chain antibodies.

[0053] The "variable region" or "variable domain" of an antibody is the amino-terminal domain of either the heavy or light chain of an antibody. The heavy and light chain variable domains are called "VH" and "VL", respectively. These domains are usually the most variable parts of an antibody (relative to other antibodies of the same type) and contain the antigen-binding site.

[0054] The term "variable" refers to the ubiquity of antibody sequence differences in several segments in the variable domain. The variable domain mediates antigen binding and defines the specificity of a particular antibody to its particular antigen. However, variability is not uniformly distributed across all amino acids in the variable domain. Instead, it is concentrated in three segments (in both light and heavy chain variable domains) called hypervariable regions (HVRs), namely, HCDR1, HCDR2, HCDR3 (which may be abbreviated as CDR1, CDR2, CDR3 in heavy chain antibodies) in the heavy chain variable region and LCDR1, LCDR2, LCDR3 in the light chain variable region. The more highly conserved parts of the variable domain are called framework regions (FRs). Natural heavy and light chain variable domains each contain four FR regions (FR1, FR2, FR3, and FR4), which are usually connected by adopting a β-pleated sheet structure to form a ring, but are occasionally connected by three HVRs that form part of the β-pleated sheet structure. The HVRs in each chain are held in close proximity by the FR regions and, together with the HVRs in the other chain, promote the formation of the antigen-binding site of the antibody. Usually, the structure of the light chain variable region is FR1-LCDR1-FR2-LCDR2-FR3-LCDR3-FR4, and the structure of the heavy chain variable region is FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4. The constant domain is not directly involved in binding the antibody to the antigen, but exhibits various effector functions, such as the participation of the antibody in antibody-dependent cell-mediated cytotoxicity. There are several variable region numbering schemes for antibodies, including Chothia, Kabat, IMGT and Contact. This specification uses the IMGT numbering scheme as an example.

[0055] The "Fc region" (fragment crystallizable region) or "Fc domain" or "Fc" is the C-terminal region of an antibody heavy chain that mediates the binding of the immunoglobulin to host tissues or factors, including binding to Fc receptors on various cells of the immune system (e.g., effector cells) or to the first component (C1q) of the classical complement system. In IgG, IgA and IgD antibody isotypes, the Fc region is composed of two identical protein fragments derived from the CH2 and CH3 domains of the two heavy chains of the antibody, while the Fc region of IgM and IgE contains three heavy chain constant domains (CH domains 2-4) in each polypeptide chain. Although the boundaries of the Fc region of an immunoglobulin heavy chain may vary, the Fc region of a human IgG heavy chain is generally defined, e.g., as in Kabat, as the segment of the sequence from an amino acid residue at position C226 or P230 of the heavy chain to the carboxyl terminus, with numbering according to the EU index. As used herein, the Fc region may be a native sequence Fc or a variant Fc.

[0056] An "antibody fragment" comprises a portion of an intact antibody, preferably the antigen-binding and / or variable regions of the intact antibody. The antibody fragment is preferably an antigen-binding fragment of an antibody. Examples of antibody fragments include Fab, Fab', F(ab'), F(ab')2, Fd, Fv fragments, Fvs linked by disulfide bonds, diabodies, linear antibodies, single-chain antibody molecules, scFv-Fc fragments, multispecific antibodies formed from antibody fragments, and any fragment whose half-life can be increased by chemical modification or incorporation into liposomes. Antigen-binding fragments can be produced by a variety of techniques, including, but not limited to, hydrolytic digestion of the complete antibody protein and expression by a host cell containing the antigen-binding fragment.

[0057] An "Fv" is the smallest antibody fragment that contains a complete antigen recognition and binding site. The fragment consists of a dimer of one heavy chain variable domain and one light chain variable domain in tight non-covalent association. The pleated sheet of the two domains gives rise to six hypervariable loops (three loops each from the heavy and light chains) that provide the amino acid residues for antigen binding and confer antigen specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three HVRs specific for an antigen) still has the ability to recognize and bind to an antigen, albeit with a lower affinity than a complete binding site. A "single-chain Fv", abbreviated as "sFv" or "scFv", is an antibody fragment that contains antibody VH and VL domains and is linked to a single polypeptide chain. Preferably, the sFv polypeptide further comprises a polypeptide linker between the VH and VL domains, allowing the sFv to form the desired antigen-binding structure. For heavy chain antibodies or nanobodies, the scFv is a VHH.

[0058] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., each antibody in the population is similar except for possible naturally occurring mutations and / or post-translational modifications (e.g., isomerization, amidation) that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In contrast to polyclonal antibody preparations (which typically contain different antibodies directed against different determinants (epitopes)), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage that they are synthesized by hybridoma culture and are therefore uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody obtained from a population of substantially homogeneous antibodies and is not to be construed as requiring production of the antibody by any particular method. For example, monoclonal antibodies for use in accordance with the present invention may be produced by a variety of techniques including, for example, hybridoma methods, phage display methods, recombinant DNA methods, and techniques for producing human or human-like antibodies from animals having partial or entire human immunoglobulin loci or genes encoding human immunoglobulin sequences, single cell sequencing methods.

[0059] As used herein, monoclonal antibodies also include "chimeric" antibodies in which a portion of the heavy and / or light chains are identical to or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, and the remainder of the chains are identical to or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, and also includes fragments of such antibodies, so long as they exhibit the desired biological activity.

[0060] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequences derived from non-human immunoglobulins. Thus, a "humanized antibody" is typically a non-human antibody in which the variable domain framework regions have been replaced with sequences found in a human antibody. Typically, in a humanized antibody, the entire antibody (except for the CDRs) is encoded by a polynucleotide of human origin or is identical to such an antibody (except for the CDRs). CDRs (partially or entirely encoded by nucleic acids derived from a non-human organism) are grafted onto the beta-pleated sheet framework of a human antibody variable region to produce an antibody whose specificity is determined by the grafted CDRs. Methods for producing such antibodies are well known in the art, for example, by using mice with genetically engineered immune systems. In the present invention, antibodies, single domain antibodies, heavy chain antibodies, etc. all include humanized variants of the respective antibodies.

[0061] A "human antibody" is an antibody having an amino acid sequence which corresponds to that of an antibody produced from a human and / or which is produced using any technique disclosed herein for producing human antibodies. This definition of a human antibody expressly excludes humanized antibodies which contain non-human antigen-binding residues. Human antibodies may be produced using a variety of techniques known in the art, including phage display libraries.

[0062] In some embodiments, the invention further provides Nanobodies, heavy chain antibodies, antibodies or antigen-binding fragments thereof (e.g. single domain antibody VHHs) that have the same epitope on human CD70 that binds to the antigen-binding region of any of the anti-CD70 Nanobodies of the invention, i.e. Nanobodies, heavy chain antibodies, antibodies or antigen-binding fragments thereof that can compete with each other for binding to CD70 for the antigen-binding region of any of the Nanobodies of the invention.

[0063] In the present invention, the anti-CD70 single domain antibody has a CDR1 shown in any one of SEQ ID NOs: 1-7, a CDR2 shown in any one of SEQ ID NOs: 8-13, and a CDR3 shown in any one of SEQ ID NOs: 14-20. Preferably, the anti-CD70 single domain antibody comprises a CDR1, CDR2 and CDR3 shown in any one of sets (a)-(h).

[0064] (a) CDR1, the sequence of which is shown in SEQ ID NO:1, CDR2, the sequence of which is shown in SEQ ID NO:8, CDR3, the sequence of which is shown in SEQ ID NO:14; (b) CDR1, the sequence of which is shown in SEQ ID NO:2, CDR2, the sequence of which is shown in SEQ ID NO:8, CDR3, the sequence of which is shown in SEQ ID NO:14; (c) CDR1, the sequence of which is shown in SEQ ID NO:3, CDR2, the sequence of which is shown in SEQ ID NO:8, CDR3, the sequence of which is shown in SEQ ID NO:15; (d) CDR1, the sequence of which is shown in SEQ ID NO:4, CDR2, the sequence of which is shown in SEQ ID NO:9, CDR3, the sequence of which is shown in SEQ ID NO:16; (e) CDR1, the sequence of which is shown in SEQ ID NO:5, CDR2, the sequence of which is shown in SEQ ID NO:10, CDR3, the sequence of which is shown in SEQ ID NO:14; (f) CDR1, the sequence of which is shown in SEQ ID NO:6, CDR2, the sequence of which is shown in SEQ ID NO:11, CDR3, the sequence of which is shown in SEQ ID NO:18; (g) CDR1, the sequence of which is shown in SEQ ID NO:6, CDR2, the sequence of which is shown in SEQ ID NO:12, CDR3, the sequence of which is shown in SEQ ID NO:19; (h) CDR1, the sequence of which is shown in SEQ ID NO:7, CDR2, the sequence of which is shown in SEQ ID NO:13, and CDR3, the sequence of which is shown in SEQ ID NO:20.

[0065] The FR1, FR2, FR3 and FR4 of the anti-CF70 single domain antibodies herein are each independently selected from the FR1, FR2, FR3 and FR4 of the single domain antibodies set forth in any one of SEQ ID NOs: 21-28. Preferably, the amino acid sequence of the anti-CF70 single domain antibody is set forth in any one of SEQ ID NOs: 21-28.

