HK40088437B - Anti-cd70 nanoantibody and use thereof - Google Patents

Description

Anti-CD70 nanobodies and their applications

Technical Field

This invention relates to the field of bioimmunotherapy technology, specifically to anti-CD70 nanobodies and their uses.

Background Technology

CD70 belongs to the tumor necrosis factor (TNF) superfamily. In normal tissues, CD70 is a type II transmembrane glycoprotein, transiently expressed only in activated T cells, B cells, and mature dendritic cells, with CD27 as its receptor. Under pathological conditions, studies have found that CD70 is highly expressed in various tumor cells. Currently, immunotherapies targeting CD70 are already in preclinical studies.

Studies have shown that CD70 is abnormally elevated in a variety 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, making it a promising target for tumor immunotherapy.

Chimeric antigen receptor-T cells (CAR-T) are a novel immunotherapy approach that targets specific antigens on the surface of tumor cells. Currently, many researchers are developing CAR-T cells for the treatment of solid tumors.

Antibodies, as a crucial component of CARs, play a decisive role in the specific and efficient killing effect and reduced toxicity of CAR-T cells. Therefore, antibodies that can bind to target antigens with high specificity while exhibiting low immunogenicity are key to the development of CAR-T products. Alpacas can produce specific, highly affinity VHH antibodies. Furthermore, due to the inherent characteristics of VHH antibodies, they can recognize antigenic epitopes that traditional scFv antibodies cannot. In addition, the alpaca VHH gene has high homology with the human VH gene, resulting in relatively low immunogenicity and facilitating humanization. Currently, there are no reports in this field of CAR-T cells containing anti-CD70 nanobodies.

Summary of the Invention

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, wherein CDR1 comprises any of the sequences shown in SEQ ID NO:1-7, CDR2 comprises any of the sequences shown in SEQ ID NO:8-13, and CDR3 comprises any of the sequences shown in SEQ ID NO:14-20.

In one or more embodiments, the heavy chain variable region sequence of the anti-CD70 nanobody is shown in any of SEQ ID NO:21-28.

In one or more embodiments, the FR1 of the anti-CD70 nanobody may be selected from FR1 of any of the VHHs shown in SEQ ID NO:21-28, the FR2 may be selected from FR2 of any of the VHHs shown in SEQ ID NO:21-28, the FR3 may be selected from FR3 of any of the VHHs shown in SEQ ID NO:21-28, and the FR4 may be selected from FR4 of any of the VHHs shown in SEQ ID NO:21-28.

In one or more embodiments, 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 their antigen-binding fragments.

In one or more embodiments, the multivalent or multispecific binding molecule is linked to multiple anti-CD70 nanobodies or their antigen-binding fragments via a linker. The linker consists of 1-15 amino acids selected from G and S.

In one or more embodiments, the nanobody is a camel heavy chain antibody or a cartilaginous fish heavy chain antibody.

In one or more embodiments, the nanobody further comprises a heavy chain constant region.

In one or more embodiments, the heavy chain constant region is a constant region of a camel heavy chain antibody, comprising CH2 and CH3. In one or more embodiments, CH2 and CH3 are CH2 and CH3 of human IgG Fc, such as CH2 and CH3 of IgG1. Preferably, the heavy chain constant region is as shown in SEQ ID NO:37.

In one or more embodiments, the heavy chain constant region is a constant region of a cartilaginous fish heavy chain antibody, comprising CH1, CH2, CH3, CH4 and CH5.

In one or more embodiments, the CD70 binding molecule described in any embodiment of the present invention is a chimeric antibody or a fully human antibody; preferably a fully human antibody.

Another aspect of the present invention provides a chimeric antigen receptor comprising an optional signal peptide sequence, a CD70 binding molecule as described in any embodiment herein, a hinge region, a transmembrane region, and an intracellular region.

In one or more embodiments, the intracellular region includes an intracellular co-stimulatory domain and/or an intracellular signaling domain.

In one or more embodiments, from the N-terminus to the C-terminus, the chimeric antigen receptor sequentially comprises a signal peptide, a CD70 binding molecule as described in any of the embodiments herein, a hinge region, a transmembrane region, an intracellular co-stimulatory domain, and an intracellular signaling domain.

The present invention also provides a nucleic acid molecule having a sequence selected from any of the following:

(1) The coding sequence of the CD70 binding molecule or chimeric antigen receptor described in any of the embodiments herein;

(2) and (1) are complementary sequences;

A 5-50bp fragment of any sequence from (3), (1), or (2).

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

The present invention also provides a nucleic acid construct comprising the nucleic acid molecules described herein.

In one or more embodiments, the nucleic acid construct is a cloning vector, an expression vector, or an integration vector.

The present invention also provides a host cell selected from:

(1) Expressing and/or secreting the CD70 binding molecule or chimeric antigen receptor as described in any of the embodiments herein;

(2) Contains the nucleic acid molecules described herein; and/or

(3) Includes the nucleic acid constructs described in this article.

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

The present invention also provides a method for generating a CD70 binding molecule according to any embodiment herein, comprising: culturing the host cells described herein under conditions suitable for generating CD70 binding molecules (e.g., nanobodies or their antigen-binding fragments, monovalent or multivalent nanobodies or single-domain antibodies, or multispecific nanobodies or single-domain antibodies), and optionally purifying the CD70 binding molecule from the culture.

The present invention also provides a pharmaceutical composition comprising a CD70 binding molecule, a nucleic acid molecule, a nucleic acid construct or a host cell as described in any embodiment herein, and pharmaceutically acceptable excipients.

In one or more embodiments, the pharmaceutical composition is used to treat diseases or conditions related to CD70 expression.

The present invention also provides the use of the CD70 binding molecule, chimeric antigen receptor, nucleic acid molecule, nucleic acid construct or host cell described in any embodiment herein in the preparation of activated immune cells (e.g. T cells).

The present invention also provides the use of the CD70 binding molecule, chimeric antigen receptor, nucleic acid molecule, nucleic acid construct or host cell described in any embodiment herein in the preparation of a medicament for the prevention or treatment of diseases or conditions associated with CD70 expression.

In one or more embodiments, the disease or condition is selected from one or more of the following: 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.

The present invention also provides a method for treating or preventing diseases or conditions related to CD70 expression, the method comprising administering to a patient in need a therapeutically effective amount of the CD70 binding molecule or host cell as described in any embodiment of the present invention, or a pharmaceutical composition as described in any embodiment of the present invention.

The present invention also provides a kit for detecting CD70, for example, to evaluate the efficacy of drug treatment or to diagnose cancer, said kit comprising a CD70 binding molecule, nucleic acid molecule, nucleic acid construct or host cell as described in any embodiment herein.

In one or more embodiments, the kit further includes reagents for detecting the binding of CD70 to the CD70-binding molecule. For example, reagents for detecting the binding by an enzyme-linked immunosorbent assay (ELISA).

In one or more embodiments, the detection binding reagent is a detectable marker, such as biotin, that can be linked to a CD70-binding molecule. The detectable marker is either linked to the CD70-binding molecule or is present separately in the kit.

This invention also provides a non-diagnostic method for detecting the presence of CD70 in a sample, the method comprising: incubating the sample with the CD70 binding molecule described in any embodiment of this invention, and detecting the binding of CD70 to the CD70 binding molecule, thereby determining the presence of CD70 in the sample. The detection is performed using an enzyme-linked immunosorbent assay (ELISA).

The present invention also provides the use of the CD70 binding molecule described in any embodiment herein in the preparation of a kit for detecting CD70 in a sample, evaluating the efficacy of drug treatment, or diagnosing cancer.

The present invention has the following beneficial effects:

This invention provides a novel mini antibody that specifically recognizes CD70 and CAR-modified cells containing the antibody. The antibody and cells have good safety and therapeutic effects targeting CD70, providing a new treatment or improvement approach for diseases related to CD70 expression.

Attached Figure Description

To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

Figure 1 shows the SDS-PAGE electrophoresis results of the recombinant human CD70-huFc protein in Example 1.

Figure 2 shows the experimental results of the binding curve determination of recombinant human CD70-huFc protein and recombinant human CD27 protein in Example 1.

Figure 3 shows the DNA electrophoresis results after PCR amplification of the alpaca VH-CH2 gene in Example 2.

Figure 4 shows the DNA electrophoresis results after PCR amplification of the alpaca VHH gene in Example 2.

Figure 5 shows the DNA electrophoresis results of the VHH/pcomb3X ligation efficiency detection experiment in Example 2.

Figure 6 shows the SDS-PAGRE electrophoresis results of the recombinant VHH-huFc antibody protein in Example 3.

Figure 7 shows the binding curve determination results of recombinant VHH-huFc antibody/CD70-huFc recombinant protein in Example 4.

Figure 8 shows the results of the affinity assay for the recombinant VHH-huFc antibody in Example 5.

Figure 9 shows the experimental results of recombinant VHH-huFc blocking CD27/CD70 binding in Example 6.

Figure 10 is a schematic diagram of the CAR structure in Example 7.

Figure 11 shows the experimental results of infection efficiency of different clones of CD70 CAR-T cells in Example 8.

Figure 12 shows the results of CD107a activation experiments of different cloned CD70 CAR-T cells in Example 9.

Figure 13 shows the experimental results of IFN-γ release when different cloned CD70 CAR-T cells were co-incubated with target cells in Example 9.

Figure 14 shows the results of IL-2 release experiments when different cloned CD70 CAR-T cells were co-incubated with target cells in Example 9. REST is the NT cell control group.

Figure 15 shows the results of the killing experiment of different cloned CD70 CAR-T cells on target cells in Example 9.

Figure 16 shows the experimental results of infection efficiency of different clones of CD70 CAR-T cells in Example 10.

Figure 17 shows the results of CD107a activation experiments of different cloned CD70 CAR-T cells in Example 11.

Figure 18 shows the results of the killing experiment of different cloned CD70 CAR-T cells on target cells in Example 9.

Figure 19 shows the tumor volume monitoring results of the in vivo efficacy test of different clones of CD70 CAR-T cells in Example 11.

Figure 20 shows the T cell survival results of the in vivo efficacy test of different cloned CD70 CAR-T cells in Example 11.

Detailed Implementation

Through extensive and in-depth research and screening, the inventors have discovered a class of anti-CD70 nanobodies and their antigen-binding fragments that can specifically recognize CD70 and bind to it with high affinity, thereby blocking the binding of CD70 to CD27 and exhibiting good functional activity.

Specifically, this invention utilizes CD70 protein to immunize alpacas, obtaining a high-quality single-domain antibody gene library. Then, phage display technology is used to screen the antibody gene library, thereby obtaining CD70-specific single-domain antibody genes. These genes are then transformed into mammalian cells, resulting in antibody strains that can be efficiently expressed in mammalian cells and exhibit high specificity. High-affinity, high-specificity, and high-functional activity nanobodies are then identified using ELISA, molecular interaction analysis, and blocking assays. The antibodies or their antigen-binding fragments possess good safety and targeting properties, specifically binding to the extracellular domain of human CD70.

The present invention also provides a chimeric antigen receptor (CAR) containing the nanobody. Using a vector containing the coding sequence of the CAR to infect immune cells can yield immune effector cells with significant killing ability against tumor cells overexpressing CD70. These immune effector cells can be used to treat or improve CD70 expression-related diseases, thus laying the foundation for the treatment of CD70-positive tumors.

Antibody

In this article, "CD70 binding molecule" refers to a protein that specifically binds to CD70, including but not limited to antibodies, heavy chain antibodies, nanobodies, or their antigen-binding fragments.

In this document, the term "antibody" includes monoclonal antibodies (including full-length antibodies having the immunoglobulin Fc region), antibody compositions with multi-epitope specificity, multispecific antibodies (e.g., bispecific antibodies), biantibodies and single-chain molecules, and antibody fragments, especially antigen-binding fragments, such as Fab, F(ab’)2, and Fv. In this document, "antibody" and "immunoglobulin" are used interchangeably.

Traditional "antibodies" contain a basic four-chain antibody unit, 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 its N-terminus, followed by three (CH1, CH2, and CH3 for each α and γ chain) and four (CH1, CH2, CH3, and CH4 for μ and ε isoforms) constant domains (CH), and a hinge region located between the CH1 and CH2 domains. Each light chain has a variable domain (VL) at its N-terminus, followed by a constant domain (CL) at its other end. The paired VH and VL together form an antigen-binding site. For information on the structure and properties of different classes of antibodies, see Basic and Clinical Immunology, 8th Edition, edited by Daniel P. Sties, Abba I. Terr, and Tristram G. Parsolw, Appleton & Lange, Norwalk, CT, 1994, p. 71 and Chapter 6. Light chains from any vertebrate species can be classified into one of two distinct types, called κ and λ, based on their constant domain amino acid sequences. Based on relatively minor differences in CH sequences and functions, γ and α classes can be further subdivided into subclasses; for example, humans express the following subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1, and IgA2.

The "heavy chain antibody" described in this article refers to antibodies derived from camelid or cartilaginous fish. Compared to the aforementioned four-chain antibodies, heavy chain antibodies lack the light chain and heavy chain constant region 1 (CH1), containing only two heavy chains composed of a variable region (VHH) and other constant regions. The variable region is linked to the constant region via a hinge-like structure. Each heavy chain of camelid heavy chain antibodies contains one variable region (VHH) and two constant regions (CH2 and CH3), while each heavy chain of cartilaginous fish heavy chain antibodies contains one variable region and five constant regions (CH1-CH5). The antigen-binding fragment of heavy chain antibodies includes VHH and single-chain heavy chain antibodies. By fusing with the constant region of human IgG Fc, heavy chain antibodies can possess the CH2 and CH3 regions of human IgG Fc.

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 the variable region of a heavy chain antibody. Typically, a single-domain antibody contains three CDRs and four FRs. Preferably, the single-domain antibody of the present invention has CDR1 shown in any one of SEQ ID NO:1-7, CDR2 shown in any one of SEQ ID NO:8-13, and CDR3 shown in any one of SEQ ID NO:14-20. A single-domain antibody is the smallest functional antigen-binding fragment. Typically, an antibody lacking both the light chain and the heavy chain constant region 1 (CH1) is first 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.

In this article, "nanobody" refers to an antibody containing the VHH described herein. It can be a heavy chain antibody as described above, or a multivalent or multispecific antibody containing multiple VHHs, or a recombinant antibody obtained by recombination of VHH and antibody Fc (e.g., CH2 and CH3 or CH2, CH3 and CH4).

