Nanometer antibody combined with CD70 and application of nanometer antibody in field of cell therapy

Abstract

The invention provides a nano antibody combined with CD70 and application of the nano antibody in the field of cell therapy. Specifically, the invention provides a nano antibody (VHH) for targeted recognition of CD70 and a chimeric antigen receptor immune cell constructed based on the nano antibody. The invention also provides application of the nano antibody and the chimeric antigen receptor immune cell in treatment of CD70 related diseases.

Description

Technical Field

[0001] This invention relates to the field of biomedicine. Specifically, this invention relates to CD70-bound nanobodies and their application in cell therapy. Background Technology

[0002] Renal cell carcinoma is one of the most common malignant tumors of the urinary system. It originates from the epithelial system of the renal parenchyma and urinary tubules. The full academic term is renal cell carcinoma, also known as renal adenocarcinoma, or simply kidney cancer. The most common type of renal cell carcinoma is clear cell renal cell carcinoma (ccRCC), accounting for 70-80%. The second largest category is papillary renal cell carcinoma (pRCC).

[0003] Over 90% of kidney-related malignancies are represented by renal cell carcinoma (RCC). Although some promising treatments for renal cell carcinoma have emerged in the past decade, such as PD-1 monoclonal antibodies, immunotherapy, and combination therapy with tyrosine kinase inhibitors (ICI-TKIs), these therapies show significant efficacy in the early stages of treatment. However, their complete response (CR) rate is only 6%-16%, necessitating further exploration of alternative approaches.

[0004] CAR-T, short for Chimeric Antigen Receptor T-Cell Immunotherapy, represents a breakthrough in immunotherapy. It's a novel, precise, targeted therapy for cancer, releasing a large number of cytokines through immune responses to efficiently kill tumor cells, thus rapidly and effectively curing cancer. This therapy has shown promising results in hematologic malignancies, making solid tumors, including kidney cancer, a focal point of immunotherapy.

[0005] In recent years, CAR-T cell therapy has achieved remarkable results in the treatment of hematological malignancies. However, the efficacy of CAR-T cell immunotherapy for solid tumors is not as significant as its efficacy in treating hematological malignancies. This is because treating solid tumors presents numerous challenges compared to hematological malignancies, such as selecting the most suitable target, promoting T cell delivery and infiltration, overcoming the tumor immunosuppressive microenvironment, and avoiding treatment side effects. Nevertheless, CAR-T therapy still represents a significant clinical need in the treatment of solid tumors, which account for over 90% of all cancers. Therefore, developing innovative strategies to overcome these challenges and improve the efficacy of CAR-T cell therapy is imperative.

[0006] CD70 is a type II transmembrane protein belonging to the tumor necrosis factor family. Under pathological conditions, CD70 is highly expressed in various tumor tissues, particularly renal cell carcinoma cells. However, in normal tissues, it is not expressed or is expressed in small amounts. Therefore, CD70 represents a potential therapeutic target for renal cell carcinoma with both efficacy and safety.

[0007] Studies have reported that the tumor microenvironment of renal cell carcinoma is a highly structured ecosystem composed of multiple cell types. However, it differs from the microenvironment of other solid tumors. Research has demonstrated that renal cell carcinoma is a type of tumor tissue with high T-cell infiltration, making it suitable for T-cell immunotherapy.

[0008] The most important component of CAR is the antibody molecule, which has a significant impact on the cytotoxic function and safety of CAR-T cells. Studies have reported the discovery of a novel antibody called a nanobody in alpacas, which possesses unique advantages such as small structural domains, good stability, high solubility, and low immunogenicity. This could be helpful in developing potent, low-toxicity CAR-T cell products targeting CD70.

[0009] Therefore, there is a need in this field to develop an alpaca-based nanobody targeting CD70 nanobody and cell-related drugs for the treatment of renal cell carcinoma. Summary of the Invention

[0010] The purpose of this invention is to provide a CD70-targeting nanobody based on alpaca nanobody and cell-related drugs for the treatment of renal cell carcinoma.

[0011] In a first aspect of the invention, an anti-CD70 nanobody is provided, wherein the complementarity-determining region (CDR) of the VHH chain of the nanobody is shown below:

[0012] CDR1 shown in SEQ ID NO:2

[0013] CDR2 shown in SEQ ID NO:3, and

[0014] CDR3 as shown in SEQ ID NO:4.

[0015] In another preferred embodiment, the CDR region of the nanobody VHH chain contains an amino acid sequence having at least 80%, preferably at least 90%, more preferably at least 95% sequence similarity to any of the above sequences.

[0016] In another preferred embodiment, any of the above-mentioned amino acid sequences further includes a derived sequence that has optionally been added, deleted, modified and / or substituted at least one amino acid and is capable of retaining CD70 binding affinity.

[0017] In another preferred embodiment, the number of added, deleted, modified and / or substituted amino acids is 1-3, more preferably 1-2, and even more preferably 1.

[0018] In another preferred embodiment, the VHH chain of the nanobody further includes a framework region (FR).

[0019] In another preferred embodiment, CDR1, CDR2 and CDR3 are separated by the frame regions FR1, FR2, FR3 and FR4 of the VHH chain.

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

[0021] In another preferred embodiment, the VHH chain of the nanobody has an amino acid sequence that is ≥85%, ≥90%, ≥95%, ≥96%, ≥97%, ≥98%, or ≥99% homology to the amino acid sequence shown in SEQ ID NO:1.

[0022] In another preferred embodiment, the VHH chain of the nanobody has one or more amino acid sequences as shown in SEQ ID NO:1.

[0023] In another preferred embodiment, the nanobody is a monomer, a bivalent (bivalent antibody), or a multivalent (multivalent antibody).

[0024] In another preferred embodiment, the amino acid sequence of the VHH chain of the nanobody is shown in SEQ ID NO:1.

[0025] In another preferred embodiment, the nanobody comprises the Fc segment of an antibody, preferably the Fc segment of IgG, and more preferably the Fc segment of human IgG.

[0026] In a second aspect of the invention, a chimeric antigen receptor is provided, wherein the antigen-binding domain of the chimeric antigen receptor is the VHH chain of the nanobody described in the first aspect of the invention.

[0027] In another preferred embodiment, the structure of the chimeric antigen receptor is shown in Formula I:

[0028] LVFH-TM-C-CD3ζ(I)

[0029] In the formula,

[0030] Each "-" independently represents a linking peptide or peptide bond;

[0031] L represents the absence of a signal peptide sequence;

[0032] V represents the antigen-binding domain;

[0033] F represents unlabeled or unmarked proteins.

[0034] H represents the hinge area;

[0035] TM represents a transmembrane domain;

[0036] C is a co-stimulatory signaling molecule;

[0037] CD3ζ is a cytoplasmic signal transduction sequence derived from CD3ζ.

[0038] In another preferred embodiment, L is a signal peptide of a protein selected from the group consisting of CD8, CD28, GM-CSF, CD4, CD137, or a combination thereof.

[0039] In another preferred embodiment, H is a hinge region derived from a protein selected from the group consisting of Fc, CD8, CD28, CD137, or a combination thereof.

[0040] In another preferred embodiment, H is a hinge region derived from CD8.

[0041] In another preferred embodiment, the amino acid sequence of the hinge region is shown in SEQ ID NO:5.

[0042] In another preferred embodiment, the TM is a transmembrane region of a protein selected from the group consisting of: CD8a, ICOS, CD28, CD3epsilon, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, or a combination thereof.

[0043] In another preferred embodiment, the TM is a transmembrane region derived from CD8a.

[0044] In another preferred embodiment, the amino acid sequence of the transmembrane region is shown in SEQ ID NO:6.