[0066] Where a single domain antibody is combined with a heavy chain constant region, the nanobody is a heavy chain antibody comprising the single domain antibody according to the present specification. The heavy chain constant region is a camelid heavy chain antibody constant region and may comprise CH2 and CH3. Preferably, the antibody constant region is derived from any one of the constant regions of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE and IgD, more preferably from any one of the constant regions of IgG1, IgG2, IgG3 and IgG4. In one or more embodiments, the heavy chain constant region is the CH2 and CH3 of human IgG Fc, e.g., the CH2 and CH3 of IgG1, as set forth in SEQ ID NO: 37.

[0067] A CF70 binding molecule according to the present specification may be a monovalent or polyvalent nanobody or single domain antibody comprising one, two or more anti-CD70 nanobodies or single domain antibodies according to the present specification, or a multispecific nanobody or single domain antibody. The multispecificity may be directed against CD70 and another antigen or against two different epitopes of CD70.

[0068] The present invention further includes derivatives and analogs of the antibody. "Derivatives" and "analogs" are polypeptides that essentially retain a biological function or activity homologous to the antibody of the present invention. The derivatives or analogs of the present invention may be (i) polypeptides having substitutions at one or more amino acid residues, or (ii) polypeptides formed by fusing the mature polypeptide to other compounds (e.g., compounds that extend the half-life of the polypeptide, such as polyethylene glycol), or (iii) polypeptides formed by fusing additional amino acid sequences to the polypeptide sequence (e.g., a leader sequence, a secretory sequence, a sequence for purifying the polypeptide or a proteinogenic sequence, or a fusion protein formed with a 6His tag). As taught herein, these derivatives and analogs are within the knowledge of those skilled in the art.

[0069] A person skilled in the art can obtain a variant of the antibody or functional fragment thereof by modifying one or more (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) amino acids in the antibody sequence of the present invention, provided that the antibody activity is not substantially affected. These variants include, but are not limited to, those obtained by deleting, inserting, and / or substituting one or more (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) amino acids, and adding one or more (usually 20 or less, preferably 10 or less, and more preferably 5 or less) amino acids to the C-terminus and / or N-terminus. In the art, conservative substitution with amino acids having close or similar properties usually does not change the function of the protein. For example, amino acids having similar properties are substituted in the FR and / or Fc region. Amino acid residues that can be conservatively substituted are well known in the art. Such substituted amino acid residues may or may not be encoded by the genetic code. Furthermore, for example, the addition of one or more amino acids to the C-terminus and / or N-terminus does not usually alter the function of the protein and is considered to be within the scope of the present invention.

[0070] Variants of the antibodies according to the present invention include homologous sequences, conservative variants, allelic variants, natural variants, induced variants, proteins encoded by DNA capable of hybridizing to the coding DNA of the antibodies according to the present invention under high or low stringency conditions, and polypeptides or proteins obtained using antisera against the antibodies according to the present invention. In some embodiments, the sequence of the variant according to the present invention may have at least 95%, 96%, 97%, 98% or 99% identity with the sequence from which it is derived. Sequence identity according to the present invention can be measured using sequence analysis software, such as the computer program BLAST using default arguments, in particular BLASTP or TBLASTN. The present invention further includes molecules having an antibody heavy chain variable region with CDRs, so long as the CDRs have 90% or more (preferably 95% or more, most preferably 98% or more) homology with the CDRs identified herein.

[0071] The antibodies of the invention may be produced by methods conventional in the art, for example hybridoma technology. The Nanobodies of the invention may be produced by methods conventional in the art, for example phage display technology, well known in the art. Alternatively, the antibodies or Nanobodies of the invention may be expressed in other cell systems. Suitable mammalian host cells may be transformed with sequences encoding the antibodies of the invention, followed by culturing the host cells and purifying the antibodies. The transformation may employ any known method, including, for example, packaging the polynucleotide into a virus (or viral vector) and transducing the host cell with the virus (or vector). The transformation process used will depend on the host to be transformed. Methods for introducing heteropolynucleotides into mammalian cells are well known in the art and include dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of polynucleotides in liposomes, and direct microinjection of DNA into the cell nucleus. Mammalian cell lines available as hosts for expression are well known in the art and include, but are not limited to, the various immortalized cell lines available from the American Type Culture Collection (ATCC), including, but not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., HepG2), and the like.

[0072] CAR The present invention further provides a chimeric antigen receptor (CAR) targeting CD70. The CAR comprises an optional signal peptide sequence, an anti-CD70 binding molecule according to the present invention which is an antigen recognition region, a hinge region, a transmembrane region and an intracellular region, wherein the intracellular region comprises one or more intracellular costimulatory domains and / or one or more intracellular signal domains. The "hinge region", "transmembrane region" and "intracellular region" according to the present invention may be selected from the sequences of the hinge region, transmembrane region and intracellular region in known CAR-T technology.

[0073] Any signal peptide in a CAR can be selected as needed. In general, a signal peptide is a peptide sequence that targets a polypeptide to a required site in a cell. A signal peptide targets a polypeptide to the secretory pathway of a cell and allows the polypeptide to be integrated and anchored in a lipid bilayer. A signal peptide may also be a membrane-localized signal peptide. Exemplary signal peptides are, for example, CD8 signal peptide, CD28 signal peptide, CD4 signal peptide, or light chain signal peptide, the sequences of which are within the knowledge of those skilled in the art. The CD8 signal peptide applied in the present invention may be any of various human CD8 signal peptide sequences commonly used in the field for CARs. In some embodiments, the amino acid sequence of the human CD8 signal peptide comprises the sequence shown in SEQ ID NO:38.

[0074] The hinge region of the chimeric antigen receptor is between the extracellular antigen binding region and the transmembrane region. The hinge region is usually an amino acid segment that exists between two regions of a protein and allows the flexibility of the protein and the relative movement of the two regions. The hinge region may be a hinge region of a natural protein or a part thereof. The hinge region of an antibody (e.g., an IgG, IgA, IgM, IgE or IgD antibody) may also be used in the chimeric antigen receptor according to the present invention. A non-natural peptide may also be used in the hinge region of the chimeric antigen receptor according to the present invention. Exemplarily, the hinge region of the CAR is selected from the CD8α hinge region, the IgD hinge region, the IgG1 Fc CH2CH3 hinge region or the IgG4 Fc CH2CH3 hinge region, the sequences of which are within the knowledge of the skilled artisan. The CD8α hinge region applied to the present invention may be any of the various human CD8α hinge region sequences commonly used in the field for CARs. In some embodiments, the human CD8α hinge region comprises the sequence shown in SEQ ID NO:39.

[0075] The transmembrane region of the chimeric antibody receptor can form an α-helix, a complex of multiple α-helices, a β-barrel, or any other stable structure that can span the cellular phospholipid bilayer. The transmembrane region can be natural or synthetic. The transmembrane region can be selected from the transmembrane regions of the following proteins: CD3ε, CD4, CD5, CD8α, CD9, CD16, CD22, CD28, CD33, CD37, CD45, CD64, CD80, CD86, CD134, CD137, CD154, and the α, β, or ζ chains of the T cell receptor. The human CD8α transmembrane region applied to the present invention can be any of various human CD8α transmembrane region sequences commonly used in the art for CARs. In some embodiments, the amino acid sequence of the human CD8α transmembrane region comprises the sequence shown in SEQ ID NO:40.

[0076] The intracellular signaling domain (or intracellular signaling region) expresses activation of at least one normal effector function of the chimeric antigen receptor immune effector cell. For example, the effector function of a T cell may be a cell dissociation activity or an auxiliary activity, including secretion of cytokines. Usually, the entire intracellular signaling region can be used, but there are many cases where it is not necessary to use the entire chain. Regarding the use of a truncated portion of the intracellular signaling region, such a truncated portion can be used instead of the complete chain, so long as it transmits an effector function signal. Thus, the intracellular signaling region includes any truncated form of the intracellular signaling region that is sufficient to transmit an effector function signal. The intracellular signaling domain of the CAR can be selected as needed, and includes, but is not limited to, intracellular signaling domains derived from at least one of CD3ζ, FcRγ (FCER1G), FcRβ (FcεRib), CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, and CD66d. Preferably, the intracellular signaling domain is derived from the human CD3ζ intracellular signaling domain. Furthermore, the human CD3 zeta intracellular signal domain has the amino acid sequence shown in SEQ ID NO:41.

[0077] In addition to stimulation by antigen-specific signals, many immune effector cells require additional costimulation to promote cell proliferation, differentiation, survival, and effector functions of activated cells. A "costimulatory domain" may be the cytoplasmic portion of a costimulatory molecule. The term "costimulatory molecule" refers to the relevant binding partner in an immune cell (e.g., a T cell) that specifically binds to a costimulatory ligand and mediates a costimulatory response by the immune cell, including but not limited to proliferation and survival. For example, an appropriate intracellular costimulatory domain can be selected as needed, including an intracellular domain having a costimulatory signal molecule, such as at least one of the intracellular domains derived from 4-1BB, CARD11, CD2, CD7, CD27, CD28, CD30, CD40, CD54, CD83, OX40, CD137, CD134, CD150, CD152, CD223, CD270, PD-L2, PD-L1, CD278, DAP10, LAT, NKD2C, SLP76, TRIM, FcεRIγ, MyD88, and 41BBL. In some embodiments, the amino acid sequence of the 4-1BB costimulatory domain comprises the sequence shown in SEQ ID NO:42.