A binding molecule containing two or more single-domain antibodies is a multivalent single-domain antibody; a binding molecule containing two or more single-domain antibodies with different specificities is a multispecific single-domain antibody. Multivalent or multispecific single-domain antibodies are linked together by a linker. The linker typically consists of 1-15 amino acids selected from G and S.

In this article, heavy chain antibodies and antibodies (traditional four-chain antibodies) are used to distinguish different combinations of antibodies. Due to their structural similarities, the structural descriptions of antibodies below, except for those involving light chains, also apply to heavy chain antibodies.

The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of either the heavy or light chain. The variable domains of the heavy and light chains are referred to as "VH" and "VL," respectively. These domains are typically the most variable parts of the antibody (relative to other antibodies of the same type) and contain antigen-binding sites.

The term "variable" refers to the wide variation in certain segments within a variable domain within an antibody sequence. Variable domains mediate antigen binding and define the specificity of a particular antibody for its specific antigen. However, variability is not uniformly distributed across all amino acids spanned by the variable domain. Instead, it is concentrated in three segments called hypervariable regions (HVRs) (present in both light and heavy chain variable domains): HCDR1, HCDR2, and HCDR3 in the heavy chain variable domain (simply referred to as CDR1, CDR2, and CDR3 in heavy chain antibodies) and LCDR1, LCDR2, and LCDR3 in the light chain variable domain. The more highly conserved portions of the variable domain are called framework regions (FRs). The variable domains of both the natural heavy and light chains each contain four FR regions (FR1, FR2, FR3, and FR4), which mostly adopt a β-sheet conformation and are linked by three HVRs that form a ring connection and, in some cases, part of a β-sheet structure. The HVRs in each chain are held together very closely by the FR regions and, together with the HVRs of the other chain, contribute to the formation of the antibody's antigen-binding site. Typically, the structure of the variable region in the light chain is FR1-LCDR1-FR2-LCDR2-FR3-LCDR3-FR4, and the structure of the variable region in the heavy chain is FR1-HCDR1-FR2-HCDR2-FR3-HCDR3-FR4. Constant domains do not directly participate in antibody-antigen binding but exhibit various effector functions, such as antibody involvement in antibody-dependent cell-mediated cytotoxicity. Several variable region labeling schemes exist for antibodies, including Chothia, Kabat, IMGT, and Contact. This article uses the IMGT labeling scheme as an example.

The “Fc region” (crystallizable fragment region), “Fc domain”, or simply “Fc” refers to the C-terminal region of an antibody heavy chain that mediates the binding of immunoglobulins 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 consists of two identical protein fragments from the CH2 and CH3 domains of the two antibody heavy chains; the Fc regions of IgM and IgE contain three heavy chain constant domains (CH domains 2–4) in each polypeptide chain. Although the boundaries of the Fc region of the immunoglobulin heavy chain can vary, the human IgG heavy chain Fc region is generally defined as the sequence segment from the amino acid residue at position C226 or P230 of the heavy chain to the carboxyl terminus, where the numbering is based on the EU index, as in Kabat. As used herein, the Fc region can be a native sequence Fc or a variant Fc.

An "antibody fragment" comprises a portion of a complete antibody, preferably the antigen-binding region and/or variable region of the complete antibody. The antibody fragment is preferably an antigen-binding fragment of the antibody. Examples of antibody fragments include Fab, Fab', F(ab'), F(ab')2, Fd, and Fv fragments; disulfide-linked Fv fragments; biantibodies; linear antibodies; single-chain antibody molecules; scFv-Fc fragments; multispecific antibodies formed from antibody fragments; and any fragment whose half-life should be increased by chemical modification or by incorporation into liposomes. Antigen-binding fragments can be prepared using a variety of techniques, including but not limited to hydrolyzing and digesting complete antibody proteins, and by expression in host cells containing the antigen-binding fragment.

"Fv" is the smallest antibody fragment containing a complete antigen recognition and binding site. This fragment consists of a dimer of a tightly bound, non-covalently linked heavy chain variable domain and a light chain variable domain. Six hypervariable rings (three from the heavy chain and three from the light chain) protrude from the folds of these two domains, contributing the amino acid residues for antigen binding and conferring antigen-binding specificity to the antibody. However, even a single variable domain (or half an Fv containing only the three antigen-specific HVRs) can recognize and bind antigens, although with lower affinity than a complete binding site. A "single-chain Fv," also abbreviated as "sFv" or "scFv," is an antibody fragment containing antibody VH and VL domains linked together into a single polypeptide chain. Preferably, the sFv polypeptide also contains 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, scFv is VHH.

In this document, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous group of antibodies, meaning that the individual antibodies constituting the group are identical except for possible naturally occurring mutations and/or post-translational modifications (e.g., isomerization, amidation) that may be present in small amounts. Monoclonal antibodies are highly specific, targeting a single antigenic site. Compared to polyclonal antibody formulations (which typically consist of different antibodies targeting different determinants (epitopes), each monoclonal antibody targets a single determinant on the antigen. In addition to their specificity, monoclonal antibodies have the advantage that they are synthesized through hybridoma culture, free from contamination by other immunoglobulins. The modifier "monoclonal" indicates the characteristic that the antibody is obtained from a substantially homogeneous group of antibodies and should not be interpreted as requiring the production of the antibody by any particular method. For example, the monoclonal antibodies to be used according to the invention can be generated by a variety of techniques, including, for example, hybridoma methods, phage display methods, recombinant DNA methods, and techniques for generating human or human-like antibodies from animals having partial or whole human immunoglobulin loci or genes encoding human immunoglobulin sequences, single-cell sequencing methods.

Monoclonal antibodies also include “chimeric” antibodies in this article, wherein a portion of the heavy chain and/or light chain is identical or homologous to the corresponding sequence in an antibody derived from a particular species or belonging to a particular antibody class or subclass, while the remaining portion of the chain is identical or homologous to the corresponding sequence in an antibody derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, provided they exhibit the desired biological activity.

The “humanized” form of a non-human (e.g., mouse) antibody refers to a chimeric antibody that minimally contains sequences derived from non-human immunoglobulins. Therefore, a “humanized antibody” generally refers to a non-human antibody with a variable domain framework region that exchanges sequences found in human antibodies. Typically, in a humanized antibody, the entire antibody (except for the CDR) is encoded by a human-derived polynucleotide or is identical to that antibody (except for the CDR). The CDR (some or all of which are encoded by nucleic acids derived from non-human organisms) is transplanted into the β-sheet backbone of the variable region of the human antibody to produce an antibody whose specificity is determined by the transplanted CDR. Methods for producing such antibodies are well known in the art, for example, using mice with genetically engineered immune systems. In this invention, antibodies, single-domain antibodies, heavy-chain antibodies, etc., all include humanized variants of the aforementioned antibodies.

"Human antibody" refers to an antibody having an amino acid sequence corresponding to that of antibodies generated by humans and/or produced using any of the techniques disclosed herein for generating human antibodies. This definition of human antibody explicitly excludes humanized antibodies containing non-human antigen-binding residues. Human antibodies can be generated using a variety of techniques known in the art, including phage display libraries.

In some embodiments, the present invention also provides nanobodies, heavy chain antibodies, antibodies or antigen-binding fragments thereof (e.g., single-domain antibody VHH) that bind to the same epitope on human CD70 as the antigen-binding region of any anti-CD70 nanobody of the present invention, i.e., nanobodies, heavy chain antibodies, antibodies or antigen-binding fragments thereof that can cross-compete with the antigen-binding region of any nanobody of the present invention for binding to CD70.

In this invention, the anti-CD70 single-domain antibody has CDR1 as shown in any one of SEQ ID NO:1-7, CDR2 as shown in any one of SEQ ID NO:8-13, and CDR3 as shown in any one of SEQ ID NO:14-20. Preferably, the anti-CD70 single-domain antibody contains CDR1, CDR2, and CDR3 as shown in any group of (a)-(h):

(a) CDR1 as shown in SEQ ID NO:1, CDR2 as shown in SEQ ID NO:8, and CDR3 as shown in SEQ ID NO:14;

(b) CDR1 with sequence as shown in SEQ ID NO:2, CDR2 with sequence as shown in SEQ ID NO:8, and CDR3 with sequence as shown in SEQ ID NO:14;

(c) CDR1 with sequence as shown in SEQ ID NO:3, CDR2 with sequence as shown in SEQ ID NO:8, and CDR3 with sequence as shown in SEQ ID NO:15;

(d) CDR1 as shown in SEQ ID NO:4, CDR2 as shown in SEQ ID NO:9, and CDR3 as shown in SEQ ID NO:16;

(e) CDR1 with sequence as shown in SEQ ID NO:5, CDR2 with sequence as shown in SEQ ID NO:10, and CDR3 with sequence as shown in SEQ ID NO:14;

(f) CDR1 with sequence as shown in SEQ ID NO:6, CDR2 with sequence as shown in SEQ ID NO:11, and CDR3 with sequence as shown in SEQ ID NO:18;

(g) CDR1 as shown in SEQ ID NO:6, CDR2 as shown in SEQ ID NO:12, and CDR3 as shown in SEQ ID NO:19;

(h) CDR1 with sequence as shown in SEQ ID NO:7, CDR2 with sequence as shown in SEQ ID NO:13, and CDR3 with sequence as shown in SEQ ID NO:20.

The FR1, FR2, FR3, and FR4 of the anti-CF70 single-domain antibody described herein can be independently selected from the FR1, FR2, FR3, and FR4 of any of the single-domain antibodies shown in SEQ ID NO:21-28. Preferably, the amino acid sequence of the anti-CF70 single-domain antibody is as shown in any of SEQ ID NO:21-28.

When a single-domain antibody is attached to a heavy chain constant region, the nanobody is a heavy chain antibody comprising the single-domain antibody described herein. The heavy chain constant region may be a constant region of a camel heavy chain antibody, comprising CH2 and CH3. Preferably, the antibody constant region is derived from the constant regions of any one of IgG1, IgG2, IgG3, IgG4, IgA, IgM, IgE, and IgD; more preferably, it is derived from the constant regions of any one of IgG1, IgG2, IgG3, and IgG4. In one or more embodiments, the heavy chain constant region is CH2 and CH3 of human IgG Fc, such as CH2 and CH3 of IgG1, as shown in SEQ ID NO:37.

The CF70 binding molecules described herein can be monovalent or multivalent nanobodies or single-domain antibodies, or multispecific nanobodies or single-domain antibodies, comprising one, two, or more of the anti-CD70 nanobodies or single-domain antibodies described herein. Multispecificity can be against CD70 and another antigen, or against two different epitopes of CD70.

This invention also includes the antibody derivatives and analogs described herein. “Derivatives” and “analytes” refer to polypeptides that substantially retain the same biological function or activity as the antibodies of this invention. The derivatives or analogs of this invention may be (i) polypeptides having substituents in one or more amino acid residues, or (ii) polypeptides formed by fusing a mature polypeptide with another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol), or (iii) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (e.g., a leader sequence, a secretory sequence, a sequence used to purify this polypeptide, or a proteogenic sequence, or a fusion protein formed with a 6His tag). Based on the teachings herein, these derivatives and analogs are within the scope well known to those skilled in the art.

Without substantially affecting antibody activity, those skilled in the art can modify the antibody sequence of the present invention by one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 or more) to obtain variants of the antibody or its functional fragment sequence. These variants include (but are not limited to): deletions, insertions, and/or substitutions of one or more amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10), and the addition of one or more amino acids (typically up to 20, preferably up to 10, more preferably up to 5) at the C-terminus and/or N-terminus. In the art, conservative substitutions with amino acids of similar or comparable properties generally do not alter protein function. For example, substitutions of amino acids with similar properties in the FR and/or Fc regions. 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. For example, adding one or more amino acids to the C-terminus and/or N-terminus usually does not change the function of the protein. These are all considered to be included within the scope of protection of this invention.

The variants of the antibodies described herein include: homologous sequences, conserved variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of the antibodies of the present invention under high or low stringency conditions, and polypeptides or proteins obtained using antiserum against the antibodies of the present invention. In some embodiments, the sequences of the variants described herein may have at least 95%, 96%, 97%, 98%, or 99% homology with their source sequences. The sequence homology described herein can be measured using sequence analysis software, such as the computer program BLAST with default parameters, especially BLASTP or TBLASTN. The present invention also includes molecules having antibody heavy chain variable regions with CDRs, provided that their CDRs have at least 90% (preferably at least 95%, most preferably at least 98%) homology with the CDRs identified herein.

The antibodies of the present invention can be prepared using methods conventional in the art, such as hybridoma techniques. The nanobodies of the present invention can be prepared using methods conventional in the art, such as phage display techniques well known in the art. Alternatively, the antibodies or nanobodies of the present invention can be expressed in other cell lines. Suitable mammalian host cells can be transformed with sequences encoding the antibodies of the present invention, followed by culturing the host cells and purifying the antibodies. Transformation can be performed using any known method, including, for example, packaging polynucleotides in a virus (or viral vector) and transducing host cells with the virus (or vector). The transformation procedure used depends on the host to be transformed. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art, including dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of polynucleotides in liposomes, and direct microinjection of DNA into the nucleus. Mammalian cell lines that can be used as hosts for expression are well known in the art, including but not limited to a variety of immortalized cell lines available from the American Type Culture Collection (ATCC), including but not limited to Chinese hamster ovary (CHO) cells, HeLa cells, young hamster kidney (BHK) cells, monkey kidney cells (COS) cells, human hepatocellular carcinoma cells (e.g., HepG2).

CAR

This invention also provides a chimeric antigen receptor (CAR) targeting CD70. The CAR contains an optional signal peptide sequence, an antigen recognition region (i.e., the anti-CD70 binding molecule described herein), a hinge region, a transmembrane region, and an intracellular region. The intracellular region includes one or more intracellular co-stimulatory domains and/or one or more intracellular signaling domains. The terms "hinge region," "transmembrane region," and "intracellular region" as used herein can all be selected from sequences of the hinge region, transmembrane region, and intracellular region in known CAR-T technologies.

The signal peptide, optionally selected for the CAR, can be chosen as needed. Generally, a signal peptide is a peptide sequence that directs the polypeptide to a desired site within the cell. The signal peptide directs the polypeptide to the cell's secretory pathway and allows the polypeptide to integrate and anchor to the lipid bilayer; the signal peptide can also be a membrane-localizing signal peptide. Exemplary signal peptides include 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 suitable for use in this invention can be any of the various human CD8 signal peptide sequences commonly used in CARs in the art. In some embodiments, the amino acid sequence of said human CD8 signal peptide comprises the sequence shown in SEQ ID NO:38.