[0045] In another preferred embodiment, C is a co-stimulatory signaling molecule selected from the group consisting of: 4-1BB (CD137), ICOS, OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, PD1, Dap10, CDS, ICAM-1, LFA-1 (CD11a / CD18), ICOS (CD278), NKG2D, GITR, TLR2, or combinations thereof.

[0046] In another preferred embodiment, C is a co-stimulatory signaling molecule derived from 4-1BB.

[0047] In another preferred embodiment, the amino acid sequence of the co-stimulatory signaling molecule is shown in SEQ ID NO:7.

[0048] In another preferred embodiment, the amino acid sequence of CD3ζ is shown in SEQ ID NO:8.

[0049] In a third aspect of the invention, a polynucleotide is provided that encodes a protein selected from the group consisting of: anti-CD70 nanobodies as described in the first aspect of the invention, chimeric antigen receptors as described in the second aspect of the invention, or combinations thereof.

[0050] In another preferred embodiment, the polynucleotide includes DNA, RNA, or cDNA.

[0051] In a fourth aspect of the invention, an expression vector is provided, the expression vector containing the polynucleotide as described in the third aspect of the invention.

[0052] In another preferred embodiment, the expression vector is selected from the group consisting of DNA, RNA, viral vectors, plasmids, transposons, other gene transfer systems, or combinations thereof.

[0053] In another preferred embodiment, the expression vector includes a viral vector, such as a lentivirus, adenovirus, AAV virus, or retrovirus.

[0054] In a fifth aspect of the invention, a host cell is provided, the host cell containing an expression vector as described in the fourth aspect of the invention, or having a genome containing polynucleotides as described in the third aspect of the invention.

[0055] In another preferred embodiment, the host cell includes a prokaryotic cell or a eukaryotic cell.

[0056] In another preferred embodiment, the host cell is selected from the group consisting of Escherichia coli, yeast cells, mammalian cells, bacteriophages, or combinations thereof.

[0057] In a sixth aspect of the invention, an engineered immune cell is provided, said immune cell expressing a chimeric antigen receptor as described in the second aspect of the invention.

[0058] In another preferred embodiment, the immune cells are T cells, NK cells, or NKT cells.

[0059] In another preferred embodiment, the engineered immune cells are chimeric antigen receptor T cells (CAR-T cells) or chimeric antigen receptor NK cells (CAR-NK cells).

[0060] In a seventh aspect of the invention, a method for generating anti-CD70 nanobodies is provided, comprising the steps of:

[0061] (a) Culturing host cells as described in the fifth aspect of the invention under conditions suitable for generating nanobodies, thereby obtaining a culture containing the anti-CD70 nanobodies; and

[0062] (b) Isolating or recovering the anti-CD70 nanobody from the culture; and

[0063] (c) Optionally, purify and / or modify the anti-CD70 nanobody obtained in step (b).

[0064] In an eighth aspect of the invention, an immunoconjugate is provided, the immunoconjugate comprising:

[0065] (a) the anti-CD70 nanobody as described in the first aspect of the present invention; and

[0066] (b) The conjugation portion of the nanobody, wherein the conjugation portion is selected from the group consisting of: detectable markers, drugs, or combinations thereof.

[0067] In another preferred embodiment, the immunoconjugate is an antibody-drug conjugate.

[0068] In another preferred embodiment, the antibody portion is coupled to the coupling portion via a chemical bond or a linker.

[0069] In another preferred embodiment, the detectable marker is a chemical marker, a biological marker, or a combination thereof.

[0070] In another preferred embodiment, the chemical label is an isotope, an immunotoxin, and / or a chemical drug.

[0071] In another preferred embodiment, the biomarker is biotin, avidin, or an enzyme label.

[0072] In another preferred embodiment, the drug is a small molecule drug, a biological factor, or a combination thereof.

[0073] In another preferred embodiment, the drug is a cytotoxic drug (toxin).

[0074] In another preferred embodiment, the cytotoxic drug is selected from the group consisting of: anti-tubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, folic acid antagonists, antimetabolites, chemosensitizers, topoisomerase inhibitors, vinca alkaloids, or combinations thereof.

[0075] In another preferred embodiment, examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and microtubule inhibitors. Typical cytotoxic drugs include, for example, auristatins, camptothecins, duocarmycins, etoposides, maytansines and maytansinoids (e.g., DM1 and DM4), taxanes, benzodiazepines or benzodiazepine-containing drugs (e.g., pyrrolo[1,4]benzodiazepines (PBDs), indolinobenzodiazepines and oxazolidinobenzodiazepines), vinca alkaloids, or combinations thereof.

[0076] In another preferred embodiment, the toxin is selected from the group consisting of: ostatins (e.g., ostatin E, ostatin F, MMAE, and MMAF), chlortetracycline, methamphetamine, pyrine, pyrine A-chain, cobustatin, docalimicin, dolalastatin, doxorubicin, daunorubicin, paclitaxel, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, and dihydroxychloroquine. Anthraxone, actinomycin, diphtheria toxin, Pseudomonas exotoxin (PE)A, PE40, abrin, abrin A chain, saccharin A chain, α-Dacococcus, white tree toxin, mitogellin, retstrictocin, phenolmycin, enoxacin, curicin, croton toxin, chachomycin, Sapaonaria officinalis inhibitor, glucocorticoids, or combinations thereof.

[0077] In another preferred embodiment, the toxin is MMAE.

[0078] In another preferred embodiment, the coupling portion is a detectable marker.

[0079] In another preferred embodiment, the immunoconjugate comprises: a multivalent (e.g., bivalent) anti-CD70 nanobody as described in the first aspect of the invention. The multivalent meaning refers to the presence of a plurality of repeating anti-CD70 nanobodies as described in the first aspect of the invention in the amino acid sequence of the immunoconjugate.

[0080] In another preferred embodiment, the immunoconjugate has the following molecular formula:

[0081]

[0082] in:

[0083] nAb is an anti-CD70 nanobody as described in the first aspect of the present invention;

[0084] LU represents a chemical bond or linker;

[0085] D is a drug;

[0086] p is the average number of drugs conjugated in the antibody-drug conjugate, and p is a value selected from 1 to 10.

[0087] In a ninth aspect of the invention, a multispecific antibody is provided, the multispecific antibody comprising the anti-CD70 nanobody as described in the first aspect of the invention.

[0088] In another preferred embodiment, the multispecific antibody comprises a heavy chain constant region.

[0089] In another preferred embodiment, the heavy chain constant region is derived from the Fc segment of IgG, preferably the Fc segment of human IgG.

[0090] In a tenth aspect of the present invention, a recombinant protein is provided, said recombinant protein having:

[0091] (i) sequences of nanobodies as described in the first aspect of the present invention, chimeric antigen receptors as described in the second aspect of the present invention, and multispecific antibodies as described in the ninth aspect of the present invention; and

[0092] (ii) Tag sequences that assist in expression and / or purification.

[0093] In another preferred embodiment, the tag sequence includes a 6His tag and an HA tag.

[0094] In another preferred embodiment, the recombinant protein specifically binds to the CD70 protein.

[0095] In an eleventh aspect of the present invention, a pharmaceutical composition is provided comprising:

[0096] (i) the anti-CD70 nanobody as described in the first aspect of the present invention, or the chimeric antigen receptor as described in the second aspect of the present invention, or the immunoconjugate as described in the eighth aspect of the present invention, or the multispecific antibody as described in the ninth aspect of the present invention, or the recombinant protein as described in the tenth aspect of the present invention, or a combination thereof; and

[0097] (ii) Pharmaceutically acceptable carriers.

[0098] In another preferred embodiment, the pharmaceutical composition is an injectable dosage form.

[0099] In another preferred embodiment, the pharmaceutical composition is used to prepare a drug for treating tumors.