[0078] For example, the above-mentioned moieties forming the chimeric antigen receptor of the present invention, such as the CD8 signal peptide, the anti-MSLN nanobody, the CD8 hinge region, the CD28 transmembrane region, the CD28 costimulatory domain, and the CD3ζ intracellular signal domain, can be directly linked to each other or can be linked by a linker sequence. The linker sequence may be a linker sequence applied to antibodies well known in the art, such as a linker sequence containing G and S. Typically, the linker comprises one or more motifs of a back-and-forth repeat. For example, the motifs may be GGGS, GGGGS, SSSSG, GSGSA, and GGSGG. Preferably, the motifs are adjacent in the linker sequence, and no amino acid residues are inserted between the repeats. The linker sequence may comprise 1, 2, 3, 4, or 5 repeat motifs. The length of the linker may be 3 to 25 amino acid residues, such as 3 to 15, 5 to 15, 10 to 20 amino acid residues. In some embodiments, the linker sequence is a polyglycine linker sequence. The number of glycines in the linker sequence is not particularly limited and is usually 2 to 20, for example, 2 to 15, 2 to 10, or 2 to 8. In addition to glycine and serine, the linker may further contain other known amino acid residues such as alanine (A), leucine (L), threonine (T), glutamic acid (E), phenylalanine (F), arginine (R), and glutamine (Q). In some embodiments, the linker sequence is a (GGGGS)n bond, where n is an integer of 1 to 5.

[0079] In an exemplary embodiment, the CAR comprises, in order from N-terminus to C-terminus, a CD8 signal peptide, an anti-CD70 Nanobody described herein or an antigen-binding fragment thereof, a CD8α hinge region, a CD8α transmembrane region, a CD3ζ intracellular signaling domain, and a 4-1BB costimulatory domain. In a specific example, an exemplary CAR having the above structure is depicted in any one of SEQ ID NOs:29-36.

[0080] In gene cloning, it is always necessary to design a suitable restriction enzyme cleavage site, so that one or more extra residues are introduced at the end of the expressed amino acid sequence, but it should be understood that this does not affect the activity of the target sequence. In order to construct a fusion protein, promote recombinant protein expression, obtain a recombinant protein that is automatically secreted outside the host cell, or aid in recombinant protein purification, some amino acids are always required to be added to the N-terminus, C-terminus, or other suitable region of the protein. Other suitable regions of the protein include, but are not limited to, suitable linker peptides, signal peptides, leader peptides, terminal extensions, etc. Therefore, the amino-terminus or carboxyl-terminus of the CAR of the present invention may further comprise one or more polypeptide fragments as a protein tag. Any suitable tag can be used herein. For example, the tag may be FLAG, HA, HA1, c-Myc, Poly-His, Poly-Arg, Strep-TagII, AU1, EE, T7, 4A6, ε, B, gE, and Ty1. These tags can be used for protein purification.

[0081] The antigen recognition region of the CAR of the present invention may be a variant of the sequence of the anti-CD70 nanobody or a functional fragment thereof. In addition, other parts of the CAR may have sequence changes, so that the resulting variant has at least 80%, preferably at least 85%, preferably at least 90%, preferably at least 95%, preferably at least 97% sequence identity with the CAR and retains the biological activity of the CAR (e.g., activated T cells). For example, the sequence identity of two aligned sequences can be calculated using BLASTp from NCBI.

[0082] Variants further include amino acid sequences having one or more mutations (insertion, deletion or substitution) in the amino acid sequence of the CAR of any one of the embodiments, while retaining the biological activity of the CAR. The number of mutations is usually within 1-10, for example, 1-8, 1-5 or 1-3. Conservative substitutions are preferred. For example, conservative substitutions with amino acids with close or similar properties in the art usually do not change the function of the protein or polypeptide. "Amino acids with close or similar properties" include, for example, families of amino acid residues with similar side chains, including amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, substitution of one or more sites of a polypeptide of the invention with another amino acid residue from the same side chain type does not substantially affect its activity.

[0083] nucleic acid The present invention further provides a polynucleotide encoding said antibody or CAR. The polynucleotide of the present invention may be in DNA or RNA form. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand. The present invention includes degenerate variants of the polynucleotide sequence encoding the fusion protein, i.e., encoding nucleotide sequences with the same amino acid sequence but different nucleotide sequences.

[0084] Therefore, the present invention also relates to polynucleotides that hybridize with the above polynucleotide sequences and where the two sequences have at least 50%, preferably at least 70%, more preferably at least 80% identity. The present invention particularly relates to polynucleotides that can hybridize with the polynucleotides of the present invention under stringent conditions. In the present invention, "stringent conditions" refers to (1) hybridization and elution at low ionic strength and high temperature, e.g., 0.2xSSC, 0.1% SDS, 60°C, or (2) hybridization with a denaturing agent, e.g., 50% (v / v) formamide, 0.1% calf serum / 0.1% Ficoll, 42°C, or (3) hybridization occurs only if the identity between the two sequences is at least 90% or more, more preferably 95% or more. Furthermore, the polypeptides encoded by the hybridizable polynucleotides have the same biological functions and activities as the mature polypeptides.

[0085] The full-length nucleotide sequence of the antibody of the present invention or a fragment thereof is usually obtained by PCR amplification, recombinant methods, or artificial synthesis. In particular, when the fragment is short, the relevant sequence may be synthesized by artificial synthesis. Usually, several small fragments are synthesized and then linked to obtain a fragment of a long sequence. Alternatively, the coding sequence of the heavy chain can be fused to an expression tag (such as 6His) to form a fusion protein. The sequence of the CAR can be obtained as described above. Alternatively, the sequence of each part of the CAR (signal peptide, antigen recognition region, hinge region, transmembrane region, or intracellular region) can be obtained as described above, and then the full-length CAR can be obtained by linking.

[0086] Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant methods. It is usually cloned into a vector, introduced into a cell, and then isolated from the host cell grown by conventional methods to obtain the relevant sequence. The biomolecules (nucleic acids, proteins, etc.) of the present invention also include biomolecules that exist in isolated form. Currently, DNA sequences encoding the proteins of the present invention (or fragments thereof, or derivatives thereof) can be obtained only by chemical synthesis. The DNA sequences are then introduced into various existing DNA molecules (e.g., vectors) and cells known in the art. Mutations may also be introduced into the protein sequences of the present invention by chemical synthesis. Each part of the CAR is cloned sequentially into a vector, or integrated into the full-length CAR and then cloned.

[0087] The present invention also relates to a nucleic acid construct comprising the polynucleotide sequences according to the present invention and one or more regulatory sequences operably linked to these sequences. The polynucleotide sequences according to the present invention may be manipulated in various ways to ensure the expression of said antibody or CAR. Before inserting the nucleic acid construct into a vector, the nucleic acid construct may be manipulated according to the differences or requirements of the expression vector. Techniques for modifying polynucleotide sequences by recombinant DNA methods are known in the art.

[0088] The regulatory sequence may be a suitable promoter sequence. The promoter sequence is usually operably linked to a coding sequence whose protein is to be expressed. The promoter may be any nucleotide sequence that exhibits transcriptional activity in the selected host cell, including mutant, truncated, and hybrid promoters, and may be derived from a gene encoding an extracellular or intracellular polypeptide that is homologous or heterologous to the host cell. One example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. The promoter sequence is a strong constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operably linked thereto. Another example of a suitable promoter is elongation factor-1 alpha (EF-1 alpha). However, other constitutive promoter sequences may also be used, including, but not limited to, Simian Virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, EB virus immediate early promoter, Rous sarcoma virus promoter, and human gene promoters. The human gene promoter may be, for example, an actin promoter, a myosin promoter, a protoheme promoter, or a creatine kinase promoter. In addition, the use of an inducible promoter is also contemplated. The use of an inducible promoter provides a molecular switch that can turn on the expression of a polynucleotide sequence operably linked to the inducible promoter when expression is desired, and turn off the expression when expression is not desired. Examples of inducible promoters include, but are not limited to, a metallothionein promoter, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.

[0089] The regulatory sequence may be a suitable transcription terminator sequence, i.e., a sequence recognized by a host cell to terminate transcription. The terminator sequence is operably linked to the 3' end of the nucleotide sequence encoding the polypeptide. Any terminator functional in the selected host cell can be used in the present invention. The regulatory sequence may be a suitable leader sequence, which is a non-translated region of an mRNA that is essential for host cell translation. The leader sequence is operably linked to the 5' end of the nucleotide sequence encoding the polypeptide. Any terminator functional in the selected host cell can be used in the present invention.

[0090] In some embodiments, the nucleic acid construct is a vector, such as, for example, a cloning vector, an expression vector, and an integrating vector. Typically, the expression of the polynucleotide sequence of the present invention is achieved by operably linking the polynucleotide sequence of the present invention to an expression vector. Exemplary cloning vectors include transcription and translation terminators, initiation sequences, and promoters that are used to regulate the expression of the desired nucleic acid sequence. Integrating vectors include components that integrate the target sequence into the cell genome. These vectors can be used to transform suitable host cells to express proteins. Vectors typically include sequences that are used for plasmid maintenance and cloning and expressing exogenous nucleotide sequences. The sequences (collectively referred to in some embodiments as "flanking sequences") typically include one or more of the following nucleotide sequences: a promoter, one or more enhancer sequences, an origin of replication, a transcription termination sequence, a complete intron sequence including donor and acceptor splice sites, a sequence encoding a leader sequence used for polypeptide secretion, a ribosome binding site, a polyadenylation sequence, a multiconnexon region for inserting a nucleic acid encoding an antibody to be expressed, and any tagging elements.