The hinge region of a chimeric antigen receptor is located between the extracellular antigen-binding region and the transmembrane region. The hinge region is an amino acid segment that typically exists between two domains of a protein and allows for protein flexibility and relative movement between the two domains. The hinge region can be a hinge region of a naturally occurring protein or a portion thereof. The hinge region of an antibody (such as IgG, IgA, IgM, IgE, or IgD antibodies) can also be used in the chimeric antigen receptor described herein. Non-naturally occurring peptides can also be used as the hinge region of the chimeric antigen receptor described herein. Exemplarily, the hinge region of a CAR is selected from the CD8α hinge region, the IgD hinge region, the IgG1 FcCH2CH3 hinge region, or the IgG4 FcCH2CH3 hinge region, the sequences of which are within the knowledge of those skilled in the art. The CD8α hinge region suitable for use in this invention can be any of the various human CD8α hinge region sequences commonly used in CARs in the art. In some embodiments, the human CD8α hinge region comprises the sequence shown in SEQ ID NO:39.

The transmembrane region of a chimeric antibody receptor can form an α-helix, a complex of more than one α-helix, a β-barrel, or any other stable structure capable of translocating the cellular phospholipid bilayer. The transmembrane region can be of natural or synthetic origin. It 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, or the α, β, or ζ chains of T-cell receptors. The human CD8α transmembrane region suitable for this invention can be any of the 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.

Intracellular signaling regions (or intracellular signal transduction regions) are responsible for activating at least one normal effector function of immune effector cells expressing chimeric antigen receptors. For example, the effector function of T cells can be lytic activity or helper activity, including cytokine secretion. While the entire intracellular signal transduction region can generally be used, in many cases, using the whole chain is unnecessary. Regarding the use of truncated portions of intracellular signal transduction regions, such truncated portions can be used instead of the whole chain as long as they transduce effector function signals. Therefore, intracellular signal transduction regions include any truncated form of intracellular signal transduction regions sufficient to transduce effector function signals. The intracellular signaling domain of a CAR can be selected as needed, including but 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 region is derived from the human CD3ζ intracellular signaling region. Furthermore, the human CD3ζ intracellular signaling region has the amino acid sequence shown in SEQ ID NO:41.

In addition to stimulation by antigen-specific signals, many immune effector cells require co-stimulation to promote cell proliferation, differentiation, and survival, as well as to activate effector functions. The "co-stimulatory domain" can be the cytoplasmic portion of a co-stimulatory molecule. The term "co-stimulatory molecule" refers to an associated binding chaperone on immune cells (such as T cells) that specifically binds to a co-stimulatory ligand, thereby enabling the immune cell to mediate a co-stimulatory response, such as, but not limited to, proliferation and survival. Suitable intracellular co-stimulatory domains can be selected as needed, including intracellular domains containing co-stimulatory signaling molecules, 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 co-stimulatory domain comprises the sequence shown in SEQ ID NO:42.

The aforementioned portions 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 co-stimulatory domain, and the CD3ζ intracellular signaling domain, can be directly linked to each other or linked via adapter sequences. The adapter sequence can be a known antibody-compatible adapter sequence, such as a G and S-containing adapter sequence. Typically, the adapter contains one or more repeating motifs. For example, the motif can be GGGS, GGGGS, SSSSG, GSGSA, and GGSGG. Preferably, the motifs are adjacent in the adapter sequence, with no inserted amino acid residues between the repeats. The adapter sequence can consist of 1, 2, 3, 4, or 5 repeating motifs. The length of the adapter can be 3 to 25 amino acid residues, for example, 3 to 15, 5 to 15, or 10 to 20 amino acid residues. In some embodiments, the adapter sequence is a polyglycine adapter sequence. The number of glycine residues in the linker sequence is not particularly limited, typically ranging from 2 to 20, for example, 2 to 15, 2 to 10, or 2 to 8. Besides glycine and serine, the linker may also 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 (GGGGS)n-linked, where n is an integer from 1 to 5.

In an exemplary embodiment, the CAR contains, from the N-terminus to the C-terminus, a CD8 signal peptide, the anti-CD70 nanobody described herein or its antigen-binding fragment, a CD8α hinge region, a CD8α transmembrane region, a CD3ζ intracellular signaling domain, and a 4-1BB co-stimulatory domain. In a specific embodiment, an exemplary CAR having the above structure is shown in any of SEQ ID NO: 29-36.

It should be understood that in gene cloning, it is often necessary to design suitable restriction enzyme sites, which inevitably introduces one or more irrelevant residues at the end of the expressed amino acid sequence, without affecting the activity of the target sequence. To construct fusion proteins, promote the expression of recombinant proteins, obtain recombinant proteins that are automatically secreted outside the host cell, or facilitate the purification of recombinant proteins, it is often necessary to add some amino acids to the N-terminus, C-terminus, or other suitable regions within the recombinant protein, such as, but not limited to, suitable adaptor peptides, signal peptides, leader peptides, and terminal extensions. Therefore, the amino or carboxyl terminus of the CAR of the present invention may also contain one or more polypeptide fragments as protein tags. Any suitable tag can be used herein. For example, the tags 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.

The antigen recognition region in the CAR of the present invention can be a variant of the aforementioned anti-CD70 nanobody or its functional fragment sequence. Furthermore, other parts of the CAR can also undergo sequence changes, resulting in a mutant with 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 retaining the biological activity of the CAR (e.g., activation of T cells). Sequence identity between two aligned sequences can be calculated using, for example, NCBI's BLASTp.

The mutant also includes an amino acid sequence having one or more mutations (insertions, deletions, or substitutions) in the amino acid sequence of the CAR described in any embodiment, while still retaining the biological activity of the CAR. The number of mutations typically refers to 1-10, for example 1-8, 1-5, or 1-3. Substitution is preferably conserved. For example, in the art, conserved substitution with amino acids of similar or comparable properties generally does not alter the function of the protein or peptide. "Amino acids with similar or comparable properties" includes, for example, families of amino acid residues having similar side chains. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), amino acids with acidic side chains (e.g., aspartic acid, glutamic acid), amino acids with uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), amino acids with nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), amino acids with β-branched side chains (e.g., threonine, valine, isoleucine), and amino acids with aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Therefore, replacing one or more sites in the polypeptide of the present invention with another amino acid residue from the same side chain class will not substantially affect its activity.

Nucleic acid

This invention also provides polynucleotides encoding the aforementioned antibodies or CARs. The polynucleotides of this invention can be in DNA or RNA form. DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand. This invention also includes degenerate variants of polynucleotide sequences encoding fusion proteins, i.e., nucleotide sequences encoding the same amino acid sequence but with different nucleotide sequences.

Therefore, the present invention also relates to polynucleotides that hybridize with the above-mentioned polynucleotide sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that are hybridizable with the polynucleotides described herein under stringent conditions. In the present invention, “stringent conditions” means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with a denaturing agent, such as 50% (v/v) formamide, 0.1% fetal bovine serum/0.1% Ficoll, 42°C, etc.; or (3) hybridization only occurs when the identity between the two sequences is at least 90%, preferably at least 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

The full-length nucleotide sequence or fragments of the antibody of the present invention can generally be obtained by PCR amplification, recombinant methods, or artificial synthesis. One feasible method is to synthesize the relevant sequence artificially, especially when the fragment length is short. Typically, a long fragment can be obtained by first synthesizing multiple small fragments and then ligating them. Furthermore, the coding sequence of the heavy chain and an expression tag (such as 6His) can be fused together to form a fusion protein. The CAR sequence can also be obtained as described above. Alternatively, the sequences of the various parts of the CAR (signal peptide, antigen recognition region, hinge region, transmembrane region, or intracellular region) can be obtained as described above and then ligated to obtain the full-length CAR.

Once the relevant sequence is obtained, it can be obtained in large quantities using recombination methods. This typically involves cloning it into a vector, transforming it into cells, and then isolating the sequence from the proliferated host cells using conventional methods. The biomolecules (nucleic acids, proteins, etc.) involved in this invention include biomolecules existing in isolated forms. Currently, DNA sequences encoding the proteins of this invention (or fragments thereof, or derivatives thereof) can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors, etc.) and cells known in the art. Furthermore, mutations can be introduced into the protein sequences of this invention through chemical synthesis. The CAR portions can be sequentially cloned into a vector or integrated into a full-length CAR before cloning.

This invention also relates to nucleic acid constructs containing the polynucleotide sequences described herein, and one or more regulatory sequences operatively linked to these sequences. The polynucleotide sequences described herein can be manipulated in various ways to ensure the expression of the antibody or CAR. The nucleic acid constructs can be manipulated depending on the expression vector or requirements before insertion into a vector. Techniques for altering polynucleotide sequences using recombinant DNA methods are known in the art.

The regulatory sequence can be a suitable promoter sequence. Promoter sequences are typically operatively linked to the coding sequence of the protein to be expressed. A promoter can be any nucleotide sequence that exhibits transcriptional activity in the chosen host cell, including mutant, truncated, and heterozygous promoters, and can be obtained from a gene encoding an extracellular or intracellular polypeptide that is homologous or heterologous to that host cell. An example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strongly constitutive promoter sequence capable of driving high-level expression of any polynucleotide sequence operatively linked to it. Another example of a suitable promoter is elongation growth factor-1α (EF-1α). However, other constitutive promoter sequences may also be used, including but not limited to the early promoter of simian virus 40 (SV40), mouse mammary cancer virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, avian leukemia virus promoter, Epstein-Barr virus immediate early promoter, Russ's sarcoma virus promoter, and human gene promoters, such as, but not limited to, actin promoter, myosin promoter, heme promoter, and creatine kinase promoter. Furthermore, the use of inducible promoters may also be considered. The use of inducible promoters provides a molecular switch that can turn on the expression of the polynucleotide sequence operatively linked to the inducible promoter during time-limited expression and turn off expression when expression is undesirable. Examples of inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

The regulatory sequence can also be a suitable transcription terminator sequence, a sequence recognized by the host cell to terminate transcription. The terminator sequence is operatively linked to the 3' end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in a selected host cell can be used in this invention. The regulatory sequence can also be a suitable leader sequence, the untranslated region of mRNA important for translation by the host cell. The leader sequence is operatively linked to the 5' end of the nucleotide sequence encoding the polypeptide. Any terminator that is functional in a selected host cell can be used in this invention.

In some embodiments, the nucleic acid construct is a vector, such as a cloning vector, an expression vector, and an integration vector. Expression of the polynucleotide sequences of the present invention is typically achieved by operably linking the polynucleotide sequences of the present invention to an expression vector. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters that can be used to regulate the expression of the desired nucleic acid sequence. Integration vectors contain components for integrating the target sequence into the cellular genome. These vectors can be used to transform appropriate host cells to enable them to express proteins. Vectors typically contain sequences for plasmid maintenance and for cloning and expressing exogenous nucleotide sequences. These sequences (collectively referred to as “flanking sequences” in some embodiments) 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 containing donor and acceptor splicing sites, a sequence encoding a leader sequence for polypeptide secretion, a ribosome binding site, a polyadenylated sequence, a multi-connector region for inserting a nucleic acid encoding an antibody to be expressed, and optional marker elements.

Furthermore, the type of vector is not limited; for example, plasmids, phage particles, phage derivatives, animal viruses, and entrapments can be modified depending on the host cell to be introduced. Viral vector technology is well known in the art and has been described in, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York) and other virology and molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses.

To assess the expression of CAR peptides or portions thereof, the vector introduced into cells may also contain one or both of an optional marker gene or reporter gene to facilitate the identification and selection of expressing cells from a population of cells seeking transfection or infection via a viral vector.

cell

The host cells suitable for introducing the nucleic acid constructs described herein can be prokaryotic cells, such as bacterial cells; lower eukaryotic cells, such as yeast cells; or higher eukaryotic cells, such as mammalian cells, especially immune cells, preferably immune effector cells. Representative examples include: Escherichia coli, Streptomyces; Salmonella typhimurium bacterial cells; fungal cells such as yeast; Drosophila S2 or Sf9 insect cells; and animal cells such as CHO, COS7, and 293 cells.

"Immune effector cells" are immune cells capable of performing immune effector functions. In some embodiments, immune effector cells express at least FcγRIII and perform ADCC effector functions. Examples of immune effector cells mediating ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, neutrophils, and eosinophils. Preferably, immune effector cells are selected from at least one of: 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 cells are T lymphocytes (same as T cells). In some embodiments, T cells can be CD4+/CD8-, CD4-/CD8+, CD4+/CD8+, CD4-/CD8-, or combinations thereof. In some embodiments, T cells produce IL-2, IFN, and/or TNF when expressing a chimeric antigen receptor and binding to target cells. In some implementations, CD8+ T cells lyse antigen-specific target cells when expressing chimeric antigen receptors and binding to target cells.

The T cells suitable for use in this invention can be of various types and origins. For example, T cells can be derived from PBMCs of patients with B-cell malignancies. In some embodiments, after obtaining T cells, they can be first stimulated and activated with an appropriate amount (e.g., 30–80 ng/ml, such as 50 ng/ml) of CD3 antibody, and then cultured in IL2 medium containing an appropriate amount (e.g., 30–80 IU/ml, such as 50 IU/ml) for later use.

Methods for introducing nucleic acids or vectors into mammalian cells are known in the art, and the vectors can be transferred into cells by physical, chemical, or biological methods. When the host is a prokaryote such as *Escherichia coli*, competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated with _CaCl2 , the steps of which are well known in the art. When the host is a eukaryote, DNA transfection methods such as calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, and liposome packaging can be used. In some embodiments, transduced or transfected immune effector cells multiply in vitro after the introduction of nucleic acids or vectors.

The obtained transformants can be cultured using conventional methods to express the antibody or CAR encoded by the gene of this invention. Depending on the host cells used, the culture medium can be selected from various conventional media. Culture is carried out under conditions suitable for host cell growth. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using a suitable method (such as temperature adjustment or chemical induction), and the cells are cultured for a further period.

The peptides used in the methods described above can be expressed intracellularly, on the cell membrane, or secreted extracellularly. If desired, the recombinant proteins can be separated and purified using various separation methods based on their physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to: conventional refolding treatment, treatment with protein precipitants (salting out), centrifugation, permeation, ultrafiltration, 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.

Uses and methods

By constructing a nanobody library, the inventors screened nanobodies and their variants that could bind to CD70. The binding ability of these antibodies to the antigen was verified through protein-level binding assays, affinity assays, competitive blocking experiments, and tissue cross-reactivity studies. Using these nanobodies, the inventors constructed CARs and CAR-T cells. Molecular and cellular experiments verified that the CAR-T cells exhibited strong immune function, superior CD107a expression, IFN-γ and IL-2 secretion, and specific killing function against target cells, demonstrating significant in vivo efficacy.