[0100] In another preferred embodiment, the tumor is a tumor that highly expresses CD70.

[0101] In another preferred embodiment, the tumor is selected from the group consisting of: renal cell carcinoma, colon cancer, mesothelioma, pancreatic cancer, prostate cancer, ovarian germ cell cancer, thyroid cancer, gastric cancer, esophageal cancer, lung cancer (such as lung adenocarcinoma and non-small cell lung cancer), breast cancer (such as triple-negative breast cancer), malignant glioma, liver cancer, bladder cancer, endometrial cancer, cervical cancer, leukemia, bone marrow cancer, osteosarcoma, angiosarcoma, or combinations thereof.

[0102] In another preferred embodiment, the tumor is kidney cancer.

[0103] In another preferred embodiment, the pharmaceutical composition further contains other drugs for treating immune system diseases or tumor diseases.

[0104] In a twelfth aspect of the invention, there is provided a use of an active ingredient selected from the group consisting of: anti-CD70 nanobodies as described in the first aspect of the invention, chimeric antigen receptors as described in the second aspect of the invention, immunoconjugates as described in the eighth aspect of the invention, multispecific antibodies as described in the ninth aspect of the invention, recombinant proteins as described in the tenth aspect of the invention, pharmaceutical compositions as described in the eleventh aspect of the invention, or combinations thereof, wherein the active ingredient is used for (a) preparing diagnostic reagents, diagnostic plates, or kits; and / or (b) preparing medicaments for the prevention and / or treatment of diseases.

[0105] In another preferred embodiment, the detection reagent, detection plate, or kit is used for:

[0106] (1) Detect CD70 protein in the sample; and / or

[0107] (2) Detection of tumor cells expressing CD70 protein.

[0108] In another preferred embodiment, the detection reagent, detection plate, or kit is used to diagnose CD70-related diseases.

[0109] In another preferred embodiment, the detection includes flow cytometry and cell immunofluorescence detection.

[0110] In another preferred embodiment, the disease is a tumor.

[0111] In another preferred embodiment, the tumor is a tumor that highly expresses CD70.

[0112] In another preferred embodiment, the tumor is selected from the group consisting of: renal cell carcinoma, colon cancer, mesothelioma, pancreatic cancer, prostate cancer, ovarian germ cell cancer, thyroid cancer, gastric cancer, esophageal cancer, lung cancer (such as lung adenocarcinoma and non-small cell lung cancer), breast cancer (such as triple-negative breast cancer), malignant glioma, liver cancer, bladder cancer, endometrial cancer, cervical cancer, leukemia, bone marrow cancer, osteosarcoma, angiosarcoma, or combinations thereof.

[0113] In another preferred embodiment, the tumor is kidney cancer.

[0114] In a thirteenth aspect of the present invention, a method for detecting CD70 protein in a sample is provided, the method comprising the steps of:

[0115] (1) Contact the sample with the anti-CD70 nanobody as described in the first aspect of the present invention;

[0116] (2) Detect whether an antigen-antibody complex is formed, where the formation of a complex indicates the presence of CD70 protein in the sample.

[0117] In a fourteenth aspect of the present invention, a CD70 protein detection reagent is provided, the detection reagent comprising:

[0118] (i) the anti-CD70 nanobody as described in the first aspect of the present invention, or the immunoconjugate as described in the eighth aspect of the present invention, or the recombinant protein as described in the tenth aspect of the present invention; and

[0119] (ii) A detectable carrier.

[0120] In another preferred embodiment, the coupling portion of the immunoconjugate is a diagnostic isotope.

[0121] In another preferred embodiment, the detection-acceptable carrier is a non-toxic, inert aqueous carrier medium.

[0122] In another preferred embodiment, the detection reagent is one or more reagents selected from the group consisting of isotope tracers, contrast agents, flow cytometry reagents, cell immunofluorescence reagents, magnetic nanoparticles, and imaging agents.

[0123] In another preferred embodiment, the detection reagent is used for in vivo detection.

[0124] In another preferred embodiment, the test reagent is in liquid or powder form (such as aqueous solution, injection, lyophilized powder, tablet, lozenge, or inhaler).

[0125] In a fifteenth aspect of the present invention, a CD70 protein detection kit is provided, the kit comprising an immunoconjugate as described in an eighth aspect of the present invention or a detection reagent as described in a fourteenth aspect of the present invention, and an instruction manual.

[0126] In another preferred embodiment, the instruction manual states that the kit is used for non-invasive detection of CD70 expression in a test subject.

[0127] In a sixteenth aspect of the invention, a method for treating a CD70-related disease is provided, the method comprising administering to a desired subject an anti-CD70 nanobody as described in a first aspect of the invention, an immunoconjugate as described in an eighth aspect of the invention, a recombinant protein as described in a tenth aspect of the invention, or a pharmaceutical composition as described in an eleventh aspect of the invention.

[0128] In another preferred embodiment, the object includes a human or a non-human mammal.

[0129] In another preferred embodiment, the non-human mammals include rodents (such as mice and rabbits) and non-human primates (such as monkeys).

[0130] It should be understood that, within the scope of this invention, the above-described technical features of this invention and the technical features specifically described below (such as in the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, they will not be described in detail here. Attached Figure Description

[0131] The following figures are used to illustrate specific embodiments of the present invention and are not intended to limit the scope of the invention as defined by the claims.

[0132] Figure 1A The technical route for expressing recombinant CD70 VHH-huFc antibody protein is shown.

[0133] Figure 1B The SDS-PAGE electrophoresis results of the recombinant CD70 VHH-huFc antibody protein are shown.

[0134] Figure 2A The results of the affinity assay for the binding of recombinant CD70 VHH-huFc antibody to tumor cells 786-O are shown.

[0135] Figure 2B The results of the affinity assay for the binding of recombinant CD70 VHH-huFc antibody to tumor cells 769-P are shown.

[0136] Figure 3A A schematic diagram of the CD70 CAR structure is shown.

[0137] Figure 3B A schematic diagram of the CD70 CAR-T lentiviral expression vector plasmid structure is shown.

[0138] Figure 4 The results of flow cytometry analysis of the CAR positivity rate in the in vitro functional validation test are shown.

[0139] Figure 5 The results of repeated stimulation experiments with different clones of CD70 CAR-T cells are shown.

[0140] Figure 6 The results of the CAR-T cell killing experiment on 786-O cells were shown after 5 rounds of repeated stimulation experiments.

[0141] Figure 7 The experimental results show that recombinant CD70 VHH-huFc blocks CD27 / CD70 binding.

[0142] Figure 8A The results of the CAR-T cell killing experiment on 786-O-CD70KO cells are shown.

[0143] Figure 8B The results of the CAR-T cell killing experiment on 786-O cells were shown.

[0144] Figure 9A The results of the CAR-T cell killing experiment on 786-O cells were shown.

[0145] Figure 9B The results of the CAR-T cell killing experiment on A-498 cells were shown.

[0146] Figure 10A The results of cytokine release assays were shown when CAR-T cells were co-incubated with 786-O.

[0147] Figure 10B The results of cytokine release assays were shown when CAR-T cells were co-incubated with A-498.

[0148] Figure 11 The results of flow cytometry analysis of the CAR positivity rate in in vivo pharmacodynamic trials are shown.

[0149] Figure 12 A schematic diagram of the anti-tumor experiment of CAR-T in mice is shown.

[0150] Figure 13 The results of tumor volume monitoring in the in vivo efficacy assay of CD70 CAR-T cells are shown.