[0091] In addition, the type of vector is not limited, and may be, for example, a plasmid, a phagemid, a phage derivative, an animal virus, or a cosmid, and may be changed according to the host cell to be introduced. Viral vector technology is well known in the art, and is described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other handbooks on virology and molecular biology. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses.

[0092] To assess expression of a CAR polypeptide or a portion thereof, the vector introduced into the cells may contain either one or both of a selectable marker gene or a reporter gene, allowing for identification and selection of expressing cells from a population of cells transfected or infected with the viral vector.

[0093] cell Host cells suitable for introduction of the nucleic acid constructs according to the present specification may include prokaryotic cells such as bacterial cells, or lower eukaryotic cells such as yeast cells, or higher eukaryotic cells such as mammalian cells, in particular immune cells, preferably immune effector cells. Representative examples include bacterial cells such as E. coli, Streptomyces, Salmonella typhimurium, fungal cells such as yeast, insect cells such as fruit fly S2 or Sf9, and animal cells such as CHO, COS7, 293 cells, etc.

[0094] An "immune effector cell" is an immune cell capable of performing an immune effector function. In some embodiments, the immune effector cell expresses at least FcγRIII and performs an ADCC effector function. Examples of immune effector cells that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, neutrophils, and eosinophils. Preferably, the immune effector cell is at least one selected from immune cells cultured and differentiated from pluripotent stem cells or embryonic stem cells, T lymphocytes, NK cells, peripheral blood mononuclear cells (PBMCs), and hematopoietic stem cells. More preferably, the immune effector cell is a T lymphocyte (i.e., a T cell). In some embodiments, the T cell may be CD4+ / CD8-, CD4- / CD8+, CD4+ / CD8+, CD4- / CD8-, or a combination thereof. In some embodiments, the T cells produce IL-2, IFN and / or TNF upon expressing the chimeric antigen receptor and binding to the target cell. In some embodiments, the CD8+ T cells dissociate the antigen-specific target cell upon expressing the chimeric antigen receptor and binding to the target cell.

[0095] The T cells applied in the present invention may be of various origins and types. For example, the T cells may be derived from PBMCs of a patient with a B cell malignancy. In some embodiments, after obtaining the T cells, they are first stimulated and activated with an appropriate amount (e.g., 30-80 ng / ml, e.g., 50 ng / ml) of CD3 antibody, and then cultured in a medium containing an appropriate amount (e.g., 30-80 IU / ml, e.g., 50 IU / ml) of IL2 for use.

[0096] Methods for introducing a nucleic acid or vector into a mammalian cell are known in the art, and the vector can be introduced into the cell by physical, chemical, or biological means. If the host is a prokaryotic organism, such as E. coli, competent cells capable of taking up DNA can be harvested after exponential growth phase and treated with CaCl2 using procedures well known in the art. If the host is a eukaryotic organism, DNA transfection methods such as calcium phosphate co-precipitation, or conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc. may be selected. In some embodiments, transduced or transfected immune effector cells are introduced with a nucleic acid or vector and then isolated and propagated.

[0097] The obtained transformants can be cultured in a conventional manner and express the antibody or CAR encoded by the gene of the present invention. The medium used for the culture may be selected from various conventional media depending on the host cells used. The culture is performed under conditions suitable for the growth of the host cells. After the host cells have grown to an appropriate cell density, the selected promoter is induced by an appropriate method (such as temperature change or chemical induction), and the cells are further cultured for a certain period of time.

[0098] The polypeptide in the above method is expressed intracellularly or on the cell membrane, or secreted extracellularly. If necessary, the recombinant protein may be isolated and purified by various isolation methods based on its physical, chemical, and other properties. These methods are well known to those skilled in the art. These methods include, but are not limited to, conventional renaturation, treatment with protein precipitants (salting out method), centrifugation, osmotic sterilization, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and various other liquid chromatography techniques, as well as combinations of these methods.

[0099] Applications and Methods The inventors constructed a nanobody library to select nanobodies and their variants that can bind to CD70. The antibodies were verified for their ability to bind to antigens through protein-level binding detection, affinity detection, competitive inhibition experiments, and tissue cross-reactivity. The inventors used these nanobodies to construct CAR and CAR-T cells, and verified through molecular and cellular experiments that the CAR-T has strong immune function, better CD107a expression, IFN-γ and L-2 secretion, and specific killing function against target cells, with significant in vivo efficacy.

[0100] All aspects of the antibodies, CARs, coding sequences, nucleic acid constructs, and cells described herein can be used to manufacture a medicament for preventing or treating various conditions and disorders described herein, including diseases or conditions related to CD70 expression, diseases directly or indirectly caused by aberrant CD70 expression, typically diseases caused by overexpression of CD70, such as cancer, including but not limited to renal cell carcinoma, acute myeloid leukemia, non-Hodgkin's lymphoma, multiple myeloma, mantle cell lymphoma, diffuse large cell lymphoma, follicular lymphoma, pancreatic cancer, breast cancer, and glioblastoma.

[0101] The present invention further includes cell therapy comprising expressing a CAR according to the present disclosure in immune cells (e.g., T cells) and administering a therapeutically effective amount of the cells to a subject in need thereof, where the cells are capable of killing tumor cells in the subject. Compared to antibody therapy, CAR-T cells can replicate in the body and have long-term persistence leading to sustained tumor suppression. The anti-tumor immune response by CAR-T cells can be a proactive or passive immune response. The immune response mediated by CAR can also be part of an adoptive immunotherapy process, in which CAR-T cells induce a specific immune response against the antigen-binding site on the CAR.

[0102] The antibody, nucleic acid or CAR-modified cell of the present invention may be administered alone or in combination with other components such as diluents and / or associated cytokines or cell populations as a pharmaceutical composition. In this embodiment, the pharmaceutical composition may be prepared in the form of a lyophilized formulation or an aqueous solution by mixing an active formulation having a predetermined purity with any pharma- ceutically acceptable carrier. The pharma-ceutically acceptable carrier is non-toxic to subjects at the dose and concentration employed, and may include at least one of a buffer (e.g., neutral buffered saline, sulfate buffered saline), an antioxidant, a preservative, an isotonicity agent, a stabilizer, a chelating agent (e.g., EDTA or glutathione), an adjuvant (e.g., aluminum hydroxide) and a surfactant. In addition, the pharmaceutical composition must be sterile so that it can be used for administration to the body. The pharmaceutical composition can be sterilized by filtration through a sterile filtration membrane.

[0103] In some embodiments, the pharmaceutical composition may include at least one additive of cytotoxic agents, chemotherapeutic agents, cytokines, immunosuppressants, growth inhibitors, and active ingredients required for the specific indications to be treated. The specific amount of additives may be adjusted according to actual needs.

[0104] The pharmaceutical composition of the present invention may be administered in an "immunologically effective amount", "antitumor effective amount", "tumor-inhibitory effective amount" or "therapeutic amount". "Treatment" refers to administering a treatment regimen according to the present specification to a subject to obtain at least one positive therapeutic effect (e.g., reduction in the number of cancer cells, reduction in tumor volume, slowing down the rate of cancer cell invasion into surrounding organs, or slowing down the rate of tumor metastasis or tumor growth). When an "immunologically effective amount", "antitumor effective amount", "tumor-inhibitory effective amount" or "therapeutic amount" is specified, the exact amount of the composition of the present invention to be administered may be determined by a physician taking into account the age, weight, tumor size, degree of infection or metastasis, and individual differences in the disease of the patient (subject). Typically, the pharmaceutical composition comprising T cells according to the present specification is administered in an amount of 10 4 ~10 9 Dose of 10 cells / kg body weight, preferably 10 5 ~10 6The dose may be in units of cells / kg body weight. The T cell compositions may be administered multiple times at these doses. The cells may be administered by injection techniques well known in immunotherapy (see, e.g., Rosenberg et al. New Eng. J. of Med. 319:1676, 1988). The optimal dosage and treatment regimen for a particular patient may be readily determined by one of ordinary skill in the medical arts by monitoring the patient's condition and adjusting the treatment accordingly.

[0105] The compositions may be administered by any convenient method, including spray, injection, oral, infusion, implantation or transplantation. The compositions herein may be administered to a patient by subcutaneous, intradermal, intratumoral, intranodal, intraspinal, intramuscular, intravenous or intraperitoneal injection. In one embodiment, the T cell compositions of the present invention may be administered to a patient by intradermal or subcutaneous injection. In another embodiment, the T cell compositions of the present invention are preferably administered by intravenous injection. The T cell compositions may be injected directly into a tumor, lymph node or infection.

[0106] In some embodiments of the present invention, the CAR-T cells of the present invention or compositions thereof may be combined with other therapies known in the art, including but not limited to chemotherapy, radiation therapy, and immunosuppressants. For example, the CAR-T cells of the present invention may be combined with radiation therapy or chemotherapy agents for treating mesothelin-mediated diseases known in the art.