All aspects of the antibodies, CARs, coding sequences, nucleic acid constructs, and cells described herein can be used to prepare drugs for the prevention or treatment of the various conditions and diseases described herein, which are diseases or conditions related to CD70 expression, referring to diseases directly or indirectly caused by abnormal CD70 expression, usually referring to diseases caused by CD70 overexpression, 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.

This invention also includes a class of cell therapies comprising expressing the CAR described herein in immune cells (e.g., T cells) and administering a therapeutically effective amount of the cells to a recipient who requires them, the cells being capable of killing the recipient's tumor cells. Compared to antibody therapies, CAR-T cells can replicate in vivo, producing long-lasting durability that can lead to sustained tumor control. The anti-tumor immune response induced by CAR-T cells can be an active or passive immune response. Additionally, CAR-mediated immune responses can be part of an adoptive immunotherapy step, wherein CAR-T cells induce an immune response specific to the antigen-binding portion of the CAR.

The antibodies, nucleic acids, or CAR-modified cells of the present invention can be administered alone or as a pharmaceutical composition in combination with a diluent and/or other components such as associated cytokines or cell populations. In this regard, the pharmaceutical compositions can be prepared by mixing an active pharmaceutical agent of desired purity with an optional pharmaceutically acceptable carrier in the form of a lyophilized formulation or an aqueous solution. Pharmaceutically acceptable carriers are non-toxic to the recipient at the dose and concentration used and may include at least one of buffers (e.g., neutral buffered saline, sulfate buffered saline), antioxidants, preservatives, isotonic agents, stabilizers, chelating agents (e.g., EDTA or glutathione), adjuvants (e.g., aluminum hydroxide), and surfactants. Furthermore, in order for the pharmaceutical compositions to be usable for in vivo administration, they must be sterile. The pharmaceutical compositions can be sterilized by filtration through a sterile filter membrane.

In some embodiments, the pharmaceutical composition may contain at least one additive selected from: a cytotoxic agent, a chemotherapeutic agent, a cytokine, an immunosuppressant, a growth inhibitor, and an active pharmaceutical agent required for the specific indication to be treated. The specific amount of the additive may be adjusted as needed.

The pharmaceutical compositions of the present invention can be administered in amounts described as “immunologically effective,” “antitumor effective,” “tumor-suppressive effective,” or “therapeutic.” “Therapeutic” refers to a subject receiving the treatment regimen described herein to achieve at least one positive therapeutic effect (e.g., a reduction in the number of cancer cells, a reduction in tumor volume, a decrease in the rate of cancer cell invasion into surrounding organs, or a decrease in the rate of tumor metastasis or tumor growth). When “immunologically effective,” “antitumor effective,” “tumor-suppressive effective,” or “therapeutic” is specified, the precise amount of the composition of the present invention to be administered can be determined by a physician, taking into account individual differences in the patient’s (subject’s) age, weight, tumor size, degree of infection or metastasis, and disease. Typically, pharmaceutical compositions comprising T cells described herein can be administered at doses of ^10⁴ to ^10⁹ cells/kg body weight, preferably ^10⁵ to ^10⁶ cells/kg body weight. The T-cell compositions can also be administered multiple times at these doses. Cells can be administered using infusion techniques known in immunotherapy (see, for example, Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dosage and treatment regimen for a specific patient can be easily determined by medical professionals by monitoring the patient's disease signs and adjusting the treatment accordingly.

The composition can be administered in any convenient manner, including by spraying, injection, swallowing, infusion, implantation, or transplantation. The composition described herein can be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intraspinally, intramuscularly, intravenously, or intraperitoneally. In one embodiment, the T-cell composition of the present invention is administered to a patient by intradermal or subcutaneous injection. In another embodiment, the T-cell composition of the present invention is preferably administered by intravenous injection. The T-cell composition can be injected directly into the tumor, lymph node, or site of infection.

In some embodiments of the present invention, the CAR-T cells or compositions thereof of the present invention can be combined with other therapies known in the art. These therapies include, but are not limited to, chemotherapy, radiotherapy, and immunosuppressants. For example, treatment may be combined with radiotherapy or chemotherapy agents known in the art for treating mesothelin-mediated diseases.

In this article, "anti-tumor effect" refers to a biological effect that can be represented by a reduction in tumor volume, a reduction in the number of tumor cells, a reduction in the number of metastases, an increase in life expectancy, or an improvement in various cancer-related physiological symptoms.

The terms "patient," "subject," and "individual" are used interchangeably in this article to refer to a living organism, such as a mammal, that can elicit an immune response. Examples include, but are not limited to, humans, dogs, cats, mice, rats, and their transgenic species.

The present invention is described in further detail with reference to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. Therefore, the invention should not be construed as limited to the following examples, but should be interpreted as including any and all variations that become apparent from the teachings provided herein. The methods and reagents used in the examples, unless otherwise stated, are conventional methods and reagents in the art.

Diagnostics, tests and kits

The binding molecules of this invention, due to their high affinity for CD70, can be used for assays, such as binding assays, to detect and/or quantify CD70 expressed in tissues or cells. Binding molecules, such as single-domain antibodies, can be used in studies further investigating the role of CD70 in disease. The method for detecting CD70 generally involves obtaining cell and/or tissue samples; detecting the level of CD70 in the samples.

The CD70 binding molecule of this invention can be used for diagnostic purposes to detect, diagnose, or monitor CD70-related diseases and/or conditions. This invention provides methods for detecting the presence of CD70 in samples using classical immunohistochemical methods known to those skilled in the art. CD70 detection can be performed in vivo or in vitro. Examples of methods suitable for detecting the presence of CD70 include ELISA, FACS, RIA, etc.

For diagnostic applications, binding molecules such as single-domain antibodies are typically labeled with detectable labeling groups. 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), fluorescent groups (e.g., FITC, rhodamine, lanthanide phosphors), enzymatic groups (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), chemiluminescent groups, biotinylated groups, or predetermined polypeptide epitopes recognized by secondary reporter molecules (e.g., leucine zipper pairs, binding sites for secondary antibodies, metal-binding domains, epitope tags), MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents. Various methods for labeling proteins are known in the art and can be used in carrying out this invention.

Another aspect of the invention provides a method for detecting the presence of a test molecule that competes with the antibody of the invention for binding to CD70. An example of such a determination would involve detecting the amount of free antibody in a solution containing a certain amount of CD70, in the presence or absence of a test molecule. An increase in the amount of free antibody (i.e., antibody not bound to CD70) would indicate that the test molecule is able to compete with the antibody for binding to CD70. 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.

This invention also provides a detection kit for detecting CD70 levels, comprising an antibody that recognizes the CD70 protein, a lysis medium for dissolving samples, and universal reagents and buffers required for detection, such as various buffers, detection labels, and detection substrates. This detection kit can be used as an in vitro diagnostic device.

The present invention will be described below by way of specific embodiments. It should be understood that these embodiments are merely illustrative and are not intended to limit the scope of the invention. Unless otherwise stated, the methods and materials used in the embodiments are conventional materials and methods in the art.

Example

Example 1: Construction and eukaryotic expression of recombinant human CD70 protein expression vector.

1. Synthesis of the gene sequence of amino acid range 39 to 193 of CD70 and construction of the protein expression vector.

The amino acid sequence from positions 39 to 193 of CD70 (uniprot accession No: P32970-1) was imported into an online codon optimization tool (http://www.jcat.de/#opennewwindow) to obtain the codon-optimized nucleic acid sequence. The gene sequence was then obtained through chemical synthesis, and the coding sequence for alpaca IgG1-Fc (amino acid sequence as shown in SEQ ID NO: 51) was added to the 3' end of this gene sequence. The spliced product was cloned into pCDNA3.1 (Thermo) using the TaKaRa seamless cloning kit to obtain the expression vector.

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

Five days after transfecting 293T cells (ATCC) with the obtained expression vector, the culture supernatant was collected, and the recombinant human CD70-alFc protein was purified using AKTA explorer100 (GE). Due to glycosylation modifications and other reasons, the recombinant human CD70-alFc protein, after reducing SDS-PAGE electrophoresis and Coomassie Brilliant Blue staining, showed a size of approximately 50 kilodaltons, as shown in Figure 1.

The activity of recombinant human CD70-alFc protein was determined by ELISA using commercially available CD27: Recombinant human CD70-alFc protein was serially diluted 4-fold in an ELISA plate, with 100 ng in the first well, followed by subsequent 4-fold dilutions (100 μL/well). The plates were incubated overnight at 4°C. The next day, the plates were washed three times with 200 μL PBST and blocked for 1 hour with 1% BSA/PBS. After removing the blocking buffer, 100 μL of 0.1 μg/mL recombinant human CD27-his protein was added to each well, and the plates were incubated at 37°C for 1 hour. After washing three times with 200 μL PBST, 100 μL of horseradish peroxidase-labeled rabbit anti-his-tag antibody (1:4000 dilution) was added to each well, and the plates were incubated at 37°C for 1 hour. After washing three times with 200 μL PBST, 100 μL TMB chromogenic solution was added, and the mixture was incubated at 37 °C for 10 minutes. The incubation was then stopped by adding 100 μL 1M hydrochloric acid, and the OD450 was read. The analytical results are shown in Figure 2 and Table 1.

Table 1 Analysis Results

EC50 (ng/mL) 8.689 R square 0.9962

Example 2: Preparation of anti-human CD70 alpaca VHH antibody

1. Immunize alpacas

A mixture of 2 mg/mL CD70-alIgG1 Fc fusion protein as antigen and an equal volume of Sigma-Aldrich complete adjuvant was emulsified and used for subcutaneous immunization of adult alpacas, with each alpaca receiving 500 μg of antigen. After the initial immunization, booster immunizations were administered every twenty days for a total of four subcutaneous immunizations. Seven days after the fourth immunization, 100 mL of whole blood was collected intravenously for the isolation of PBMCs.

2. Serum titer testing

Before each booster immunization, 10 mL of blood was drawn intravenously, centrifuged to remove cells, and serum was retained. 50 ng/well of CD70-his protein (ACRO Biosystems) was added to each well of an ELISA microplate, and the plate was incubated overnight at 4°C. The plate was washed three times with PBS, and 200 μL/well of 1% BSA/PBS was added, with the plate blocked at 37°C for 1 hour. Serially diluted alpaca serum was added, and the plate was incubated at 37°C for 1 hour. The plate was washed three times with PBST, and 100 μL of 1:5000 diluted HRP-goat anti-alpaca IgG1-Fc (Jackson Immuno Research) was added, with the plate incubated at 37°C for 1 hour. The plate was washed three times with PBST, and 100 μL/well of TMB chromogenic buffer was added, with the plate incubated at 37°C for 10 minutes. 100 μL/well of ELISA stop solution was added, and the OD450 value was read using a microplate reader. Serum titer results are shown in Table 2.

Table 2. Serum titer detection after alpaca immunization

Dilution factor Negative Secondary immunization Three immunizations Four immunizations 1:2K 0.043 1.2551 1.411 2.1077 1:4K 0.0423 0.6624 0.9337 1.2117 1:8K 0.0418 0.391 0.5955 0.8136 1:16K 0.0429 0.2568 0.276 0.3979 1:32K 0.0444 0.1311 0.152 0.1965 1:64K 0.0443 0.1056 0.1077 0.1505 1:128K 0.0439 0.0733 0.0728 0.0858 blank 0.0461 0.0442 0.0434 0.0508

3. Constructing an immune library

3.1 Obtaining total cDNA from alpaca PBMCs

Total RNA was extracted from alpaca PBMCs using the Trizol RNA Extraction Kit. First-strand cDNA was synthesized using the SuperScript ^™ IV First-Strand Synthesis System kit, using the extracted RNA as a template.

3.2 VHH gene amplification

Using the cDNA as a template, the heavy chain gene was amplified by PCR using upstream primers for the variable domain of the heavy chain and downstream constant region CH2 primers (VH-F, CH2-R). In a 50 μL reaction system, 25 μL of PrimeSTAR MAX master mix (Takara), 2.5 μL (25 pmol) of upstream primer, 2.5 μL (25 pmol) of downstream primer, 1.5 μL of DMSO, 0.5 μL of cDNA, and 18 μL of ddH2O were added. The PCR reaction was performed according to the following program: pre-denaturation at 98 °C for 1 minute, followed by temperature cycling: 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 a final extension at 72 °C for 10 minutes.

The amplified VHH-CH2 gene was recovered using a DNA gel extraction kit, and the electrophoresis results are shown in Figure 3. Using 100 ng of VHH-CH2 as a template, the VHH gene was amplified by PCR using upstream primer VH-F and downstream primer VH-R. In a 50 μL reaction system, 25 μL of PrimeSTAR MAX master mix (Takara), 2.5 μL (25 pmol) of upstream primer, 2.5 μL (25 pmol) of downstream primer, 1.5 μL of DMSO, 0.5 μL of VH-CH2 DNA, and 18 μL of ddH2O were added, respectively. The PCR reaction was performed according to the following program: pre-denaturation at 98℃ for 1 minute, followed by temperature cycling: denaturation at 98℃ for 110 seconds, annealing at 60℃ for 15 seconds, extension at 72℃ for 30 minutes, for 25 cycles, and a final extension at 72℃ for 10 minutes. The amplified VHH gene fragment was recovered using a gel extraction kit, and the electrophoresis results are shown in Figure 4.

3.3 Construction of immune libraries

The VHH gene fragment and the pcomb3X-TT vector (Scripps Research Institute, USA) were digested separately using SfiI DNA endonuclease. In a 50 μL reaction mixture, 2 μL of SfiI, 5 μL of 10x buffer, and 3 μg of DNA were added, followed by ddH2O to a final volume of 50 μL. After thorough mixing, the mixture was incubated at 50°C for 3 hours.

The digested VHH gene fragment and pcomb3X vector were recovered using a DNA gel extraction kit. The digested VHH gene fragment and pcomb3X vector were then cyclized using T4 ligase. In a 50 μL reaction mixture, 1 μL of T4 ligase, 5 μL of 10x buffer, 150 ng of VHH gene, and 1000 ng of pComb3X vector were added, followed by ddH2O to a final volume of 50 μL. After thorough mixing, the mixture was incubated at 4°C for 16 hours. A small amount of the product was taken and agarose gel electrophoresis was performed to verify the ligation efficiency. The electrophoresis results are shown in Figure 5.