[0151] Figure 14The graph shows the change in body weight during the in vivo efficacy study of CD70 CAR-T cells. The vertical axis represents body weight, and the horizontal axis represents the date. Detailed Implementation

[0152] Through extensive and in-depth research, the inventors have developed, for the first time, a CD70-binding nanobody and its application in cell therapy. This invention provides a novel CD70-targeting nanobody (VHH). The VHH of this invention exhibits high affinity and specificity. In vitro functional validation experiments and in vivo efficacy experiments show that the VHH of this invention has a significant killing effect on CD70-positive tumor cells and in vivo tumor-clearing ability. Based on these findings, this invention was completed.

[0153] the term

[0154] To facilitate understanding of the invention, certain technical and scientific terms are specifically defined below. Unless otherwise expressly defined herein, all other technical and scientific terms used herein have the meanings commonly understood by one of ordinary skill in the art to which this invention pertains. Before describing the invention, it should be understood that the invention is not limited to the specific methods and experimental conditions described, as such methods and conditions can vary. It should also be understood that the terminology used herein is intended only to describe particular embodiments and is not intended to be restrictive; the scope of the invention will be limited only by the appended claims.

[0155] As used herein, when referring to a specific enumerated value, the term “about” means that the value can vary by no more than 1% from the enumerated values. For example, as used herein, the expression “about 100” includes all values ​​between 99 and 101 (e.g., 99.1, 99.2, 99.3, 99.4, etc.).

[0156] As used herein, the terms “comprising,” “including,” and “containing” are used interchangeably and include not only closed definitions but also semi-closed and open definitions. In other words, the terms include “consisting of” and “substantially consisting of”.

[0157] As used herein, the term "pharmaceutically acceptable carrier" refers to a substance that is suitable for use in humans and / or animals without excessive adverse side effects (such as toxicity, irritation, and allergic reactions), i.e., a reasonable benefit / risk ratio.

[0158] As used herein, the term "therapeutic effective amount" refers to an amount that is functional or active in humans and / or animals and is acceptable to humans and / or animals. Those skilled in the art will understand that the "therapeutic effective amount" can vary depending on the form of the pharmaceutical composition, the route of administration, the excipients used, the severity of the disease, and whether it is used in combination with other drugs.

[0159] Nanobody

[0160] As used herein, the terms "anti-CD70 nanobody of the present invention" and "anti-CD70 nanobody" are used interchangeably and both refer to the nanobody that specifically recognizes and binds to CD70 according to the first aspect of the present invention, and particularly preferred are nanobodies with the amino acid sequence of the VHH chain as shown in SEQ ID NO:1.

[0161] As used herein, the terms "antibody" or "immunoglobulin" refer to isotetraglycoproteins of approximately 150,000 Daltons with identical structural features, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to the heavy chain by a covalent disulfide bond, although the number of disulfide bonds between heavy chains varies among different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end, followed by multiple constant regions. Each light chain has a variable region (VL) at one end and a constant region at the other; the constant regions of the light chains are opposite the first constant region of the heavy chains, and the variable regions of the light chains are opposite the variable regions of the heavy chains. Specific amino acid residues form interfaces between the variable regions of the light and heavy chains.

[0162] As used herein, the terms "single-domain antibody," "nanobody," and "VHH" have the same meaning, referring to the variable region of the heavy chain of a monoclonal antibody. A nanobody (VHH) is the smallest antigen-binding fragment with complete function. Typically, an antibody lacking both the light chain and the constant region 1 (CH1) of the heavy chain is first obtained, and then the variable region of the antibody heavy chain is cloned to construct a nanobody (VHH) consisting of only one heavy chain variable region.

[0163] As used herein, the term "heavy chain antibody" refers to an antibody containing only the heavy chain. Some antibodies found in the blood of camels are "heavy chain antibodies" lacking the light chain. The heavy chain antibody of this invention comprises a heavy chain variable region (VHH) and heavy chain constant regions CH2 and CH3. The heavy chain antibody of this invention can be an antibody derived from an animal (e.g., camel-derived) that is naturally lacking both the light chain and heavy chain constant region 1 (CH1); or it can be a recombinant antibody obtained by recombination of the nanobody (VHH) of this invention with the heavy chain constant region. The heavy chain antibody of this invention may contain a constant region derived from, for example, IgG1, IgG2, IgG3, or IgG4, preferably derived from the constant region of IgG1.

[0164] As used herein, the term "variable" refers to the fact that certain portions of the variable region of an antibody differ sequentially, contributing to the binding and specificity of various specific antibodies to their specific antigens. However, variability is not uniformly distributed throughout the entire variable region of an antibody. It is concentrated in three segments within the variable regions of the light and heavy chains, known as complementarity-determining regions (CDRs) or hypervariable regions. The more conserved portions of the variable region are called framework regions (FRs). The variable regions of the native heavy and light chains each contain four FRs, which are generally β-sheet configurations linked by three CDRs forming a linking loop, and in some cases, partially β-sheet structures. The CDRs in each chain are tightly packed together by the FR regions and, together with the CDRs of the other chain, form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)). Constant regions do not directly participate in antibody-antigen binding, but they exhibit different effector functions, such as participating in antibody-dependent cytotoxicity.

[0165] In a preferred embodiment of the present invention, the heavy chain variable region of the antibody includes three complementarity-determining regions (CDR1, CDR2, and CDR3). Various methods exist for CDR partitioning, including the IMGT method, Kabat method, Chothia method, and VBASE2 method. In one embodiment, the IMGT method is used for all CDR partitioning methods mentioned in this invention.

[0166] In a preferred embodiment of the present invention, the heavy chain of the antibody includes the aforementioned heavy chain variable region and heavy chain constant region.

[0167] The present invention also provides other proteins or fusion expression products having the antibodies of the present invention. Specifically, the present invention includes any protein or protein conjugate and fusion expression product (i.e., immunoconjugate and fusion expression product) having a heavy chain containing a variable region, provided that the variable region is the same as or has at least 90% homology with the heavy chain variable region of the antibody of the present invention, preferably at least 95% homology.

[0168] In this invention, the terms "recombinant protein of the present invention," "fusion protein of the present invention," or "peptide of the present invention" are used interchangeably and all refer to peptides that specifically bind to the CD70 protein, such as proteins or peptides having the VHH chain of the nanobody of the present invention. They may or may not contain initiating methionine.

[0169] The heavy chain variable regions of the antibodies of the present invention are of particular interest because at least a portion of them are involved in binding antigens. Therefore, the present invention 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 to the CDRs identified herein.

[0170] This invention includes not only complete antibodies, but also fragments of immunologically active antibodies or fusion proteins formed by antibodies and other sequences. Therefore, this invention also includes fragments, derivatives, and analogs of said antibodies.

[0171] As used herein, the terms “fragment,” “derivative,” and “analyte” refer to polypeptides that substantially retain the same biological function or activity as the antibodies of the present invention. The polypeptide fragments, derivatives, or analogs of the present invention may be (i) polypeptides in which one or more conserved or non-conserved amino acid residues (preferably conserved amino acid residues) are substituted, and such substituted amino acid residues may or may not be encoded by the genetic code; or (ii) polypeptides having substituent groups in one or more amino acid residues; or (iii) 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 (iv) polypeptides formed by fusing an additional amino acid sequence to this polypeptide sequence (e.g., a leader sequence or secretion sequence, or 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 fragments, derivatives, and analogs are within the scope well known to those skilled in the art.

[0172] The antibody of this invention refers to a polypeptide containing the aforementioned CDR region and possessing CD70 protein-binding activity. This term also includes variants of the polypeptide containing the aforementioned CDR region that have the same function as the antibody of this invention. These variants include (but are not limited to): deletions, insertions, and / or substitutions of one or more amino acids (typically 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10), and the addition of one or more amino acids (typically less than 20, preferably less than 10, more preferably less than 5) to the C-terminus and / or N-terminus. For example, in the art, substitution with amino acids of similar or comparable properties generally does not alter the function of the protein. Similarly, the addition of one or more amino acids to the C-terminus and / or N-terminus generally does not alter the function of the protein. This term also includes active fragments and active derivatives of the antibody of this invention.