[0107] As used herein, the term "antitumor effect" refers to a biological effect manifested as a reduction in tumor volume, a reduction in tumor cell number, a reduction in the number of metastases, an increase in life expectancy, or an improvement in various physiological symptoms associated with cancer.

[0108] The terms "patient," "subject," "individual," and the like, which may be used interchangeably herein, refer to a living organism in which an immune response can be generated, such as, for example, a mammal, including, but not limited to, humans, dogs, cats, mice, rats, and transgenic species thereof.

[0109] The present invention is described in more detail with reference to the following experimental examples. These examples are presented for illustrative purposes and are not limiting unless otherwise specified. Therefore, the present invention is not limited to the following examples, and includes any and all variations made clear by the teachings provided by this specification. The methods and reagents used in the examples are conventional methods and reagents in the art unless otherwise specified.

[0110] Diagnostic, Detection and Reagent Kits The binding molecules of the present invention have high affinity for CD70 and can therefore be used in assays, for example to detect and / or quantify CD70 expressed in tissues or cells. Binding molecules such as single domain antibodies can be used to study the effects of CD70 in diseases. Methods for detecting CD70 include roughly obtaining a cell and / or tissue sample and detecting the level of CD70 in the sample.

[0111] The CD70 binding molecules of the present invention can be used for diagnostic purposes to detect, diagnose or monitor diseases and / or conditions associated with CD70. The present invention provides for detecting the presence of CD70 in a sample by representative immunohistological methods well known to those skilled in the art. CD70 can be detected in vivo or in vitro. Examples of suitable methods for detecting the presence of CD70 include ELISA, FACS, RIA, etc.

[0112] For diagnostic applications, the binding molecule, such as a single domain antibody, is usually labeled with a detectable labeling group. Suitable labeling groups include, but are not limited to, the following: radioisotopes or radionuclides (e.g., 3H, 14C, 15N, 35S, 90Y, 99Tc, 111In, 125I, 131I), fluorophores (e.g., FITC, rhodamine, lanthanide phosphors), enzyme groups (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminophores, biotin groups or predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper sequences, binding sites utilized for secondary antibodies, metal binding domains, epitope tags), MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents. Each method for labeling proteins is well known in the art and can be used to practice the present invention.

[0113] Another aspect of the invention provides a method for detecting the presence of a test molecule that binds competitively to CD70 with an antibody of the invention. The illustrative measurement involves detecting the amount of free antibody in a solution containing a given amount of CD70 in the presence or absence of the test molecule. An increase in the amount of free antibody (i.e., antibody that is not bound to CD70) indicates that the test molecule is capable of binding competitively to CD70 with respect to the antibody. In one embodiment, the antibody is labeled with a labeling group. Alternatively, the test molecule is labeled and the amount of free test molecule is monitored in the presence or absence of the antibody.

[0114] The present invention further provides a detection kit for detecting the level of CD70, the kit comprising an antibody that recognizes the CD70 protein, a dissociation medium for lysing the sample, and general reagents and buffers required for detection, such as various buffers, detection labels, detection substrates, etc. The detection kit may be an in vitro diagnostic device.

[0115] The present invention will be described below with reference to specific examples. These examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The methods and materials used in the examples are well known in the art unless otherwise specified.

[0116] Working Example Example 1: Construction of expression vector and eukaryotic expression of recombinant human CD70 protein.

[0117] 1. Synthesis of the gene sequence of the CD70 amino acid stretch from amino acid 39 to 193 and construction of the protein expression vector.

[0118] The amino acid sequence from 39th to 193rd amino acid of CD70 (uniprot accession No: P32970-1) was introduced into an online codon optimization tool (http: / / www.jcat.de / #opennewwindow) to obtain a codon-optimized nucleic acid sequence, and then the gene sequence was obtained by chemical synthesis, and the coding sequence of alpaca IgG1-Fc (the amino acid sequence is shown in SEQ ID NO: 51) was added to the 3' end of the gene sequence. The splicing product was cloned into pCDNA3.1 (Thermo) by molecular cloning using a TaKaRa seamless cloning kit to obtain an expression vector.

[0119] 2. Expression, purification and activity identification of recombinant human CD70 protein.

[0120] The expression vector was transfected into 293T cells (ATCC) for 5 days, and the culture supernatant was collected and the recombinant human CD70-alFc protein was purified using AKTA explorer 100 (GE). Due to glycosylation modification and other reasons, the recombinant human CD70-alFc protein showed a size of about 50 kilodaltons after CBB staining by reducing SDS-PAGE electrophoresis, and the results are shown in Figure 1.

[0121] ELISA activity identification was performed on recombinant human CD70-alFc protein with commercialized CD27. Recombinant human CD70-alFc protein was coated on ELISA plate with 4-fold gradient dilution, 100ng coated in the first well, 4-fold serial dilution (100uL / well), incubated at 4℃ overnight, washed 3 times with 200uL PBST the next day, blocked for 1 hour by adding 1% BSA / PBS. Blocking buffer was removed, and 100μL 0.1μg / mL recombinant human CD27-his protein was added per well and incubated for 1 hour at 37℃. After washing 3 times with 200μL PBST, rabbit anti-his-tag antibody (100μL per well) diluted 1:4000 and labeled with horseradish peroxidase was added and incubated for 1 hour at 37℃. After washing three times with 200 μL PBST, 100 μL TMB developing solution was added and developed at 37° C. for 10 minutes, 100 μL 1M hydrochloric acid was added to terminate the development, and OD450 was read. The analytical results are shown in FIG. 2 and Table 1.

[0122] [Table 1]

[0123] Example 2: Production of anti-human CD70 alpaca VHH antibodies 1. Immunized Alpacas 2mg / mL CD70-alIgG1 Fc fusion protein was mixed with an equal volume of complete Freund's adjuvant (Sigma-Aldrich) to emulsify the antigen, and adult alpacas were subcutaneously immunized with 500μg of antigen per animal. After the first immunization, booster immunization was performed once every 20 days, for a total of four subcutaneous immunizations. Seven days after the fourth immunization, 100mL of whole blood was collected from the vein and PBMCs were isolated.

[0124] 2. Serum titer detection Before each booster immunization, 10 mL of blood was drawn from the vein, cells were removed by centrifugation, and serum was retained. ELISA microwell plates were coated overnight at 4°C with 50 ng / well of CD70-his protein (ACRO Biosystems). After washing three times with PBS, 200 μL / well of 1% BSA / PBS was added and blocked for 1 hour at 37°C. Gradient-diluted alpaca serum was added and bound for 1 hour at 37°C. After washing three times with PBST, 100 μL of 1:5000 diluted HRP-goat anti-alpaca IgG1-Fc (Jackson ImmunoResearch) was added and bound for 1 hour at 37°C. After washing three times with PBST, 100 μL / well of TMB development solution was added and developed for 10 minutes at 37°C, 100 μL / well of ELISA stop solution was added, and OD450 values ​​were read using a microplate reader. The serum titer detection results are shown in Table 2.

[0125] [Table 2]

[0126] 3. Construction of immune libraries 3.1 Obtaining alpaca PBMC total cDNA Total RNA from alpaca PBMCs was extracted using a Trizol RNA extraction kit. First-strand cDNA was synthesized using the RNA as a template using the SuperScript® IV First-Strand Synthesis System reagent kit.

[0127] 3.2 Amplification of VHH genes Using the cDNA as a template, the heavy chain gene was PCR amplified with an upstream primer for the heavy chain variable region and a downstream constant region CH2 primer (VH-F, CH2-R). 25 μL PrimeSTAR MAX master mix (Takara), 2.5 μL (25 pmol) of the upstream primer, 2.5 μL (25 pmol) of the downstream primer, 1.5 μL DMSO, 0.5 μL cDNA, and 18 μL ddH2O were added to a 50 μL reaction system. The PCR reaction was carried out in the following steps. After pre-denaturation at 98 ° C for 1 minute, temperature cycling was started, denaturation at 98 ° C for 110 seconds, annealing at 60 ° C for 15 seconds, extension at 72 ° C for 30 minutes, repeated 25 times, and finally extension at 72 ° C for 10 minutes.

[0128] The VHH-CH2 gene obtained by amplification was recovered with a DNA gel recovery reagent kit, and the electrophoresis results are shown in Figure 3. 100 ng VHH-CH2 was taken as a template, and the VHH gene was PCR amplified with the upstream primer VH-F and downstream primer VH-R. 25 μL PrimeSTAR MAX master mix (Takara), 2.5 μL (25 pmol) of the upstream primer, 2.5 μL (25 pmol) of the downstream primer, 1.5 μL DMSO, 0.5 μL VH-CH2 DNA, and 18 μL ddH2O were added to a 50 μL reaction system. The PCR reaction was carried out in the following steps. After pre-denaturation at 98 ° C for 1 minute, temperature cycling was started, followed by denaturation at 98 ° C for 110 seconds, annealing at 60 ° C for 15 seconds, extension at 72 ° C for 30 minutes, repeated 25 times, and finally extension at 72 ° C for 10 minutes. The VHH gene fragments obtained by amplification were recovered using a gel recovery reagent kit, and the electrophoretic results are shown in FIG.