10 μL of the above-mentioned ligation and cyclization product was added to self-made TG1 electroporation competent cells, and then electroporation was performed using an electroporator. 10 μL of the electroporated bacteria was taken out, diluted appropriately, and streaked onto a plate containing ampicillin to count the bacteria and determine the size of the phage antibody library. The remaining electroporated bacteria were added to 2xYT medium containing 100 μg/mL ampicillin and 2% glucose and incubated in a heated incubator. After incubation, the culture was centrifuged at 4000G for 10 minutes at 4°C, and an appropriate amount of glycerol was added to the precipitate. The culture was then stored at -80°C as an antibody culture library. Through multiple electroporations, an scFv immunoglobulin library with a capacity exceeding 9E+9 was obtained.

4. Screening for CD70 antibodies

4.1 Recombinant human CD70 protein coupled to streptavidin magnetic beads

Using a biotinylation kit (EasyBio) following the kit instructions, the avi-tag of recombinant human CD70 protein was biotinylated to obtain biotinylated CD70 protein. 10 μg of the biotinylated recombinant protein was added to 100 μL of streptavidin magnetic beads (DynaBeads 280) that had been washed three times with PBS. The mixture was placed on a rotary shaker at 18 rpm and coupled at room temperature for 30 minutes, followed by three washes with PBS.

4.2 Blocking phage libraries and magnetic beads.

Add 0.5 mL of 1% BSA/PBS to 0.5 mL of phage library, place on a rotary shaker at 18 rpm, and incubate at room temperature for 1 hour. These phages are designated Input1. Simultaneously, take 100 μL of unconjugated DynaBeads 280, wash three times with PBS, add 1 mL of 1% BSA/PBS, and incubate under the same conditions for 1 hour. Separately, add 1 mL of 1% BSA/PBS to the CD70-conjugated magnetic beads and incubate under the same conditions for 1 hour.

4.3 Negative screening.

To remove antibodies that interact with the magnetic beads, negative panning is necessary. A BSA-blocked phage library and unconjugated magnetic beads are mixed and incubated under the conditions described above for 1 hour. After incubation, the phage-magnetic bead mixture is placed on a magnetic rack. Once the beads have adhered to the walls, the supernatant is transferred to a new EP tube.

4.4 Positive screening.

The blocked magnetic beads conjugated with CD70 protein were added to the phage supernatant after negative selection for positive selection, and incubated at room temperature for 1 hour under the conditions described above. After incubation, the magnetic beads were washed with 1 mL of PBST (0.1% Tween-20 in PBS), and the washing was repeated 10 times. After washing, 1 mL of 100 mM glycine (pH 2.0) was added, and the mixture was placed on a rotary shaker at 18 rpm for elution for 10 minutes. After elution, the EP tube was placed on a magnetic rack, and after the magnetic beads adhered to the wall, the eluent was transferred to a new EP tube. 0.2 mL of 1 M Tris-HCl solution (pH 8.0) was added to the eluent for neutralization. The neutralized eluent was added to 30 mL of TG1 bacterial culture with an OD600 of approximately 0.6 and allowed to stand for 30 minutes for infection. Then, 20 times the number of M13KO7 bacteriophages were added and allowed to stand for another 30 minutes for infection. Finally, 100 mL of 2YT medium and ampicillin and kanamycin at a final concentration of 100 μg/mL were added, and the mixture was incubated overnight at 30°C and 220 rpm. The next day, bacteriophages were harvested using the same method described above for harvesting a bacteriophage library. The bacteriophage obtained at this point was Input2.

4.5 Repeated positive screening.

The above panning method was repeated twice, that is, Input2 was subjected to another round of negative and positive panning to obtain Input3. The difference is that after the eluent obtained from Input3 was infected with TG1, M13KO7 was not added. Instead, 10 μL of bacterial culture was serially diluted, and 100 μL of each of the three dilutions ( ^10³ , ^10⁴ , and ^10⁵ ) was spread on 2YT/amp plates and incubated overnight at 30°C; the remaining bacterial culture was incubated overnight at 30°C and 220 rpm.

4.6 ELISA screening for positive antibodies.

Randomly pick TG1 monoclonal antibodies from the above plates with a toothpick and transfer them to a deep-well plate containing 600 μL of 2 YT/amp. Cover the deep-well plate with a breathable membrane and incubate at 37°C and 220 rpm for 3 hours. After incubation, remove the breathable membrane and add IPTG to a final concentration of 1 mM. Incubate overnight at 30°C and 220 rpm. Coat each well of the ELISA plate with 100 ng of recombinant human CD70 protein. The next day, centrifuge the deep-well plate at 4000 rpm for 10 minutes, remove the culture medium from the wells, and retain the bacterial pellet. Add 100 μL of TES solution (20% sucrose, 0.1 mM EDTA, 50 mM Tris-HCl, pH 8.0) to each well, shake to resuspend the bacterial pellet, incubate on ice for 30 minutes, add 200 μL of ultrapure water, shake to mix, and centrifuge at 4000 rpm for 10 minutes. The supernatant in the deep-well plate at this point is the periplasmic extract containing the antibody. Wash the ELISA plate three times with a plate washer, then add 200 μL of 1% BSA/PBS and block at 37°C for 1 hour. Remove the blocking solution from the ELISA plate, add 100 μL of the above periplasmic extract, incubate at 37°C for 1 hour, wash three times with a plate washer, add HRP-conjugated-Goat antiHA solution, incubate at 37°C for 1 hour, wash three times with a plate washer, add 100 μL of TMB chromogenic buffer, incubate at 37°C for 10 minutes, and stop the reaction with 100 μL of 1M hydrochloric acid. Read the OD450 value using a microplate reader. Perform Sanger sequencing on clones with OD450 values three times higher than the background value to obtain the antibody gene sequence.

4.7 Verify positive clones.

Based on the sequencing results, clones with significant amino acid sequence differences in antibody CDR3 were selected, re-inoculated, and induced overnight. The ELISA method described above was then used to verify whether the selected clones could bind to CD70. Ultimately, eight VHH antibody sequences were obtained: CD70-11C9, CD70-8E1, CD70-8F9, CD70-9D8, CD70-2A5, CD70-5C10, CD70-8A6, and CD70-8B4.

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

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

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

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

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

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

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

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

Example 3: Expression of recombinant VHH antibody

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 via homologous recombination to obtain recombinant VHH-huIgG1-Fc. The above vector was transiently transfected into 293F cells for eukaryotic expression. After harvesting the supernatant, the protein was purified using HiTrap Protein A HP/AKTA pure100. The purified protein was concentrated using ultrafiltration, and the buffer was replaced with PBS. SDS-PAGE electrophoresis was used to detect protein purity. The electrophoresis results are shown in Figure 6.

Example 4: Determination of binding curves between recombinant human CD70 protein and eight VHH antibodies

The specific procedures for the ELISA experiment are as follows: Add 100 ng/well of the recombinant human CD70 protein prepared above to the microplate and coat overnight at 4°C. Wash three times with PBS, add 200 μL/well of 1% BSA/PBS, and block at 37°C for 1 hour. After washing with 100 μL of PBS, add serially diluted 4 scFv proteins mentioned above and bind at 37°C for 1 hour. Wash three times with PBST, add 100 μL of 1:5000 diluted HRP-goat anti-human IgG (Fab-specific) and bind at 37°C for 1 hour. Wash three times with PBST, add 100 μL/well of TMB chromogenic buffer, incubate at 37°C for 10 minutes, add 100 μL/well of ELISA stop solution, and read the OD450 value using a microplate reader. The results are shown in Figure 7 and Table 3.

Table 3, Test Results

Example 5: Affinity Measurement

The affinity of four VHH-huFc antibodies (CD70-2A5, CD70-8B4, CD70-9D8, and CD70-11C9) for human CD70 was analyzed using an Octet K2 molecular interaction analyzer. SA probes were immobilized using 200 μL of 100 nM biotinylated recombinant human CD70 protein, with an immobilization height of 1 nM. The purified VHH-huFc antibodies were used as analytes, and affinity was determined at four concentrations: 200 nM, 100 nM, 50 nM, and 25 nM. The results are shown in Figure 8 and Table 4.

Table 4. Results of VHH-huFc affinity determination

name KD(M) Kon(1/Ms) Koff(1/s) CD70-2A5 <1E-12 3.67E+4 <1.0E-7 CD70-8B4 <1E-12 4.55E+4 <1.0E-7 CD70-9D8 1.83E-9 1.29E+5 2.35E-4 CD70-11C9 <1E-12 2.64E+5 <1.0E-7

Example 6: VHH-huFc blocks CD27/CD70 binding

The specific procedures for the ELISA experiment are as follows: Add 100 ng/well of recombinant human CD27 protein to the microplate and incubate overnight at 4°C. Wash three times with PBS, add 200 μL/well of 1% BSA/PBS, and block at 37°C for 1 hour. Incubate 100 ng of biotinylated recombinant CD70 protein with 1 μg, 500 ng, 250 ng, and 125 ng of the above-mentioned VHH-huFc antibody for 1 hour, respectively. After washing with 200 μL of PBS, add the above-mentioned CD70/VHH-Fc mixture and bind at 37°C for 1 hour. Wash three times with PBST, add 100 μL of 1:200 diluted SA-HRP, and bind at 37°C for 1 hour. Wash three times with PBST, add 100 μL/well of TMB chromogenic buffer, incubate at 37°C for 10 minutes, add 100 μL/well of ELISA stop solution, and read the OD450 value using a microplate reader. The results are shown in Figure 9 and Table 5.

Example 7: Preparation of retroviral stock solution containing anti-human CD70 chimeric antigen receptor element.

1. Preparation of chimeric antigen receptors targeting human CD70 antigen

A chimeric antigen receptor sequence containing a single-chain antibody (scFv) against human CD70 antigen was synthesized or cloned, comprising the hinge region, transmembrane region, and intracellular signaling segment, as shown in Figure 10. Based on the different VHH loading sites, the chimeric antigen receptors were 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. Their amino acid sequences are shown in SEQ ID NO:29-36, and their nucleotide sequences are shown in SEQ ID NO:42-49, respectively. Simultaneously, a chimeric antigen receptor was constructed from the scFv of a known CD70 antibody (clone number: ARGX110) reported in the literature as a control, named ARGX-BBz, with its nucleic acid sequence shown in SEQ ID NO:52.

Using the reverse transcription vector MSGV as the backbone, chimeric antigen receptor retroviral plasmids expressing clones of CD70-2A5-BBz, CD70-5C10-BBz, CD70-8A6-BBz, CD70-8B4-BBz, CD70-8E1-BBz, CD70-8F9-BBz, CD70-9D8-BBz, CD70-11C9-BBz, and ARGX-BBz were constructed. Clones with correct sequencing were selected, inoculated into 300 mL of LB medium, and cultured overnight. Plasmid extraction was then performed according to the NucleoBond Xtra Maxi EF kit instructions.

2. Retroviral packaging.

The retrovirus was packaged using the cationic polymer PEI (Polyplus) as follows: PEI and the retrovirus packaging plasmids (main viral plasmid, Gag-pol, 10A1) were diluted separately with serum-free DMEM; then, PEI/DMEM was added to the plasmid/DMEM mixture, vortexed to mix, and incubated at room temperature for 15 minutes; the plasmid-PEI complex was then added to pre-coated 293T cells. The medium was changed 16 hours after transfection, and the viral supernatant was collected after 48 hours, filtered through a 0.45µm filter, aliquoted into 15mL centrifuge tubes, and stored at -80℃ for later use.

Example 8: Preparation of CD70 CAR-T cells and determination of CAR positivity rate

1. PBMC isolation and activation.

Peripheral blood was collected from volunteers, and PBMCs were isolated using Ficoll separation medium. The cell density was adjusted to 1 x ^10⁶ /mL using X-VIVO (LONZA) medium containing 5% AB serum. TC-treated 6-well plates were pre-coated with 1 mL of coating solution containing 50 ng/mL anti-human CD3 antibody (Beijing Tongli Haiyuan) and 50 ng/mL CD28 antibody (Beijing Tongli Haiyuan) at 37°C for 2 h. The coating solution was removed before use. Cells were seeded at 1 mL/well into the antibody-coated 6-well plates, followed by the addition of 100 IU/mL IL-2 (Beijing Shuanglu). After 48 hours of stimulation and culture, viral infection was performed.

2. Virus stock solution infection and culture.

Activated T cells were adjusted to a density of 5 x ^10⁵ /mL. 1 mL of T cells and 1 mL of viral stock solution were added to each well of a 24-well plate, along with 1 μL of polybrene. The plates were centrifuged at 32°C and 2500 rpm for 1.5 h. The supernatant was discarded, and 1 mL of T cell culture medium (containing 100 IU/mL IL-2) was added to each well. The plates were incubated at 37°C in a 5% _CO₂ incubator. 24 h post-infection, the cells were transferred to 6-well plates. Cell density was monitored daily, and T cell culture medium containing 100 IU/mL IL-2 was added as needed to maintain a T cell density of approximately 1 x ^10⁶ /mL, promoting cell proliferation.

3. CAR positivity rate detection.

The CAR positivity rate of retrovirally infected T lymphocytes was detected 72 h after viral infection. Chimeric antigen receptor groups containing clones of CD70-2A5-BBz, CD70-5C10-BBz, CD70-8A6-BBz, CD70-8B4-BBz, CD70-8E1-BBz, CD70-8F9-BBz, CD70-9D8-BBz, CD70-11C9-BBz, and ARGX-BBz, and negative uninfected control groups (NT), were collected at 1× ^10⁶ cells per cell. After centrifugation to remove the culture medium, the cells were washed once with PBS and resuspended in 100 μL in flow cytometry tubes (BD). Fc-labeled CD70 antigen (1:100) was added and incubated at °C for 30 min. After washing the cells once with PBS, secondary antibody PE-Fc was added at the recommended ratio and incubated at °C for 30 min in the dark. After washing the cells once with PBS, the cells were resuspended in 200 μL of PBS and analyzed by flow cytometry. The results of the CAR-T positivity rate analysis are shown in Figure 11.

Example 9: Functional analysis based on anti-human CD70 CAR-T cells.

1. Analysis of CD107a expression in anti-human CD70 CAR-T cells.

CAR-T cells containing different antibody clones and NT cells were co-incubated with target cells (CD70-positive cell line MOLM13) at a 1:1 effector-target ratio (3 x 10⁵ effector cells and ³ x 10⁵ target cells each) by flow cytometry to evaluate the degranulation response of CAR-T cells after stimulation by target cells. After co-incubating effector cells and target cells together at 37°C in a 5% _CO₂ incubator for 4 hours, the proportion of CD107a-expressing cells to CD3+ cells in each group was determined by flow cytometry. The flow cytometry analysis results of CD107a expression are shown in Figure 12.