[0173] The variant forms of the polypeptide include: homologous sequences, conserved variants, allelic variants, natural mutants, induced mutants, proteins encoded by DNA that can hybridize with the encoding DNA of the antibody of the present invention under high or low severity conditions, and polypeptides or proteins obtained using antiserum against the antibody of the present invention.

[0174] The present invention also provides other polypeptides, such as fusion proteins comprising nanobodies or fragments thereof. In addition to nearly full-length polypeptides, the present invention also includes fragments of the nanobodies of the present invention. Typically, the fragment has at least about 50 consecutive amino acids of the antibody of the present invention, preferably at least about 50 consecutive amino acids, more preferably at least about 80 consecutive amino acids, and most preferably at least about 100 consecutive amino acids.

[0175] In this invention, "a conserved variant of the antibody of the present invention" refers to a polypeptide formed by replacing up to 10, preferably up to 8, more preferably up to 5, and most preferably up to 3 amino acids with amino acids of similar or analogous properties compared to the amino acid sequence of the antibody of the present invention. These conserved variant polypeptides are preferably generated by amino acid substitutions according to Table A.

[0176] Table A

[0177] The initial residues Representative substitution Preferred replacement Ala(A) Val; Leu; Ile Val Arg(R) Lys;Gln;Asn Lys Asn(N) Gln; His; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg;Gln;Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala Leu

[0178] Chimeric antigen receptor (CAR)

[0179] The chimeric antigen receptor (CAR) of this invention comprises an extracellular domain, a transmembrane domain, and an intracellular domain. The extracellular domain includes a target-specific binding element (also referred to as an antigen-binding domain). The intracellular domain includes a co-stimulatory signaling region and a ζ-chain portion. The co-stimulatory signaling region refers to a portion of the intracellular domain containing a co-stimulatory molecule. The co-stimulatory molecule is a cell surface molecule required for an effective lymphocyte response to an antigen, rather than an antigen receptor or its ligands.

[0180] A linker may be incorporated between the extracellular and transmembrane domains of the CAR, or between the cytoplasmic and transmembrane domains of the CAR. As used herein, the term "linker" generally refers to any oligopeptide or polypeptide that functions to link the transmembrane domain to the extracellular or cytoplasmic domain of the polypeptide chain. Linkers may comprise 0-300 amino acids, preferably 2 to 100 amino acids, and most preferably 3 to 50 amino acids.

[0181] In a preferred embodiment of the present invention, the extracellular domain of the CAR provided by the present invention includes an antigen-binding domain targeting CD70. When expressed in T cells, the CAR of the present invention is capable of antigen recognition based on antigen-binding specificity. When it binds to its associated antigen, it affects tumor cells, causing them to stop growing, be induced to die, or otherwise be affected, leading to a reduction or elimination of the patient's tumor burden. The antigen-binding domain is preferably fused with an intracellular domain derived from one or more of the co-stimulatory molecule and the ζ chain. Preferably, the antigen-binding domain is fused with an intracellular domain combining a 4-1BB signaling domain and a CD3ζ signaling domain.

[0182] As used herein, "antigen-binding domain" refers to a Fab fragment, Fab' fragment, F(ab')2 fragment, scFv fragment, or VHH fragment that has antigen-binding activity. In one embodiment of the invention, the antigen-binding domain of the CAR is the VHH fragment of the anti-CD70 nanobody described in the first aspect of the invention.

[0183] In this invention, CD70 was used to immunize alpacas, and antibody screening was performed to obtain anti-CD70 single-domain antibodies. A CAR vector containing one or more single-domain antibodies that specifically recognize CD70 was constructed, and CAR-T cells were then prepared.

[0184] Polynucleotides, vectors and host cells

[0185] The present invention also provides a polynucleotide molecule encoding the aforementioned antibody or a fragment thereof or a fusion protein thereof, or a chimeric antigen receptor comprising the antibody. The polynucleotide of the present invention may be in DNA or RNA form. The DNA form includes cDNA, genomic DNA, or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be a coding strand or a non-coding strand.

[0186] The polynucleotide encoding the mature polypeptide of the present invention includes: a coding sequence that encodes only the mature polypeptide; a coding sequence of the mature polypeptide and various additional coding sequences; a coding sequence of the mature polypeptide (and optional additional coding sequences) and a non-coding sequence.

[0187] The term "polynucleotide encoding a polypeptide" can refer to a polynucleotide that includes the polypeptide, or it can also include additional coding and / or non-coding sequences.

[0188] The present invention also relates to polynucleotides that hybridize with the above-described sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% identity between the two sequences. The present invention particularly relates to polynucleotides that hybridize with the polynucleotides described herein under stringent conditions. In the present invention, “stringent conditions” means: (1) hybridization and elution at lower ionic strength and higher temperatures, such as 0.2×SSC, 0.1% SDS, 60°C; or (2) hybridization with a denaturing agent, such as 50% (v / v) formamide, 0.1% fetal bovine serum / 0.1% Ficoll, 42°C, etc.; or (3) hybridization only occurs when the identity between the two sequences is at least 90%, more preferably at least 95%. Furthermore, the polypeptide encoded by the hybridizable polynucleotide has the same biological function and activity as the mature polypeptide.

[0189] The full-length nucleotide sequence or fragments of the antibody of the present invention can generally be obtained by PCR amplification, recombinant methods, or artificial synthesis. One feasible method is to synthesize the relevant sequence artificially, especially when the fragment length is short. Typically, long fragments can be obtained by first synthesizing multiple small fragments and then ligating them. Furthermore, the coding sequence of the heavy chain and an expression tag (such as 6His) can be fused together to form a fusion protein.

[0190] 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.

[0191] Currently, the DNA sequence encoding the protein of this invention (or a fragment thereof, or a derivative thereof) can be obtained entirely through chemical synthesis. This DNA sequence can then be introduced into various existing DNA molecules (or vectors) and cells known in the art. Furthermore, mutations can be introduced into the protein sequence of this invention through chemical synthesis.

[0192] The present invention also relates to vectors comprising the aforementioned suitable DNA sequences and suitable promoters or control sequences. These vectors can be used to transform suitable host cells to enable them to express proteins.

[0193] The host cell can be a prokaryotic cell, such as a bacterial cell; a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a mammalian cell. Representative examples include: Escherichia coli, Streptomyces; bacterial cells of Salmonella typhimurium; fungal cells such as yeast; insect cells of Drosophila S2 or Sf9; and animal cells of CHO, COS7, and 293 cells.

[0194] Transformation of host cells with recombinant DNA can be performed using conventional techniques well known to those skilled in the art. When the host is a prokaryote such as *E. coli*, competent cells capable of uptake DNA can be harvested after the exponential growth phase and treated with CaCl2, the steps of which are well known in the art. Another method is to use MgCl2. If desired, transformation can also be performed using electroporation. When the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, etc.

[0195] The obtained transformants can be cultured using conventional methods to express the polypeptide 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.

[0196] The recombinant 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.

[0197] application

[0198] As described above, the nanobody of the present invention has broad biological and clinical application value, and its applications involve multiple fields such as the diagnosis and treatment of CD70-related diseases, basic medical research, and biological research. A preferred application is for the clinical diagnosis and targeted therapy of CD70.

[0199] The main advantages of this invention include:

[0200] (a) This invention provides a novel nanobody (VHH) that targets and recognizes CD70. The nanobody (VHH) of this invention has high affinity and specificity. Compared with existing CD70 antibodies (BM1, BM2), the CD70 nanobody sequence of this invention has specific targeting and recognition of CD70-positive tumor cells and stronger blocking activity against CD27-CD70 receptor ligand binding.