[0129] 3.3 Construction of immune library The VHH gene fragment and the pcomb3X-TT vector were each digested with SfiI DNA endonuclease (Scripps Institute, USA). 2 μL of SfiI, 5 μL of 10x buffer, and 3 μg of DNA were added to a 50 μL reaction system, and ddH2O was added up to 50 μL. After thorough homogeneity, the mixture was incubated at 50°C for 3 hours.

[0130] The digested VHH gene fragments and pcomb3X vector were collected using a DNA gel collection reagent kit. The digested VHH gene fragments and the digested pcomb3X vector were cyclized with T4 ligase. 1 μL of T4 ligase, 5 μL of 10x buffer, 150 ng of VHH gene, and 1000 ng of pComb3X vector were added to a 50 μL reaction system, and ddH2O was added up to 50 μL. After thorough homogeneous mixing, the mixture was incubated at 4°C for 16 hours. A small amount of the product was taken and the ligation efficiency was verified by agarose gel electrophoresis, and the electrophoretic results are shown in Figure 5.

[0131] 10 μL of the above ligated cyclized product was added to the homemade TG1 electrocompetence, and then electroporation was performed using an electroporator. 10 μL of the bacteria after electroporation was taken, appropriately diluted, and lined on a plate containing ampicillin, and then counted to statistically determine the size of the phage antibody library. The remaining bacteria after electroporation were added to 2xYT medium containing 100 μg / mL ampicillin and 2% glucose, and cultured in a heated incubator. After the culture was completed, the mixture was centrifuged at 4°C and 4000G for 10 minutes, and the precipitated bacteria were supplemented with an appropriate amount of glycerol and stored at -80°C to obtain the antibody species library. The scFv immune library was accumulated through multiple electroporations and exceeded the 9E+9 library capacity.

[0132] 4. Selection of CD70 Antibody 4.1 Coupling of recombinant human CD70 protein to streptavidin magnetic beads Biotinylated CD70 protein was obtained by biotinylating the avi-tag of recombinant human CD70 protein using a biotinylation reagent kit (iGeneBio) according to the instructions in the kit. 10 μg of the biotinylated recombinant protein was added to 100 μL streptavidin magnetic beads (DynaBeads 280) that had been washed three times with PBS, placed on a rotary shaker, and coupled for 30 minutes at room temperature at a speed of 18 rpm, then washed three times with PBS.

[0133] 4.2 Blocking of phage libraries and magnetic beads Add 0.5mL 1% BSA / PBS to 0.5mL phage library, place on a rotary shaker, rotate at 18 rpm and room temperature for 1 hour to block, these phages are Input1. At the same time, take 100μL of uncoupled protein DynaBeads 280, wash with PBS three times, add 1mL 1% BSA / PBS, and incubate with rotation under the above conditions for 1 hour. Also, add 1mL 1% BSA / PBS to the above coupled CD70 magnetic beads, rotate under the above conditions for 1 hour to block.

[0134] 4.3 Negative selection Negative selection is required to remove antibodies that interact with the magnetic beads. The BSA-blocked phage library and uncoupled antigen magnetic beads were mixed and incubated under rotation for 1 hour under the above conditions. After incubation, the phage-magnetic bead mixture was placed on a grid magnet to allow the magnetic beads to adhere, and the supernatant was then transferred to a new EP tube.

[0135] 4.4 Positive selection The magnetic beads coupled with the blocked CD70 protein were added to the negatively selected phage supernatant to perform positive selection, and incubated under the above conditions at room temperature for 1 hour with rotation. After incubation, the magnetic beads were washed with 1 mL PBST (0.1% Tween-20 in PBS) and washed 10 times. After washing, 1 mL 100 mM glycine (pH 2.0) was added, placed on a rotary shaker, set the speed to 18 rotations / min, and rotated for 10 minutes to elute. After elution, the EP tube was placed on a grid magnet to attach the magnetic beads, and the eluate was transferred to a new EP tube. The eluate was neutralized by adding 0.2 mL 1 M Tris-HCl solution (pH 8.0). The neutralized eluate was added to 30 mL of TG1 bacterial solution with an OD600 of approximately 0.6, and left to infect for 30 minutes. Next, 20 times the number of bacteria, M13KO7 phages, were added and left to infect for 30 minutes. Finally, 100 mL of 2YT medium and ampicillin and kanamycin at a final concentration of 100 μg / mL were added and cultured overnight at 30° C. and 220 rpm. The next day, the phages were harvested using the above-mentioned phage library harvesting method, and the phages obtained at this time are Input2.

[0136] 4.5 Repeated positive selection The above selection method was repeated twice, and the following negative and positive selections were performed on Input 2 to obtain Input 3. The difference is that after infecting TG1 with the eluate obtained by selecting Input 3, M13KO7 was not added, and 10 μL of the bacterial solution was taken and gradient diluted to 10 3 , 10 4 , 10 5 Three dilution gradients were taken, and 100 μL of each bacterial solution was applied to a 2YT / amp plate and cultured at 30° C. overnight. The remaining bacterial solution was cultured at 30° C. and 220 rpm overnight.

[0137] 4.6 Selection of positive antibodies by ELISA TG1 was randomly taken from the above plate with a toothpick, monocloned into a deep well plate containing 600μL 2YT / amp, a gas permeable membrane was attached to the deep well plate, and the plate was cultured at 37℃ and 220rpm for 3 hours, after which the gas permeable membrane was peeled off, IPTG was added to the well at a final concentration of 1mM, and the plate was cultured overnight at 30℃ and 220rpm. ELISA plates were coated with recombinant human CD70 protein at 100ng per well. The next day, the deep well plate was centrifuged at 4000rpm for 10 minutes, the medium in the well was removed, the bacterial cell precipitate was retained, 100μL TES solution (20% sucrose, 0.1mM EDTA, 50mM Tris-HCl, pH 8.0) was added per well, the bacterial cells were resuspended by shaking, and the plate was placed in an ice bath for 30 minutes, 200μL ultrapure water was added, the mixture was mixed uniformly by shaking, and the plate was centrifuged at 4000rpm for 10 minutes. The supernatant in the deep well plate is the periplasmic space extract containing the antibody. After washing the ELISA plate three times with a microplate washer, 200μL 1% BSA / PBS was added and blocked at 37℃ for 1 hour. The blocking buffer in the ELISA plate was removed, 100μL of the above periplasmic space extract was added, incubated at 37℃ for 1 hour, washed three times with a microplate washer, HRP-conjugated-Goat anti HA (horseradish peroxidase-labeled sheep anti-HA antibody) solution was added, incubated at 37℃ for 1 hour, washed three times with a microplate washer, 100μL TMB development solution was added, developed at 37℃ for 10 minutes, and 100μL 1M hydrochloric acid was added to terminate the process. The OD450 value was read with a microplate reader, and Sanger sequencing was performed on the clones with a reading value higher than 3 times the background value to obtain the gene sequence of the antibody.

[0138] 4.7 Verification of positive cloning According to the sequencing results, the clones with the largest difference in the antibody CDR3 amino acid sequence were selected and re-inoculated, induced overnight, and the selected clones were re-tested by the above ELISA method to see whether they could bind to CD70. Finally, eight VHH antibody sequences were obtained: CD70-11C9, CD70-8E1, CD70-8F9, CD70-9D8, CD70-2A5, CD70-5C10, CD70-8A6, and CD70-8B4.

[0139] The heavy chain variable region amino acid sequence of CD70-2A5 is shown in SEQ ID NO:21.

[0140] The heavy chain variable region amino acid sequence of CD70-5C10 is shown in SEQ ID NO:22.

[0141] The heavy chain variable region amino acid sequence of CD70-8A6 is shown in SEQ ID NO:23.

[0142] The heavy chain variable region amino acid sequence of CD70-8B4 is shown in SEQ ID NO:24.

[0143] The heavy chain variable region amino acid sequence of CD70-8E1 is shown in SEQ ID NO:25.

[0144] The heavy chain variable region amino acid sequence of CD70-8F9 is shown in SEQ ID NO:26.

[0145] The heavy chain variable region amino acid sequence of CD70-9D8 is shown in SEQ ID NO:27.

[0146] The heavy chain variable region amino acid sequence of CD70-11C9 is shown in SEQ ID NO:28.

[0147] Example 3: Expression of recombinant VHH antibodies Eight VHH antibody genes, CD70-2A5, CD70-5C10, CD70-8A6, CD70-8B4, CD70-8E1, CD70-8F9, CD70-9D8, and CD70-11C9, were constructed into pcDNA3.1-huIgG1-Fc by homologous recombination to obtain recombinant VHH-huIgG1-Fc. 293F cells were transiently transfected with the above vector to perform eukaryotic expression, and the supernatant was harvested and then purified with HiTrap Protein A HP / AKTA pure100. The purified protein was concentrated with an ultrafiltration device, the buffer was replaced with PBS, and the purity of the protein was detected by SDS-PAGE electrophoresis. The electrophoresis results are shown in Figure 6.

[0148] Example 4: Measurement of binding curves between recombinant human CD70 protein and eight VHH antibodies The ELISA experiment is specifically performed as follows: 100ng / well of the recombinant human CD70 protein prepared above was added to a microplate and coated overnight at 4°C. After washing with PBS three times, 200μL / well of 1% BSA / PBS was added and blocked at 37°C for 1 hour. After washing the plate with 100μL PBS, the above four scFv proteins were added in a gradient dilution and allowed to bind at 37°C for 1 hour. After washing three times with PBST, 100μL of 1:5000 diluted HRP-goat anti-human IgG (Fab specific) was added and allowed to bind at 37°C for 1 hour. After washing three times with PBST, 100μL / well of TMB developing solution was added and developed at 37°C for 10 minutes, 100μL / well of ELISA stopping solution was added, and the OD450 value was read using a microplate reader, and the results are shown in Figure 7 and Table 3.