2. Detection of cytokine secretion capacity of anti-human CD70 CAR-T cells.

CAR-T cells containing different antibody clones were co-incubated with target cells (CD70-positive cell line MOLM13) at a 1:1 effector-target ratio (1 x 10⁵ effector cells and ¹ x 10⁵ target cells) for 24 hours. The supernatant was collected, and the secretion of IFN-γ and IL-2 was detected using ELISA (enzyme-linked immunosorbent assay). IFN-γ was detected using the BD IFN-γ and IL-2 kit, and the experimental procedures were performed according to the product instructions. The results of IFN-γ secretion are shown in Figure 13. The results of IL-2 secretion are shown in Figure 14.

3. Anti-human CD70 CAR-T cell toxicity experiment.

The CAR-T cell cytotoxicity assay evaluated the in vitro function of CAR-T cells by detecting their cytotoxic effect on target cells. T cells were co-cultured with CD70-positive target cells (MOLM13-LUC-GFP) stably expressing firefly luciferase at different effector-to-target ratios (based on ³ x 10⁴ target cells, with effector-to-target ratios of 10:1, 5:1, and 2.5:1). A negative control group (NT) consisting of target cells and untransfected CAR element T cells was also included. After overnight incubation, luciferase reaction substrate was added to the culture system, and fluorescence values were measured. The cytotoxicity was calculated using the following formula: Cytotoxicity = (1 - fluorescence value of experimental wells / fluorescence value of control wells) x 100%. The experimental groups and analysis results are shown in Figure 15.

Example 10: Intra-liquid efficacy test of anti-human CD70 CAR-T cell injection.

CAR-T cells of four clones (CD70-2A5/8B4/9D8/11C9) were prepared according to the method described in Example 8 of this invention for in vivo pharmacodynamic testing in animals. The CAR-T positivity rate and the in vitro killing efficiency of the prepared CAR-T cells against ACHN cells (human renal cancer cell line expressing CD70) were detected. MOLM13 cells were used as a control. The details are as follows:

1. CAR positivity rate detection

The CAR positivity rate of retrovirally infected T lymphocytes was detected 72 h after viral infection. For chimeric antigen receptor groups containing clones of CD70-2A5-BBz, CD70-8B4-BBz, CD70-9D8-BBz, CD70-11C9-BBz, and ARGX-BBz, and for the negative uninfected control group (NT), 1× ^10⁶ cells were collected, centrifuged to remove the culture medium, washed once with PBS, and resuspended in 100 μL in flow cytometry tubes (BD). Fc-labeled CD70 antigen (1:100) was added and incubated at °C for 30 min. After washing once with PBS, secondary antibody PE-Fc was added at the recommended ratio and incubated at °C in the dark for 30 min. After washing once with PBS, the cells were resuspended in 200 μL of PBS and analyzed by flow cytometry. The results of CAR-T positivity rate analysis are shown in Figure 16.

2. Analysis of CD107a expression in anti-human CD70 CAR-T cells.

CAR-T cells containing CD70-2A5, CD70-8B4, CD70-9D8, and CD70-11C9, as well as NT cells, were co-incubated with ACHN and MOLM13 cells (renal cancer cell line ACHN and promyelocytic leukemia cell line MOLM13) at a 1:1 effector-target ratio (3 x 10⁵ effector cells and ³ x 10⁵ target cells each) by flow cytometry to evaluate the degranulation response of CAR-T cells after stimulation by target cells. After co-incubating effector and target cells together at 37°C in a 5% _CO₂ incubator for 4 hours, the proportion of CD107a-expressing cells to CD3+ cells in each group was determined by flow cytometry. The flow cytometry results of CD107a expression are shown in Figure 17.

3. Experiments on the killing of ACHN and MOLM13 cells by anti-human CD70 CAR-T cells.

CAR-T cells containing CD70-2A5, CD70-8B4, CD70-9D8, and CD70-11C9, as well as NT cells, were co-cultured with CD70-positive ACHN and MOLM13 target cells stably expressing firefly luciferase (ACHN-LUC-GFP and MOLM13-LUC-GFP) at different effector-to-target ratios (based on ³ x 10⁴ target cells, effector-to-target ratios were 10:1, 5:1, and 2.5:1). After overnight incubation, luciferase reaction substrate was added to the culture system, and fluorescence values were detected. The killing efficiency was calculated using the following formula: Killing efficiency = (1 - fluorescence value of experimental wells / fluorescence value of control wells) x 100%. The experimental groups and analysis results are shown in Figure 18.

4. In vivo efficacy experiment in mice

Forty-eight NOG-bearing female mice (subcutaneously inoculated with human renal cell carcinoma line ACHN cells) were used in the experiment. When the tumor volume reached approximately 100 ^mm³ , they were randomly divided into six groups according to tumor size. These groups received intravenous injections of NT cell therapy and five different clones of CD70 CAR-T cells (codes: P376, 2A5, 11C9, 8B4, and 9D8). Each group received 200 μL of the drug per mouse, with a dose of 3 × ^10⁷ total cells per mouse. Clinical observation was conducted daily during the experiment. Approximately 100 μL of anticoagulated blood was collected from each group via the orbital sinus on days 1, 7, 16, and 22 for flow cytometry analysis to detect T cell survival. Tumor volume was measured approximately twice weekly, and mouse survival status was recorded (mice with tumor volumes reaching 2000 ^mm³ were euthanized). The tumor volume monitoring results are shown in Figure 19, and the T cell percentage results are shown in Figure 20.

The above results indicate that the CD70-targeting CAR-T cells constructed based on the novel VHH antibody that specifically binds to CD70 provided by this invention have strong immune functions. Compared with control CAR-T cells (clone number ARGX110), they exhibit superior CD107a expression, IFN-γ and IL-2 secretion, and specific killing function against target cells, demonstrating significant in vivo efficacy.

The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

SEQUENCE LISTING

<110> Shanghai Hengrunda Biotechnology Co., Ltd.

<120> Anti-CD70 nanobody and its applications

<130> 219639

<160> 52

<170> PatentIn version 3.5

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1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Ser Met Asn

20 25 30

Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Ala Ile Thr Ser Gly Gly Ser Thr Asn Tyr Val Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Ala Gly Ala Gly Arg Ala Phe Pro Gly Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Gln Val Thr Val Ser Ser

115

<210> 23

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH

<400> 23

Glu Val Gln Val Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Ser Ile Phe Ser Ile Asn

20 25 30

Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Ala Ile Thr Ser Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Ser Gly Ala Gly Arg Ala Phe Pro Gly Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 24

<211> 123

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH

<400> 24

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Phe Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Gly Tyr

20 25 30

Ala Val Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

Ser Cys Ile Ser Ser Ser Asp Gly Ser Thr Tyr Tyr Ile Asp Ser Val

50 55 60

Gln Gly Arg Phe Thr Ile Thr Arg Asn Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Thr Asp Val Leu Thr Ser Cys Arg Ser Asp Arg Trp Leu Glu Val

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120

<210> 25

<211> 118

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH

<400> 25

Gln Val Gln Leu Val Glu Thr Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asn Ile Phe Ser Asn Asn

20 25 30

Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Ala Ile Thr Ser Asp Gly Ser Thr Asn Tyr Val Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Leu Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Ala Gly Ala Gly Arg Ala Phe Pro Gly Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser

115

<210> 26

<211> 126

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH

<400> 26

Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Arg Thr Ser Ser Thr Tyr

20 25 30

Ala Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Ile Val

35 40 45

Ala Val Ile Gly Trp Ser Gly Gly Arg Thr Ala Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ala Lys Asp Asn Ala Lys Asn Thr Val Ser

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Gly Val Tyr Tyr Cys

85 90 95

Ala Ala Asp Ser Glu Gln Ser Ser Ser Ser Glu Gly Gln Ala Tyr Gly

100 105 110

Tyr Glu Asp Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser

115 120 125

<210> 27

<211> 125

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH

<400> 27

Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Arg Thr Ser Ser Thr Tyr

20 25 30

Ala Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Ile Val

35 40 45

Ala Val Ile Thr Trp Ser Gly Gly Arg Thr Ala Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ala Lys Asp Ser Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Asp Ser Glu Val Ser Ser Ser Ser Glu Gly Gln Tyr Gly Tyr

100 105 110

Glu Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser

115 120 125

<210> 28

<211> 124

<212> PRT

<213> Artificial Sequence

<220>

<223> VHH

<400> 28

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Tyr Tyr

20 25 30

Ala Val Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

Ser Cys Ile Ser Ser Ser Ser Asp Gly Ser Thr Tyr Tyr Val Asp Ser

50 55 60

Val Leu Gly Arg Phe Thr Ile Ser Arg Asn Asn Ala Glu Asn Thr Val

65 70 75 80

Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ser Thr Asp Val Leu Thr Ser Cys Arg Ser Asp Arg Tyr Leu Glu

100 105 110

Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Gly

115 120

<210> 29

<211> 346

<212> PRT

<213> Artificial Sequence

<220>

<223> CAR

<400> 29

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Ser Ile Asn

20 25 30

Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Ala Ile Thr Ser Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Ala Gly Ala Gly Arg Ala Phe Pro Gly Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Gln Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr

115 120 125

Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala

130 135 140

Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe

145 150 155 160

Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val

165 170 175

Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Arg Phe Ser Val Val

180 185 190

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

195 200 205

Arg Pro Val Gln Thr Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

210 215 220

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg

225 230 235 240

Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn

245 250 255

Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg

260 265 270

Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro

275 280 285

Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala

290 295 300

Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His

305 310 315 320

Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp

325 330 335

Ala Leu His Met Gln Ala Leu Pro Pro Arg

340 345

<210> 30

<211> 346

<212> PRT

<213> Artificial Sequence

<220>

<223> CAR

<400> 30

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Ile Phe Ser Met Asn

20 25 30

Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Ala Ile Thr Ser Gly Gly Ser Thr Asn Tyr Val Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Ala Gly Ala Gly Arg Ala Phe Pro Gly Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Gln Val Thr Val Ser Ser Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr

115 120 125

Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala

130 135 140

Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe

145 150 155 160

Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val

165 170 175

Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Arg Phe Ser Val Val

180 185 190

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

195 200 205

Arg Pro Val Gln Thr Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

210 215 220

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg

225 230 235 240

Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn

245 250 255

Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg

260 265 270

Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro

275 280 285

Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala

290 295 300

Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His

305 310 315 320

Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp

325 330 335

Ala Leu His Met Gln Ala Leu Pro Pro Arg

340 345

<210> 31

<211> 346

<212> PRT

<213> Artificial Sequence

<220>

<223> CAR

<400> 31

Glu Val Gln Val Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Arg Ser Ile Phe Ser Ile Asn

20 25 30

Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Ala Ile Thr Ser Gly Gly Ser Thr Asn Tyr Ala Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Tyr Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Ser Gly Ala Gly Arg Ala Phe Pro Gly Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Thr Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr

115 120 125

Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala

130 135 140

Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe

145 150 155 160

Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val

165 170 175

Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Arg Phe Ser Val Val

180 185 190

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

195 200 205

Arg Pro Val Gln Thr Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

210 215 220

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg

225 230 235 240

Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn

245 250 255

Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg

260 265 270

Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro

275 280 285

Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala

290 295 300

Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His

305 310 315 320

Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp

325 330 335

Ala Leu His Met Gln Ala Leu Pro Pro Arg

340 345

<210> 32

<211> 351

<212> PRT

<213> Artificial Sequence

<220>

<223> CAR

<400> 32

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Phe Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Gly Tyr

20 25 30

Ala Val Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

Ser Cys Ile Ser Ser Ser Asp Gly Ser Thr Tyr Tyr Ile Asp Ser Val

50 55 60

Gln Gly Arg Phe Thr Ile Thr Arg Asn Asn Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Thr Thr Asp Val Leu Thr Ser Cys Arg Ser Asp Arg Trp Leu Glu Val

100 105 110

Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Thr Thr Thr Pro Ala

115 120 125

Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser

130 135 140

Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr

145 150 155 160

Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala

165 170 175

Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys

180 185 190

Arg Phe Ser Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe

195 200 205

Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly

210 215 220

Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg

225 230 235 240

Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln

245 250 255

Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp

260 265 270

Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro

275 280 285

Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp

290 295 300

Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg

305 310 315 320

Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr

325 330 335

Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

340 345 350

<210> 33

<211> 346

<212> PRT

<213> Artificial Sequence

<220>

<223> CAR

<400> 33

Gln Val Gln Leu Val Glu Thr Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Asn Ile Phe Ser Asn Asn

20 25 30

Ala Met Gly Trp Tyr Arg Gln Ala Pro Gly Lys Gln Arg Glu Leu Val

35 40 45

Ala Ala Ile Thr Ser Asp Gly Ser Thr Asn Tyr Val Asp Ser Val Lys

50 55 60

Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Val Leu Leu

65 70 75 80

Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys Asn

85 90 95

Ala Gly Ala Gly Arg Ala Phe Pro Gly Asp Tyr Trp Gly Gln Gly Thr

100 105 110

Leu Val Thr Val Ser Ser Thr Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr

115 120 125

Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala

130 135 140

Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe

145 150 155 160

Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val

165 170 175

Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Arg Phe Ser Val Val

180 185 190

Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met

195 200 205

Arg Pro Val Gln Thr Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe

210 215 220

Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg

225 230 235 240

Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln Asn Gln Leu Tyr Asn

245 250 255

Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg

260 265 270

Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro

275 280 285

Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala

290 295 300

Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His

305 310 315 320

Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp

325 330 335

Ala Leu His Met Gln Ala Leu Pro Pro Arg

340 345

<210> 34

<211> 354

<212> PRT

<213> Artificial Sequence

<220>

<223> CAR

<400> 34

Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Ala Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Arg Thr Ser Ser Thr Tyr

20 25 30

Ala Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Ile Val

35 40 45

Ala Val Ile Gly Trp Ser Gly Gly Arg Thr Ala Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ala Lys Asp Asn Ala Lys Asn Thr Val Ser

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Gly Val Tyr Tyr Cys

85 90 95

Ala Ala Asp Ser Glu Gln Ser Ser Ser Ser Glu Gly Gln Ala Tyr Gly

100 105 110

Tyr Glu Asp Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Thr Thr

115 120 125

Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln

130 135 140

Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala

145 150 155 160

Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala

165 170 175

Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr

180 185 190

Leu Tyr Cys Arg Phe Ser Val Val Lys Arg Gly Arg Lys Lys Leu Leu

195 200 205

Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Thr Gln Glu

210 215 220

Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys

225 230 235 240

Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln

245 250 255

Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu

260 265 270

Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly

275 280 285

Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu

290 295 300

Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly

305 310 315 320

Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser

325 330 335

Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro

340 345 350

Pro Arg

<210> 35

<211> 353

<212> PRT

<213> Artificial Sequence

<220>

<223> CAR

<400> 35

Gln Leu Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Val Ser Gly Arg Thr Ser Ser Thr Tyr