[0201] (b) The nanobody (VHH) of the present invention exhibits low immunogenicity, structural stability, and good drug-like properties. After reinfusion into mice, the mice's body weight remained stable without significant decrease, demonstrating its good safety and absence of adverse reactions.

[0202] (c) The present invention provides nanobodies (VHH) with high efficacy in tumor treatment. In vitro functional validation experiments and in vivo efficacy experiments show that they have significant killing effects on CD70-positive tumor cells and in vivo tumor-clearing capabilities.

[0203] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods in the following embodiments, unless otherwise specified, are generally performed under conventional conditions, such as those described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. Unless otherwise stated, percentages and parts are weight percentages and parts by weight.

[0204] Example 1: Immunization of alpacas with hCD70-i1-his protein

[0205] Alpaca immunization was conducted according to the immunization strategy in Table 1. The first immunization used Freund's complete adjuvant, the second and third immunizations used Freund's incomplete adjuvant, and the fourth and fifth immunizations used FAMA adjuvant.

[0206] Table 1. Alpaca Immune Strategy

[0207]

[0208]

[0209] Experiment Example 2: Construction and Screening of Alpaca Nanobody Library

[0210] High serum titers were obtained after immunization. Then, 100 mL of peripheral blood was collected from the jugular vein of the alpaca; PBMCs were then isolated from the peripheral blood. Alpaca RNA was then extracted from the PBMCs and reverse transcribed into cDNA.

[0211] Alpaca RNA extraction was performed using a commercial kit (brand: TaKaRa, catalog number: 740984.50), and all RNA extraction steps were performed according to the kit's instructions.

[0212] cDNA preparation was performed using PrimeScript. TM The 1st Strand cDNA Synthesis Kit (brand: Takara, catalog number: 6110A) was used to prepare the cDNA according to the kit instructions.

[0213] Example 3: Preparation of Nanobodies using a mammalian cell expression system

[0214] According to the technical route Figure 1A The clone 717 nanobody obtained was expressed in a 20 mL HEK293 cell system. After purification, the nanobody was obtained. The SDS-PAGE electrophoresis results are shown in the figure. Figure 1BInformation on the purified nanobodies is shown in Table 2.

[0215] Table 2. Nanobody Expression and Purification

[0216]

[0217] Experiment Example 4: Affinity Determination of Recombinant CD70 VHH-huFc Antibody

[0218] Antibody affinity was determined for clone 717VHH antibody. The statistical information of the control group used in this invention is shown in Table 3.

[0219] Table 3. Control Group Information

[0220] Serial Number name source Reference Number 1 4F11 US20220387572A1 BMK1 2 2A5(NO21) CN116496396A BMK2 3 CD27z Tim Sauer, et al. Blood 2021 BMK3

[0221] The specific steps for antibody affinity assay are as follows:

[0222] 1) Pre-experiment preparation: Prepare 786-O and 769-P cells in the logarithmic growth phase in advance;

[0223] 2) Cell treatment: Digest and collect cells, resuspend cells in PBS and adjust the cell count to a concentration of 2.00E+06 / mL;

[0224] 3) Cell plating: Add 100 μL of cell suspension per well to a 96-well (U-bottom) cell culture plate;

[0225] 4) Antibody preparation: The antibody was diluted in advance with PBS + 1% BSA buffer, 30 μg / mL for the first well, and then serially diluted 3-fold for a total of 12 concentration gradients;

[0226] 5) Incubation with primary antibody: Add 80 μL of antibody dilution buffer per well to the cell plate containing cell suspension. After adding, mix well by pipetting with a pipette and incubate at 4°C in the dark for 60 min.

[0227] 6) Washing: After incubation, centrifuge at 500g for 5 min and discard the supernatant. Wash the cells twice with 200 μL / well of PBS + 1% BSA;

[0228] 7) Incubation with secondary antibody: Add 100 μL / well of PE anti-IgG Fc antibody (brand: BioLegend, catalog number: 410708) dilution buffer (1:100 dilution), mix well by pipetting, and incubate at 4°C in the dark for 30 min;

[0229] 8) Washing: After incubation, centrifuge at 500g for 5 min and discard the supernatant; add 200μL / well of PBS + 1% BSA to wash the cells twice;

[0230] 9) Resuspend: Resuspend cells in 200 μL of PBS + 1% BSA in each well;

[0231] 10) Flow cytometry detection: Median fluorescence intensity (Median-PE) of the expression in the test cells, and a curve is generated based on the fluorescence intensity.

[0232] See results Figure 2A (786-O) and Figure 2B (769-P) 717VHH antibody was obtained and it bound to tumor cells 786-O and 769-P, and the affinity was significantly higher than that of the control BMK1 and BMK2.

[0233] Table 4 Affinity to 786-O

[0234] 717 BMK1 BMK2 EC50 0.04674 0.07128 0.1579

[0235] Table 5 Affinity to 769-P

[0236] 717 BMK1 BMK2 EC50 0.05389 0.08911 0.07646

[0237] Experiment 5: Construction of CD70 CAR plasmid

[0238] Design as Figure 3A The CAR structure shown was validated using the target tag HA. The obtained CD70 VHH antibody was ligated, transformed, and single clones were selected. The resulting vector is shown below. Figure 3B As shown. The amino acid sequence is shown in SEQ ID NO:1. The CAR structural element also includes a hinge region as shown in SEQ ID NO:5; a transmembrane domain as shown in SEQ ID NO:6; a co-stimulatory domain as shown in SEQ ID NO:7; and an activation domain as shown in SEQ ID NO:8.

[0239] Experiment 6: Preparation of Lentiviral Viruses and Infection of T Lymphocytes

[0240] use Lentiviral cells were packaged using the HD transfection reagent (Promega) kit. 293T cells were cultured in DMEM medium containing 10% FBS until optimal condition. The packaging plasmid and expression plasmid from the third-generation lentiviral packaging system were added to 15 mL centrifuge tubes in a specific ratio. Then, 3 volumes of [unspecified substance] were added. HD transfection reagent. Mix well and let stand at room temperature for 15 min before adding to prepared 293T cells. Collect cell supernatant after 48 h and 72 h, purify the virus, and freeze for later use.

[0241] Experiment 7: Detection of CAR-T cell infection efficiency and phenotype

[0242] T cells were isolated using immunomagnetic beads: Peripheral blood mononuclear cells (PBMCs) were resuscitated from a volunteer donor (Donor ID: P123070213C) and resuspended in 5 volumes of X-VIVO 15. The cells were then centrifuged at 300g for 10 min. The supernatant was discarded, and the cells were resuspended at a specific ratio, with an appropriate volume of CD3 magnetic beads added. After mixing, the cells were incubated at 4℃ for 30 min. After washing and resuspending the cells, they were separated using a MACS column. Positively isolated cells were identified as T cells. These were cultured in X-VIVO 15 medium containing 10% FBS. Simultaneously, TransAct activator was added for 48 h, followed by the addition of lentivirus (MOI = 3). CAR positivity was detected by flow cytometry 5 days after infection using the antibody: PE anti-HA.11Epitope Tag (Brand: BioLegend, Catalog No.: 901518).

[0243] Results: Flow cytometry results are shown below. Figure 4 The CAR-T cell positivity rate prepared from clone 717 was 68.4%. This demonstrates that lentiviruses packaged with plasmids containing the CD70CAR structure can effectively infect T cells.