[0149] [Table 3]

[0150] Example 5: Affinity Measurements The affinity of four VHH-huFc antibodies, CD70-2A5, CD70-8B4, CD70-9D8, and CD70-11C9, to human CD70 was analyzed using an Octet K2 biomolecular interaction analyzer. SA probe clots were performed with 200 μL 100 nM biotinylated recombinant human CD70 protein, and the clot height was 1 nM. The four VHH-huFc antibodies were purified and used as the analysis material. The affinity measurements were performed at four concentrations: 200 nM, 100 nM, 50 nM, and 25 nM. The results are shown in Figure 8 and Table 4.

[0151] [Table 4]

[0152] Example 6: Blockade of CD27 / CD70 binding by VHH-huFc The ELISA experiment was carried out as follows: 100ng / well of recombinant human CD27 protein was added to the microplate and coated overnight at 4℃. After washing with PBS three times, 200μL / well of 1% BSA / PBS was added and blocked at 37℃ for 1 hour. 100ng of biotinylated CD70 recombinant protein was incubated with 1μg, 500ng, 250ng, and 125ng of the above VHH-huFc antibody for 1 hour. After washing the plate with 200μL PBS, the above CD70 / VHH-Fc mixture was added and allowed to bind at 37℃ for 1 hour. After washing with PBST three times, 100μL of 1:200 diluted SA-HRP was added and allowed to bind at 37℃ for 1 hour. After washing three times with PBST, 100 μL / well of TMB developing solution was added, and the plate was developed at 37° C. for 10 minutes. ELISA stopping solution was added at 100 μL / well, and the OD450 value was read using a microplate reader. The results are shown in FIG. 9 and Table 5.

[0153] Example 7: Preparation of retroviral stocks containing anti-human CD70 chimeric antigen receptor elements 1. Preparation of chimeric antigen receptors targeting human CD70 antigen The chimeric antigen receptor sequences of single chain antibody scFv containing anti-human CD70 antigen, hinge region, transmembrane region and intracellular signal domain were gene synthesized or cloned, and their structures are shown in Figure 10. Depending on the VHH to be assembled, the chimeric antigen receptors are named CD70-2A5-BBz, CD70-5C10-BBz, CD70-8A6-BBz, CD70-8B4-BBz, CD70-8E1-BBz, CD70-8F9-BBz, CD70-9D8-BBz and CD70-11C9-BBz, respectively, and their amino acid sequences are shown in SEQ ID NOs: 29-36, respectively, and their nucleotide sequences are shown in SEQ ID NOs: 42-49, respectively. At the same time, as a control, a scFv of a known CD70 antibody (clone number: ARGX110) reported in the literature was selected to construct a chimeric antigen receptor, named ARGX-BBz, whose nucleic acid sequence is shown in SEQ ID NO:52.

[0154] Using the retrovector MSGV as a backbone vector, chimeric antigen receptor retroviral plasmids expressing CD70-2A5-BBz, CD70-5C10-BBz, CD70-8A6-BBz, CD70-8B4-BBz, CD70-8E1-BBz, CD70-8F9-BBz, CD70-9D8-BBz and CD70-11C9-BBz and ARGX-BBz clones were constructed. The clones with accurate sequencing were selected, and the bacterial liquid was inoculated into 300 mL LB medium, shaken uniformly overnight, and the large-scale production of plasmids was completed according to the NucleoBond Xtra Maxi EF Reagent Kit instruction manual.

[0155] 2. Retroviral packaging The retrovirus was packaged with cationic polymer PEI (Polyplus), and the process was as follows: PEI and retrovirus packaging plasmids (viral main plasmid, Gag-pol, 10A1) were diluted in serum-free DMEM, respectively, and then PEI / DMEM was added to the plasmid / DMEM mixture, vortexed evenly, and allowed to stand at room temperature for 15 minutes, and the plasmid-PEI complex was added to the pre-plated 293T cells. 16 hours after transfection, the liquid was exchanged, and 48 hours later, the virus supernatant was collected, filtered through a 0.45um filter, and the stock solution was dispensed into 15mL centrifuge tubes and stored at -80℃ for use.

[0156] Example 8: Production of CD70 CAR-T cells and measurement of CAR positive rate 1. Isolation and activation of PBMCs Peripheral blood was collected from volunteers and isolated with Ficoll isolation solution to obtain PBMCs. The cells were cultured in X-VIVO (LONZA) medium containing 5% AB serum at a cell density of 1x10 6 The concentration was adjusted to 1 mL / well. A TC-coated 6-well plate was incubated at 37°C for 2 h using 1 mL of coating solution containing 50 ng / mL anti-human CD3 antibody (T&L Biotechnology) and 50 ng / mL CD28 antibody (T&L Biotechnology), and the coating solution was removed before use. Cells were seeded at 1 mL / well on the antibody-coated 6-well plate, and 100 IU / mL IL2 (SL PHARM) was added to stimulate and culture for 48 hours, after which they were infected with the virus.

[0157] 2. Infection and culture of virus stock 5x10 activated T cells 51 mL of T cells and 1 mL of virus stock solution were added to a 24-well plate, 1 μL of polybrene was added per well, and the mixture was centrifuged at 32°C and 2500 rpm for 1.5 h. The supernatant was discarded, and 1 mL of T cell medium (containing IL-2 100 IU / mL) was added per well. The culture plate was placed in a 37°C, 5% CO2 incubator for culture. 24 hours after infection, the cells were transferred to a 6-well plate, and the cell density was observed every day. The T cell culture medium containing IL-2 100 IU / mL was replenished as appropriate, and the T cell density was adjusted to approximately 1 x 10 6 / mL and the cells were expanded.

[0158] 3. Detection of CAR positivity The retrovirus-infected T lymphocytes were used to detect CAR positivity 72 h after viral infection. The chimeric antigen receptor groups including CD70-2A5-BBz, CD70-5C10-BBz, CD70-8A6-BBz, CD70-8B4-BBz, CD70-8E1-BBz, CD70-8F9-BBz, CD70-9D8-BBz, and CD70-11C9-BBz, as well as the ARGX-BBz clone, and the negative uninfected control group NT were each 1 × 10 6 The individual cells were taken, the medium was removed by centrifugation, the cells were washed once with PBS, and then resuspended in 100 μL in a flow cytometry sample tube (BD). Fc-labeled CD70 antigen (1:100) was added and incubated at 4 °C for 30 minutes. The cells were washed once with PBS, and then secondary antibody PE-Fc was added at the recommended ratio and incubated at 4 °C for 30 minutes in the dark. The cells were washed once with PBS, and then resuspended in 200 μL PBS and detected by the device. The flow cytometry analysis results of CAR-T positive rate are shown in Figure 11.

[0159] Example 9: Functional analysis based on anti-human CD70 CAR-T cells 1. Analysis of CD107a expression on anti-human CD70 CAR-T cells CAR-T cells and NT cells containing different antibody clones were incubated with target cells (CD70-positive cell line MOLM13) at an ET ratio of 1:1 (3x10 effector cells and 3x10 target cells). 5 The effector cells and target cells were co-incubated with each other and then the CD107a expression status was detected by flow cytometry to evaluate the degranulation response of CAR-T cells after stimulation with target cells. The effector cells and target cells were mixed and co-incubated at 37°C in a 5% CO2 incubator for 4 hours, and the ratio of cells expressing CD107a to the number of CD3+ cells in each group sample was detected by flow cytometry. The flow cytometry analysis results of CD107a expression are shown in Figure 12.

[0160] 2. Detection of anti-human CD70 CAR-T cell cytokine secretion ability CAR-T cells containing different antibody clones were cultured with target cells (CD70-positive cell line MOLM13) at an ET ratio of 1:1 (1x10 effector cells and 1x10 target cells). 5 After co-incubation with 100 μg / ml of 10 ... for 24 hours, the supernatant was collected and the secretion status of IFN-γ and IL-2 was detected by ELISA (enzyme-linked immunosorbent assay). IFN-γ was detected using BD IFN-γ and IL-2 reagent kit, and the experimental procedure was performed according to the product instruction manual. The detection results of IFN-γ secretion are shown in Figure 13. The detection results of IL-2 secretion are shown in Figure 14.

[0161] 3. Anti-human CD70 CAR-T cytotoxicity experiment The CAR-T cytotoxicity experiment evaluated the in vitro function of CAR-T cells by detecting the in vitro cytotoxic effect of CAR-T cells on target cells. 4Based on target cells, T cells were co-cultured with CD70-positive target cells MOLM13-LUC-GFP stably expressing firefly luciferase at ET ratios of 10:1, 5:1, and 2.5:1, respectively, and a negative control group (NT) consisting of a mixture of target cells and T cells not transfected with the CAR element was also set up. After overnight incubation, a luciferase reaction substrate was added to the culture system, the fluorescence value was detected, and the killing efficiency was calculated according to the following formula: Killing efficiency = (1 - experimental well fluorescence value / control well fluorescence value) x 100%. The experimental grouping and analysis results are shown in Figure 15.