20 25 30

Ala Met Ala Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Ile Val

35 40 45

Ala Val Ile Thr Trp Ser Gly Gly Arg Thr Ala Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ala Lys Asp Ser Ala Lys Asn Thr Val Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Ala Asp Ser Glu Val Ser Ser Ser Ser Glu Gly Gln Tyr Gly Tyr

100 105 110

Glu Ser Trp Gly Gln Gly Thr Gln Val Thr Val Ser Ser Thr Thr Thr

115 120 125

Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro

130 135 140

Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val

145 150 155 160

His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro

165 170 175

Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu

180 185 190

Tyr Cys Arg Phe Ser Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr

195 200 205

Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu

210 215 220

Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu

225 230 235 240

Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln

245 250 255

Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu

260 265 270

Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly

275 280 285

Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln

290 295 300

Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu

305 310 315 320

Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr

325 330 335

Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro

340 345 350

Arg

<210> 36

<211> 352

<212> PRT

<213> Artificial Sequence

<220>

<223> CAR

<400> 36

Gln Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Leu Asp Tyr Tyr

20 25 30

Ala Val Gly Trp Phe Arg Gln Ala Pro Gly Lys Glu Arg Glu Gly Val

35 40 45

Ser Cys Ile Ser Ser Ser Ser Asp Gly Ser Thr Tyr Tyr Val Asp Ser

50 55 60

Val Leu Gly Arg Phe Thr Ile Ser Arg Asn Asn Ala Glu Asn Thr Val

65 70 75 80

Tyr Leu Gln Met Asn Ser Leu Lys Pro Glu Asp Thr Ala Val Tyr Tyr

85 90 95

Cys Ser Thr Asp Val Leu Thr Ser Cys Arg Ser Asp Arg Tyr Leu Glu

100 105 110

Val Trp Gly Gln Gly Thr Leu Val Thr Val Ser Gly Thr Thr Thr Pro

115 120 125

Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu

130 135 140

Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His

145 150 155 160

Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu

165 170 175

Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr

180 185 190

Cys Arg Phe Ser Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile

195 200 205

Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp

210 215 220

Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Gly Gly Cys Glu Leu

225 230 235 240

Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly

245 250 255

Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr

260 265 270

Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys

275 280 285

Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys

290 295 300

Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg

305 310 315 320

Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala

325 330 335

Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

340 345 350

<210> 37

<211> 231

<212> PRT

<213> Artificial Sequence

<220>

<223> CH

<400> 37

Pro Lys Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro

1 5 10 15

Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys

20 25 30

Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val

35 40 45

Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp

50 55 60

Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr

65 70 75 80

Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp

85 90 95

Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu

100 105 110

Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg

115 120 125

Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys

130 135 140

Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp

145 150 155 160

Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys

165 170 175

Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser

180 185 190

Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser

195 200 205

Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser

210 215 220

Leu Ser Leu Ser Pro Gly Lys

225 230

<210> 38

<211> 21

<212> PRT

<213> Artificial Sequence

<220>

<223> CD8 SP

<400> 38

Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu

1 5 10 15

His Ala Ala Arg Pro

20

<210> 39

<211> 47

<212> PRT

<213> Artificial Sequence

<220>

<223> CD8 Hinge

<400> 39

Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala

1 5 10 15

Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly

20 25 30

Gly Ala Val His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr

35 40 45

<210> 40

<211> 22

<212> PRT

<213> Artificial Sequence

<220>

<223> CD8™

<400> 40

Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu

1 5 10 15

Val Ile Thr Leu Tyr Cys

20

<210> 41

<211> 111

<212> PRT

<213> Artificial Sequence

<220>

<223> CD3zeta cytoplasmic domain

<400> 41

Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Gln Gln Gly Gln

1 5 10 15

Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp

20 25 30

Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro

35 40 45

Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp

50 55 60

Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg

65 70 75 80

Arg Gly Lys Gly His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr

85 90 95

Lys Asp Thr Tyr Asp Ala Leu His Met Gln Ala Leu Pro Pro Arg

100 105 110

<210> 42

<211> 48

<212> PRT

<213> Artificial Sequence

<220>

<223> 41BB cytoplasmic domain

<400> 42

Arg Phe Ser Val Val Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe

1 5 10 15

Lys Gln Pro Phe Met Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly

20 25 30

Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg

35 40 45

<210> 43

<211> 1038

<212> DNA

<213> Artificial Sequence

<220>

<223> CAR

<400> 43

gaggtgcagc tggtggagtc tgggggaggc ttggtgcagg ctggggggtc tctgagactc 60

tcctgtgcag cctctggaag catcttcagt atcaatgcca tgggctggta ccgccaggct 120

ccagggaagc agcgcgagtt ggtcgcagct attactagtg gtggtagcac aaactatgca 180

gactccgtga aggggccgatt caccatctcc agagacaacg ccaagaacac ggtgtatctg 240

caaatgaaca gcctgaaacc tgaggacaca gccgtctatt actgtaatgc aggagcgggg 300

cgggcctttc ccggggacta ctggggccag gggacccagg tcaccgtctc ctcaactaca 360

actccagcac ccagaccccc tacacctgct ccaactatcg caagtcagcc cctgtcactg 420

cgccctgaag cctgtcgccc tgctgccggg ggagctgtgc atactcgggg actggacttt 480

gcctgtgata tctacatctg ggcgcccttg gccgggactt gtggggtcct tctcctgtca 540

ctggttatca ccctttatactg caggttcagt gtcgtgaaga gaggccggaa gaagctgctg 600

tacatcttca agcagccttt catgaggccc gtgcagacta cccaggagga agatggatgc 660

agctgtagat tccctgaaga ggaggaagga ggctgtgagc tgagagtgaa gttctcccga 720

agcgcagatg ccccagccta tcagcaggga cagaatcagc tgtacaacga gctgaacctg 780

ggaagacggg aggaatacga tgtgctggac aaaaggcggg gcagagatcc tgagatgggc 840

ggcaaaccaa gacggaagaa ccccaggaa ggtctgtata atgagctgca gaaagacaag 900

atggctgagg cctactcaga aatcgggatg aagggcgaaa gaaggagagg aaaaggccac 960

gacggactgt accaggggct gagtacagca acaaaagaca cctatgacgc tctgcacatg 1020

caggctctgc caccaaga 1038

<210> 44

<211> 1038

<212> DNA

<213> Artificial Sequence

<220>

<223> CAR

<400> 44

caggtgcagc tcgtggagtc tgggggaggc ttggtgcagg ctggggggtc tctgagactc 60

tcctgtgcag cctctggaag catcttcagt atgaatgcca tgggctggta ccgccaggct 120

ccagggaagc agcgcgagtt ggtcgcagct attactagtg gtggtagcac aaactatgta 180

gactccgtga aggggccgatt caccatctcc agagacaacg ccaagaacac ggtgtatctg 240

caaatgaaca gcctgaaacc tgaggacaca gccgtctatt actgtaatgc aggagcgggg 300

cgggcctttc ccggggacta ctggggccag gggacccagg tcaccgtctc ctcaactaca 360

actccagcac ccagaccccc tacacctgct ccaactatcg caagtcagcc cctgtcactg 420

cgccctgaag cctgtcgccc tgctgccggg ggagctgtgc atactcgggg actggacttt 480

gcctgtgata tctacatctg ggcgcccttg gccgggactt gtggggtcct tctcctgtca 540

ctggttatca ccctttatactg caggttcagt gtcgtgaaga gaggccggaa gaagctgctg 600

tacatcttca agcagccttt catgaggccc gtgcagacta cccaggagga agatggatgc 660

agctgtagat tccctgaaga ggaggaagga ggctgtgagc tgagagtgaa gttctcccga 720

agcgcagatg ccccagccta tcagcaggga cagaatcagc tgtacaacga gctgaacctg 780

ggaagacggg aggaatacga tgtgctggac aaaaggcggg gcagagatcc tgagatgggc 840

ggcaaaccaa gacggaagaa ccccaggaa ggtctgtata atgagctgca gaaagacaag 900

atggctgagg cctactcaga aatcgggatg aagggcgaaa gaaggagagg aaaaggccac 960

gacggactgt accaggggct gagtacagca acaaaagaca cctatgacgc tctgcacatg 1020

caggctctgc caccaaga 1038

<210> 45

<211> 1038

<212> DNA

<213> Artificial Sequence

<220>

<223> CAR

<400> 45

gaggtgcagg tcgtggagtc tgggggaggc ttggtgcagc ctggggggtc tctgagactc 60

tcctgtgcag cctctcgaag catcttcagt atcaatgcca tgggctggta ccgccaggct 120

ccagggaaac agcgcgagtt ggtcgcagct attactagtg gtggtagcac aaactatgca 180

gactccgtga aggggccgatt caccatctcc agagacaacg ccaagaacac ggtgtatctg 240

caaatgaaca gcctgaaacc tgaggacaca gccgtctatt actgtaattc aggagcgggg 300

cgggcctttc ccggggacta ctggggccag gggacctgg tcaccgtctc ctcaactaca 360

actccagcac ccagaccccc tacacctgct ccaactatcg caagtcagcc cctgtcactg 420

cgccctgaag cctgtcgccc tgctgccggg ggagctgtgc atactcgggg actggacttt 480

gcctgtgata tctacatctg ggcgcccttg gccgggactt gtggggtcct tctcctgtca 540

ctggttatca ccctttatactg caggttcagt gtcgtgaaga gaggccggaa gaagctgctg 600

tacatcttca agcagccttt catgaggccc gtgcagacta cccaggagga agatggatgc 660

agctgtagat tccctgaaga ggaggaagga ggctgtgagc tgagagtgaa gttctcccga 720

agcgcagatg ccccagccta tcagcaggga cagaatcagc tgtacaacga gctgaacctg 780

ggaagacggg aggaatacga tgtgctggac aaaaggcggg gcagagatcc tgagatgggc 840

ggcaaaccaa gacggaagaa ccccaggaa ggtctgtata atgagctgca gaaagacaag 900

atggctgagg cctactcaga aatcgggatg aagggcgaaa gaaggagagg aaaaggccac 960

gacggactgt accaggggct gagtacagca acaaaagaca cctatgacgc tctgcacatg 1020

caggctctgc caccaaga 1038

<210> 46

<211> 1053

<212> DNA

<213> Artificial Sequence

<220>

<223> CAR

<400> 46

gaggtgcagc tcgtggagtc tgggggaggc tttgtgcagc cgggggggtc tctgagactc 60

tcctgtgcag cctctggatt cactttggat ggctatgccg tagcctggtt ccgccaggcc 120

ccagggaaag agcgcgaggg ggtctcatgt attagtagta gtgatggtag cacatactat 180

atagactccg tacagggccg attcaccatc acaagaaaca atgccaagaa cacggtgtat 240

ctgcaaatga acagcctgaa acctgaggac acggccgttt attactgtac gacagatgtc 300

cttacgagct gccggagcga caggtggctc gaagtttggg gccagggcac cctggtcact 360

gtctcctcaa ctacaactcc agcacccaga ccccctacac ctgctccaac tatcgcaagt 420

cagcccctgt cactgcgccc tgaagcctgt cgccctgctg ccgggggagc tgtgcatact 480

cggggactgg actttgcctg tgatatctac atctgggcgc ccttggccgg gacttgtggg 540

gtccttctcc tgtcactggt tatcaccctt tactgcaggt tcagtgtcgt gaagagaggc 600

cggaagaagc tgctgtacat cttcaagcag cctttcatga ggcccgtgca gactacccag 660

gaggaagatg gatgcagctg tagattccct gaagaggagg aaggaggctg tgagctgaga 720

gtgaagttct cccgaagcgc agatgcccca gcctatcagc agggacagaa tcagctgtac 780

aacgagctga acctgggaag acggggaggaa tacgatgtgc tggacaaaag gcggggcaga 840

gatcctgaga tgggcggcaa accaagacgg aagaaccccc aggaaggtct gtataatgag 900

ctgcagaaag acaagatggc tgaggcctac tcagaaatcg ggatgaaggg cgaaagaagg 960

agaggaaaag gccacgacgg actgtaccag gggctgagta cagcaacaaa agacacctat 1020

gacgctctgc acatgcaggc tctgccacca aga 1053

<210> 47

<211> 1038

<212> DNA

<213> Artificial Sequence

<220>

<223> CAR

<400> 47

caagtgcagc tggtggagac tgggggaggc ttggtgcagg ctggggggtc tctgagactc 60

tcctgtgcag cctctggaaa catcttcagt aacaatgcca tgggctggta ccgccaggct 120

ccagggaagc agcgcgagtt ggtcgcagct attactagtg atggtagcac aaactatgta 180

gactccgtga aggggccgatt caccatctcc agagacaacg ccaagaacac ggtgcttctg 240

caaatgaaca gcctgaaacc tgaggacaca gccgtctatt actgtaatgc aggagcgggg 300

cgggcctttc ccggggacta ctggggccag gggaccctgg tcactgtctc ctcaactaca 360

actccagcac ccagaccccc tacacctgct ccaactatcg caagtcagcc cctgtcactg 420

cgccctgaag cctgtcgccc tgctgccggg ggagctgtgc atactcgggg actggacttt 480

gcctgtgata tctacatctg ggcgcccttg gccgggactt gtggggtcct tctcctgtca 540

ctggttatca ccctttatactg caggttcagt gtcgtgaaga gaggccggaa gaagctgctg 600

tacatcttca agcagccttt catgaggccc gtgcagacta cccaggagga agatggatgc 660

agctgtagat tccctgaaga ggaggaagga ggctgtgagc tgagagtgaa gttctcccga 720