[0244] Table 6. Positive rate of cloned CD70 CAR

[0245] Serial Number CAR-T CAR% 1 717 68.4％

[0246] Experiment Example 8: Repeated Stimulation Experiment of CAR-T Cells

[0247] Repeated stimulation experiments were conducted using CAR-T cells with different clones to verify their expansion capacity under repeated stimulation conditions. The specific steps of repeated stimulation are as follows:

[0248] 1) Target cell preparation: Collect target cells (786-O-Luc) in good growth condition into 15 mL centrifuge tubes, centrifuge at 300 g for 4 min, and discard the supernatant; resuspend the cells in culture medium to a density of 1.00E+06 / mL, and treat overnight with Mitomycin C (1 μg / mL). The next day, wash the cells twice with PBS buffer, centrifuge at 300 g for 4 min, and discard the supernatant; resuspend the cells in X-VIVO-15 medium containing 1% FBS, count the cells using a Count Star cell counter and check cell viability, and finally dilute the cell density to a concentration of 1.00E+06 / mL for later use.

[0249] 2) Effector cell preparation: Take the prepared CD70 CAR-T cells, centrifuge at 200g for 10min; resuspend the cells in X-VIVO-15 medium, count them with Count Star, and adjust the cell density to be greater than 2.00E+06 / mL for later use.

[0250] 3) Co-culture of effector cells and target cells: Add the treated effector cells and target cells to a 12-well plate at an effector-to-target ratio of 2:1. Add 500 μL of target cells (0.50E+06 / mL) and 1000 μL of CAR-T cells (1.00E+06 / mL, calculated based on CAR-T positive cells) to each well. After adding the samples, gently shake the 12-well plate to mix the cells evenly. Incubate the cells in a 37°C, 5% CO2 incubator.

[0251] 4) Multiple rounds of tumor cell stimulation for proliferation: Cells were co-cultured for 3 days. After complete lysis of tumor cells, CAR-T cells were harvested, centrifuged at 200g for 10 minutes, and the supernatant was discarded. After resuspending, the CAR-T cells were counted. At the same time, the above steps were repeated for 5 rounds of stimulation.

[0252] Results of the repetitive stimulus test are shown in Figure 5 Clone 717 exhibits excellent CAR-T cell targeting and proliferation capabilities, with an expansion factor exceeding 681.

[0253] 5) After five rounds of stimulation, CAR-T cells were co-incubated with 786-O cells for a killing experiment.

[0254] First, tumor cells 786-O were collected, washed once with PBS buffer, and centrifuged at 300g for 4 min. The supernatant was discarded, and the cells were resuspended in X-VIVO™ 15 containing 1% FBS, counted, and finally diluted to a concentration of 1.00E+05 / mL.

[0255] Then, CAR-T cells were collected after five rounds of repeated stimulation, washed once with PBS buffer, centrifuged at 200g for 10 min, and the supernatant was discarded. Cells were resuspended in X-VIVO 15 complete medium, counted, and diluted to concentrations of 8.00E+05 / mL, 4.00E+05 / mL, 2.00E+05 / mL, 1.00E+05 / mL, and 0.50E+05 / mL, with effector-to-target ratios of 8:1, 4:1, 2:1, 1:1, and 1:2 (calculated based on CAR-T positive cells). After cell preparation, 50 μL of target cells and 50 μL of CAR-T cells were mixed 1:1 and added to 96-well plates. After incubation at 37℃ and 5% CO2 for 24 h, 100 μL of ONE-Glo was added to each well. TM The substrate was detected using the luciferase Assay System. After mixing and standing for 5 minutes, the sample was placed on a TECAN microplate reader to detect fluorescence (wavelength 560 nm).

[0256] The lethality is calculated based on the OD value.

[0257] After five rounds of repeated stimulation, all CD70 CAR-T cells retained varying degrees of cytotoxicity, as shown in the following figures. Figure 6 At E:T ratios of 8:1, 4:1, and 2:1, the CAR-T kill rate of clone 717 was almost 100%.

[0258] Experimental Example 9: Recombinant CD70 VHH-huFc blocks the CD27 / CD70 binding reaction.

[0259] The VHH antibody of clone 717 was subjected to a CD27 / CD70 binding blocking assay to verify its functional activity in binding to the CD70 target. The specific steps are as follows:

[0260] 1) Pre-experiment preparation: Pre-resuscitate 786-O cells

[0261] 2) Cell treatment: Collect and wash cells, and adjust the cell number to a concentration of 2.00E+06 / mL in ice-cold PBS + 1% BSA buffer.

[0262] 3) Plating: Add 100 μL of cell suspension to each well of a 96-well plate. First, add the self-made protein CD27-IgG1-FC-HA (50 μg / mL) and incubate for 30 min. After incubation, wash with PBS + 1% BSA buffer.

[0263] 4) Antibody preparation: The antibody to be tested was diluted with PBS + 1% BSA buffer to 100 μg / mL, and then diluted 5-fold to 8 concentration gradients.

[0264] 5) Incubation of primary antibody: Add 100 μL of antibody dilution buffer per well, mix well, and incubate at 4°C in the dark for 60 min.

[0265] 6) Washing: Add 100 μL / well of PBS + 1% BSA buffer, centrifuge at 500g for 5 min, and discard the supernatant. Repeat once.

[0266] 7) Incubation with secondary antibody: Add 100 μL / well PE anti-HA.11Epitope Tag (secondary antibody 1:100) (brand: BioLegend, catalog number: 901518). Incubate at 4℃ in the dark for 30 min, centrifuge at 500g for 5 min, and discard the supernatant.

[0267] 8) Washing: Add 100 μL / well of PBS + 1% BSA buffer, centrifuge at 500g for 5 min, and discard the supernatant. Repeat once.

[0268] 9) Resuspend: Resuspend the cells in 100 μL PBS + 1% BSA buffer.

[0269] 10) Calculation: Detect the median fluorescence intensity (Median-PE) expressed in the test cells using FACS, construct a curve based on the fluorescence intensity, and calculate EC50.

[0270] See results Figure 7 All clones of 717 effectively blocked CD27 / CD70 binding with VHH, and their blocking ability was significantly better than that of the controls BMK1 and BMK2.

[0271] Table 7 CD70 CAR blocking ability

[0272] 717 BMK1 BMK2 EC50 0.3126 1.341 0.8704

[0273] Experiment Example 10: CAR-T cell killing experiment on 786-O cells

[0274] Simultaneously, CAR-T cells from the D9 cryopreserved 717 clone were revived for in vitro killing experiments. The specific steps are as follows:

[0275] 1) Target cell preparation: Collect tumor cells 786-O and 786-O-CD70KO (786-O cell line with CD70 gene knocked out, as a negative control), wash the cells once with PBS buffer, and centrifuge at 300g for 4 min. Discard the supernatant, resuspend the cells in X-VIVO™ 15 containing 1% FBS, count the cells, and finally dilute the cells to a concentration of 1.00E+05 / mL;

[0276] 2) Effector cell preparation: Resuscitate frozen CAR-T cells and untransduced T cells (Mock T). The volunteer donor's ID is P123070213C. Wash the cells once with PBS buffer, centrifuge at 200g for 10 min, and discard the supernatant. Resuspend the cells in X-VIVO 15 complete medium, count them, and dilute them to concentrations of 8.00E+05 / mL, 4.00E+05 / mL, 2.00E+05 / mL, 1.00E+05 / mL, and 0.50E+05 / mL, with effector-to-target ratios of 8:1, 4:1, 2:1, 1:1, and 1:2, respectively (calculated based on CAR-T positive cells).

[0277] 3) Co-culture of effector cells and target cells: After preparing the cells, mix 50 μL of target cells and 50 μL of CAR-T cells 1:1 and add them to a 96-well plate. Incubate at 37℃ and 5% CO2 for 24 h. Then, add 100 μL of ONE-Glo™ luciferase Assay System (Brand: Promega, Catalog No.: E6120) to each well to detect the substrate. Mix well and let stand for 5 min before placing the plate in a TECAN microplate reader to detect fluorescence (wavelength 560 nm).