[0162] Example 10: In vivo efficacy test of anti-human CD70 CAR-T cell injection The method according to Example 8 of the present invention was used to produce CAR-T cells of four clones, CD70-2A5 / 8B4 / 9D8 / 11C9, which were used in animal in vivo efficacy testing, and the CAR-T positive rate was detected, and the in vitro killing efficiency of the produced CAR-T cells against ACHN cells (human renal cancer cell line, CD70 expression) was detected, and MOLM13 cells were used as a control.

[0163] 1. Detection of CAR positivity The retrovirus-infected T lymphocytes were used to detect CAR positivity 72 h after viral infection. The chimeric antigen receptor groups containing CD70-2A5-BBz, CD70-8B4-BBz, CD70-9D8-BBz, CD70-11C9-BBz and ARGX-BBz clones and the negative uninfected control group NT were each 1 × 10 6 The individual cells were taken, the medium was removed by centrifugation, the cells were washed once with PBS, and then resuspended in 100 μL in a flow cytometry sample tube (BD). Fc-labeled CD70 antigen (1:100) was added and incubated at 4 °C for 30 minutes. The cells were washed once with PBS, and then secondary antibody PE-Fc was added at the recommended ratio and incubated at 4 °C for 30 minutes in the dark. The cells were washed once with PBS, and then resuspended in 200 μL PBS and detected by the device. The flow cytometry analysis results of CAR-T positive rate are shown in Figure 16.

[0164] 2. Analysis of CD107a expression on anti-human CD70 CAR-T cells CAR-T cells and NT cells containing CD70-2A5, CD70-8B4, CD70-9D8, and CD70-11C9 were incubated with ACHN and MOLM13 cells (renal cancer cell line ACHN and promyelocytic leukemia cell line MOLM13), respectively, at an ET ratio of 1:1 (effector cells and target cells were both 3 × 10 5 The effector cells and target cells were co-incubated with each other and then the CD107a expression status was detected by flow cytometry to evaluate the degranulation response of CAR-T cells after stimulation with target cells. The effector cells and target cells were mixed and co-incubated at 37°C in a 5% CO2 incubator for 4 hours, and the ratio of cells expressing CD107a to the number of CD3+ cells in each group sample was detected by flow cytometry. The flow cytometry analysis results of CD107a expression are shown in Figure 17.

[0165] 3. Cytotoxicity of anti-human CD70 CAR-T cells against ACHN and MOLM13 cells CAR-T cells and NT cells containing CD70-2A5, CD70-8B4, CD70-9D8, and CD70-11C9 were cultured at different ET ratios (3×10 4 The target cells were co-cultured with CD70-positive ACHN and MOLM13 target cells (ACHN-LUC-GFP and MOLM13-LUC-GFP) that stably express firefly luciferase at ET ratios of 10:1, 5:1, and 2.5:1, respectively. After overnight incubation, a luciferase reaction substrate was added to the culture system, the fluorescence value was detected, and the killing efficiency was calculated according to the following formula: Killing efficiency = (1 - fluorescence value of experimental well / fluorescence value of control well) x 100%. The experimental grouping and analysis results are shown in Figure 18.

[0166] 4. Drug efficacy experiments in mice Forty-eight female mice bearing experimental NOG tumors (subcutaneously inoculated with human renal cell line ACHN cells) were examined for tumor volumes of approximately 100 mm 3When the tumors grew to 100x, they were randomly divided into 6 groups according to tumor volume and intravenously administered NT cell injections and 5 different clones of CD70 CAR-T cells, the codes of which are P376, 2A5, 11C9, 8B4, and 9D8, respectively. Each group was administered 200μL / mouse at a dose of 3×10 7 Total cells / mouse. Clinical observation was performed once a day during the test period. Approximately 100 μL of anticoagulated blood was taken from the orbit of each mouse on Day 1, Day 7, Day 16, and Day 22, and the residual T cell status in the mouse body was detected by flow cytometry. The tumor volume was measured approximately twice a week, and the survival status of the mouse was recorded (when the mouse tumor volume reached 2000 mm 3 (If the mice reach 0.5 mg / kg / day, they are immediately euthanized.) The study was continued until D24, and the results of monitoring the tumor volume are shown in FIG. 19, and the results of detecting the percentage of T cells are shown in FIG.

[0167] According to the above results, the CD70-targeting CAR-T cells provided by the present invention, constructed with a new VHH antibody that specifically binds to CD70, have strong immune functions, and compared with the control CAR-T (clone number ARGX110) cells, they show superior CD107a expression, IFN-γ and IL-2 secretion, and specific killing function against target cells, with significant in vivo efficacy.

[0168] The above description is merely a preferred embodiment of the present invention, and does not limit the present invention. The present invention may have various modifications and changes for those skilled in the art. Any modifications, equivalent replacements, improvements, etc. that do not deviate from the spirit and principles of the present invention are included in the scope of the claims of the present invention.

Claims

1. 1. A CD70 binding molecule comprising an anti-CD70 Nanobody or an antigen-binding fragment thereof, wherein the complementarity determining regions (CDRs) of said anti-CD70 Nanobody comprise CDR1, CDR2 and CDR3, in which CDR1 comprises a sequence as set forth in any one of SEQ ID NOs: 1-7, CDR2 comprises a sequence as set forth in any one of SEQ ID NOs: 8-13, and CDR3 comprises a sequence as set forth in any one of SEQ ID NOs: 14-20, Preferably, the CD70 binding molecule further comprises the following characteristics: the heavy chain variable region sequence of said anti-CD70 Nanobody is set forth in any one of SEQ ID NOs:21-28; the CD70 binding molecule is a monovalent or multivalent nanobody or single domain antibody, or a multispecific nanobody or single domain antibody, comprising one, two or more anti-CD70 nanobodies or antigen-binding fragments thereof; the nanobody is a camelid heavy chain antibody or a cartilaginous fish heavy chain antibody; the Nanobody further comprises a heavy chain constant region; The CD70 binding molecule has one or more characteristics selected from the following: the CD70 binding molecule is a chimeric antibody or a fully human antibody.

2. 1. A chimeric antigen receptor comprising an optional signal peptide sequence, the CD70 binding molecule of claim 1, a hinge region, a transmembrane region, and an intracellular region, Preferably, the intracellular region comprises an intracellular costimulatory domain and / or an intracellular signaling domain; Preferably, the chimeric antigen receptor comprises, from the N-terminus to the C-terminus, a signal peptide, the CD70 binding molecule of claim 1, a hinge region, a transmembrane region, an intracellular costimulatory domain and an intracellular signaling domain, in that order.

3. The following sequence: (1) The CD70-binding molecule according to claim 1 or A coding sequence for the chimeric antigen receptor of claim 2. (2) the complementary sequence of (1), (3) A nucleic acid molecule having any one of the sequences selected from the group consisting of a 5-50 bp fragment of any one of the sequences of (1) or (2), Preferably, the nucleic acid molecule, wherein said fragment is a primer.

4. A nucleic acid construct comprising the nucleic acid molecule of claim 3, Preferably, said nucleic acid construct is a cloning vector, an expression vector or an integration vector.

5. (1) The CD70-binding molecule according to claim 1 or One that expresses and / or secretes the chimeric antigen receptor of claim 2. (2) A nucleic acid molecule according to claim 3, and / or (3) A host cell comprising the nucleic acid construct according to claim 4, Preferably, said host cell is an immune effector cell, more preferably a T cell.

6. 10. A method for producing a CD70 binding molecule or a chimeric antigen receptor according to claim 1, comprising culturing a host cell according to claim 5 under conditions suitable for the production of the CD70 binding molecule, and optionally purifying said CD70 binding molecule or chimeric antigen receptor from the culture.

3. A method for generating a chimeric antigen receptor according to claim 2.

7. 10. A pharmaceutical composition comprising the CD70 binding molecule of claim 1, the chimeric antigen receptor of claim 2, the nucleic acid molecule of claim 3, the nucleic acid construct of claim 4 or the host cell of claim 5, and a pharma- ceutical acceptable auxiliary.

8. 13. The use of a CD70 binding molecule according to claim 1, a chimeric antigen receptor according to claim 2, a nucleic acid molecule according to claim 3, a nucleic acid construct according to claim 4 or a host cell according to claim 5 in the manufacture of an activated immune cell or in the manufacture of a medicament for the prevention or treatment of a disease or condition related to CD70 expression, Preferably, the disease or condition is one or more selected from renal cell carcinoma, acute myeloid leukemia, non-Hodgkin's lymphoma, multiple myeloma, mantle cell lymphoma, diffuse large cell lymphoma, follicular lymphoma, pancreatic cancer, breast cancer, and glioblastoma.

9. A reagent kit for detecting CD70 comprising a CD70 binding molecule according to claim 1, a nucleic acid molecule according to claim 3, a nucleic acid construct according to claim 4 or a host cell according to claim 5, Preferably, the reagent kit further comprises a reagent for detecting binding between CD70 and the CD70 binding molecule, more preferably, the reagent for detecting binding is a detectable label capable of binding to the CD70 binding molecule.

10. 13. Use of the CD70 binding molecule according to claim 1 in the manufacture of a reagent kit for detecting CD70 in a sample, for evaluating the efficacy of drug therapy, or for diagnosing cancer.

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