agcgcagatg ccccagccta tcagcaggga cagaatcagc tgtacaacga gctgaacctg 780

ggaagacggg aggaatacga tgtgctggac aaaaggcggg gcagagatcc tgagatgggc 840

ggcaaaccaa gacggaagaa ccccaggaa ggtctgtata atgagctgca gaaagacaag 900

atggctgagg cctactcaga aatcgggatg aagggcgaaa gaaggagagg aaaaggccac 960

gacggactgt accaggggct gagtacagca acaaaagaca cctatgacgc tctgcacatg 1020

caggctctgc caccaaga 1038

<210> 48

<211> 1062

<212> DNA

<213> Artificial Sequence

<220>

<223> CAR

<400> 48

cagttgcagc tcgtggagtc tgggggagga ttggtgcagg ctgggggctc tctaagactc 60

tcctgtgcag tttctggacg cacctcaagt acttatgcca tggcctggtt ccgccaggct 120

ccaggcaagg agcgtgagat tgtagcagtt attggatgga gtggtggtag gacggcctat 180

gcagactccg tgaagggccg attcactatc gccaaagaca acgccaagaa cacggtgtct 240

ctgcaaatga acagtctgaa acctgaggac acgggcgttt attactgtgc agcagattct 300

gaacaaagta gctcgtccga aggccaggcg tatggctatg aggactgggg ccaggggacc 360

ctggtcactg tctcctcaac tacaactcca gcacccagac cccctacacc tgctccaact 420

atcgcaagtc agcccctgtc actgcgcct gaagcctgtc gccctgctgc cgggggagct 480

gtgcatactc ggggactgga ctttgcctgt gatatctaca tctgggcgcc cttggccggg 540

acttgtgggg tccttctcct gtcactggtt atcacccttt actgcaggtt cagtgtcgtg 600

aagagaggcc ggaagaagct gctgtacatc ttcaagcagc ctttcatgag gcccgtgcag 660

actacccagg aggaagatgg atgcagctgt agattccctg aagaggagga aggaggctgt 720

gagctgagag tgaagttctc ccgaagcgca gatgccccag cctatcagca gggacagaat 780

cagctgtaca acgagctgaa cctgggaaga cgggaggaat acgatgtgct ggacaaaagg 840

cggggcagag atcctgagat gggcggcaaa ccaagacgga agaaccccca ggaaggtctg 900

tataatgagc tgcagaaaga caagatggct gaggcctact cagaaatcgg gatgaagggc 960

gaaagaagga gaggaaaagg ccacgacgga ctgtaccagg ggctgagtac agcaacaaaa 1020

gacacctatg acgctctgca catgcaggct ctgccaccaa ga 1062

<210> 49

<211> 1059

<212> DNA

<213> Artificial Sequence

<220>

<223> CAR

<400> 49

cagttgcagc tcgtggagtc tgggggaggc ttggtgcagc ctggggggtc cctgagactc 60

tcctgtgcag tttctggacg cacctcaagt acttatgcca tggcctggtt ccgccaggct 120

ccaggcaagg agcgtgagat tgtagcagtt attacatgga gtggtggtag gacggcctat 180

gcagactccg tgaagggccg atcactatc gccaaagaca gcgccaagaa cacggtgtat 240

ctgcaaatga acagcctgaa acctgaggat acggccgttt attactgtgc agcagattct 300

gaagtaagca gctcgtccga aggccagtat ggctatgagt cctggggcca ggggacccag 360

gtcaccgtct cctcaactac aactccagca cccagacccc ctacacctgc tccaactatc 420

gcaagtcagc ccctgtcact gcgccctgaa gcctgtcgcc ctgctgccgg gggagctgtg 480

catactcggg gactggactt tgcctgtgat atctacatct gggcgccctt ggccgggact 540

tgtggggtcc ttctcctgtc actggttatc accctttact gcaggttcag tgtcgtgaag 600

agaggccgga agaagctgct gtacatcttc aagcagcctt tcatgaggcc cgtgcagact 660

acccaggagg aagatggatg cagctgtaga ttccctgaag aggaggaagg aggctgtgag 720

ctgagagtga agttctcccg aagcgcagat gccccagcct atcagcaggg agagaatcag 780

ctgtacaacg agctgaacct gggaagacgg gaggaatacg atgtgctgga caaaaggcgg 840

ggcagagatc ctgagatggg cggcaaacca agacggaaga accccagga aggtctgtat 900

aatgagctgc agaaagacaa gatggctgag gcctactcag aaatcggggat gaagggcgaa 960

agaaggagag gaaaaggcca cgacggactg taccaggggc tgagtacagc aacaaaagac 1020

acctatgacg ctctgcacat gcaggctctg ccaccaaga 1059

<210> 50

<211> 1056

<212> DNA

<213> Artificial Sequence

<220>

<223> CAR

<400> 50

caggtgcagc tggtggagtc tgggggaggc ttggtgcagc cgggggggtc tctgagactc 60

tcctgtgcag cctctggatt cactttggat tattatgccg taggctggtt ccgccaggcc 120

ccagggaagg agcgcgaggg ggtctcatgt attagtagta gtagtgatgg tagcacatac 180

tatgtagact ccgtgctggg ccgattcacc atctccagaa acaatgccga gaacacggtg 240

tatctgcaaa tgaacagcct gaaacctgag gacacggccg tttattactg ttcgacagat 300

gtccttacga gctgccggag cgacaggtat ctcgaagttt ggggccaggg caccctggtc 360

actgtctcag gcactacaac tccagcaccc agaccccccta cacctgctcc aactatcgca 420

agtcagcccc tgtcactgcg ccctgaagcc tgtcgccctg ctgccggggg agctgtgcat 480

actcggggac tggactttgc ctgtgatatc tacatctggg cgcccttggc cgggacttgt 540

ggggtccttc tcctgtcact ggttatcacc ctttactgca ggttcagtgt cgtgaagaga 600

ggccggaaga agctgctgta catcttcaag cagcctttca tgaggcccgt gcagactacc 660

caggaggaag atggatgcag ctgtagattc cctgaagagg aggaaggagg ctgtgagctg 720

agagtgaagt tctcccgaag cgcagatgcc ccagcctatc agcagggaca gaatcagctg 780

tacaacgagc tgaacctggg aagacgggag gaatacgatg tgctggacaa aaggcggggc 840

agagatcctg agatgggcgg caaaccaaga cggaagaacc cccaggaagg tctgtataat 900

gagctgcaga aagacaagat ggctgaggcc tactcagaaa tcggggatgaa gggcgaaaga 960

aggagaggaa aaggccacga cggactgtac caggggctga gtacagcaac aaaagacacc 1020

tatgacgctc tgcacatgca ggctctgcca ccaaga 1056

<210> 51

<211> 238

<212> PRT

<213> Artificial Sequence

<220>

<223> IgG1Fc

<400> 51

Glu Leu Lys Thr Pro Gln Pro Gln Ser Gln Pro Glu Cys Arg Cys Pro

1 5 10 15

Lys Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro Ser Val Phe Ile Phe

20 25 30

Pro Pro Lys Pro Lys Asp Val Leu Ser Ile Ser Gly Arg Pro Glu Val

35 40 45

Thr Cys Val Val Val Asp Val Gly Gln Glu Asp Pro Glu Val Ser Phe

50 55 60

Asn Trp Tyr Ile Asp Gly Ala Glu Val Arg Thr Ala Asn Thr Lys Pro

65 70 75 80

Lys Glu Glu Gln Phe Asn Ser Thr Tyr Arg Val Val Ser Val Leu Pro

85 90 95

Ile Arg His Gln Asp Trp Leu Thr Gly Lys Glu Phe Lys Cys Lys Val

100 105 110

Asn Asn Lys Ala Leu Pro Ala Pro Ile Glu Arg Thr Ile Ser Lys Ala

115 120 125

Lys Gly Gln Thr Arg Glu Pro Gln Val Tyr Ala Leu Ala Pro His Arg

130 135 140

Glu Glu Leu Ala Lys Asp Thr Val Ser Val Thr Cys Leu Val Lys Asp

145 150 155 160

Phe Tyr Pro Val Asp Ile Asn Ile Glu Trp Gln Arg Asn Gly Gln Pro

165 170 175

Glu Ser Glu Gly Thr Tyr Ala Thr Thr Pro Pro Gln Leu Asp Asn Asp

180 185 190

Gly Thr Tyr Phe Leu Tyr Ser Lys Leu Ser Val Gly Lys Asn Thr Trp

195 200 205

Gln Arg Gly Glu Thr Phe Thr Cys Val Val Met His Glu Ala Leu Pro

210 215 220

Asn His Tyr Thr Gln Lys Ser Ile Thr Gln Ser Ser Gly Lys

225                 230                 235

<210> 52

<211> 1410

<212> DNA

<213> Artificial Sequence

<220>

<223> ARGXBBz

<400> 52

gttgtgaccc aggagccttc cctgacagtg tctccaggag ggacggtcac gctcacctgc 60

ggcctcaaat ctgggtctgt cacttccgat aacttcccca cttggtacca gcagacacca 120

ggccaggctc cccgattgct tatctacaac acaaacaccc gtcactctgg cgtccccgac 180

cgcttctccg gatccatcct gggcaacaaa gccgccctca ccatcacggg ggcccaggcc 240

gacgacgagg ccgaatattt ctgtgctctg ttcataagta atcctagtgt tgagttcggc 300

ggagggaccc aactgaccgt cctaggtggc agcaccagcg gctccggcaa gcctggatct 360

ggcgagggca gcaccaaggg cgaggtgcag ctcgtggagt ctgggggagg cttggtgcag 420

cctggggggt ctctgagact ctcctgtgca gcctctggat tcaccttcag tgtctactac 480

atgaactggg tccgccaggc tccagggaag gggctcgagt gggtctcaga tattaataat 540

gaaggtggta ctacatacta tgcagactcc gtgaagggcc gattcaccat ctccagagac 600

aactctaaga acagcctgta tctgcaaatg aacagcctgc gcgccgagga cacggccgtg 660

tactactgcg cgagagatgc cggatatagc aaccatgtac ccatctttga ttcttggggc 720

cagggcacta caactccagc accgaccc cctacacctg ctccaactat cgcaagtcag 780

cccctgtcac tgcgccctga agcctgtcgc cctgctgccg ggggagctgt gcatactcgg 840

ggactggact ttgcctgtga tatctacatc tgggcgccct tggccgggac ttgtggggtc 900

cttctcctgt cactggttat caccctttac tgcaggttca gtgtcgtgaa gagaggccgg 960

aagaagctgc tgtacatctt caagcagcct ttcatgaggc ccgtgcagac tacccaggag 1020

gaagatggat gcagctgtag attccctgaa gaggaggaag gaggctgtga gctgagagtg 1080

aagttctccc gaagcgcaga tgccccagcc tatcagcagg gacagaatca gctgtacaac 1140

gagctgaacc tgggaagacg ggaggaatac gatgtgctgg acaaaaggcg gggcagagat 1200

cctgagatgg gcggcaaacc aagacggaag aacccccagg aaggtctgta taatgagctg 1260

cagaaagaca agatggctga ggcctactca gaaatcggga tgaagggcga aagaaggaga 1320

ggaaaaggcc acgacggact gtaccagggg ctgagtacag caacaaaaga cacctatgac 1380

gctctgcaca tgcaggctct gccaccaaga 1410

Claims (20)

1. 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 or the antigen-binding fragment thereof comprises CDR1, CDR2, and CDR3, and 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, wherein, CDR1 is shown as SEQ ID NO:4, CDR2 as shown as SEQ ID NO:9, and CDR3 as shown as SEQ ID NO:16, or CDR1 is shown in SEQ ID NO:7, CDR2 is shown in SEQ ID NO:13, and CDR3 is shown in SEQ ID NO:20. 2. The CD70-binding molecule as claimed in claim 1, characterized in that the CD70-binding molecule further has one or more of the following features: The heavy chain variable region sequence of the anti-CD70 nanobody is shown in SEQ ID NO:24 or 28. The nanobody is a camel heavy chain antibody. The nanobody also contains a heavy chain constant region. 3. A chimeric antigen receptor comprising the CD70 binding molecule, hinge region, transmembrane region, and intracellular region as described in claim 1 or 2. 4. The chimeric antigen receptor as claimed in claim 3, wherein the chimeric antigen receptor comprises a signal peptide sequence, the CD70 binding molecule as claimed in claim 1 or 2, a hinge region, a transmembrane region, and an intracellular region. 5. The chimeric antigen receptor as described in claim 3 or 4, wherein the intracellular region comprises an intracellular co-stimulatory domain and/or an intracellular signaling domain. 6. The chimeric antigen receptor as described in claim 3 or 4, characterized in that, from the N-terminus to the C-terminus, the chimeric antigen receptor sequentially comprises a signal peptide, the CD70 binding molecule as described in claim 1 or 2, a hinge region, a transmembrane region, an intracellular co-stimulatory domain, and an intracellular signaling domain. 7. A nucleic acid molecule whose sequence is selected from any of the following: (1) The coding sequence of the CD70 binding molecule of claim 1 or 2 or the chimeric antigen receptor of any one of claims 3-6; (2) and (1) are complementary sequences. 8. A nucleic acid construct comprising the nucleic acid molecule of claim 7. 9. The nucleic acid construct according to claim 8, wherein the nucleic acid construct is a cloning vector, an expression vector, or an integration vector. 10. A host cell, selected from: (1) Expressing and/or secreting the CD70 binding molecule of claim 1 or 2 or the chimeric antigen receptor of any one of claims 3-6; (2) Contains the nucleic acid molecule as described in claim 7; and/or (3) Contains the nucleic acid construct as described in claim 8 or 9. 11. The host cell according to claim 10, wherein the host cell is an immune effector cell. 12. The host cell as claimed in claim 11, wherein the host cell is a T cell. 13. A method for generating a CD70 binding molecule as described in claim 1 or 2 or a chimeric antigen receptor as described in any one of claims 3-6, comprising: culturing a host cell as described in any one of claims 10-12 under conditions suitable for generating a CD70 binding molecule. 14. The method of claim 13, wherein the method comprises: purifying the CD70 binding molecule or chimeric antigen receptor from the culture. 15. A pharmaceutical composition comprising: a chimeric antigen receptor or its coding sequence as described in any one of claims 3-6, a nucleic acid construct or host cell comprising the coding sequence, and a pharmaceutically acceptable excipient, wherein the host cell expresses and/or secretes the chimeric antigen receptor, the coding sequence, or the nucleic acid construct. 16. Use of the CD70 binding molecule of claim 1 or 2, the chimeric antigen receptor of any one of claims 3-6, the nucleic acid molecule of claim 7, the nucleic acid construct of claim 8 or 9, or the host cell of any one of claims 10-12 in the preparation of activated immune cells, or in the preparation of a medicament for the prevention or treatment of CD70-positive tumors, wherein the CD70-positive tumors are selected from one or more of the following: 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. 17. A kit for detecting CD70, the kit comprising the CD70 binding molecule of claim 1 or 2. 18. The kit of claim 17, wherein the kit further comprises a reagent for detecting the binding of CD70 to the CD70 binding molecule. 19. The kit of claim 18, wherein the reagent for detection binding is a detectable marker that can be linked to a CD70 binding molecule. 20. Use of the CD70 binding molecule of claim 1 or 2 in the preparation of a kit for detecting CD70 in a sample, a kit for evaluating the efficacy of drug treatment, or a kit for diagnosing CD70-positive tumors.