[0278] The lethality is calculated based on the OD value.

[0279] See results Figure 8A (786-O-CD70KO) and Figure 8B (786-O). Co-incubation with 786-O tumor cells at E:T ratios of 8:1 and 4:1 showed that cloned 717CAR-T cells exhibited strong killing ability against tumor cells. Mock T served as a negative control. However, co-incubation with negative tumor cells 786-O-CD70KO did not cause any killing of these cells, demonstrating that the 717VHH antibody specifically targets and recognizes the CD70 target without any off-target reactions.

[0280] Experiment Example 11: Killing Experiment of CAR-T Cells on 786-O and A-498 Cells

[0281] To replicate and validate the functional stability of clone 717 CAR-T cells, another batch of CAR-T cells was prepared. The positive control was CD27z CAR-T (from a previously reported literature in this field, Tim Sauer, et al. Blood 2021), and the negative control was untransduced T cells (Mock T). The volunteer blood donor's donor number was XW0801200. The experimental procedures are as shown in Experiment 10.

[0282] See results Figure 9A (786-O) and 9B(A-498). At E:T = 8:1 and 4:1, CD70 CAR-T cells of clone 717 showed strong killing ability against tumor cells when co-incubated with tumor cells 786-O and A-498, and were significantly better than the control CD27z.

[0283] Experiment 12: Detection of the release of cytokines IL-2, TNF-α and IFN-γ when CAR-T cells are co-incubated with target cells 786-O and A-498.

[0284] CAR-T cells (Donor ID XW0801200) were co-incubated with tumor cells (786-O and A-498) at an effector-target ratio of 2:1. The release levels of cytokines IL-2, TNF-α, and IFN-γ were then measured. The specific steps are as follows:

[0285] CAR-T cells (717 and CD27z) and target cells (786-O and A-498) were collected, washed once with dilution buffer, centrifuged at 300g for 10 min, and the supernatant was discarded. Cells were then diluted with X-VIVO containing 10% FBS. TMCells were resuspended in 15 wells and counted. The final cell count was diluted to 1.00E+06 cells / mL for later use. CAR-T cells were counted based on the number of CAR-positive cells. Then, 50 μL of target cells and 100 μL of CAR-T cells were mixed 1:2 and added to a 96-well plate. The plates were co-cultured at 37°C and 5% CO2 for 24 h. After centrifugation at 500g for 5 min, the supernatant was collected. Cytokines were detected using the Human TH1 / TH2 Cytokine CBA Assay Kit (BD, catalog number: 551809).

[0286] See results Figure 10A (786-O) and 10B (A-498). As shown in the figure, all CAR-T cells released large amounts of cytokines IL-2, TNF-α, and IFN-γ when stimulated by target cells 786-O and A-498. More importantly, when CAR-T cells of clone 717 were co-incubated with target cells 786-O and A-498, the release of cytokines IL-2, TNF-α, and IFN-γ was significantly higher than that of the control CD27z.

[0287] Experiment Example 13: Anti-tumor experiment of CAR-T cells in mice

[0288] Anti-CD70 CAR-T cells for in vivo efficacy testing in animals were prepared using clone 717 according to the method described in Experimental Example 6 of this invention. The volunteer blood donor's number was P123070213C, and the CAR-T positivity rate was detected, as detailed below:

[0289] 1) CAR positivity rate detection

[0290] The CAR positivity rate was measured 72 hours after the preparation of CD70 CAR-T cells. Uninfected lentivirus T lymphocytes (MockT) served as a negative control. The detection method was flow cytometry, and the antibody was PE anti-HA.11Epitope Tag (brand: BioLegend, catalog number: 901518).

[0291] See results Figure 11 The CAR-T cell positivity rate of clone 717 was 68.1%, while the positivity rate of the control BMK2 was 59.7%. All CAR positivity rates exceeded 20%, meeting the requirements for in vivo efficacy experiments in mice.

[0292] 2) In vivo efficacy experiment in mice

[0293] Thirty NCG mice (6-8 week old female mice) were used in the experiment, with five mice in each group. 1.00E+07 tumor cells (786-O) were subcutaneously injected. Ten days later, when the tumor volume reached approximately 400 mmHg... 3At that time, CAR-T cells were injected intravenously, with cell numbers including low dose (3.00E+06) and high dose (8.00E+06), 200 μL / animal per group. The in vivo experimental procedure is as follows: Figure 12 The experimental groups included 717, BMK2, Mock T cells (T cells without lentivirus transduction), and a solvent control group. Tumor volume was measured and mouse survival status was recorded every 3-4 days during the experiment. All mice were euthanized after the tumors in the experimental groups had been almost completely cleared.

[0294] Results of mouse tumor volume monitoring are shown in Figure 13 Compared to the Mock T group, both low-dose and high-dose CAR-T therapy showed significant tumor-suppressing effects in mice. By day 28, the tumors were almost completely cleared. Conversely, tumors in the solvent group and the Mock T group continued to grow. Furthermore, after administration, the mice in all experimental groups maintained stable weight gain without significant weight loss, reflecting the safety and reliability of CAR-T cell immunotherapy with clone 717 and the absence of adverse reactions. (See attached figures). Figure 14 .

[0295] Table 8 CDR Sequences

[0296]

[0297] Table 9 VHH Sequences

[0298]

[0299]

[0300] Table 10 CAR Component Sequence

[0301]

[0302] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. An anti-CD70 Nanobody, characterized in that, The complementarity determining regions CDR of the VHH chain of the nanobody are as follows: CDR1 as set forth in SEQ ID NO: 2, CDR2 as set forth in SEQ ID NO: 3, and CDR3 as set forth in SEQ ID NO:

4.

2. The Nanobody of claim 1, wherein The VHH chain of the nanobody has an amino acid sequence with ≥ 85%, ≥ 90%, ≥ 95%, ≥ 96%, ≥ 97%, ≥ 98%, or ≥ 99% homology to the amino acid sequence set forth in SEQ ID NO:

1.

3. The Nanobody of claim 1, wherein The VHH chain of the nanobody has one or more amino acid sequences as set forth in SEQ ID NO:

1.

4. The nanobody of claim 1, wherein The VHH chain of the nanobody has an amino acid sequence as set forth in SEQ ID NO:

1.

5. A chimeric antigen receptor, the antigen binding domain of the chimeric antigen receptor being the VHH chain of the nanobody of claim 1.

6. The chimeric antigen receptor of claim 5, wherein, The chimeric antigen receptor has the following structure as shown in Formula I: L-V-F-H-TM-C-CD3ζ (I) In the formula, each “-” is independently a linker peptide or a peptide bond; L is nothing or a signal peptide sequence; V is an antigen binding domain; F is nothing or a marker protein H is a hinge region; TM is a transmembrane domain; C is a costimulatory signaling molecule; CD3ζ is a cytoplasmic signaling sequence derived from CD3ζ.

7. A polynucleotide comprising a nucleic acid sequence encoding a polypeptide of any one of claims 1-6. The polynucleotide encodes a protein selected from the group consisting of the anti-CD70 nanobody of claim 1, the chimeric antigen receptor of claim 5, or a combination thereof.

8. An expression vector, characterized by, The expression vector contains the polynucleotide of claim 7.

9. A host cell, characterized in that, The host cell contains the expression vector of claim 8, or the genome of which is integrated with the polynucleotide of claim 7.

10. An engineered immune cell, characterized in that, The immune cell expresses the chimeric antigen receptor of claim 5.

Images